ER exit pathways and the control of proteostasis: Crucial role of the UPR,COPII, and ER-phagy in the secretory pathway

The endoplasmic reticulum (ER) is the site of entry of all proteins that function in the secretory pathway including the extracellular environment. Because it controls the folding of newly synthesized secretory proteins, the ER is indispensable for the maintenance of proteostasis in the secretory pathway. Within the ER and, in part, in post-ER compartments, the quality control of protein folding is under the regulation of the unfolded protein response (UPR) pathways. The UPR strategy is to enhance protein folding, increase the ER degradation pathway of misfolded proteins, and allow the exit from the ER of only correctly folded proteins. The latter is controlled by the multimeric complex COPII, which also provides some of the components for ER-phagy the only route for the disposal of protein aggregates. In this overview, we wish to contribute to the introduction of new perspectives in the study of the mechanisms underlying the control of proteostasis within the secretory pathway.     

Inhibitory of EV-A71 virus-induced apoptosis by ZVAD through ROS mediated signaling pathways

Emerging evidence that Enterovirus A 71 (EV-A71) infection closely related to apoptosis. The ZVAD is a caspase inhibitor that can prevent apoptosis. The aims of this project were to evaluate the mechanism of the ZVAD inhibited EV-A71 virus and to provide experiment basis for finding new antiviral drugs. In this study, after treated with ZVAD in EV-A71 infected Vero cells, the viral replication was reduced, and the cell viability was higher than EV-A71 group. Additionally, ZVAD decreased the cell apoptosis and the level of inflammatory cytokines induced by EV-A71 in the infected Vero cells. ZVAD inhibited cell apoptosis by regulating ROS mediated signaling pathway and inflammation cytokines to achieve antiviral.     

Nucleotide and protein distribution in BrdU-labelled polytene chromosomes revealed by ion probe mass spectrometry

Detailed chemical maps of BrdU-labelled polytene chromosomes of Drosophila melanogaster, obtained by imaging secondary ion mass spectrometry, reveal separately the distribution of DNA and proteins in the chromosomes. The thymidine-analogue BrdU within the chromosomal DNA is localized by detecting the Br^? secondary ion signal, while both nucleic acid and protein content are mapped through the abundantly emitted CN^? signal. This novel approach supercedes, and helps explain the origin of, the banding patterns that are observed by conventional staining techniques. The high spatial resolution and chemical and isotopic sensitivity of this technique should enhance the localization of specific genes by in situ hybridization in mitotic chromosomes.     

On the size of small protein particles determined by electron microscopy of unidirectionally shadowed freeze-etched preparations

The influence exerted by the thickness of the deposited metal layer and the ionic strength of the solution on the apparent size of particles of bovine serum albumin in unidirectionally shadowed freeze-etch preparations of spray-frozen specimens was investigated. It appeared that the size increase due to shadowing is nearly twice the thickness of the deposited metal layer. Apparent particle size was shown to increase linearly with the inverse square root of the ionic strength of the solution. At ionic strength 0·001 the particles appeared about 30% larger than at infinite ionic strength.     

Imaging the dynamics of intracellular protein translocation by photoconversion of phamret-cybr/ROM

Cybr/Reduced On-random Motile (ROM) is a scaffold protein, containing a postsynaptic density protein-95/discs-large/ZO-1 (PDZ) domain, a LEU region and a PDZ domain binding region at the C-terminus. In the immune system, Cybr/ROM was found to localize in vesicles and at the plasma membrane, through interactions with cytohesin-1. In this investigation, we reported Cybr/ROM as occurring in vesicles, the cytoplasm and at membrane ruffles of H1299 lung cancer cells. Its localization at the ruffles was dependent on intact actin structures as indicated by latrunculin A treatment, which abrogated ruffle formation and staining of Cybr/ROM at the cells' periphery. Transfection of truncation mutants consisting of either the PDZ or LEU domain showed that the LEU domain of ROM was localized to membrane ruffles, vesicles and the cytoplasm, whereas, the PDZ domain localized to the membrane ruffles and cytoplasm only. There was therefore, domain/molecular segregation of Cybr/ROM in different cellular compartments. Cybr/ROM was subcloned into a plasmid carrying the photoactivation-mediated resonance energy transfer (Phamret) protein. The photoconversion experiments demonstrated the diffusion of ROM from the cytoplasm to the membrane ruffling sites and conversely from membrane ruffles to the cytoplasm. Large variances in the transport velocity of Cybr/ROM in the cytoplasm suggested that its movements were facilitated by other mechanisms in addition to diffusion.     

Investigation of intact protein complexes by mass spectrometry

Mass spectrometry has grown in recent years to a well-accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non-covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non-covalent protein-protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights.     

Expression of the male reproduction‐related gene in spermatic ducts of the blue swimming crab,Portunus pelagicus,and transfer of modified protein to the sperm acrosome

Expression of a sex‐specific gene in Macrobrachium rosenbergii (Mr‐Mrr), encoding a male reproduction‐related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp‐Mrr) and Scylla serrata (Ss‐Mrr). We then investigated expression of Pp‐Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp‐Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp‐Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti‐Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp‐Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs. Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

凯氏定氮法测定乳粉中蛋白质的不确定度分析

测量不确定度定义为表征合理地赋予被测量之值的分散性[1]。一个完整的测量结果,除了应该给出被测量的最佳估计值之外,还应同时给出测量结果的不确定度。本文依据凯氏定氮法,对奶粉中的蛋白质含量的不确定度各分量进行计算合成,找出影响不确定度的因素,进行评定。     

Large fluctuations in the disassembly rate of microtubules revealed by atomic force microscopy

Atomic force microscopy (AFM) in situ has been used to observe the cold disassembly dynamics of microtubules at a previously unrealised spatial resolution. Microtubules either electrostatically or covalently bound to aminosilane surfaces disassembled at room temperature under buffer solutions with no free tubulin present. This process was followed by taking sequential tapping-mode AFM images and measuring the change in the microtubule end position as a function of time, with an spatial accuracy down to +/-20nm and a temporal accuracy of +/-1s. As well as giving average disassembly rates on the order of 1-10 tubulin monomers per second, large fluctuations in the disassembly rate were revealed, indicating that the process is far from smooth and linear under these experimental conditions. The surface bound rates measured here are comparable to the rates for GMPCPP-tubulin microtubules free in solution, suggesting that inhibition of tubulin curvature through steric hindrance controls the average, relatively low disassembly rate. The large fluctuations in this rate are thought to be due to multiple pathways in the kinetics of disassembly with differing rate constants and/or stalling due to defects in the microtubule lattice. Microtubules that were covalently bound to the surface left behind the protofilaments covalently cross-linked to the aminosilane via glutaraldehyde during the disassembly process. Further work is needed to quantitatively assess the effects of surface binding on protofibril disassembly rates, reveal any differences in disassembly rates between the plus and minus ends and to enable assembly as well as disassembly to be imaged in the microscope fluid cell in real-time.     

粗蛋白质测定仪测奶制品蛋白质比较分析

目的：预期掌握目前部分大理产奶制品中蛋白质含量以及非蛋白质氮的含量情况,并与外包装指示含量进行比较与奶制品是否一致。方法：粗蛋白测定法,根据微量凯氏定氮法的原理,测定奶制品中总凝固沉淀物重量,及奶制品中非沉淀物经三氯乙酸处理后取沉淀再采用沉淀重量法测定蛋白质含量并与国家规定标准（简称＂国标＂）进行比较。结果：将调味牛奶、酸牛奶、纯牛奶、奶粉中总含氮物质与真蛋白质的含量进行比较,差异没有统计学意义,蛋白质含量与外包装上标注的蛋白质含量接近一致,蛋白质含量均达到国标以上。结论：目前部分大理产调味牛奶、酸牛奶、纯牛奶、奶粉中不含有非食用氮物质,蛋白质含量与外包装上标注的蛋白质含量接近一致,所测量的部分大理产奶制品的蛋白质含量符合国家奶制品蛋白质含量标准。     

Improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime imaging microscopy data

The deconvolution of fluorescence lifetime imaging microscopy (FLIM) data that were processed with global analysis techniques is described. Global analysis of FLIM data enables the determination of relative numbers of molecules in different protein reaction states on a pixel-by-pixel basis in cells. The three-dimensional fluorescence distributions of each protein state can then be calculated and deconvolved. High-resolution maps of the relative concentrations of each state are then obtained from the deconvolved images. We applied these techniques to quantitatively image the phosphorylation state of ErbB1 receptors tagged with green fluorescent protein in MCF7 cells.     

MRT letter: Nanoscopy of protein colocalization in living cells by STED and GSDIM

We report the use of superresolution fluorescence microscopy for studying the nanoscale distribution of protein colocalization in living mammalian cells. Nanoscale imaging is attained both by a targeted and a stochastic fluorescence on-off switching superresolution method, namely by stimulated emission depletion (STED) and ground state depletion microscopy followed by individual molecular return (GSDIM), respectively. Analysis of protein colocalization is performed by bimolecular fluorescence complementation (BiFC). Specifically, a nonfluorescent fragment of the yellow fluorescent protein Citrine is fused to tubulin while a counterpart nonfluorescent fragment is fused to the microtubulin-associated protein MAP2 such that fluorescence is reconstituted on contact of the fragment-carrying proteins. Images with resolution down to 65 nm prove a powerful new way for studying protein colocalization in living cells at the nanoscale.     

Hydrogen exchange mass spectrometry for the analysis of protein dynamics

Hydrogen exchange coupled to mass spectrometry (MS) has become a valuable analytical tool for the study of protein dynamics. By combining information about protein dynamics with more classical functional data, a more thorough understanding of protein function can be obtained. In many cases, protein dynamics are directly related to specific protein functions such as conformational changes during enzyme activation or protein movements during binding. The method is made possible because labile backbone hydrogens in a protein will exchange with deuterium atoms when the protein is placed in a D2O solution. The subsequent increase in protein mass over time is measured with high-resolution MS. The location of the deuterium incorporation is determined by monitoring deuterium incorporation in peptic fragments that are produced after the labeling reaction. In this review, we will summarize the general principles of the method, discuss the latest variations on the experimental protocol that probe different types of protein movements, and review other recent work and improvements in the field.     

Assessment of protein expression by means of 2-D gel electrophoresis with and without mass spectrometry

Careful examination of current literature, particularly over the last 5 years, reveals a wide range of approaches for the relative quantification of protein expression in cells, tissues, and body fluids. In view of such an observation, it is reasonable to ask whether researchers need new methods, or whether it is more productive to optimize and tune already existing ones. It is generally agreed that none of the existing methodologies on its own can give a full account of protein expression in a complex medium; this limitation, however, has not prevented the use of existing methods to provide valuable information on a wide range of proteins, where their expression has been correlated to certain pathologies and/or to pharmacological, genetic, or environmental factors. In the present work, an attempt is made to review the application of one of these methodologies, namely two-dimensional polyacrylamide gel electrophoresis on its own or in conjunction with mass spectrometry, to assess protein expression, particularly when such expression can be correlated to certain pathologies.     

Detection of Brucella abortus by a platform functionalized with protein A and specific antibodies IgG

Oriented immobilization of antibodies on a sensor surface is critical for enhancing both the antigen‐binding capacity and the sensitivity of immunosensors. In this study, we describe a strategy to adsorb immunoglobulin G (IgG) anti‐Brucella antibodies onto a silicon surface, oriented by protein A obtained from Staphylococcus aureus (SpA). X‐ray photoelectron spectroscopy and atomic force microscopy were used to characterize topographically, morphologically, and chemical changes of the sensor functionalization. The activity of the biosensor was assessed by confocal microscopy, scanning electronic microscopy, and bacteria capture assays (BCA). According to the BCA, the efficiency of Brucella abortus detection with the SpA‐IgG anti Brucella biosensor was three‐fold higher than that of the random orientated IgG anti Brucella biosensor. The limit of detection was 1 × 10⁶ CFU/ml. These data show that the orientation of antibodies immobilization is crucial to developing immunosensors for bacterial antigen detection as Brucella spp and improve its sensibility level. Functionalization with protein A increases Brucella detection by an antibody‐coated surface. Functionalized silicon surface for Brucella detection was characterized by atomic force microscopy, X‐ray photoelectron spectroscopy and confocal microscopy.     

近红外光谱技术检测牛奶中脂肪及蛋白质含量校正模型的建立

为实现牛奶成分的快速检测,研究了近红外光谱法在牛奶主要成分分析中的应用,重点对比了不同近红外区域的检测结果。研究中利用偏最小二乘法(PLS)建立校正模型,探讨了不同光谱区域和数据预处理对模型准确性的影响。模型结果表明在长波段(1700nm~2500nm)检测牛奶中脂肪及蛋白质含量的准确性最高。     

Atypical cells in the normal guinea pig organ of Corti as revealed by micromanipulation in SEM.

Inner ear tissue of the normal guinea pig was conductively stained (OTOTO-method) for SEM investigations. The Hensen's cells of the organ of Corti were removed using a micromanipulator inside the SEM. By this method atypical bodies of sensory and supporting cells were revealed in the apical turns of the cochlea. Atypical sensory cells showed great variations in size and shape. Several had no contact to Deiter's cells and no or only one nerve supply at their basal end. Atypical Deiter's cells showed alterations in shape and in the form of their phalangeal processes. Additionally altered parts of the organ of Corti were isolated by micromanipulation and embedded for correlative TEM-investigations.