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Sample records for subcellular location partial

  1. Multitask learning for protein subcellular location prediction.

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    Xu, Qian; Pan, Sinno Jialin; Xue, Hannah Hong; Yang, Qiang

    2011-01-01

    Protein subcellular localization is concerned with predicting the location of a protein within a cell using computational methods. The location information can indicate key functionalities of proteins. Thus, accurate prediction of subcellular localizations of proteins can help the prediction of protein functions and genome annotations, as well as the identification of drug targets. Machine learning methods such as Support Vector Machines (SVMs) have been used in the past for the problem of protein subcellular localization, but have been shown to suffer from a lack of annotated training data in each species under study. To overcome this data sparsity problem, we observe that because some of the organisms may be related to each other, there may be some commonalities across different organisms that can be discovered and used to help boost the data in each localization task. In this paper, we formulate protein subcellular localization problem as one of multitask learning across different organisms. We adapt and compare two specializations of the multitask learning algorithms on 20 different organisms. Our experimental results show that multitask learning performs much better than the traditional single-task methods. Among the different multitask learning methods, we found that the multitask kernels and supertype kernels under multitask learning that share parameters perform slightly better than multitask learning by sharing latent features. The most significant improvement in terms of localization accuracy is about 25 percent. We find that if the organisms are very different or are remotely related from a biological point of view, then jointly training the multiple models cannot lead to significant improvement. However, if they are closely related biologically, the multitask learning can do much better than individual learning.

  2. Objective Clustering of Proteins Based on Subcellular Location Patterns

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    Xiang Chen

    2005-01-01

    Full Text Available The goal of proteomics is the complete characterization of all proteins. Efforts to characterize subcellular location have been limited to assigning proteins to general categories of organelles. We have previously designed numerical features to describe location patterns in microscope images and developed automated classifiers that distinguish major subcellular patterns with high accuracy (including patterns not distinguishable by visual examination. The results suggest the feasibility of automatically determining which proteins share a single location pattern in a given cell type. We describe an automated method that selects the best feature set to describe images for a given collection of proteins and constructs an effective partitioning of the proteins by location. An example for a limited protein set is presented. As additional data become available, this approach can produce for the first time an objective systematics for protein location and provide an important starting point for discovering sequence motifs that determine localization.

  3. Predicting subcellular location of proteins using integrated-algorithm method.

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    Cai, Yu-Dong; Lu, Lin; Chen, Lei; He, Jian-Feng

    2010-08-01

    Protein's subcellular location, which indicates where a protein resides in a cell, is an important characteristic of protein. Correctly assigning proteins to their subcellular locations would be of great help to the prediction of proteins' function, genome annotation, and drug design. Yet, in spite of great technical advance in the past decades, it is still time-consuming and laborious to experimentally determine protein subcellular locations on a high throughput scale. Hence, four integrated-algorithm methods were developed to fulfill such high throughput prediction in this article. Two data sets taken from the literature (Chou and Elrod, Protein Eng 12:107-118, 1999) were used as training set and test set, which consisted of 2,391 and 2,598 proteins, respectively. Amino acid composition was applied to represent the protein sequences. The jackknife cross-validation was used to test the training set. The final best integrated-algorithm predictor was constructed by integrating 10 algorithms in Weka (a software tool for tackling data mining tasks, http://www.cs.waikato.ac.nz/ml/weka/ ) based on an mRMR (Minimum Redundancy Maximum Relevance, http://research.janelia.org/peng/proj/mRMR/ ) method. It can achieve correct rate of 77.83 and 80.56% for the training set and test set, respectively, which is better than all of the 60 algorithms collected in Weka. This predicting software is available upon request.

  4. LocateP : genome-scale subcellular-location predictor for bacterial proteins

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    Zhou, M.; Boekhorst, J.; Francke, C.; Siezen, R.J.

    2008-01-01

    BACKGROUND: In the past decades, various protein subcellular-location (SCL) predictors have been developed. Most of these predictors, like TMHMM 2.0, SignalP 3.0, PrediSi and Phobius, aim at the identification of one or a few SCLs, whereas others such as CELLO and Psortb.v.2.0 aim at a broader

  5. Mutational analyses of the signals involved in the subcellular location of DSCR1

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    Henrique-Silva Flávio

    2002-09-01

    Full Text Available Abstract Background Down syndrome is the most frequent genetic disorder in humans. Rare cases involving partial trisomy of chromosome 21 allowed a small chromosomal region common to all carriers, called Down Syndrome Critical Region (DSCR, to be determined. The DSCR1 gene was identified in this region and is expressed preferentially in the brain, heart and skeletal muscle. Recent studies have shown that DSCR1 belongs to a family of proteins that binds and inhibits calcineurin, a serine-threonine phosphatase. The work reported on herein consisted of a study of the subcellular location of DSCR1 and DSCR1-mutated forms by fusion with a green fluorescent protein, using various cell lines, including human. Results The protein's location was preferentially nuclear, independently of the isoform, cell line and insertion in the GFP's N- or C-terminal. A segment in the C-terminal, which is important in the location of the protein, was identified by deletion. On the other hand, site-directed mutational analyses have indicated the involvement of some serine and threonine residues in this event. Conclusion In this paper, we discuss the identification of amino acids which can be important for subcellular location of DSCR1. The involvement of residues that are prone to phosphorylation suggests that the location and function of DSCR1 may be regulated by kinases and/or phosphatases.

  6. Predicting protein subcellular location using digital signal processing.

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    Pan, Yu-Xi; Li, Da-Wei; Duan, Yun; Zhang, Zhi-Zhou; Xu, Ming-Qing; Feng, Guo-Yin; He, Lin

    2005-02-01

    The biological functions of a protein are closely related to its attributes in a cell. With the rapid accumulation of newly found protein sequence data in databanks, it is highly desirable to develop an automated method for predicting the subcellular location of proteins. The establishment of such a predictor will expedite the functional determination of newly found proteins and the process of prioritizing genes and proteins identified by genomic efforts as potential molecular targets for drug design. The traditional algorithms for predicting these attributes were based solely on amino acid composition in which no sequence order effect was taken into account. To improve the prediction quality, it is necessary to incorporate such an effect. However, the number of possible patterns in protein sequences is extremely large, posing a formidable difficulty for realizing this goal. To deal with such difficulty, a well-developed tool in digital signal processing named digital Fourier transform (DFT) [1] was introduced. After being translated to a digital signal according to the hydrophobicity of each amino acid, a protein was analyzed by DFT within the frequency domain. A set of frequency spectrum parameters, thus obtained, were regarded as the factors to represent the sequence order effect. A significant improvement in prediction quality was observed by incorporating the frequency spectrum parameters with the conventional amino acid composition. One of the crucial merits of this approach is that many existing tools in mathematics and engineering can be easily applied in the predicting process. It is anticipated that digital signal processing may serve as a useful vehicle for many other protein science areas.

  7. Robust classification of subcellular location patterns in high resolution 3D fluorescence microscope images.

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    Chen, Xiang; Murphy, Robert

    2004-01-01

    Knowledge of a protein's subcellular location is essential to a complete understanding of its functions. Automated interpretation methods for protein location patterns are needed for proteomics projects, and we have previously described systems for classifying the major subcellular patterns in cultured mammalian cells. We describe here the calculation of improved 3D Haralick texture features, which yielded a near-perfect classification accuracy when combined with 3D morphological and edge features. In particular, a set of 7 features achieved 98% overall accuracy for classifying 10 major subcellular location patterns in HeLa cells.

  8. SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations.

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    Hooper, Cornelia M; Castleden, Ian R; Tanz, Sandra K; Aryamanesh, Nader; Millar, A Harvey

    2017-01-04

    The SUBcellular location database for Arabidopsis proteins (SUBA4, http://suba.live) is a comprehensive collection of manually curated published data sets of large-scale subcellular proteomics, fluorescent protein visualization, protein-protein interaction (PPI) as well as subcellular targeting calls from 22 prediction programs. SUBA4 contains an additional 35 568 localizations totalling more than 60 000 experimental protein location claims as well as 37 new suborganellar localization categories. The experimental PPI data has been expanded to 26 327 PPI pairs including 856 PPI localizations from experimental fluorescent visualizations. The new SUBA4 user interface enables users to choose quickly from the filter categories: 'subcellular location', 'protein properties', 'protein-protein interaction' and 'affiliations' to build complex queries. This allows substantial expansion of search parameters into 80 annotation types comprising 1 150 204 new annotations to study metadata associated with subcellular localization. The 'BLAST' tab contains a sequence alignment tool to enable a sequence fragment from any species to find the closest match in Arabidopsis and retrieve data on subcellular location. Using the location consensus SUBAcon, the SUBA4 toolbox delivers three novel data services allowing interactive analysis of user data to provide relative compartmental protein abundances and proximity relationship analysis of PPI and coexpression partners from a submitted list of Arabidopsis gene identifiers. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. LocateP: Genome-scale subcellular-location predictor for bacterial proteins

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    Zhou Miaomiao

    2008-03-01

    Full Text Available Abstract Background In the past decades, various protein subcellular-location (SCL predictors have been developed. Most of these predictors, like TMHMM 2.0, SignalP 3.0, PrediSi and Phobius, aim at the identification of one or a few SCLs, whereas others such as CELLO and Psortb.v.2.0 aim at a broader classification. Although these tools and pipelines can achieve a high precision in the accurate prediction of signal peptides and transmembrane helices, they have a much lower accuracy when other sequence characteristics are concerned. For instance, it proved notoriously difficult to identify the fate of proteins carrying a putative type I signal peptidase (SPIase cleavage site, as many of those proteins are retained in the cell membrane as N-terminally anchored membrane proteins. Moreover, most of the SCL classifiers are based on the classification of the Swiss-Prot database and consequently inherited the inconsistency of that SCL classification. As accurate and detailed SCL prediction on a genome scale is highly desired by experimental researchers, we decided to construct a new SCL prediction pipeline: LocateP. Results LocateP combines many of the existing high-precision SCL identifiers with our own newly developed identifiers for specific SCLs. The LocateP pipeline was designed such that it mimics protein targeting and secretion processes. It distinguishes 7 different SCLs within Gram-positive bacteria: intracellular, multi-transmembrane, N-terminally membrane anchored, C-terminally membrane anchored, lipid-anchored, LPxTG-type cell-wall anchored, and secreted/released proteins. Moreover, it distinguishes pathways for Sec- or Tat-dependent secretion and alternative secretion of bacteriocin-like proteins. The pipeline was tested on data sets extracted from literature, including experimental proteomics studies. The tests showed that LocateP performs as well as, or even slightly better than other SCL predictors for some locations and outperforms

  10. Use of correspondence discriminant analysis to predict the subcellular location of bacterial proteins.

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    Perrière, Guy; Thioulouse, Jean

    2003-02-01

    Correspondence discriminant analysis (CDA) is a multivariate statistical method derived from discriminant analysis which can be used on contingency tables. We have used CDA to separate Gram negative bacteria proteins according to their subcellular location. The high resolution of the discrimination obtained makes this method a good tool to predict subcellular location when this information is not known. The main advantage of this technique is its simplicity. Indeed, by computing two linear formulae on amino acid composition, it is possible to classify a protein into one of the three classes of subcellular location we have defined. The CDA itself can be computed with the ADE-4 software package that can be downloaded, as well as the data set used in this study, from the Pôle Bio-Informatique Lyonnais (PBIL) server at http://pbil.univ-lyon1.fr.

  11. Quantification of subcellular glycogen in resting human muscle: granule size, number, and location.

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    Marchand, I; Chorneyko, K; Tarnopolsky, M; Hamilton, S; Shearer, J; Potvin, J; Graham, T E

    2002-11-01

    A few qualitative investigations suggested that location of muscle glycogen (G) granules in specific sites may be associated with distinct metabolic roles. Similarly, it has been suggested that the acid-soluble and -insoluble G fractions (macro- and proglycogen, respectively) are different metabolic pools and also could exist as separate entities. We employed a transmission electron microscopic technique to quantify subcellular G particle size, number, and location in human vastus lateralis biopsies of 11 resting men. The intra- and interobserver variability for the various measures was generally etam and followed a continuous, normal distribution. This implies that proglycogen is not a distinct entity, but rather that pro- and macroglycogen are divisions of smaller and larger molecules. These results demonstrate a compartmentalized pattern of subcellular G deposition in human skeletal muscle for both the size and density of granules.

  12. Subcellular location of the enzymes of purine breakdown in the yeast Candida famata grown on uric acid

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    Large, Peter J.; Waterham, Hans R.; Veenhuis, Marten

    1990-01-01

    The subcellular location of the enzymes of purine breakdown in the yeast Candida famata, which grows on uric acid as sole carbon and nitrogen source, has been examined by subcellular fractionation methods. Uricase was confirmed as being peroxisomal, but the other three enzymes, allantoinase,

  13. Automated interpretation of subcellular patterns in fluorescence microscope images for location proteomics.

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    Chen, Xiang; Velliste, Meel; Murphy, Robert F

    2006-07-01

    Proteomics, the large scale identification and characterization of many or all proteins expressed in a given cell type, has become a major area of biological research. In addition to information on protein sequence, structure and expression levels, knowledge of a protein's subcellular location is essential to a complete understanding of its functions. Currently, subcellular location patterns are routinely determined by visual inspection of fluorescence microscope images. We review here research aimed at creating systems for automated, systematic determination of location. These employ numerical feature extraction from images, feature reduction to identify the most useful features, and various supervised learning (classification) and unsupervised learning (clustering) methods. These methods have been shown to perform significantly better than human interpretation of the same images. When coupled with technologies for tagging large numbers of proteins and high-throughput microscope systems, the computational methods reviewed here enable the new subfield of location proteomics. This subfield will make critical contributions in two related areas. First, it will provide structured, high-resolution information on location to enable Systems Biology efforts to simulate cell behavior from the gene level on up. Second, it will provide tools for Cytomics projects aimed at characterizing the behaviors of all cell types before, during, and after the onset of various diseases. Copyright 2006 International Society for Analytical Cytology.

  14. A novel approach for protein subcellular location prediction using amino acid exposure

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    2013-01-01

    Background Proteins perform their functions in associated cellular locations. Therefore, the study of protein function can be facilitated by predictions of protein location. Protein location can be predicted either from the sequence of a protein alone by identification of targeting peptide sequences and motifs, or by homology to proteins of known location. A third approach, which is complementary, exploits the differences in amino acid composition of proteins associated to different cellular locations, and can be useful if motif and homology information are missing. Here we expand this approach taking into account amino acid composition at different levels of amino acid exposure. Results Our method has two stages. For stage one, we trained multiple Support Vector Machines (SVMs) to score eukaryotic protein sequences for membership to each of three categories: nuclear, cytoplasmic and extracellular, plus extra category nucleocytoplasmic, accounting for the fact that a large number of proteins shuttles between those two locations. In stage two we use an artificial neural network (ANN) to propose a category from the scores given to the four locations in stage one. The method reaches an accuracy of 68% when using as input 3D-derived values of amino acid exposure. Calibration of the method using predicted values of amino acid exposure allows classifying proteins without 3D-information with an accuracy of 62% and discerning proteins in different locations even if they shared high levels of identity. Conclusions In this study we explored the relationship between residue exposure and protein subcellular location. We developed a new algorithm for subcellular location prediction that uses residue exposure signatures. Our algorithm uses a novel approach to address the multiclass classification problem. The algorithm is implemented as web server 'NYCE’ and can be accessed at http://cbdm.mdc-berlin.de/~amer/nyce. PMID:24283794

  15. Prediction of subcellular location apoptosis proteins with ensemble classifier and feature selection.

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    Gu, Quan; Ding, Yong-Sheng; Jiang, Xiao-Ying; Zhang, Tong-Liang

    2010-04-01

    Apoptosis proteins have a central role in the development and the homeostasis of an organism. These proteins are very important for understanding the mechanism of programmed cell death. The function of an apoptosis protein is closely related to its subcellular location. It is crucial to develop powerful tools to predict apoptosis protein locations for rapidly increasing gap between the number of known structural proteins and the number of known sequences in protein databank. In this study, amino acids pair compositions with different spaces are used to construct feature sets for representing sample of protein feature selection approach based on binary particle swarm optimization, which is applied to extract effective feature. Ensemble classifier is used as prediction engine, of which the basic classifier is the fuzzy K-nearest neighbor. Each basic classifier is trained with different feature sets. Two datasets often used in prior works are selected to validate the performance of proposed approach. The results obtained by jackknife test are quite encouraging, indicating that the proposed method might become a potentially useful tool for subcellular location of apoptosis protein, or at least can play a complimentary role to the existing methods in the relevant areas. The supplement information and software written in Matlab are available by contacting the corresponding author.

  16. Detrecting and Locating Partial Discharges in Transformers

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    Shourbaji, A.; Richards, R.; Kisner, R. A.; Hardy, J.

    2005-02-04

    A collaborative research between the Oak Ridge National Laboratory (ORNL), the American Electric Power (AEP), the Tennessee Valley Authority (TVA), and the State of Ohio Energy Office (OEO) has been formed to conduct a feasibility study to detect and locate partial discharges (PDs) inside large transformers. The success of early detection of the PDs is necessary to avoid costly catastrophic failures that can occur if the process of PD is ignored. The detection method under this research is based on an innovative technology developed by ORNL researchers using optical methods to sense the acoustical energy produced by the PDs. ORNL researchers conducted experimental studies to detect PD using an optical fiber as an acoustic sensor capable of detecting acoustical disturbances at any point along its length. This technical approach also has the potential to locate the point at which the PD was sensed within the transformer. Several optical approaches were experimentally investigated, including interferometric detection of acoustical disturbances along the sensing fiber, light detection and ranging (LIDAR) techniques using frequency modulation continuous wave (FMCW), frequency modulated (FM) laser with a multimode fiber, FM laser with a single mode fiber, and amplitude modulated (AM) laser with a multimode fiber. The implementation of the optical fiber-based acoustic measurement technique would include installing a fiber inside a transformer allowing real-time detection of PDs and determining their locations. The fibers are nonconductive and very small (core plus cladding are diameters of 125 μm for single-mode fibers and 230 μm for multimode fibers). The research identified the capabilities and limitations of using optical technology to detect and locate sources of acoustical disturbances such as in PDs in large transformers. Amplitude modulation techniques showed the most promising results and deserve further research to better quantify the technique’s sensitivity

  17. Subcellular location of PKA controls striatal plasticity: stochastic simulations in spiny dendrites.

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    Rodrigo F Oliveira

    2012-02-01

    Full Text Available Dopamine release in the striatum has been implicated in various forms of reward dependent learning. Dopamine leads to production of cAMP and activation of protein kinase A (PKA, which are involved in striatal synaptic plasticity and learning. PKA and its protein targets are not diffusely located throughout the neuron, but are confined to various subcellular compartments by anchoring molecules such as A-Kinase Anchoring Proteins (AKAPs. Experiments have shown that blocking the interaction of PKA with AKAPs disrupts its subcellular location and prevents LTP in the hippocampus and striatum; however, these experiments have not revealed whether the critical function of anchoring is to locate PKA near the cAMP that activates it or near its targets, such as AMPA receptors located in the post-synaptic density. We have developed a large scale stochastic reaction-diffusion model of signaling pathways in a medium spiny projection neuron dendrite with spines, based on published biochemical measurements, to investigate this question and to evaluate whether dopamine signaling exhibits spatial specificity post-synaptically. The model was stimulated with dopamine pulses mimicking those recorded in response to reward. Simulations show that PKA colocalization with adenylate cyclase, either in the spine head or in the dendrite, leads to greater phosphorylation of DARPP-32 Thr34 and AMPA receptor GluA1 Ser845 than when PKA is anchored away from adenylate cyclase. Simulations further demonstrate that though cAMP exhibits a strong spatial gradient, diffusible DARPP-32 facilitates the spread of PKA activity, suggesting that additional inactivation mechanisms are required to produce spatial specificity of PKA activity.

  18. Human cell structure-driven model construction for predicting protein subcellular location from biological images.

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    Shao, Wei; Liu, Mingxia; Zhang, Daoqiang

    2016-01-01

    The systematic study of subcellular location pattern is very important for fully characterizing the human proteome. Nowadays, with the great advances in automated microscopic imaging, accurate bioimage-based classification methods to predict protein subcellular locations are highly desired. All existing models were constructed on the independent parallel hypothesis, where the cellular component classes are positioned independently in a multi-class classification engine. The important structural information of cellular compartments is missed. To deal with this problem for developing more accurate models, we proposed a novel cell structure-driven classifier construction approach (SC-PSorter) by employing the prior biological structural information in the learning model. Specifically, the structural relationship among the cellular components is reflected by a new codeword matrix under the error correcting output coding framework. Then, we construct multiple SC-PSorter-based classifiers corresponding to the columns of the error correcting output coding codeword matrix using a multi-kernel support vector machine classification approach. Finally, we perform the classifier ensemble by combining those multiple SC-PSorter-based classifiers via majority voting. We evaluate our method on a collection of 1636 immunohistochemistry images from the Human Protein Atlas database. The experimental results show that our method achieves an overall accuracy of 89.0%, which is 6.4% higher than the state-of-the-art method. The dataset and code can be downloaded from https://github.com/shaoweinuaa/. dqzhang@nuaa.edu.cn Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Protein subcellular location pattern classification in cellular images using latent discriminative models.

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    Li, Jieyue; Xiong, Liang; Schneider, Jeff; Murphy, Robert F

    2012-06-15

    Knowledge of the subcellular location of a protein is crucial for understanding its functions. The subcellular pattern of a protein is typically represented as the set of cellular components in which it is located, and an important task is to determine this set from microscope images. In this article, we address this classification problem using confocal immunofluorescence images from the Human Protein Atlas (HPA) project. The HPA contains images of cells stained for many proteins; each is also stained for three reference components, but there are many other components that are invisible. Given one such cell, the task is to classify the pattern type of the stained protein. We first randomly select local image regions within the cells, and then extract various carefully designed features from these regions. This region-based approach enables us to explicitly study the relationship between proteins and different cell components, as well as the interactions between these components. To achieve these two goals, we propose two discriminative models that extend logistic regression with structured latent variables. The first model allows the same protein pattern class to be expressed differently according to the underlying components in different regions. The second model further captures the spatial dependencies between the components within the same cell so that we can better infer these components. To learn these models, we propose a fast approximate algorithm for inference, and then use gradient-based methods to maximize the data likelihood. In the experiments, we show that the proposed models help improve the classification accuracies on synthetic data and real cellular images. The best overall accuracy we report in this article for classifying 942 proteins into 13 classes of patterns is about 84.6%, which to our knowledge is the best so far. In addition, the dependencies learned are consistent with prior knowledge of cell organization. http://murphylab.web.cmu.edu/software/.

  20. Synthesis and subcellular location of peroxisomal membrane proteins in a peroxisome-deficient mutant of the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Sulter, G.J.; Vrieling, E.G.; Harder, W.; Veenhuis, M.

    We have studied the synthesis and subcellular location of peroxisomal membrane proteins (PMPs) in cells of a peroxisome-deficient (per) mutant of the methylotrophic yeast Hansenula polymorpha. Western blot analysis of methanol-induced cells of the per mutant, which had been growing in a continuous

  1. Using the concept of Chou's pseudo amino acid composition to predict apoptosis proteins subcellular location: an approach by approximate entropy.

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    Jiang, Xiaoying; Wei, Rong; Zhang, Tongliang; Gu, Quan

    2008-01-01

    The function of protein is closely correlated with it subcellular location. Prediction of subcellular location of apoptosis proteins is an important research area in post-genetic era because the knowledge of apoptosis proteins is useful to understand the mechanism of programmed cell death. Compared with the conventional amino acid composition (AAC), the Pseudo Amino Acid composition (PseAA) as originally introduced by Chou can incorporate much more information of a protein sequence so as to remarkably enhance the power of using a discrete model to predict various attributes of a protein. In this study, a novel approach is presented to predict apoptosis protein solely from sequence based on the concept of Chou's PseAA composition. The concept of approximate entropy (ApEn), which is a parameter denoting complexity of time series, is used to construct PseAA composition as additional features. Fuzzy K-nearest neighbor (FKNN) classifier is selected as prediction engine. Particle swarm optimization (PSO) algorithm is adopted for optimizing the weight factors which are important in PseAA composition. Two datasets are used to validate the performance of the proposed approach, which incorporate six subcellular location and four subcellular locations, respectively. The results obtained by jackknife test are quite encouraging. It indicates that the ApEn of protein sequence could represent effectively the information of apoptosis proteins subcellular locations. It can at least play a complimentary role to many of the existing methods, and might become potentially useful tool for protein function prediction. The software in Matlab is available freely by contacting the corresponding author.

  2. Rice DB: an Oryza Information Portal linking annotation, subcellular location, function, expression, regulation, and evolutionary information for rice and Arabidopsis.

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    Narsai, Reena; Devenish, James; Castleden, Ian; Narsai, Kabir; Xu, Lin; Shou, Huixia; Whelan, James

    2013-12-01

    Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or 'expressology', thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au). © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  3. Different subcellular locations of secretome components of Gram-positive bacteria

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    Buist, Girbe; Ridder, Anja N. J. A.; Kok, Jan; Kuipers, Oscar P.

    2006-01-01

    Gram-positive bacteria contain different types of secretion systems for the transport of proteins into or across the cytoplasmic membrane. Recent studies on subcellular localization of specific components of these secretion systems and their substrates have shown that they can be present at various

  4. Subcellular and cellular locations of nitric-oxide synthase isoforms as determinants of health and disease

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    Villanueva, Cleva; Giulivi, Cecilia

    2010-01-01

    The effects of nitric oxide in biological systems depend on its steady-state concentration and where it is being produced. The organ where nitric oxide is produced is relevant, and within the organ, which types of cells are actually contributing to this production seem to play a major determinant of its effect. Subcellular compartmentalization of specific nitric-oxide synthase enzymes has been shown to play a major role in health and disease. Pathophysiological conditions affect the cellular expression and localization of nitric oxide synthases, which in turn alter organ cross talk. In this study, we described the compartmentalization of nitric oxide in organs, cells and subcellular organelles, and how its localization relates to several relevant clinical conditions. Understanding the complexity of the compartmentalization of nitric oxide production and the implications of this compartmentalization in terms of cellular targets and downstream effects will eventually contribute toward the development of better strategies for treating or preventing pathological events associated with the increase, inhibition or mislocalization of nitric oxide production. PMID:20388537

  5. The impact of subcellular location on the near infrared-mediated thermal ablation of cells by targeted carbon nanotubes

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    Murali, Vasanth S.; Wang, Ruhung; Mikoryak, Carole A.; Pantano, Paul; Draper, Rockford K.

    2016-10-01

    Single-walled carbon nanotubes (SWNTs) are used in the near infrared (NIR)-mediated thermal ablation of tumor cells because they efficiently convert absorbed NIR light into heat. Despite the therapeutic potential of SWNTs, there have been no published studies that directly quantify how many SWNTs need be associated with a cell to achieve a desired efficiency of killing, or what is the most efficient subcellular location of SWNTs for killing cells. Herein we measured dose response curves for the efficiency of killing correlated to the measured amounts of folate-targeted SWNTs that were either on the surface or within the vacuolar compartment of normal rat kidney cells. Folate-targeted SWNTs on the cell surface were measured after different concentrations of SWNTs in medium were incubated with cells for 30 min at 4 °C. Folate-targeted SWNTs within the vacuolar compartments were measured after cells were incubated with different concentrations of SWNTs in medium for 6 h at 37 °C. It was observed that a SWNT load of ∼13 pg/cell when internalized was sufficient to kill 90% of the cells under standardized conditions of NIR light irradiation. When ∼3.5 pg/cell of SWNTs were internalized within the endosomal/lysosomal compartments, ∼50% of the cells were killed, but when ∼3.5 pg/cell of SWNTs were confined to the cell surface only ∼5% of the cells were killed under the same NIR irradiation conditions. The SWNT subcellular locations were verified using Raman imaging of SWNTs merged with fluorescence images of known subcellular markers. To our knowledge, this is the first time that SWNT amounts at known subcellular locations have been correlated with a dose-normalized efficacy of thermal ablation and the results support the idea that SWNTs confined to the plasma membrane are not as effective in NIR-mediated cell killing as an equivalent amount of SWNTs when internalized within the endosomal/lysosomal vesicles.

  6. HybridGO-Loc: mining hybrid features on gene ontology for predicting subcellular localization of multi-location proteins.

    Directory of Open Access Journals (Sweden)

    Shibiao Wan

    Full Text Available Protein subcellular localization prediction, as an essential step to elucidate the functions in vivo of proteins and identify drugs targets, has been extensively studied in previous decades. Instead of only determining subcellular localization of single-label proteins, recent studies have focused on predicting both single- and multi-location proteins. Computational methods based on Gene Ontology (GO have been demonstrated to be superior to methods based on other features. However, existing GO-based methods focus on the occurrences of GO terms and disregard their relationships. This paper proposes a multi-label subcellular-localization predictor, namely HybridGO-Loc, that leverages not only the GO term occurrences but also the inter-term relationships. This is achieved by hybridizing the GO frequencies of occurrences and the semantic similarity between GO terms. Given a protein, a set of GO terms are retrieved by searching against the gene ontology database, using the accession numbers of homologous proteins obtained via BLAST search as the keys. The frequency of GO occurrences and semantic similarity (SS between GO terms are used to formulate frequency vectors and semantic similarity vectors, respectively, which are subsequently hybridized to construct fusion vectors. An adaptive-decision based multi-label support vector machine (SVM classifier is proposed to classify the fusion vectors. Experimental results based on recent benchmark datasets and a new dataset containing novel proteins show that the proposed hybrid-feature predictor significantly outperforms predictors based on individual GO features as well as other state-of-the-art predictors. For readers' convenience, the HybridGO-Loc server, which is for predicting virus or plant proteins, is available online at http://bioinfo.eie.polyu.edu.hk/HybridGoServer/.

  7. A graphical model approach to automated classification of protein subcellular location patterns in multi-cell images

    Directory of Open Access Journals (Sweden)

    Murphy Robert F

    2006-02-01

    Full Text Available Abstract Background Knowledge of the subcellular location of a protein is critical to understanding how that protein works in a cell. This location is frequently determined by the interpretation of fluorescence microscope images. In recent years, automated systems have been developed for consistent and objective interpretation of such images so that the protein pattern in a single cell can be assigned to a known location category. While these systems perform with nearly perfect accuracy for single cell images of all major subcellular structures, their ability to distinguish subpatterns of an organelle (such as two Golgi proteins is not perfect. Our goal in the work described here was to improve the ability of an automated system to decide which of two similar patterns is present in a field of cells by considering more than one cell at a time. Since cells displaying the same location pattern are often clustered together, considering multiple cells may be expected to improve discrimination between similar patterns. Results We describe how to take advantage of information on experimental conditions to construct a graphical representation for multiple cells in a field. Assuming that a field is composed of a small number of classes, the classification accuracy can be improved by allowing the computed probability of each pattern for each cell to be influenced by the probabilities of its neighboring cells in the model. We describe a novel way to allow this influence to occur, in which we adjust the prior probabilities of each class to reflect the patterns that are present. When this graphical model approach is used on synthetic multi-cell images in which the true class of each cell is known, we observe that the ability to distinguish similar classes is improved without suffering any degradation in ability to distinguish dissimilar classes. The computational complexity of the method is sufficiently low that improved assignments of classes can be

  8. The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

    Directory of Open Access Journals (Sweden)

    Frank Voncken

    Full Text Available Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3. Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90% of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

  9. The Phosphoarginine Energy-Buffering System of Trypanosoma brucei Involves Multiple Arginine Kinase Isoforms with Different Subcellular Locations

    Science.gov (United States)

    Wadforth, Cath; Harley, Maggie; Colasante, Claudia

    2013-01-01

    Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3). Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide ‘SNL’. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90%) of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed. PMID:23776565

  10. Predicting human protein subcellular locations by the ensemble of multiple predictors via protein-protein interaction network with edge clustering coefficients.

    Directory of Open Access Journals (Sweden)

    Pufeng Du

    Full Text Available One of the fundamental tasks in biology is to identify the functions of all proteins to reveal the primary machinery of a cell. Knowledge of the subcellular locations of proteins will provide key hints to reveal their functions and to understand the intricate pathways that regulate biological processes at the cellular level. Protein subcellular location prediction has been extensively studied in the past two decades. A lot of methods have been developed based on protein primary sequences as well as protein-protein interaction network. In this paper, we propose to use the protein-protein interaction network as an infrastructure to integrate existing sequence based predictors. When predicting the subcellular locations of a given protein, not only the protein itself, but also all its interacting partners were considered. Unlike existing methods, our method requires neither the comprehensive knowledge of the protein-protein interaction network nor the experimentally annotated subcellular locations of most proteins in the protein-protein interaction network. Besides, our method can be used as a framework to integrate multiple predictors. Our method achieved 56% on human proteome in absolute-true rate, which is higher than the state-of-the-art methods.

  11. The Subcellular Location of Selenoproteins and the Impact on Their Function

    Directory of Open Access Journals (Sweden)

    Alan M. Diamond

    2015-05-01

    Full Text Available Most human selenium containing proteins contain selenium in the form of the amino acid selenocysteine, which is encoded in the corresponding mRNA as a UGA codon. Only a few non-selenocysteine containing selenoproteins are present and the nature of the association with selenium is not well understood. This review focuses on two selenocysteine-containing proteins that are members of the glutathione peroxidase family, GPx-1 and GPx-4, and the selenium-associated protein referred to as Selenium Binding Protein 1. Each of these proteins have been described to reside in two or more cellular compartments, and in the case of GPx-1 and SBP1, interact with each other. The enzymatic activity of GPx-1 and GPx-4 have been well described, but it is less clear how their cellular location impacts the health related phenotypes associated with activities, while no catalytic function is assigned to SBP1. The distribution of these proteins is presented as is the possible consequences of that compartmentalization.

  12. Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development.

    Directory of Open Access Journals (Sweden)

    Mohd A Mohd Ridzuan

    Full Text Available An actomyosin motor complex assembled below the parasite's plasma membrane drives erythrocyte invasion by Plasmodium falciparum merozoites. The complex is comprised of several proteins including myosin (MyoA, myosin tail domain interacting protein (MTIP and glideosome associated proteins (GAP 45 and 50, and is anchored on the inner membrane complex (IMC, which underlies the plasmalemma. A ternary complex of MyoA, MTIP and GAP45 is formed that then associates with GAP50. We show that full length GAP45 labelled internally with GFP is assembled into the motor complex and transported to the developing IMC in early schizogony, where it accumulates during intracellular development until merozoite release. We show that GAP45 is phosphorylated by calcium dependent protein kinase 1 (CDPK1, and identify the modified serine residues. Replacing these serine residues with alanine or aspartate has no apparent effect on GAP45 assembly into the motor protein complex or its subcellular location in the parasite. The early assembly of the motor complex suggests that it has functions in addition to its role in erythrocyte invasion.

  13. Sequences that direct subcellular traffic of the Drosophila methoprene-tolerant protein (MET) are located predominantly in the PAS domains.

    Science.gov (United States)

    Greb-Markiewicz, Beata; Orłowski, Marek; Dobrucki, Jerzy; Ożyhar, Andrzej

    2011-10-15

    Methoprene-tolerant protein (MET) is a key mediator of antimetamorphic signaling in insects. MET belongs to the family of bHLH-PAS transcription factors which regulate gene expression and determine essential physiological and developmental processes. The ability of many bHLH-PAS proteins to carry out their functions is related to the patterns of intracellular trafficking, which are determined by specific sequences and indicate that a nuclear localization signal (NLS) or a nuclear export signal (NES) is present and active. Therefore, the identification of NLS and NES signals is fundamental in order to understand the intracellular signaling role of MET. Nevertheless, data on the intracellular trafficking of MET are inconsistent, and until now there hasn't been any data on potential NLS and NES sequences. To analyze the trafficking of MET we designed a number of expression vectors encoding full-length MET, as well as various derivatives, that were fused to yellow fluorescent protein (YFP). Confocal microscopy analysis of the subcellular distribution of YFP-MET indicated that while this protein was localized mainly in the nucleus, it was also observed in the cytoplasm. This suggested the presence of both an NLS and NES in MET. Our work has shown that each of the two PAS domains of MET (PAS-A and PAS-B, respectively) contain one NLS and one NES sequence. Additional NES activity was present in the C-terminal fragment. The NLS activity located in PAS-B was dependent on the presence of juvenile hormone (JH), the potential ligand for MET. In contrast to this, JH didn't seem to be required for the NLS in PAS-A to be active. However, on the basis of current knowledge about the function of PAS-A in other bHLH-PAS proteins, we suggest there might be other proteins that control the activity of the NLS and possibly the NES located in the PAS-A of MET. Thus, the intracellular trafficking of MET seems to be regulated by a rather complicated network of different factors. Copyright

  14. A multiple information fusion method for predicting subcellular locations of two different types of bacterial protein simultaneously.

    Science.gov (United States)

    Chen, Jing; Xu, Huimin; He, Ping-An; Dai, Qi; Yao, Yuhua

    2016-01-01

    Subcellular localization prediction of bacterial protein is an important component of bioinformatics, which has great importance for drug design and other applications. For the prediction of protein subcellular localization, as we all know, lots of computational tools have been developed in the recent decades. In this study, we firstly introduce three kinds of protein sequences encoding schemes: physicochemical-based, evolutionary-based, and GO-based. The original and consensus sequences were combined with physicochemical properties. And elements information of different rows and columns in position-specific scoring matrix were taken into consideration simultaneously for more core and essence information. Computational methods based on gene ontology (GO) have been demonstrated to be superior to methods based on other features. Then principal component analysis (PCA) is applied for feature selection and reduced vectors are input to a support vector machine (SVM) to predict protein subcellular localization. The proposed method can achieve a prediction accuracy of 98.28% and 97.87% on a stringent Gram-positive (Gpos) and Gram-negative (Gneg) dataset with Jackknife test, respectively. At last, we calculate "absolute true overall accuracy (ATOA)", which is stricter than overall accuracy. The ATOA obtained from the proposed method is also up to 97.32% and 93.06% for Gpos and Gneg. From both the rationality of testing procedure and the success rates of test results, the current method can improve the prediction quality of protein subcellular localization. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. On models for continuous facility location with partial coverage

    DEFF Research Database (Denmark)

    Brimberg, Jack; Juel, Henrik; Körner, Mark-Christoph

    2014-01-01

    This paper presents a new concept of partial coverage distance, where demand points within a given threshold distance of a new facility are covered in the traditional sense, while non-covered demand points are penalized an amount proportional to their distance to the covered region. Two single...

  16. Location of. gamma. -carboxyglutamyl residues in partially carboxylated prothrombin preparations

    Energy Technology Data Exchange (ETDEWEB)

    Liska, D.J.; Suttie, J.W.

    1988-11-15

    Prothrombin contains 10-..gamma..-carboxylglutamyl (Gla) residues in the N-terminal (fragment 1) domain of the protein. Following anticoagulant administration, a spectrum of undercarboxylated, physiologically less active forms of prothrombin is secreted into bovine or human plasma. The sites of undercarboxylation in these prothrombin species have now been investigated. Plasma containing a mixture of partially carboxylated forms of prothombin was obtained from a dicoumarol-treated bovine, and three pools of partially carboxylated (four, six, or eight Gla) species were purified by adsorption onto barium citrate and barium oxalate, ammonium sulfate fractionation, and chromatography. Fragment 1 obtained from these variants was equilibrated with /sup 3/H/sub 2/O and heated in a dry state to decarboxylate Gla and incorporate /sup 3/H into the resulting Glu residues. This peptide was then sequenced by Edman degradation, and the specific radioactivity of PTH-Glu was determined for each potential Gla-containing site. Data obtained from normal prothrombin fragment 1 fit a linear model when the log of specific activity of PTH-Glu was plotted against the cycle number. Analysis of the 80% variant showed a decrease in carboxylation only in the last two Gla residues, while data obtained from the 60% variant indicated a general decrease in carboxylation from the most amino-terminal of the Gla residues. In the 40% Gla variant, all but the most amino-terminal of the Gla residues appeared to be undercarboxylated. These data indicate that the ..gamma..-carboxylation of glutamyl residues in prothrombin does not occur randomly but instead with preferential carboxylation of the most amino-terminal Gla residues. When carboxylation is limited, the impairment of carboxylation is more severe at the more carboxyl-terminal residues.

  17. Predict subcellular locations of singleplex and multiplex proteins by semi-supervised learning and dimension-reducing general mode of Chou's PseAAC.

    Science.gov (United States)

    Pacharawongsakda, Eakasit; Theeramunkong, Thanaruk

    2013-12-01

    Predicting protein subcellular location is one of major challenges in Bioinformatics area since such knowledge helps us understand protein functions and enables us to select the targeted proteins during drug discovery process. While many computational techniques have been proposed to improve predictive performance for protein subcellular location, they have several shortcomings. In this work, we propose a method to solve three main issues in such techniques; i) manipulation of multiplex proteins which may exist or move between multiple cellular compartments, ii) handling of high dimensionality in input and output spaces and iii) requirement of sufficient labeled data for model training. Towards these issues, this work presents a new computational method for predicting proteins which have either single or multiple locations. The proposed technique, namely iFLAST-CORE, incorporates the dimensionality reduction in the feature and label spaces with co-training paradigm for semi-supervised multi-label classification. For this purpose, the Singular Value Decomposition (SVD) is applied to transform the high-dimensional feature space and label space into the lower-dimensional spaces. After that, due to limitation of labeled data, the co-training regression makes use of unlabeled data by predicting the target values in the lower-dimensional spaces of unlabeled data. In the last step, the component of SVD is used to project labels in the lower-dimensional space back to those in the original space and an adaptive threshold is used to map a numeric value to a binary value for label determination. A set of experiments on viral proteins and gram-negative bacterial proteins evidence that our proposed method improve the classification performance in terms of various evaluation metrics such as Aiming (or Precision), Coverage (or Recall) and macro F-measure, compared to the traditional method that uses only labeled data.

  18. Site fidelity and individual variation in winter location in partially migratory European shags

    OpenAIRE

    Hannah Grist; Francis Daunt; Sarah Wanless; Nelson, Emily J.; Harris, Mike P.; Mark Newell; Sarah Burthe; Reid, Jane M.

    2014-01-01

    In partially migratory populations, individuals from a single breeding area experience a range of environments during the non-breeding season. If individuals show high within- and among- year fidelity to specific locations, any annual environmental effect on individual life histories could be reinforced, causing substantial demographic heterogeneity. Quantifying within- and among- individual variation and repeatability in non-breeding season location is therefore key to predicting broad-scale...

  19. Design parameter based method of partial discharge detection and location in power transformers

    Directory of Open Access Journals (Sweden)

    Kumar Santosh Annadurai

    2009-01-01

    Full Text Available Insulation defect detection in time ensures higher operational reliability of power system assets. Power transformers are the most critical unit of power systems both from economical and operational front. Hence it becomes necessary to have knowledge of the actual insulation condition of transformer to increase dependability of the system. The performance and ageing of the transformer insulation is mainly affected by Partial discharges (PD. Proper diagnosis in terms of amplitude and location of partial discharge in a power transformer enables us to predict well in advance, with much confidence, the defect in insulation system, which avoids large catastrophic failures. In this work a 20kVA, 230/50kV single phase core type transformer is used for evaluation of the transfer function-based partial discharge detection and location using modeling of the winding, using design data. The simulation of capturing on-line PD pulses across the bushing tap capacitor is done for various tap positions. Standard PD source model is used to inject PD pulse signal at 10 tap locations in the winding and corresponding response signatures are captured at the bushing tap end (across 1000pF. The equivalent high frequency model of the winding is derived from the design parameters using analytical calculations and simulations in packages such as MAGNET and ANSOFT. The test conditions are simulated using ORCAD-9 and the results are evaluated for location accuracy using design parameter based PD monitoring method. .

  20. Short-term retrospective case series of implant-assisted removable partial dentures with locator abutments.

    Science.gov (United States)

    Ortiz-Puigpelat, Octavi; Gargallo-Albiol, Jordi; Hernández-Alfaro, Federico; Cabratosa-Termes, Josep

    2014-01-01

    The purpose of this retrospective case series was to report on the clinical performance of implant-assisted removable partial dentures (IARPDs) with Locator abutments in different partial edentulism situations, with a mean follow-up period of 28.6 months. Twelve consecutive patients were treated with IARPDs. A total of 24 implants were placed in the edentulous area. Minimum follow-up period was 12 months. Overall patient satisfaction, health of peri-implant tissues, survival of implants and abutments, and prosthetic complications were reported. Overall implant survival was 91.6%; two implants failed. No major complications were reported-only one IARPD metal framework broke. No Locator abutment loosening was reported. Within the limitations of this retrospective study, treatment with IARPDs can improve the patient's function, phonetics, and esthetics without the need for extensive bone regeneration surgeries and prosthodontic rehabilitations. However, well-designed prospective clinical studies on IARPDs are needed to support their long-term use.

  1. Site fidelity and individual variation in winter location in partially migratory European shags.

    Directory of Open Access Journals (Sweden)

    Hannah Grist

    Full Text Available In partially migratory populations, individuals from a single breeding area experience a range of environments during the non-breeding season. If individuals show high within- and among- year fidelity to specific locations, any annual environmental effect on individual life histories could be reinforced, causing substantial demographic heterogeneity. Quantifying within- and among- individual variation and repeatability in non-breeding season location is therefore key to predicting broad-scale environmental impacts on the dynamics of partially migratory populations. We used field resightings of colour-ringed adult European shags known to have bred on the Isle of May, Scotland, to quantify individual variation and repeatability in winter location within and among three consecutive winters. In total, 3797 resightings of 882 individuals were recorded over 622 km of coastline, including the Isle of May. These individuals comprised over 50% of the known breeding population, and encompassed representative distributions of ages and sexes. The distances from the Isle of May at which individuals were resighted during winter varied substantially, up to 486 km and 136 km north and south respectively and including the breeding colony on the Isle of May. However, resighting distances were highly repeatable within individuals; within- and among-winter repeatabilities were >0.72 and >0.59 respectively across the full September-March observation period, and >0.95 and >0.79 respectively across more restricted mid-winter periods. Repeatability did not differ significantly between males and females or among different age classes, either within or among winters. These data demonstrate that the focal shag population is partially migratory, and moreover that individuals show highly repeatable variation in winter location and hence migration strategy across consecutive winters. Such high among-individual variation and within-individual repeatability, both within and

  2. Study of cross-shaped ultrasonic array sensor applied to partial discharge location in transformer oil.

    Science.gov (United States)

    Li, Jisheng; Xin, Xiaohu; Luo, Yongfen; Ji, Haiying; Li, Yanming; Deng, Junbo

    2013-11-01

    A conformal combined sensor is designed and it is used in Partial Discharge (PD) location experiments in transformer oil. The sensor includes a cross-shaped ultrasonic phased array of 13 elements and an ultra-high-frequency (UHF) electromagnetic rectangle array of 2 × 2 elements. Virtual expansion with high order cumulants, the ultrasonic array can achieve the effect of array with 61 elements. This greatly improves the aperture and direction sharpness of original array and reduces the cost of follow-up hardware. With the cross-shaped ultrasonic array, the results of PD location experiments are precise and the maximum error of the direction of arrival (DOA) is less than 5°.

  3. Partial Breast Reconstruction Using Various Oncoplastic Techniques for Centrally Located Breast Cancer

    Directory of Open Access Journals (Sweden)

    Hyo Chun Park

    2014-09-01

    Full Text Available BackgroundAs the breast cancer incidence has increased, breast-conserving surgery has replaced total mastectomy as the predominant procedure. However, centrally located breast cancers pose significant challenges to successful breast-conserving surgeries. Therefore, we performed partial mastectomy and oncoplastic procedures on centrally located breast cancer as a means of partial breast reconstruction. The authors examined and evaluated the functional and aesthetic usefulness of this reconstruction method.MethodsFrom January 2007 to June 2011, 35 patients with centrally located breast cancers who underwent various oncoplastic procedures based on the breast size and resection volume. The oncoplastic procedures performed included volume displacement surgical techniques such as purse-string suture, linear suture, and reduction mammaplasty. Other oncoplastic procedures included volume replacement procedures with an adipofascial, thoracoepigastric, intercostal artery perforator, thoracodorsal artery perforator, or latissimus dorsi flap.ResultsMean patient age was 49 years, and mean follow-up period was 11 months. In cases of small to moderate-sized breasts and resection volumes 50 g, volume replacement procedures were performed. In cases of larger breasts and smaller resection volumes, glandular reshaping was performed. Finally, in cases of larger breasts and larger resection volumes, reduction mammaplasty was performed. This reconstruction method also elicits a high patient satisfaction rate with no significant complications.ConclusionsIn centrally located breast cancer, oncoplastic surgery considering breast size and resection volume is safe and provides appropriate aesthetic outcomes. Therefore, our method is advisable for breast cancer patients who elect to conserve their breasts and retain a natural breast shape.

  4. Radar Determination of Fault Slip and Location in Partially Decorrelated Images

    Science.gov (United States)

    Parker, Jay; Glasscoe, Margaret; Donnellan, Andrea; Stough, Timothy; Pierce, Marlon; Wang, Jun

    2017-06-01

    Faced with the challenge of thousands of frames of radar interferometric images, automated feature extraction promises to spur data understanding and highlight geophysically active land regions for further study. We have developed techniques for automatically determining surface fault slip and location using deformation images from the NASA Uninhabited Aerial Vehicle Synthetic Aperture Radar (UAVSAR), which is similar to satellite-based SAR but has more mission flexibility and higher resolution (pixels are approximately 7 m). This radar interferometry provides a highly sensitive method, clearly indicating faults slipping at levels of 10 mm or less. But interferometric images are subject to decorrelation between revisit times, creating spots of bad data in the image. Our method begins with freely available data products from the UAVSAR mission, chiefly unwrapped interferograms, coherence images, and flight metadata. The computer vision techniques we use assume no data gaps or holes; so a preliminary step detects and removes spots of bad data and fills these holes by interpolation and blurring. Detected and partially validated surface fractures from earthquake main shocks, aftershocks, and aseismic-induced slip are shown for faults in California, including El Mayor-Cucapah (M7.2, 2010), the Ocotillo aftershock (M5.7, 2010), and South Napa (M6.0, 2014). Aseismic slip is detected on the San Andreas Fault from the El Mayor-Cucapah earthquake, in regions of highly patterned partial decorrelation. Validation is performed by comparing slip estimates from two interferograms with published ground truth measurements.

  5. Development and verification of a software tool for the acoustic location of partial discharge in a power transformer

    Directory of Open Access Journals (Sweden)

    Polužanski Vladimir

    2014-01-01

    Full Text Available This paper discusses the development and verification of software tool for determining the location of partial discharge in a power transformer with the acoustic method. Classification and systematization of physical principles and detection methods and tests of partial discharge in power transformers are shown at the beginning of this paper. The most important mathematical models, features, algorithms, and real problems that affect measurement accuracy are highlighted. This paper describes the development and implementation of a software tool for determining the location of partial discharge in a power transformer based on a no iterative mathematical algorithm. Verification and accuracy of measurement are proved both by computer simulation and experimental results available in the literature.

  6. Vibration reduction in helicopter rotors using an actively controlled partial span trailing edge flap located on the blade

    Science.gov (United States)

    Millott, T. A.; Friedmann, P. P.

    1994-06-01

    This report describes an analytical study of vibration reduction in a four-bladed helicopter rotor using an actively controlled, partial span, trailing edge flap located on the blade. The vibration reduction produced by the actively controlled flap (ACF) is compared with that obtained using individual blade control (IBC), in which the entire blade is oscillated in pitch. For both cases a deterministic feedback controller is implemented to reduce the 4/rev hub loads. For all cases considered, the ACF produced vibration reduction comparable with that obtained using IBC, but consumed only 10-30% of the power required to implement IBC. A careful parametric study is conducted to determine the influence of blade torsional stiffness, spanwise location of the control flap, and hinge moment correction on the vibration reduction characteristics of the ACF. The results clearly demonstrate the feasibility of this new approach to vibration reduction. It should be emphasized than the ACF, used together with a conventional swashplate, is completely decoupled from the primary flight control system and thus it has no influence on the airworthiness of the helicopter. This attribute is potentially a significant advantage when compared to IBC.

  7. Subcellular Organization of GPCR Signaling.

    Science.gov (United States)

    Eichel, Kelsie; von Zastrow, Mark

    2018-02-01

    G protein-coupled receptors (GPCRs) comprise a large and diverse class of signal-transducing receptors that undergo dynamic and isoform-specific membrane trafficking. GPCRs thus have an inherent potential to initiate or regulate signaling reactions from multiple membrane locations. This review discusses emerging insights into the subcellular organization of GPCR function in mammalian cells, focusing on signaling transduced by heterotrimeric G proteins and β-arrestins. We summarize recent evidence indicating that GPCR-mediated activation of G proteins occurs not only from the plasma membrane (PM) but also from endosomes and Golgi membranes and that β-arrestin-dependent signaling can be transduced from the PM by β-arrestin trafficking to clathrin-coated pits (CCPs) after dissociation from a ligand-activated GPCR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. A software tool based on the Surface Evolver for precise location of tumours as a preoperative procedure to partial mastectomy

    Science.gov (United States)

    Elias Fabris, Antonio; Zanchetta do Nascimento, Marcelo; Ramos Batista, Valério

    2015-09-01

    We present a fast and reliable program that gives precise location of breast tumours for a partial mastectomy. Our program is fully implemented in the Surface Evolver, which is a general-purpose simulator of physical experiments. By starting from the mammograms that show a tumour one takes its 2D coordinates in each view (CC and MLO). These coordinates, together with some measurements of the patient's breast, are given as input to our simulator. From this point on the simulator reproduces all main steps of taking mammography with a virtual transparent breast that matches the patient's. The virtual mammography procedure is graphically displayed on the computer screen, so that users can track the virtual tumour inside the breast. As output we have the coordinates of the tumour position when the woman lies on the operating table for the surgery. With these coordinates the surgeon can make a small incision into the breast and reach the tumour for its removal. The whole structure of the breast is preserved after a simple plastic correction.

  9. Subcellular targeting strategies for drug design and delivery.

    Science.gov (United States)

    Rajendran, Lawrence; Knölker, Hans-Joachim; Simons, Kai

    2010-01-01

    Many drug targets are localized to particular subcellular compartments, yet current drug design strategies are focused on bioavailability and tissue targeting and rarely address drug delivery to specific intracellular compartments. Insights into how the cell traffics its constituents to these different cellular locations could improve drug design. In this Review, we explore the fundamentals of membrane trafficking and subcellular organization, as well as strategies used by pathogens to appropriate these mechanisms and the implications for drug design and delivery.

  10. Location, Location, Location!

    Science.gov (United States)

    Ramsdell, Kristin

    2004-01-01

    Of prime importance in real estate, location is also a key element in the appeal of romances. Popular geographic settings and historical periods sell, unpopular ones do not--not always with a logical explanation, as the author discovered when she conducted a survey on this topic last year. (Why, for example, are the French Revolution and the…

  11. GAP Activity, but Not Subcellular Targeting, Is Required for Arabidopsis RanGAP Cellular and Developmental Functions.

    Science.gov (United States)

    Boruc, Joanna; Griffis, Anna H N; Rodrigo-Peiris, Thushani; Zhou, Xiao; Tilford, Bailey; Van Damme, Daniël; Meier, Iris

    2015-07-01

    The Ran GTPase activating protein (RanGAP) is important to Ran signaling involved in nucleocytoplasmic transport, spindle organization, and postmitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis thaliana RanGAP1. A double null mutant of the two Arabidopsis RanGAP homologs is gametophyte lethal. Here, we created a series of mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, the severity of developmental phenotypes increases, but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development is differentially affected by RanGAP mutant allele combinations of increasing severity and requires the GAP activity of RanGAP, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP from the nuclear envelope. © 2015 American Society of Plant Biologists. All rights reserved.

  12. Subcellular localization prediction through boosting association rules.

    Science.gov (United States)

    Yoon, Yongwook; Lee, Gary Geunbae

    2012-01-01

    Computational methods for predicting protein subcellular localization have used various types of features, including N-terminal sorting signals, amino acid compositions, and text annotations from protein databases. Our approach does not use biological knowledge such as the sorting signals or homologues, but use just protein sequence information. The method divides a protein sequence into short $k$-mer sequence fragments which can be mapped to word features in document classification. A large number of class association rules are mined from the protein sequence examples that range from the N-terminus to the C-terminus. Then, a boosting algorithm is applied to those rules to build up a final classifier. Experimental results using benchmark datasets show our method is excellent in terms of both the classification performance and the test coverage. The result also implies that the $k$-mer sequence features which determine subcellular locations do not necessarily exist in specific positions of a protein sequence. Online prediction service implementing our method is available at http://isoft.postech.ac.kr/research/BCAR/subcell.

  13. A novel intra-molecular protein-protein interaction code based on partial complementary coding of co-locating amino acids.

    Science.gov (United States)

    Biro, Jan C

    2006-01-01

    Proteins are assumed to contain all the information necessary for unambiguous folding and specific interaction with each other. However, ab initio structure prediction is often not successful because the amino acid sequence itself is simply not sufficient to guide between endless folding possibilities. It seems to be logical to try to find the "missing" information in nucleic acids, in the redundant codon. Statistical analyses of approximately 35K amino acid co-locations in 80 different protein structures indicate the existence of a weak intra-molecular protein-protein interaction code. Co-locating amino acids are preferentially coded by codons which are complementary in reverse orientation to each other at the 1st and 3rd codon positions, but not necessarily at the 2nd. This code, called D-1 X 3/RC-3 X 1, limits the number of preferred amino acid pairs from 20 to 10.3+/-0.8 (SEM, n=20) and emphasizes the importance of "strictly" defined amino acids (those having less synonymous codons). The existence of this code does not by any means violate the known physicochemical rules of protein folding or interaction. It is suggested that the biological source of preferential (specific) amino acid co-locations is the partial complementarity of their codons. This special coding of co-locating amino acids is important to better understanding of some fundamental biochemical processes and observations such as: (a) protein folding; (b) specific and high affinity protein-protein interactions; (c) the role of the wobble bases; (d) the significance of the redundant genetic code; (e) the origin of specific protein-protein interactions. Furthermore it might be useful even in protein design.

  14. Protein Subcellular Relocalization of Duplicated Genes in Arabidopsis

    Science.gov (United States)

    Liu, Shao-Lun; Pan, An Qi; Adams, Keith L.

    2014-01-01

    Gene duplications during eukaroytic evolution, by successive rounds of polyploidy and by smaller scale duplications, have provided an enormous reservoir of new genes for the evolution of new functions. Preservation of many duplicated genes can be ascribed to changes in sequences, expression patterns, and functions. Protein subcellular relocalization (protein targeting to a new location within the cell) is another way that duplicated genes can diverge. We studied subcellular relocalization of gene pairs duplicated during the evolution of the Brassicaceae including gene pairs from the alpha whole genome duplication that occurred at the base of the family. We analyzed experimental localization data from green fluorescent protein experiments for 128 duplicate pairs in Arabidopsis thaliana, revealing 19 pairs with subcellular relocalization. Many more of the duplicate pairs with relocalization than with the same localization showed an accelerated rate of amino acid sequence evolution in one duplicate, and one gene showed evidence for positive selection. We studied six duplicate gene pairs in more detail. We used gene family analysis with several pairs to infer which gene shows relocalization. We identified potential sequence mutations through comparative analysis that likely result in relocalization of two duplicated gene products. We show that four cases of relocalization have new expression patterns, compared with orthologs in outgroup species, including two with novel expression in pollen. This study provides insights into subcellular relocalization of evolutionarily recent gene duplicates and features of genes whose products have been relocalized. PMID:25193306

  15. Comparison of fiber reinforcement placed at different locations of pontic in interim fixed partial denture to prevent fracture: An in vitro study.

    Science.gov (United States)

    Kapri, Surg Cdr Anita

    2015-01-01

    The interim restoration is an important phase in fixed prosthodontic therapy. It should provide sufficient durability to withstand the forces of mastication. A fractured interim restoration is damaging to the prosthodontic care and may lead to an unscheduled appointment for repair. Several attempts have been made to reinforce interim fixed partial dentures (FPDs). These have included the use of metal wire, a lingual cast metal reinforcement, a processed acrylic resin interim restoration, and different types of fibers, e.g., carbon, polyethylene, nylon and glass. These fibers can be placed in the occlusal, middle or cervical thirds in the FPD. There is no scientific data to evaluate the effect of fiber placement methods on the fracture resistance of clinical interim FPDs. Hence this study was designed to evaluate fracture load values of interim FPDs with different locations of fiber reinforcement. 30 interim FPD samples with polymethyl methacrylate (PMMA) reinforced with fibers at three different locations mainly occlusal, cervical and middle (10 samples each) were fabricated using a metal FPD on a master die. They were tested for fracture resistance in universal testing machine. The fracture resistance was recorded and is tabulated and analyzed statistically. The results showed that the placement of the reinforcement in the occlusal third of the pontic resulted in higher fracture resistance which was significantly higher (P fiber for reinforcing the PMMA interim restorative resin.

  16. A functional dissection of PTEN N-terminus : Implications in PTEN subcellular targeting and tumor suppressor activity

    NARCIS (Netherlands)

    Gil, Anabel; Rodríguez-Escudero, Isabel; Stumpf, Miriam; Molina, María; Cid, Víctor J.; Pulido, Rafael

    2015-01-01

    Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization

  17. Evaluation and comparison of mammalian subcellular localization prediction methods

    Directory of Open Access Journals (Sweden)

    Fink J Lynn

    2006-12-01

    Full Text Available Abstract Background Determination of the subcellular location of a protein is essential to understanding its biochemical function. This information can provide insight into the function of hypothetical or novel proteins. These data are difficult to obtain experimentally but have become especially important since many whole genome sequencing projects have been finished and many resulting protein sequences are still lacking detailed functional information. In order to address this paucity of data, many computational prediction methods have been developed. However, these methods have varying levels of accuracy and perform differently based on the sequences that are presented to the underlying algorithm. It is therefore useful to compare these methods and monitor their performance. Results In order to perform a comprehensive survey of prediction methods, we selected only methods that accepted large batches of protein sequences, were publicly available, and were able to predict localization to at least nine of the major subcellular locations (nucleus, cytosol, mitochondrion, extracellular region, plasma membrane, Golgi apparatus, endoplasmic reticulum (ER, peroxisome, and lysosome. The selected methods were CELLO, MultiLoc, Proteome Analyst, pTarget and WoLF PSORT. These methods were evaluated using 3763 mouse proteins from SwissProt that represent the source of the training sets used in development of the individual methods. In addition, an independent evaluation set of 2145 mouse proteins from LOCATE with a bias towards the subcellular localization underrepresented in SwissProt was used. The sensitivity and specificity were calculated for each method and compared to a theoretical value based on what might be observed by random chance. Conclusion No individual method had a sufficient level of sensitivity across both evaluation sets that would enable reliable application to hypothetical proteins. All methods showed lower performance on the LOCATE

  18. Subcellular location and species specificity of pipecolate degradation

    Energy Technology Data Exchange (ETDEWEB)

    Mihalik, S.J.; Rhead, W.J.

    1986-03-05

    Defects in pipecolic acid (PA) catabolism are characteristic of several inherited metabolic diseases including hyperpipecolic acidemia, Zellweger's Syndrome, neonatal-onset adrenoleukodystrophy, and infantile Refsum's disease. In the latter three diseases, peroxisomes are abnormal. The authors have studied the subcelluar distribution of the PA degradation to determine a mammalian model for the normal pathway. Crude light and heavy mitochondrial fractions (including lysosomes and peroxisomes) from kidney cortex or liver were separated on Percoll gradients. Individual fractions were then incubated at 37/sup 0/C with 3H-2,3,4,5,6 L-PA. Using ion exchange chromatography, the production of 3H ..cap alpha..-aminoadipic acid (AAA) and 3H-H2O were quantitated. AAA production paralleled the activity of the mitochondrial marker enzyme, glutamate dehydrogenase, in the rabbit, guinea pig, dog, pig, and sheep. 3H-AAA production ranged from 382 to 13,900 pmol/mg prot/h. Guinea pig kidney cortex exhibited highest specific activity. The mitochondrial enzyme was absent from human liver (n=3) and liver and kidney cortex from rat, mouse, and monkey. In these tissues, the activity followed the pattern of the peroxisomal core enzyme, urate oxidase.

  19. ALG-2 oscillates in subcellular localization, unitemporally with calcium oscillations

    DEFF Research Database (Denmark)

    la Cour, Jonas Marstrand; Mollerup, Jens; Berchtold, Martin Werner

    2007-01-01

    discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate...... localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG...

  20. Tau regulates the subcellular localization of calmodulin

    Energy Technology Data Exchange (ETDEWEB)

    Barreda, Elena Gomez de [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Avila, Jesus, E-mail: javila@cbm.uam.es [Centro de Biologia Molecular ' Severo Ochoa' , CSIC/UAM, Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); CIBER de Enfermedades Neurodegenerativas, 28031 Madrid (Spain)

    2011-05-13

    Highlights: {yields} In this work we have tried to explain how a cytoplasmic protein could regulate a cell nuclear function. We have tested the role of a cytoplasmic protein (tau) in regulating the expression of calbindin gene. We found that calmodulin, a tau-binding protein with nuclear and cytoplasmic localization, increases its nuclear localization in the absence of tau. Since nuclear calmodulin regulates calbindin expression, a decrease in nuclear calmodulin, due to the presence of tau that retains it at the cytoplasm, results in a change in calbindin expression. -- Abstract: Lack of tau expression in neuronal cells results in a change in the expression of few genes. However, little is known about how tau regulates gene expression. Here we show that the presence of tau could alter the subcellular localization of calmodulin, a protein that could be located at the cytoplasm or in the nucleus. Nuclear calmodulin binds to co-transcription factors, regulating the expression of genes like calbindin. In this work, we have found that in neurons containing tau, a higher proportion of calmodulin is present in the cytoplasm compared with neurons lacking tau and that an increase in cytoplasmic calmodulin correlates with a higher expression of calbindin.

  1. Monoterpene biosynthesis potential of plant subcellular compartments

    NARCIS (Netherlands)

    Dong, L.; Jongedijk, E.J.; Bouwmeester, H.J.; Krol, van der A.R.

    2016-01-01

    Subcellular monoterpene biosynthesis capacity based on local geranyl diphosphate (GDP) availability or locally boosted GDP production was determined for plastids, cytosol and mitochondria. A geraniol synthase (GES) was targeted to plastids, cytosol, or mitochondria. Transient expression in Nicotiana

  2. Expression and subcellular localization of antiporter regulating ...

    African Journals Online (AJOL)

    Expression and subcellular localization of antiporter regulating protein OsARP in rice induced by submergence, salt and drought stresses. Md Imtiaz Uddin, Maki Kihara, Lina Yin, Mst Farida Perveen, Kiyoshi Tanaka ...

  3. Optogenetic Tools for Subcellular Applications in Neuroscience.

    Science.gov (United States)

    Rost, Benjamin R; Schneider-Warme, Franziska; Schmitz, Dietmar; Hegemann, Peter

    2017-11-01

    The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell. Therefore, optogenetic activation often does not reflect context-dependent effects of highly localized intracellular signaling events. Subcellular targeting is required to achieve more specific optogenetic readouts and photomanipulation. Here we first provide a detailed overview of the available optogenetic tools with a focus on optogenetic actuators. Second, we review established strategies for targeting these tools to specific subcellular compartments. Finally, we discuss useful tools and targeting strategies that are currently missing from the optogenetics repertoire and provide suggestions for novel subcellular optogenetic applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Geary autocorrelation and DCCA coefficient: Application to predict apoptosis protein subcellular localization via PSSM

    Science.gov (United States)

    Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

    2017-02-01

    Apoptosis is a fundamental process controlling normal tissue homeostasis by regulating a balance between cell proliferation and death. Predicting subcellular location of apoptosis proteins is very helpful for understanding its mechanism of programmed cell death. Prediction of apoptosis protein subcellular location is still a challenging and complicated task, and existing methods mainly based on protein primary sequences. In this paper, we propose a new position-specific scoring matrix (PSSM)-based model by using Geary autocorrelation function and detrended cross-correlation coefficient (DCCA coefficient). Then a 270-dimensional (270D) feature vector is constructed on three widely used datasets: ZD98, ZW225 and CL317, and support vector machine is adopted as classifier. The overall prediction accuracies are significantly improved by rigorous jackknife test. The results show that our model offers a reliable and effective PSSM-based tool for prediction of apoptosis protein subcellular localization.

  5. Subcellular Localization of Cadmium in Chlorella vulgaris Beijerinck Strain Bt-09

    Directory of Open Access Journals (Sweden)

    P.B. Lintongan

    2004-06-01

    Full Text Available Growth response curves of Chlorella vulgaris Beijerinck strain Bt-09 to sublethal concentrations of cadmium were evaluated. The growth responses of this microalgal isolate was determined through analysis of chlorophyll a levels. Cadmium was effectively taken up by the cells as determined by Flame Atomic Absorption Spectrophotometry (F-AAS. Subcellular fractionation was undertaken to locate sites that accumulate cadmium.

  6. Expression and subcellular localization of antiporter regulating ...

    African Journals Online (AJOL)

    Md. Imtiaz Uddin

    2012-02-14

    Feb 14, 2012 ... We examined the expression and subcellular localization of antiporter regulating protein OsARP in a submergence tolerant rice (Oryza sativa L.) cultivar FR13A. In the public databases, this protein was designated as putative Os02g0465900 protein. The cDNA containing the full-length sequence of OsARP.

  7. Domains involved in TAF15 subcellular localisation

    DEFF Research Database (Denmark)

    Marko, Marija; Vlassis, Arsenios; Guialis, Apostolia

    2012-01-01

    to play important roles in the onset of specific tumours, certain forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In this study we identified the domains of TAF15 responsible for its subcellular localisation in human (HeLa) cells and experimentally confirmed...

  8. Lipidomics in tissues, cells and subcellular compartments

    National Research Council Canada - National Science Library

    Horn, Patrick J; Chapman, Kent D

    2012-01-01

    ...‐infusion MS, localization of lipids in tissues and cells by laser desorption/ionization MS, and even profiling of lipids in individual subcellular compartments by direct‐organelle MS. Applications of these approaches to achieve improved understanding of plant lipid metabolism, compartmentation and function are discussed.

  9. Detrended cross-correlation coefficient: Application to predict apoptosis protein subcellular localization.

    Science.gov (United States)

    Liang, Yunyun; Liu, Sanyang; Zhang, Shengli

    2016-12-01

    Apoptosis, or programed cell death, plays a central role in the development and homeostasis of an organism. Obtaining information on subcellular location of apoptosis proteins is very helpful for understanding the apoptosis mechanism. The prediction of subcellular localization of an apoptosis protein is still a challenging task, and existing methods mainly based on protein primary sequences. In this paper, we introduce a new position-specific scoring matrix (PSSM)-based method by using detrended cross-correlation (DCCA) coefficient of non-overlapping windows. Then a 190-dimensional (190D) feature vector is constructed on two widely used datasets: CL317 and ZD98, and support vector machine is adopted as classifier. To evaluate the proposed method, objective and rigorous jackknife cross-validation tests are performed on the two datasets. The results show that our approach offers a novel and reliable PSSM-based tool for prediction of apoptosis protein subcellular localization. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Distinct MicroRNA Subcellular Size and Expression Patterns in Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Beibei Chen

    2012-01-01

    Full Text Available Introduction. Small noncoding RNAs have important regulatory functions in different cell pathways. It is believed that most of them mainly play role in gene post-transcriptional regulation in the cytoplasm. Recent evidence suggests miRNA and siRNA activity in the nucleus. Here, we show distinct genome-wide sub-cellular localization distribution profiles of small noncoding RNAs in human breast cancer cells. Methods. We separated breast cancer cell nuclei from cytoplasm, and identified small RNA sequences using a high-throughput sequencing platform. To determine the relationship between miRNA sub-cellular distribution and cancer progression, we used microarray analysis to examine the miRNA expression levels in nucleus and cytoplasm of three human cell lines, one normal breast cell line and two breast cancer cell lines. Logistic regression and SVM were used for further analysis. Results. The sub-cellular distribution of small noncoding RNAs shows that numerous miRNAs and their isoforms (isomiR not only locate to the cytoplasm but also appeare in the nucleus. Subsequent microarray analyses indicated that the miRNA nuclear-cytoplasmic-ratio is a significant characteristic of different cancer cell lines. Conclusions. Our results indicate that the sub-cellular distribution is important for miRNA function, and that the characterization of the small RNAs sub-cellular localizome may contribute to cancer research and diagnosis.

  11. Molecular cytogenetics and DNA sequence analysis of an apomixis-linked BAC in Paspalum simplex reveal a non pericentromere location and partial microcolinearity with rice

    NARCIS (Netherlands)

    Calderini, O.; Chang, S.B.; Jong, de J.H.S.G.M.; Busti, A.; Paolocci, F.; Arcioni, S.; Vries, de S.C.; Abma-Henkens, M.H.C.; Klein Lankhorst, R.M.; Donnison, I.S.; Pupilli, F.

    2006-01-01

    Apomixis in plants is a form of clonal reproduction through seeds. A BAC clone linked to apomictic reproduction in Paspalum simplex was used to locate the apomixis locus on meiotic chromosome preparations. Fluorescent in situ hybridisation revealed the existence of a single locus embedded in a

  12. Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

    Science.gov (United States)

    Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

    2015-05-01

    Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1

  13. PSI: a comprehensive and integrative approach for accurate plant subcellular localization prediction.

    Directory of Open Access Journals (Sweden)

    Lili Liu

    Full Text Available Predicting the subcellular localization of proteins conquers the major drawbacks of high-throughput localization experiments that are costly and time-consuming. However, current subcellular localization predictors are limited in scope and accuracy. In particular, most predictors perform well on certain locations or with certain data sets while poorly on others. Here, we present PSI, a novel high accuracy web server for plant subcellular localization prediction. PSI derives the wisdom of multiple specialized predictors via a joint-approach of group decision making strategy and machine learning methods to give an integrated best result. The overall accuracy obtained (up to 93.4% was higher than best individual (CELLO by ~10.7%. The precision of each predicable subcellular location (more than 80% far exceeds that of the individual predictors. It can also deal with multi-localization proteins. PSI is expected to be a powerful tool in protein location engineering as well as in plant sciences, while the strategy employed could be applied to other integrative problems. A user-friendly web server, PSI, has been developed for free access at http://bis.zju.edu.cn/psi/.

  14. Comparison of the relative merits of port-location rearrangement and partial-feeding as the strategy for improving the performances of a three-zone simulated moving chromatography for separation of succinic acid and lactic acid.

    Science.gov (United States)

    Mun, Sungyong

    2014-05-09

    The three-zone simulated moving bed (SMB) chromatographic process for separation of succinic acid and lactic acid, which has been developed previously, was based on a classical port-location arrangement (desorbent→extract→feed→raffinate) and a classical feeding mode (full-feeding). To improve the performance of the three-zone SMB process, it is worth utilizing the strategy of either a port-location rearrangement (desorbent→feed→raffinate→extract) or a partial-feeding. To investigate which of the two strategies is more effective, the three-zone SMBs based on the port-location rearrangement (PR) and the partial-feeding (PF) were optimized each under equal conditions and then the two strategies were compared in terms of product purities or throughput. The result showed that the PR strategy led to higher purities or higher throughput than the PF strategy in regard to the previously reported three-zone SMB system. To check whether such trend is still valid in other separation systems, the above optimization works were repeated while varying only the selectivity between two feed components. It was confirmed that the PR strategy is definitely superior to the PF strategy. However, such superiority of the PR over the PF strategy is lessened as the selectivity becomes lower. If the selectivity is significantly low, the PR strategy is rather outperformed by the PF strategy. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Partial determination of the primary structure of a variant surface glycoprotein from Trypanosoma equiperdum. Composition and location of a carbohydrate moiety.

    Science.gov (United States)

    Duvillier, G; Richet, C; Briand, G; Baltz, T; Degand, P

    1983-05-01

    Salivarian trypanosomes have the ability to evade the immune response of their hosts by the sequential expression of different cell surface glycoproteins. Among the isolated specific antigens from cloned variants of Trypanosoma equiperdum, a structural study was undertaken on two immunologically cross-reacting variant surface glycoproteins, and results concerning the basic antigenic type are reported. The glycoprotein was cleaved by cyanogen bromide, and amino acids of several purified fractions obtained by gel filtration chromatography of this cleavage mixture were sequenced by automated Edman degradation. Sequencing in particular allowed the identification of the N-terminal portion of the molecule (residues 1-74). Sugar compositions of the fractions have demonstrated the presence of at least two carbohydrate moieties in the glycoprotein. Using a subsequent enzymatic subcleavage we were able to locate the first glycosylation site in position 57. An important observation was that the first oligosaccharide identified was rich in mannose and devoid of galactose.

  16. Comparison study of the partial-breast irradiation techniques: Dosimetric analysis of three-dimensional conformal radiation therapy, electron beam therapy, and helical tomotherapy depending on various tumor locations

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min-Joo; Park, So-Hyun [Department of Biomedical Engineering, The Catholic University of Korea, Seoul (Korea, Republic of); Research Institute of Biomedical Engineering, The Catholic University of Korea, Seoul (Korea, Republic of); Son, Seok-Hyun; Cheon, Keum-Seong; Choi, Byung-Ock [Department of Radiation Oncology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul (Korea, Republic of); Suh, Tae-Suk, E-mail: suhsanta@catholic.ac.kr [Department of Biomedical Engineering, The Catholic University of Korea, Seoul (Korea, Republic of); Research Institute of Biomedical Engineering, The Catholic University of Korea, Seoul (Korea, Republic of)

    2013-10-01

    The partial-breast irradiation (PBI) technique, an alternative to whole-breast irradiation, is a beam delivery method that uses a limited range of treatment volume. The present study was designed to determine the optimal PBI treatment modalities for 8 different tumor locations. Treatment planning was performed on computed tomography (CT) data sets of 6 patients who had received lumpectomy treatments. Tumor locations were classified into 8 subsections according to breast quadrant and depth. Three-dimensional conformal radiation therapy (3D-CRT), electron beam therapy (ET), and helical tomotherapy (H-TOMO) were utilized to evaluate the dosimetric effect for each tumor location. Conformation number (CN), radical dose homogeneity index (rDHI), and dose delivered to healthy tissue were estimated. The Kruskal-Wallis, Mann-Whitney U, and Bonferroni tests were used for statistical analysis. The ET approach showed good sparing effects and acceptable target coverage for the lower inner quadrant—superficial (LIQ-S) and lower inner quadrant—deep (LIQ-D) locations. The H-TOMO method was the least effective technique as no evaluation index achieved superiority for all tumor locations except CN. The ET method is advisable for treating LIQ-S and LIQ-D tumors, as opposed to 3D-CRT or H-TOMO, because of acceptable target coverage and much lower dose applied to surrounding tissue.

  17. Enhancing membrane protein subcellular localization prediction by parallel fusion of multi-view features.

    Science.gov (United States)

    Yu, Dongjun; Wu, Xiaowei; Shen, Hongbin; Yang, Jian; Tang, Zhenmin; Qi, Yong; Yang, Jingyu

    2012-12-01

    Membrane proteins are encoded by ~ 30% in the genome and function importantly in the living organisms. Previous studies have revealed that membrane proteins' structures and functions show obvious cell organelle-specific properties. Hence, it is highly desired to predict membrane protein's subcellular location from the primary sequence considering the extreme difficulties of membrane protein wet-lab studies. Although many models have been developed for predicting protein subcellular locations, only a few are specific to membrane proteins. Existing prediction approaches were constructed based on statistical machine learning algorithms with serial combination of multi-view features, i.e., different feature vectors are simply serially combined to form a super feature vector. However, such simple combination of features will simultaneously increase the information redundancy that could, in turn, deteriorate the final prediction accuracy. That's why it was often found that prediction success rates in the serial super space were even lower than those in a single-view space. The purpose of this paper is investigation of a proper method for fusing multiple multi-view protein sequential features for subcellular location predictions. Instead of serial strategy, we propose a novel parallel framework for fusing multiple membrane protein multi-view attributes that will represent protein samples in complex spaces. We also proposed generalized principle component analysis (GPCA) for feature reduction purpose in the complex geometry. All the experimental results through different machine learning algorithms on benchmark membrane protein subcellular localization datasets demonstrate that the newly proposed parallel strategy outperforms the traditional serial approach. We also demonstrate the efficacy of the parallel strategy on a soluble protein subcellular localization dataset indicating the parallel technique is flexible to suite for other computational biology problems. The

  18. Differential subcellular targeting and activity-dependent subcellular localization of diacylglycerol kinase isozymes in transfected cells.

    Science.gov (United States)

    Kobayashi, Naoki; Hozumi, Yasukazu; Ito, Tsukasa; Hosoya, Takaaki; Kondo, Hisatake; Goto, Kaoru

    2007-08-01

    Diacylglycerol kinase (DGK) plays a pivotal role in cellular signal transduction through regulating levels of the second messenger diacylglycerol (DG). Previous studies have revealed that DGK is composed of a family of isozymes that show remarkable heterogeneity in terms of molecular structure, functional domains, tissue and cellular gene expression. Recently, it has been shown that DG is produced in various subcellular compartments including the plasma membrane, internal membranes, cytoskeleton, and nucleus. However, it remains unclear how DG is regulated at distinct subcellular sites. To address this point, we have used an epitope-tag expression system in cultured cells and investigated the subcellular localization of DGK isozymes under the same experimental conditions. We show here that DGK isozymes are targeted differentially to unique subcellular sites in transfected COS7 cells, including the cytoplasm, actin stress fibers, Golgi complex, endoplasmic reticulum, and nucleus. It is also shown that among the isozymes overexpression of DGKbeta causes fragmentation of actin stress fibers while a kinase-dead mutant of DGKbeta abolishes its colocalization with actin stress fibers. These data strongly suggest that each isozyme may be responsible for the metabolism of DG that is produced upon stimulation at a different and specific subcellular site and that DGKbeta activity might have effects on the reorganization of actin stress fibers in transfected COS7 cells.

  19. Subcellular compartmentation of glutathione in dicotyledonous plants

    Science.gov (United States)

    Müller, Maria

    2010-01-01

    This study describes the subcellular distribution of glutathione in roots and leaves of different plant species (Arabidopsis, Cucurbita, and Nicotiana). Glutathione is an important antioxidant and redox buffer which is involved in many metabolic processes including plant defense. Thus information on the subcellular distribution in these model plants especially during stress situations provides a deeper insight into compartment specific defense reactions and reflects the occurrence of compartment specific oxidative stress. With immunogold cytochemistry and computer-supported transmission electron microscopy glutathione could be localized in highest contents in mitochondria, followed by nuclei, peroxisomes, the cytosol, and plastids. Within chloroplasts and mitochondria, glutathione was restricted to the stroma and matrix, respectively, and did not occur in the lumen of cristae and thylakoids. Glutathione was also found at the membrane and in the lumen of the endoplasmic reticulum. It was also associated with the trans and cis side of dictyosomes. None or only very little glutathione was detected in vacuoles and the apoplast of mesophyll and root cells. Additionally, glutathione was found in all cell compartments of phloem vessels, vascular parenchyma cells (including vacuoles) but was absent in xylem vessels. The specificity of this method was supported by the reduction of glutathione labeling in all cell compartments (up to 98%) of the glutathione-deficient Arabidopsis thaliana rml1 mutant. Additionally, we found a similar distribution of glutathione in samples after conventional fixation and rapid microwave-supported fixation. Thus, indicating that a redistribution of glutathione does not occur during sample preparation. Summing up, this study gives a detailed insight into the subcellular distribution of glutathione in plants and presents solid evidence for the accuracy and specificity of the applied method. PMID:20186447

  20. ngLOC: software and web server for predicting protein subcellular localization in prokaryotes and eukaryotes

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    King Brian R

    2012-07-01

    Full Text Available Abstract Background Understanding protein subcellular localization is a necessary component toward understanding the overall function of a protein. Numerous computational methods have been published over the past decade, with varying degrees of success. Despite the large number of published methods in this area, only a small fraction of them are available for researchers to use in their own studies. Of those that are available, many are limited by predicting only a small number of organelles in the cell. Additionally, the majority of methods predict only a single location for a sequence, even though it is known that a large fraction of the proteins in eukaryotic species shuttle between locations to carry out their function. Findings We present a software package and a web server for predicting the subcellular localization of protein sequences based on the ngLOC method. ngLOC is an n-gram-based Bayesian classifier that predicts subcellular localization of proteins both in prokaryotes and eukaryotes. The overall prediction accuracy varies from 89.8% to 91.4% across species. This program can predict 11 distinct locations each in plant and animal species. ngLOC also predicts 4 and 5 distinct locations on gram-positive and gram-negative bacterial datasets, respectively. Conclusions ngLOC is a generic method that can be trained by data from a variety of species or classes for predicting protein subcellular localization. The standalone software is freely available for academic use under GNU GPL, and the ngLOC web server is also accessible at http://ngloc.unmc.edu.

  1. Subcellular localization and internalization of the vasopressin V1B receptor.

    Science.gov (United States)

    Kashiwazaki, Aki; Fujiwara, Yoko; Tsuchiya, Hiroyoshi; Sakai, Nobuya; Shibata, Katsushi; Koshimizu, Taka-aki

    2015-10-15

    Only limited information is available on agonist-dependent changes in the subcellular localization of vasopressin V1B receptors. Our radioligand binding study of membrane preparations and intact cells revealed that a large fraction of the V1B receptor is located in the cytoplasm in unstimulated CHO cells, which is in contrast to the plasma membrane localization of the V1A and V2 receptors. Moreover, when the affinity of radiolabeled arginine-vasopressin ([3H]AVP) was compared between membrane preparations and intact cells, the affinity of [3H]AVP to the cell surface V1B receptors, but not the V1A receptors, was significantly reduced. Although the number and affinity of cell surface V1B receptors decreased, they became extensively internalized upon binding with [3H]AVP. Approximately 87% of cell surface-bound [3H]AVP was internalized and became resistant to acid wash during incubation with 1 nM [3H]AVP. By contrast, less ligand (35%) was internalized in the cells expressing the V1A receptor. Extensive internalization of the V1B receptors was partially attenuated by inhibitors of cytoskeletal proteins, siRNA against β-arrestin 2, or the removal of sodium chloride from the extracellular buffer, indicating that this internalization involves clathrin-coated pits. Together, these results indicate that the mechanism that regulates the number and affinity of V1B receptors in the plasma membrane is markedly distinct from the corresponding mechanisms for the V1A and V2 receptors and plays a critical role under stress conditions, when vasopressin release is augmented. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Reducing radiation dose to the female breast during CT coronary angiography: A simulation study comparing breast shielding, angular tube current modulation, reduced kV, and partial angle protocols using an unknown-location signal-detectability metric

    Energy Technology Data Exchange (ETDEWEB)

    Rupcich, Franco; Gilat Schmidt, Taly [Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin 53233 (United States); Badal, Andreu; Popescu, Lucretiu M.; Kyprianou, Iacovos [Center for Devices and Radiological Health, US Food and Drug Administration, Silver Spring, Maryland 20905 (United States)

    2013-08-15

    Purpose: The authors compared the performance of five protocols intended to reduce dose to the breast during computed tomography (CT) coronary angiography scans using a model observer unknown-location signal-detectability metric.Methods: The authors simulated CT images of an anthropomorphic female thorax phantom for a 120 kV reference protocol and five “dose reduction” protocols intended to reduce dose to the breast: 120 kV partial angle (posteriorly centered), 120 kV tube-current modulated (TCM), 120 kV with shielded breasts, 80 kV, and 80 kV partial angle (posteriorly centered). Two image quality tasks were investigated: the detection and localization of 4-mm, 3.25 mg/ml and 1-mm, 6.0 mg/ml iodine contrast signals randomly located in the heart region. For each protocol, the authors plotted the signal detectability, as quantified by the area under the exponentially transformed free response characteristic curve estimator (A-caret{sub FE}), as well as noise and contrast-to-noise ratio (CNR) versus breast and lung dose. In addition, the authors quantified each protocol's dose performance as the percent difference in dose relative to the reference protocol achieved while maintaining equivalent A-caret{sub FE}.Results: For the 4-mm signal-size task, the 80 kV full scan and 80 kV partial angle protocols decreased dose to the breast (80.5% and 85.3%, respectively) and lung (80.5% and 76.7%, respectively) with A-caret{sub FE} = 0.96, but also resulted in an approximate three-fold increase in image noise. The 120 kV partial protocol reduced dose to the breast (17.6%) at the expense of increased lung dose (25.3%). The TCM algorithm decreased dose to the breast (6.0%) and lung (10.4%). Breast shielding increased breast dose (67.8%) and lung dose (103.4%). The 80 kV and 80 kV partial protocols demonstrated greater dose reductions for the 4-mm task than for the 1-mm task, and the shielded protocol showed a larger increase in dose for the 4-mm task than for the 1-mm

  3. Fast subcellular localization by cascaded fusion of signal-based and homology-based methods

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2011-10-01

    Full Text Available Abstract Background The functions of proteins are closely related to their subcellular locations. In the post-genomics era, the amount of gene and protein data grows exponentially, which necessitates the prediction of subcellular localization by computational means. Results This paper proposes mitigating the computation burden of alignment-based approaches to subcellular localization prediction by a cascaded fusion of cleavage site prediction and profile alignment. Specifically, the informative segments of protein sequences are identified by a cleavage site predictor using the information in their N-terminal shorting signals. Then, the sequences are truncated at the cleavage site positions, and the shortened sequences are passed to PSI-BLAST for computing their profiles. Subcellular localization are subsequently predicted by a profile-to-profile alignment support-vector-machine (SVM classifier. To further reduce the training and recognition time of the classifier, the SVM classifier is replaced by a new kernel method based on the perturbational discriminant analysis (PDA. Conclusions Experimental results on a new dataset based on Swiss-Prot Release 57.5 show that the method can make use of the best property of signal- and homology-based approaches and can attain an accuracy comparable to that achieved by using full-length sequences. Analysis of profile-alignment score matrices suggest that both profile creation time and profile alignment time can be reduced without significant reduction in subcellular localization accuracy. It was found that PDA enjoys a short training time as compared to the conventional SVM. We advocate that the method will be important for biologists to conduct large-scale protein annotation or for bioinformaticians to perform preliminary investigations on new algorithms that involve pairwise alignments.

  4. Subcellular targeting and biosynthesis of cyclotides in plant cells.

    Science.gov (United States)

    Conlan, Brendon F; Gillon, Amanda D; Barbeta, Barbara L; Anderson, Marilyn A

    2011-12-01

    The cyclotide kalata B1 is found in the leaves of Oldenlandia affinis and is a potent insecticidal and nematocidal molecule. This peptide is cleaved from a precursor protein, Oak1, and ligation of the N- and C-termini occurs to form a continuous peptide backbone. The subcellular location of the excision and cyclization reactions is unknown, and there is debate as to which enzyme catalyzes the event. To determine where in the plant cell Oak1 is processed, we prepared constructs encoding GFP (green fluorescent protein) linked to the cyclotide precursor Oak1. The GFP constructs were transiently expressed in the leaves of Nicotiana benthamiana, and GFP fluorescence was observed in living cells using confocal microscopy. A Fei Mao (FM) styryl dye was infiltrated into whole leaves that were still growing and expressing GFP constructs, enabling the plasma membrane and the tonoplast to be highlighted for visualization of the vacuole in living cells. The full length Oak1 precursor directed GFP to the vacuole, suggesting that excision and cyclization of the cyclotide domain occurs in the vacuole where the cyclotides are then stored. The N-terminal propeptide and N-terminal repeat of Oak1 were both sufficient to target GFP to the vacuole, although the C-terminal propeptide, which is essential for cyclization, was not a targeting signal. The vacuolar location of cyclotides supports our hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.

  5. Subcellular targeting: a new frontier for drug-loaded pharmaceutical nanocarriers and the concept of the magic bullet.

    Science.gov (United States)

    D'Souza, Gerard G M; Weissig, Volkmar

    2009-11-01

    The ability of a pharmacologically active molecule selectively to find its target is closely linked with its potential as a successful therapeutic drug. It has become increasingly evident that there are several pharmacologically active molecules that exert their action on molecular targets inside cell organelles. In the case of a drug molecule with no defined specificity for a particular organelle, the molecule would either need to have sufficiently long metabolic stability to allow for random interaction with the organelle to occur, or a targeting strategy for the intended subcellular compartment would need to be devised in order to potentiate therapeutic effect. In the case of molecules with a stronger affinity for a non-target subcellular compartment, there exists even greater need for the ability to control subcellular disposition. Subcellular or organelle-specific targeting has thus emerged as a new frontier in drug delivery. In this review selected examples of recent work are discussed that the authors believe might eventually lead to the application of pharmaceutical nanocarriers to create the next generation of 'magic bullets' that are capable of delivering a drug payload to a molecular target at a subcellular location.

  6. Subcellular Localization of Class I Histone Deacetylases in the Developing Xenopus tectum.

    Science.gov (United States)

    Guo, Xia; Ruan, Hangze; Li, Xia; Qin, Liming; Tao, Yi; Qi, Xianjie; Gao, Juanmei; Gan, Lin; Duan, Shumin; Shen, Wanhua

    2015-01-01

    Histone deacetylases (HDACs) are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis, and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus, and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus, and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2, and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12) remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA) or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

  7. Subcellular localization of class I histone deacetylases in the developing Xenopus tectum

    Directory of Open Access Journals (Sweden)

    Xia eGuo

    2016-01-01

    Full Text Available Histone deacetylases (HDACs are thought to localize in the nucleus to regulate gene transcription and play pivotal roles in neurogenesis, apoptosis and plasticity. However, the subcellular distribution of class I HDACs in the developing brain remains unclear. Here, we show that HDAC1 and HDAC2 are located in both the mitochondria and the nucleus in the Xenopus laevis stage 34 tectum and are mainly restricted to the nucleus following further brain development. HDAC3 is widely present in the mitochondria, nucleus and cytoplasm during early tectal development and is mainly distributed in the nucleus in stage 45 tectum. In contrast, HDAC8 is broadly located in the mitochondria, nucleus and cytoplasm during tectal development. These data demonstrate that HDAC1, HDAC2 and HDAC3 are transiently localized in the mitochondria and that the subcellular distribution of class I HDACs in the Xenopus tectum is heterogeneous. Furthermore, we observed that spherical mitochondria accumulate in the cytoplasm at earlier stages, whereas elongated mitochondria are evenly distributed in the tectum at later stages. The activity of histone acetylation (H4K12 remains low in mitochondria during tectal development. Pharmacological blockades of HDACs using a broad spectrum HDAC inhibitor of Trichostatin A (TSA or specific class I HDAC inhibitors of MS-275 and MGCD0103 decrease the number of mitochondria in the tectum at stage 34. These findings highlight a link between the subcellular distribution of class I HDACs and mitochondrial dynamics in the developing optic tectum of Xenopus laevis.

  8. Subcellular boron and fluorine distributions with SIMS ion microscopy in BNCT and cancer research

    Energy Technology Data Exchange (ETDEWEB)

    Subhash Chandra

    2008-05-30

    The development of a secondary ion mass spectrometry (SIMS) based technique of Ion Microscopy in boron neutron capture therapy (BNCT) was the main goal of this project, so that one can study the subcellular location of boron-10 atoms and their partitioning between the normal and cancerous tissue. This information is fundamental for the screening of boronated drugs appropriate for neutron capture therapy of cancer. Our studies at Cornell concentrated mainly on studies of glioblastoma multiforme (GBM). The early years of the grant were dedicated to the development of cryogenic methods and correlative microscopic approaches so that a reliable subcellular analysis of boron-10 atoms can be made with SIMS. In later years SIMS was applied to animal models and human tissues of GBM for studying the efficacy of potential boronated agents in BNCT. Under this grant the SIMS program at Cornell attained a new level of excellence and collaborative SIMS studies were published with leading BNCT researchers in the U.S.

  9. Subcellular Compartmentalization and Trafficking of the Biosynthetic Machinery for Fungal Melanin

    Directory of Open Access Journals (Sweden)

    Srijana Upadhyay

    2016-03-01

    Full Text Available Protection by melanin depends on its subcellular location. Although most filamentous fungi synthesize melanin via a polyketide synthase pathway, where and how melanin biosynthesis occurs and how it is deposited as extracellular granules remain elusive. Using a forward genetic screen in the pathogen Aspergillus fumigatus, we find that mutations in an endosomal sorting nexin abolish melanin cell-wall deposition. We find that all enzymes involved in the early steps of melanin biosynthesis are recruited to endosomes through a non-conventional secretory pathway. In contrast, late melanin enzymes accumulate in the cell wall. Such subcellular compartmentalization of the melanin biosynthetic machinery occurs in both A. fumigatus and A. nidulans. Thus, fungal melanin biosynthesis appears to be initiated in endosomes with exocytosis leading to melanin extracellular deposition, much like the synthesis and trafficking of mammalian melanin in endosomally derived melanosomes.

  10. Characterization of RanBPM Molecular Determinants that Control Its Subcellular Localization

    Science.gov (United States)

    Salemi, Louisa M.; Loureiro, Sandra O.; Schild-Poulter, Caroline

    2015-01-01

    RanBPM/RanBP9 is a ubiquitous, nucleocytoplasmic protein that is part of an evolutionary conserved E3 ubiquitin ligase complex whose function and targets in mammals are still unknown. RanBPM itself has been implicated in various cellular processes that involve both nuclear and cytoplasmic functions. However, to date, little is known about how RanBPM subcellular localization is regulated. We have conducted a systematic analysis of RanBPM regions that control its subcellular localization using RanBPM shRNA cells to examine ectopic RanBPM mutant subcellular localization without interference from the endogenously expressed protein. We show that several domains and motifs regulate RanBPM nuclear and cytoplasmic localization. In particular, RanBPM comprises two motifs that can confer nuclear localization, one proline/glutamine-rich motif in the extreme N-terminus which has a dominant effect on RanBPM localization, and a second motif in the C-terminus which minimally contributes to RanBPM nuclear targeting. We also identified a nuclear export signal (NES) which mutation prevented RanBPM accumulation in the cytoplasm. Likewise, deletion of the central RanBPM conserved domains (SPRY and LisH/CTLH) resulted in the relocalization of RanBPM to the nucleus, suggesting that RanBPM cytoplasmic localization is also conferred by protein-protein interactions that promote its cytoplasmic retention. Indeed we found that in the cytoplasm, RanBPM partially colocalizes with microtubules and associates with α-tubulin. Finally, in the nucleus, a significant fraction of RanBPM is associated with chromatin. Altogether, these analyses reveal that RanBPM subcellular localization results from the combined effects of several elements that either confer direct transport through the nucleocytoplasmic transport machinery or regulate it indirectly, likely through interactions with other proteins and by intramolecular folding. PMID:25659156

  11. A formal ontology of subcellular neuroanatomy

    Directory of Open Access Journals (Sweden)

    Stephen D Larson

    2007-11-01

    Full Text Available The complexity of the nervous system requires high-resolution microscopy to resolve the detailed 3D structure of nerve cells and supracellular domains. The analysis of such imaging data to extract cellular surfaces and cell components often requires the combination of expert human knowledge with carefully engineered software tools. In an effort to make better tools to assist humans in this endeavor, create a more accessible and permanent record of their data, and to aid the process of constructing complex and detailed computational models, we have created a core of formalized knowledge about the structure of the nervous system and have integrated that core into several software applications. In this paper, we describe the structure and content of a formal ontology whose scope is the subcellular anatomy of the nervous system (SAO, covering nerve cells, their parts, and interactions between these parts. Many applications of this ontology to image annotation, content-based retrieval of structural data, and integration of shared data across scales and researchers are also described.

  12. Subcellular drug targeting, pharmacokinetics and bioavailability.

    Science.gov (United States)

    Leucuta, Sorin Emilian

    2014-02-01

    Effective treatment of diseases at the molecular level is possible by directing the drug substance (micromolecular, protein or peptide drugs, DNA, oligonucleotides, siRNA) with the aid of a specialized nanoparticulate carrier, for safe and effective transport to the specific site of action in the cytosol and its organelles including nuclear targeting. To achieve efficient cytosolic delivery of therapeutics or nuclear targeting, different drug delivery systems (DDS) have been developed (macromolecular drug conjugates, chemically or genetically modified proteins, and particulate drug carriers) capable of subcellular internalization overcoming the biological barriers, by passive targeting and especially by active targeting (receptor-targeted delivery). The success depends on the physicochemical nature of DDS, intracellular barriers that these systems need to overcome, the bioavailability of the bioactive drug, biodistribution, the intracellular pharmacokinetics and its influence on the pharmacodynamic effect. Models necessary for this purpose exist but they need to be more developed especially with quantitative treatments, after the development of the means of highlighting the evolution of the drug substance in biophase or at the level of the target cellular organelle by quantitative assays. It is expected that intracellularly targeted drug delivery approaches will be clinically useful using specialized DDSs belonging to the pharmaceutical nanotechnologies.

  13. cAMP signaling in subcellular compartments.

    Science.gov (United States)

    Lefkimmiatis, Konstantinos; Zaccolo, Manuela

    2014-09-01

    In the complex microcosm of a cell, information security and its faithful transmission are critical for maintaining internal stability. To achieve a coordinated response of all its parts to any stimulus the cell must protect the information received from potentially confounding signals. Physical segregation of the information transmission chain ensures that only the entities able to perform the encoded task have access to the relevant information. The cAMP intracellular signaling pathway is an important system for signal transmission responsible for the ancestral 'flight or fight' response and involved in the control of critical functions including frequency and strength of heart contraction, energy metabolism and gene transcription. It is becoming increasingly apparent that the cAMP signaling pathway uses compartmentalization as a strategy for coordinating the large number of key cellular functions under its control. Spatial confinement allows the formation of cAMP signaling "hot spots" at discrete subcellular domains in response to specific stimuli, bringing the information in proximity to the relevant effectors and their recipients, thus achieving specificity of action. In this report we discuss how the different constituents of the cAMP pathway are targeted and participate in the formation of cAMP compartmentalized signaling events. We illustrate a few examples of localized cAMP signaling, with a particular focus on the nucleus, the sarcoplasmic reticulum and the mitochondria. Finally, we discuss the therapeutic potential of interventions designed to perturb specific cAMP cascades locally. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Multimodal subcellular imaging with microcavity photoacoustic transducer.

    Science.gov (United States)

    Tan, Zhiliang; Tang, Zhilie; Wu, Yongbo; Liao, Yanfei; Dong, Wei; Guo, Lina

    2011-01-31

    Photoacoustic microscopy (PAM) is dominantly sensitive to the endogenous optical absorption compared with the confocal microscopy which images with scattering photons. PAM has similar structure such as optical transportation system, the optical scanning, and light source with the laser scanning confocal microscopy (LSCM). In order to match the PAM with LSCM, a special design microcavity photoacoustic (PA) transducer with high sensitivity is developed to detect the photoacoustic signals induced by modulated continuous wave (CW) laser. By employing a microcavity PA transducer, a PAM can be integrated with LSCM. Thus a simultaneous multimodal imaging can be obtained with the same laser source and optical system. The lateral resolutions of the PAM and the LSCM are both tested to be better than 1.25 μm. Then subcellular multimodal imaging can be achieved. Images from the two modes are corresponding with each other but functionally complementary. Combining PAM and LSCM provides more comprehensive information for the cytological test. This technique is demonstrated for imaging red-blood cells and meristematic cells.

  15. Zymogen Activation and Subcellular Activity of Subtilisin Kexin Isozyme 1/Site 1 Protease*

    Science.gov (United States)

    da Palma, Joel Ramos; Burri, Dominique Julien; Oppliger, Joël; Salamina, Marco; Cendron, Laura; de Laureto, Patrizia Polverino; Seidah, Nabil Georges; Kunz, Stefan; Pasquato, Antonella

    2014-01-01

    The proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) plays crucial roles in cellular homeostatic functions and is hijacked by pathogenic viruses for the processing of their envelope glycoproteins. Zymogen activation of SKI-1/S1P involves sequential autocatalytic processing of its N-terminal prodomain at sites B′/B followed by the herein newly identified C′/C sites. We found that SKI-1/S1P autoprocessing results in intermediates whose catalytic domain remains associated with prodomain fragments of different lengths. In contrast to other zymogen proprotein convertases, all incompletely matured intermediates of SKI-1/S1P showed full catalytic activity toward cellular substrates, whereas optimal cleavage of viral glycoproteins depended on B′/B processing. Incompletely matured forms of SKI-1/S1P further process cellular and viral substrates in distinct subcellular compartments. Using a cell-based sensor for SKI-1/S1P activity, we found that 9 amino acid residues at the cleavage site (P1–P8) and P1′ are necessary and sufficient to define the subcellular location of processing and to determine to what extent processing of a substrate depends on SKI-1/S1P maturation. In sum, our study reveals novel and unexpected features of SKI-1/S1P zymogen activation and subcellular specificity of activity toward cellular and pathogen-derived substrates. PMID:25378398

  16. Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum.

    Directory of Open Access Journals (Sweden)

    Wei Gao

    Full Text Available The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC imaging method for cadmium ions (Cd2+ was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

  17. HECTAR: a method to predict subcellular targeting in heterokonts

    National Research Council Canada - National Science Library

    Gschloessl, Bernhard; Guermeur, Yann; Cock, J Mark

    2008-01-01

    .... To understand the biology of these organisms, it is necessary to be able to predict the subcellular localisation of their proteins but this is not straightforward, particularly in photosynthetic...

  18. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    Science.gov (United States)

    Vertes, Akos; Stolee, Jessica A; Shrestha, Bindesh

    2014-12-02

    In various embodiments, a method of laser ablation electrospray ionization mass spectrometry (LAESI-MS) may generally comprise micro-dissecting a cell comprising at least one of a cell wall and a cell membrane to expose at least one subcellular component therein, ablating the at least one subcellular component by an infrared laser pulse to form an ablation plume, intercepting the ablation plume by an electrospray plume to form ions, and detecting the ions by mass spectrometry.

  19. Proteome-wide Subcellular Topologies of E. coli Polypeptides Database (STEPdb)*

    Science.gov (United States)

    Orfanoudaki, Georgia; Economou, Anastassios

    2014-01-01

    Cell compartmentalization serves both the isolation and the specialization of cell functions. After synthesis in the cytoplasm, over a third of all proteins are targeted to other subcellular compartments. Knowing how proteins are distributed within the cell and how they interact is a prerequisite for understanding it as a whole. Surface and secreted proteins are important pathogenicity determinants. Here we present the STEP database (STEPdb) that contains a comprehensive characterization of subcellular localization and topology of the complete proteome of Escherichia coli. Two widely used E. coli proteomes (K-12 and BL21) are presented organized into thirteen subcellular classes. STEPdb exploits the wealth of genetic, proteomic, biochemical, and functional information on protein localization, secretion, and targeting in E. coli, one of the best understood model organisms. Subcellular annotations were derived from a combination of bioinformatics prediction, proteomic, biochemical, functional, topological data and extensive literature re-examination that were refined through manual curation. Strong experimental support for the location of 1553 out of 4303 proteins was based on 426 articles and some experimental indications for another 526. Annotations were provided for another 320 proteins based on firm bioinformatic predictions. STEPdb is the first database that contains an extensive set of peripheral IM proteins (PIM proteins) and includes their graphical visualization into complexes, cellular functions, and interactions. It also summarizes all currently known protein export machineries of E. coli K-12 and pairs them, where available, with the secretory proteins that use them. It catalogs the Sec- and TAT-utilizing secretomes and summarizes their topological features such as signal peptides and transmembrane regions, transmembrane topologies and orientations. It also catalogs physicochemical and structural features that influence topology such as abundance

  20. Structural and functional plasticity of subcellular tethering, targeting and processing of RPGRIP1 by RPGR isoforms

    Directory of Open Access Journals (Sweden)

    Hemangi Patil

    2012-02-01

    Mutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1 interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR through a domain homologous to RCC1 (RHD, a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID of RPGRIP1 embraces multivalently the shared RHD of RPGR1–19 and RPGRORF15 isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1α1 expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α1 self-aggregation ensue. RPGR1–19 localizes to the endoplasmic reticulum, whereas RPGRORF15 presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α1. Disease mutations in RPGR1–19, RPGRORF15, or RID of RPGRIP1α1, singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α1 with RPGR1–19 or RPGRORF15 in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGRORF15, but not RPGR1–19, protects the RID of RPGRIP1α1 from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1α1, with deficits in RPGRORF15-dependent intracellular localization of RPGRIP1α1 contributing to pathomechanisms shared by etiologically distinct syndromic retinal dystrophies.

  1. Subcellular distribution of calcium during spermatogenesis of zebrafish, Danio rerio.

    Science.gov (United States)

    Golpour, Amin; Pšenička, Martin; Niksirat, Hamid

    2017-08-01

    Calcium plays a variety of vital regulatory functions in many physiological and biochemical events in the cell. The aim of this study was to describe the ultrastructural distribution of calcium during different developmental stages of spermatogenesis in a model organism, the zebrafish (Danio rerio), using a combined oxalate-pyroantimonate technique. Samples were treated by potassium oxalate and potassium pyroantimonate during two fixation stages and examined using transmission electron microscopy to detect electron dense intracellular calcium. The subcellular distribution of intracellular calcium was characterized in spermatogonium, spermatocyte, spermatid, and spermatozoon stages. The area which is covered by intracellular calcium in different stages was quantified and compared using software. Isolated calcium deposits were mainly detectable in the cytoplasm and the nucleus of the spermatogonium and spermatocyte. In the spermatid, calcium was partially localized in the cytoplasm as isolated deposits. However, most calcium was transformed from isolated deposits into an unbound pool (free calcium) within the nucleus of the spermatid and the spermatozoon. Interestingly, in the spermatozoon, calcium was mainly localized in a form of an unbound pool which was detectable as an electron-dense mass within the nucleus. Also, sporadic calcium deposits were scattered in the midpiece and flagellum. The proportional area which was covered by intracellular calcium increased significantly from early to late stages of spermatogenesis. The extent of the area which was covered by intracellular calcium in the spermatozoon was the highest compared to earlier stages. Calcium deposits were also observed in the somatic cells (Sertoli, myoid, Leydig) of zebrafish testis. The notable changes in the distribution of intracellular calcium of germ cells during different developmental stages of zebrafish spermatogenesis suggest its different homeostasis and physiological functions during the

  2. Analysis of subcellular metabolite distributions within Arabidopsis thaliana leaf tissue: a primer for subcellular metabolomics.

    Science.gov (United States)

    Krueger, Stephan; Steinhauser, Dirk; Lisec, Jan; Giavalisco, Patrick

    2014-01-01

    Every biological organism relies for its proper function on interactions between a multitude of molecular entities like RNA, proteins, and metabolites. The comprehensive measurement and the analysis of all these entities would therefore provide the basis for our functional and mechanistic understanding of most biological processes. Next to their amount and identity, it is most crucial to also gain information about the subcellular distribution and the flux of the measured compounds between the cellular compartments. That is, we want to understand not only the individual functions of cellular components but also their functional implications within the whole organism. While the analysis of macromolecules like DNA, RNA, and proteins is quite established and robust, analytical techniques for small metabolites, which are prone to diffusion and degradation processes, provide a host of unsolved challenges. The major limitations here are the metabolite conversion and relocation processes. In this protocol we describe a methodological workflow which includes a nonaqueous fractionation method, a fractionated two-phase liquid/liquid extraction protocol, and a software package, which together allow extracting and analyzing starch, proteins, and especially polar and lipophilic metabolites from a single sample towards the estimation of their subcellular distributions.

  3. Inhibition of the Secretory pathway by Foot-and-Mouth disease virus 2BC protein is reproduced by co-expression of 2B with 2C, and the site of inhibition is determined by the subcellular location of 2C

    DEFF Research Database (Denmark)

    Moffat, Katy; Knox, Caroline; Howell, Gareth

    2007-01-01

    immune responses in vivo. Foot-and-mouth disease virus (FMDV), another picornavirus, can cause persistent infection of ruminants, suggesting it too may inhibit immune responses. Endoplasmic reticulum (ER)-to-Golgi apparatus transport of proteins is blocked by the FMDV 2BC protein. The observation that 2...... blocked in FMDV-infected cells. The block could be reconstituted by coexpression of 2B and 2C, showing that processing of 2BC did not compromise the ability of FMDV to slow secretion. Under these conditions, 2C was located to the Golgi apparatus, and the block in transport also occurred in the Golgi...... apparatus. Interestingly, the block in transport could be redirected to the ER when 2B was coexpressed with a 2C protein fused to an ER retention element. Thus, for FMDV a block in secretion is dependent on both 2B and 2C, with the latter determining the site of the block....

  4. Ischemia-related subcellular redistribution of sodium channels enhances the proarrhythmic effect of class I antiarrhythmic drugs: a simulation study.

    Directory of Open Access Journals (Sweden)

    Kunichika Tsumoto

    Full Text Available Cardiomyocytes located at the ischemic border zone of infarcted ventricle are accompanied by redistribution of gap junctions, which mediate electrical transmission between cardiomyocytes. This ischemic border zone provides an arrhythmogenic substrate. It was also shown that sodium (Na+ channels are redistributed within myocytes located in the ischemic border zone. However, the roles of the subcellular redistribution of Na+ channels in the arrhythmogenicity under ischemia remain unclear.Computer simulations of excitation conduction were performed in a myofiber model incorporating both subcellular Na+ channel redistribution and the electric field mechanism, taking into account the intercellular cleft potentials.We found in the myofiber model that the subcellular redistribution of the Na+ channels under myocardial ischemia, decreasing in Na+ channel expression of the lateral cell membrane of each myocyte, decreased the tissue excitability, resulting in conduction slowing even without any ischemia-related electrophysiological change. The conventional model (i.e., without the electric field mechanism did not reproduce the conduction slowing caused by the subcellular Na+ channel redistribution. Furthermore, Na+ channel blockade with the coexistence of a non-ischemic zone with an ischemic border zone expanded the vulnerable period for reentrant tachyarrhythmias compared to the model without the ischemic border zone. Na+ channel blockade tended to cause unidirectional conduction block at sites near the ischemic border zone. Thus, such a unidirectional conduction block induced by a premature stimulus at sites near the ischemic border zone is associated with the initiation of reentrant tachyarrhythmias.Proarrhythmia of Na+ channel blockade in patients with old myocardial infarction might be partly attributable to the ischemia-related subcellular Na+ channel redistribution.

  5. Using the SUBcellular database for Arabidopsis proteins to localize the Deg protease family

    Science.gov (United States)

    Tanz, Sandra K.; Castleden, Ian; Hooper, Cornelia M.; Small, Ian; Millar, A. Harvey

    2014-01-01

    Sub-functionalization during the expansion of gene families in eukaryotes has occurred in part through specific subcellular localization of different family members. To better understand this process in plants, compiled records of large-scale proteomic and fluorescent protein localization datasets can be explored and bioinformatic predictions for protein localization can be used to predict the gaps in experimental data. This process can be followed by targeted experiments to test predictions. The SUBA3 database is a free web-service at http://suba.plantenergy.uwa.edu.au that helps users to explore reported experimental data and predictions concerning proteins encoded by gene families and to define the experiments required to locate these homologous sets of proteins. Here we show how SUBA3 can be used to explore the subcellular location of the Deg protease family of ATP-independent serine endopeptidases (Deg1–Deg16). Combined data integration and new experiments refined location information for Deg1 and Deg9, confirmed Deg2, Deg5, and Deg8 in plastids and Deg 15 in peroxisomes and provide substantial experimental evidence for mitochondrial localized Deg proteases. Two of these, Deg3 and Deg10, additionally localized to the plastid, revealing novel dual-targeted Deg proteases in the plastid and the mitochondrion. SUBA3 is continually updated to ensure that researchers can use the latest published data when planning the experimental steps remaining to localize gene family functions. PMID:25161662

  6. Partial priapism

    DEFF Research Database (Denmark)

    Hoyerup, Peter; Azawi, Nessn Htum

    2013-01-01

    With only 34 prior cases in world literature, partial priapism (PP), also called partial segmental thrombosis of the corpus cavernosum, is a rare urological condition. The aetiology and treatment of PP is still unclear, but bicycle riding, trauma, drug usage, sexual intercourse, haematological...

  7. Gram-positive and gram-negative subcellular localization using rotation forest and physicochemical-based features

    Science.gov (United States)

    2015-01-01

    Background The functioning of a protein relies on its location in the cell. Therefore, predicting protein subcellular localization is an important step towards protein function prediction. Recent studies have shown that relying on Gene Ontology (GO) for feature extraction can improve the prediction performance. However, for newly sequenced proteins, the GO is not available. Therefore, for these cases, the prediction performance of GO based methods degrade significantly. Results In this study, we develop a method to effectively employ physicochemical and evolutionary-based information in the protein sequence. To do this, we propose segmentation based feature extraction method to explore potential discriminatory information based on physicochemical properties of the amino acids to tackle Gram-positive and Gram-negative subcellular localization. We explore our proposed feature extraction techniques using 10 attributes that have been experimentally selected among a wide range of physicochemical attributes. Finally by applying the Rotation Forest classification technique to our extracted features, we enhance Gram-positive and Gram-negative subcellular localization accuracies up to 3.4% better than previous studies which used GO for feature extraction. Conclusion By proposing segmentation based feature extraction method to explore potential discriminatory information based on physicochemical properties of the amino acids as well as using Rotation Forest classification technique, we are able to enhance the Gram-positive and Gram-negative subcellular localization prediction accuracies, significantly. PMID:25734546

  8. Protein subcellular localization assays using split fluorescent proteins

    Science.gov (United States)

    Waldo, Geoffrey S [Santa Fe, NM; Cabantous, Stephanie [Los Alamos, NM

    2009-09-08

    The invention provides protein subcellular localization assays using split fluorescent protein systems. The assays are conducted in living cells, do not require fixation and washing steps inherent in existing immunostaining and related techniques, and permit rapid, non-invasive, direct visualization of protein localization in living cells. The split fluorescent protein systems used in the practice of the invention generally comprise two or more self-complementing fragments of a fluorescent protein, such as GFP, wherein one or more of the fragments correspond to one or more beta-strand microdomains and are used to "tag" proteins of interest, and a complementary "assay" fragment of the fluorescent protein. Either or both of the fragments may be functionalized with a subcellular targeting sequence enabling it to be expressed in or directed to a particular subcellular compartment (i.e., the nucleus).

  9. HECTAR: a method to predict subcellular targeting in heterokonts.

    Science.gov (United States)

    Gschloessl, Bernhard; Guermeur, Yann; Cock, J Mark

    2008-09-23

    The heterokonts are a particularly interesting group of eukaryotic organisms; they include many key species of planktonic and coastal algae and several important pathogens. To understand the biology of these organisms, it is necessary to be able to predict the subcellular localisation of their proteins but this is not straightforward, particularly in photosynthetic heterokonts which possess a complex chloroplast, acquired as the result of a secondary endosymbiosis. This is because the bipartite target peptides that deliver proteins to these chloroplasts can be easily confused with the signal peptides of secreted proteins, causing currently available algorithms to make erroneous predictions. HECTAR, a subcellular targeting prediction method which takes into account the specific properties of heterokont proteins, has been developed to address this problem. HECTAR is a statistical prediction method designed to assign proteins to five different categories of subcellular targeting: Signal peptides, type II signal anchors, chloroplast transit peptides, mitochondrion transit peptides and proteins which do not possess any N-terminal target peptide. The recognition rate of HECTAR is 96.3%, with Matthews correlation coefficients ranging from 0.67 to 0.95. The method is based on a hierarchical architecture which implements the divide and conquer approach to identify the different possible target peptides one at a time. At each node of the hierarchy, the most relevant outputs of various existing subcellular prediction methods are combined by a Support Vector Machine. The HECTAR method is able to predict the subcellular localisation of heterokont proteins with high accuracy. It also efficiently predicts the subcellular localisation of proteins from cryptophytes, a group that is phylogenetically close to the heterokonts. A variant of HECTAR, called HECTARSEC, can be used to identify signal peptide and type II signal anchor sequences in proteins from any eukaryotic organism. Both

  10. HECTAR: A method to predict subcellular targeting in heterokonts

    Directory of Open Access Journals (Sweden)

    Guermeur Yann

    2008-09-01

    Full Text Available Abstract Background The heterokonts are a particularly interesting group of eukaryotic organisms; they include many key species of planktonic and coastal algae and several important pathogens. To understand the biology of these organisms, it is necessary to be able to predict the subcellular localisation of their proteins but this is not straightforward, particularly in photosynthetic heterokonts which possess a complex chloroplast, acquired as the result of a secondary endosymbiosis. This is because the bipartite target peptides that deliver proteins to these chloroplasts can be easily confused with the signal peptides of secreted proteins, causing currently available algorithms to make erroneous predictions. HECTAR, a subcellular targeting prediction method which takes into account the specific properties of heterokont proteins, has been developed to address this problem. Results HECTAR is a statistical prediction method designed to assign proteins to five different categories of subcellular targeting: Signal peptides, type II signal anchors, chloroplast transit peptides, mitochondrion transit peptides and proteins which do not possess any N-terminal target peptide. The recognition rate of HECTAR is 96.3%, with Matthews correlation coefficients ranging from 0.67 to 0.95. The method is based on a hierarchical architecture which implements the divide and conquer approach to identify the different possible target peptides one at a time. At each node of the hierarchy, the most relevant outputs of various existing subcellular prediction methods are combined by a Support Vector Machine. Conclusion The HECTAR method is able to predict the subcellular localisation of heterokont proteins with high accuracy. It also efficiently predicts the subcellular localisation of proteins from cryptophytes, a group that is phylogenetically close to the heterokonts. A variant of HECTAR, called HECTARSEC, can be used to identify signal peptide and type II signal

  11. Correlation of N-myc downstream-regulated gene 1 subcellular localization and lymph node metastases of colorectal neoplasms

    Energy Technology Data Exchange (ETDEWEB)

    Song, Yan [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China); Lv, Liyang [Department of Health, Jinan Military Area Command, Jinan 250022 (China); Du, Juan; Yue, Longtao [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China); Cao, Lili, E-mail: cllly22@163.com [Medical Research Center, Shandong Provincial Qianfoshan Hospital, Shandong University, Jinan 250014 (China)

    2013-09-20

    Highlights: •We clarified NDRG1 subcellular location in colorectal cancer. •We found the changes of NDRG1 distribution during colorectal cancer progression. •We clarified the correlation between NDRG1 distribution and lymph node metastasis. •It is possible that NDRG1 subcellular localization may determine its function. •Maybe NDRG1 is valuable early diagnostic markers for metastasis. -- Abstract: In colorectal neoplasms, N-myc downstream-regulated gene 1 (NDRG1) is a primarily cytoplasmic protein, but it is also expressed on the cell membrane and in the nucleus. NDRG1 is involved in various stages of tumor development in colorectal cancer, and it is possible that the different subcellular localizations may determine the function of NDRG1 protein. Here, we attempt to clarify the characteristics of NDRG1 protein subcellular localization during the progression of colorectal cancer. We examined NDRG1 expression in 49 colorectal cancer patients in cancerous, non-cancerous, and corresponding lymph node tissues. Cytoplasmic and membrane NDRG1 expression was higher in the lymph nodes with metastases than in those without metastases (P < 0.01). Nuclear NDRG1 expression in colorectal neoplasms was significantly higher than in the normal colorectal mucosa, and yet the normal colorectal mucosa showed no nuclear expression. Furthermore, our results showed higher cytoplasmic NDRG1 expression was better for differentiation, and higher membrane NDRG1 expression resulted in a greater possibility of lymph node metastasis. These data indicate that a certain relationship between the cytoplasmic and membrane expression of NDRG1 in lymph nodes exists with lymph node metastasis. NDRG1 expression may translocate from the membrane of the colorectal cancer cells to the nucleus, where it is involved in lymph node metastasis. Combination analysis of NDRG1 subcellular expression and clinical variables will help predict the incidence of lymph node metastasis.

  12. Programmed subcellular release to study the dynamics of cell detachment

    Science.gov (United States)

    Wildt, Bridget

    Cell detachment is central to a broad range of physio-pathological changes however there are no quantitative methods to study this process. Here we report programmed subcellular release, a method for spatially and temporally controlled cellular detachment and present the first quantitative results of the detachment dynamics of 3T3 fibroblasts at the subcellular level. Programmed subcellular release is an in vitro technique designed to trigger the detachment of distinct parts of a single cell from a patterned substrate with both spatial and temporal control. Subcellular release is achieved by plating cells on an array of patterned gold electrodes created by standard microfabrication techniques. The electrodes are biochemically functionalized with an adhesion-promoting RGD peptide sequence that is attached to the gold electrode via a thiol linkage. Each electrode is electrically isolated so that a subcellular section of a single cell spanning multiple electrodes can be released independently. Upon application of a voltage pulse to a single electrode, RGD-thiol molecules on an individual electrode undergo rapid electrochemical desorption that leads to subsequent cell contraction. The dynamics of cell contraction are found to have characteristic induction and contraction times. This thesis presents the first molecular inhibition studies conducted using programmed subcellular release verifying that this technique can be used to study complex signaling pathways critical to cell motility. Molecular level dynamics of focal adhesion proteins and actin stress fibers provide some insight into the complexities associated with triggered cell detachment. In addition to subcellular release, the programmed release of alkanethiols provides a tool for to study the spatially and temporally controlled release of small molecules or particles from individually addressable gold electrodes. Here we report on experiments which determine the dynamics of programmed release using fluorophore

  13. Biomechanics of subcellular structures by non-invasive Brillouin microscopy

    Science.gov (United States)

    Antonacci, Giuseppe; Braakman, Sietse

    2016-11-01

    Cellular biomechanics play a pivotal role in the pathophysiology of several diseases. Unfortunately, current methods to measure biomechanical properties are invasive and mostly limited to the surface of a cell. As a result, the mechanical behaviour of subcellular structures and organelles remains poorly characterised. Here, we show three-dimensional biomechanical images of single cells obtained with non-invasive, non-destructive Brillouin microscopy with an unprecedented spatial resolution. Our results quantify the longitudinal elastic modulus of subcellular structures. In particular, we found the nucleoli to be stiffer than both the nuclear envelope (p biomechanics and its role in pathophysiology.

  14. Subcellular targeting of nine calcium-dependent protein kinase isoforms from Arabidopsis

    Science.gov (United States)

    Dammann, Christian; Ichida, Audrey; Hong, Bimei; Romanowsky, Shawn M.; Hrabak, Estelle M.; Harmon, Alice C.; Pickard, Barbara G.; Harper, Jeffrey F.; Evans, M. L. (Principal Investigator)

    2003-01-01

    Calcium-dependent protein kinases (CDPKs) are specific to plants and some protists. Their activation by calcium makes them important switches for the transduction of intracellular calcium signals. Here, we identify the subcellular targeting potentials for nine CDPK isoforms from Arabidopsis, as determined by expression of green fluorescent protein (GFP) fusions in transgenic plants. Subcellular locations were determined by fluorescence microscopy in cells near the root tip. Isoforms AtCPK3-GFP and AtCPK4-GFP showed a nuclear and cytosolic distribution similar to that of free GFP. Membrane fractionation experiments confirmed that these isoforms were primarily soluble. A membrane association was observed for AtCPKs 1, 7, 8, 9, 16, 21, and 28, based on imaging and membrane fractionation experiments. This correlates with the presence of potential N-terminal acylation sites, consistent with acylation as an important factor in membrane association. All but one of the membrane-associated isoforms targeted exclusively to the plasma membrane. The exception was AtCPK1-GFP, which targeted to peroxisomes, as determined by covisualization with a peroxisome marker. Peroxisome targeting of AtCPK1-GFP was disrupted by a deletion of two potential N-terminal acylation sites. The observation of a peroxisome-located CDPK suggests a mechanism for calcium regulation of peroxisomal functions involved in oxidative stress and lipid metabolism.

  15. Subcellular localization of casein kinase I

    DEFF Research Database (Denmark)

    Grankowski, N; Issinger, O G

    1990-01-01

    An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus....

  16. Subcellular distribution of glycogen and decreased tetanic Ca2+ in fatigued single intact mouse muscle fibres

    Science.gov (United States)

    Nielsen, Joachim; Cheng, Arthur J; Ørtenblad, Niels; Westerblad, Håkan

    2014-01-01

    In skeletal muscle fibres, glycogen has been shown to be stored at different subcellular locations: (i) between the myofibrils (intermyofibrillar); (ii) within the myofibrils (intramyofibrillar); and (iii) subsarcolemmal. Of these, intramyofibrillar glycogen has been implied as a critical regulator of sarcoplasmic reticulum Ca2+ release. The aim of the present study was to test directly how the decrease in cytoplasmic free Ca2+ ([Ca2+]i) during repeated tetanic contractions relates to the subcellular glycogen distribution. Single fibres of mouse flexor digitorum brevis muscles were fatigued with 70 Hz, 350 ms tetani given at 2 s (high-intensity fatigue, HIF) or 10 s (low-intensity fatigue, LIF) intervals, while force and [Ca2+]i were measured. Stimulation continued until force decreased to 30% of its initial value. Fibres were then prepared for analyses of subcellular glycogen distribution by transmission electron microscopy. At fatigue, tetanic [Ca2+]i was reduced to 70 ± 4% and 54 ± 4% of the initial in HIF (P fibres, respectively. At fatigue, the mean inter- and intramyofibrillar glycogen content was 60–75% lower than in rested control fibres (P fibres showed a good correlation between the fatigue-induced decrease in tetanic [Ca2+]i and the reduction in intermyofibrillar (P = 0.051) and intramyofibrillar (P = 0.0008) glycogen. In conclusion, the fatigue-induced decrease in tetanic [Ca2+]i, and hence force, is accompanied by major reductions in inter- and intramyofibrillar glycogen. The stronger correlation between decreased tetanic [Ca2+]i and reduced intramyofibrillar glycogen implies that sarcoplasmic reticulum Ca2+ release critically depends on energy supply from the intramyofibrillar glycogen pool. PMID:24591577

  17. Brain glycogen – new perspectives on its metabolic function and regulation at the subcellular level

    Directory of Open Access Journals (Sweden)

    Linea Frimodt Obel

    2012-03-01

    Full Text Available Glycogen is a complex glucose polymer found in a variety of tissues, including brain, where it is localized primarily in astrocytes. The small quantity found in brain compared to e.g. liver has led to the understanding that brain glycogen is merely used during hypoglycemia or ischemia. In this review evidence is brought forward highlighting what has been an emerging understanding in brain energy metabolism: that glycogen is more than just a convenient way to store energy for use in emergencies – it is a highly dynamic molecule with versatile implications in brain function, i.e. synaptic activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms underlying glycogen metabolism. Based on i the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP, ii alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and iii a sequential component in the intermolecular mechanisms of glycogen metabolism, we suggest that glycogen metabolism in astrocytes is compartmentalized at the subcellular level. As a consequence, the meaning and importance of conventional terms used to describe glycogen metabolism (e.g. turnover is challenged. Overall, this review represents an overview of contemporary knowledge about brain glycogen and its metabolism and function. However, it also has a sharp focus on what we do not know, which is perhaps even more important for the future quest of uncovering the roles of glycogen in brain physiology and pathology.

  18. Predicting Subcellular Localization of Proteins by Bioinformatic Algorithms

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2015-01-01

    When predicting the subcellular localization of proteins from their amino acid sequences, there are basically three approaches: signal-based, global property-based, and homology-based. Each of these has its advantages and drawbacks, and it is important when comparing methods to know which approac...

  19. Measurement of endogenous subcellular concentration of steroids in tissue

    NARCIS (Netherlands)

    Poortman, J.; Landeghem, A.A.J. van; Helmond-Agema, A.; Thussen, J.H.H.

    1984-01-01

    A reliable method for the extraction of steroid hormones from human uterine tissue and the subsequent measurement of these hormones in the subcellular compartments by radioimmunoassay is described. Extraction of radioactive steroid hormones from in vivo labelled human uterine tissue by different

  20. Mapping the subcellular localization of Fe3O4@TiO2 nanoparticles by X-ray Fluorescence Microscopy

    Science.gov (United States)

    Yuan, Y.; Chen, S.; Gleber, S. C.; Lai, B.; Brister, K.; Flachenecker, C.; Wanzer, B.; Paunesku, T.; Vogt, S.; Woloschak, G. E.

    2013-10-01

    The targeted delivery of Fe3O4@TiO2 nanoparticles to cancer cells is an important step in their development as nanomedicines. We have synthesized nanoparticles that can bind the Epidermal Growth Factor Receptor, a cell surface protein that is overexpressed in many epithelial type cancers. In order to study the subcellular distribution of these nanoparticles, we have utilized the sub-micron resolution of X-ray Fluorescence Microscopy to map the location of Fe3O4@TiO2 NPs and other trace metal elements within HeLa cervical cancer cells. Here we demonstrate how the higher resolution of the newly installed Bionanoprobe at the Advanced Photon Source at Argonne National Laboratory can greatly improve our ability to distinguish intracellular nanoparticles and their spatial relationship with subcellular compartments.

  1. Correlation profiling of brain sub-cellular proteomes reveals co-assembly of synaptic proteins and subcellular distribution

    NARCIS (Netherlands)

    Pandya, N.J. (Nikhil J.); Koopmans, F. (Frank); J.A. Slotman (Johan A.); Paliukhovich, I. (Iryna); A.B. Houtsmuller (Adriaan); A.B. Smit (August); Li, K.W. (Ka Wan)

    2017-01-01

    textabstractProtein correlation profiling might assist in defining co-assembled proteins and subcellular distribution. Here, we quantified the proteomes of five biochemically isolated mouse brain cellular sub-fractions, with emphasis on synaptic compartments, from three brain regions, hippocampus,

  2. Gene Locater

    DEFF Research Database (Denmark)

    Anwar, Muhammad Zohaib; Sehar, Anoosha; Rehman, Inayat-Ur

    2012-01-01

    UNLABELLED: Locating genes on a chromosome is important for understanding the gene function and its linkage and recombination. Knowledge of gene positions on chromosomes is necessary for annotation. The study is essential for disease genetics and genomics, among other aspects. Currently available...... software's for calculating recombination frequency is mostly limited to the range and flexibility of this type of analysis. GENE LOCATER is a fully customizable program for calculating recombination frequency, written in JAVA. Through an easy-to-use interface, GENE LOCATOR allows users a high degree...

  3. Structural and functional plasticity of subcellular tethering, targeting and processing of RPGRIP1 by RPGR isoforms.

    Science.gov (United States)

    Patil, Hemangi; Guruju, Mallikarjuna R; Cho, Kyoung-In; Yi, Haiqing; Orry, Andrew; Kim, Hyesung; Ferreira, Paulo A

    2012-02-15

    Mutations affecting the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) interactome cause syndromic retinal dystrophies. RPGRIP1 interacts with the retinitis pigmentosa GTPase regulator (RPGR) through a domain homologous to RCC1 (RHD), a nucleotide exchange factor of Ran GTPase. However, functional relationships between RPGR and RPGRIP1 and their subcellular roles are lacking. We show by molecular modeling and analyses of RPGR disease-mutations that the RPGR-interacting domain (RID) of RPGRIP1 embraces multivalently the shared RHD of RPGR(1-19) and RPGR(ORF15) isoforms and the mutations are non-overlapping with the interface found between RCC1 and Ran GTPase. RPGR disease-mutations grouped into six classes based on their structural locations and differential impairment with RPGRIP1 interaction. RPGRIP1α(1) expression alone causes its profuse self-aggregation, an effect suppressed by co-expression of either RPGR isoform before and after RPGRIP1α(1) self-aggregation ensue. RPGR(1-19) localizes to the endoplasmic reticulum, whereas RPGR(ORF15) presents cytosolic distribution and they determine uniquely the subcellular co-localization of RPGRIP1α(1). Disease mutations in RPGR(1) (-19), RPGR(ORF15), or RID of RPGRIP1α(1), singly or in combination, exert distinct effects on the subcellular targeting, co-localization or tethering of RPGRIP1α(1) with RPGR(1-19) or RPGR(ORF15) in kidney, photoreceptor and hepatocyte cell lines. Additionally, RPGR(ORF15), but not RPGR(1-19), protects the RID of RPGRIP1α(1) from limited proteolysis. These studies define RPGR- and cell-type-dependent targeting pathways with structural and functional plasticity modulating the expression of mutations in RPGR and RPGRIP1. Further, RPGR isoforms distinctively determine the subcellular targeting of RPGRIP1α(1,) with deficits in RPGR(ORF15)-dependent intracellular localization of RPGRIP1α(1) contributing to pathomechanisms shared by etiologically distinct syndromic

  4. Library Locations

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — Carnegie Library of Pittsburgh locations including address, coordinates, phone number, square footage, and standard operating hours. The map below does not display...

  5. Subcellular distribution and chemical forms of cadmium in Phytolacca americana L

    Energy Technology Data Exchange (ETDEWEB)

    Fu Xiaoping; Dou Changming [Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control, Institute of Environmental Science and Technology, Zhejiang University, Hangzhou 310029 (China); Chen Yingxu, E-mail: yingxu_chen@hotmail.com [Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control, Institute of Environmental Science and Technology, Zhejiang University, Hangzhou 310029 (China); Chen Xincai; Shi Jiyan; Yu Mingge; Xu Jie [Ministry of Agriculture Key Laboratory of Non-point Source Pollution Control, Institute of Environmental Science and Technology, Zhejiang University, Hangzhou 310029 (China)

    2011-02-15

    Phytolacca americana L. (pokeweed) is a promising species for Cd phytoextraction with large biomass and fast growth rate. To further understand the mechanisms involved in Cd tolerance and detoxification, the present study investigated subcellular distribution and chemical forms of Cd in pokeweed. Subcellular fractionation of Cd-containing tissues indicated that both in root and leaves, the majority of the element was located in soluble fraction and cell walls. Meanwhile, Cd taken up by pokeweed existed in different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of 80% ethanol in roots, followed by 1 M NaCl, d-H{sub 2}O and 2% HAc, while in leaves and stems, most of the Cd was extracted by 1 M NaCl, and the subdominant amount of Cd was extracted by 80% ethanol. It could be suggested that Cd compartmentation with organo-ligands in vacuole or integrated with pectates and proteins in cell wall might be responsible for the adaptation of pokeweed to Cd stress.

  6. iLoc-Euk: a multi-label classifier for predicting the subcellular localization of singleplex and multiplex eukaryotic proteins.

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

    Full Text Available Predicting protein subcellular localization is an important and difficult problem, particularly when query proteins may have the multiplex character, i.e., simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing protein subcellular location predictor can only be used to deal with the single-location or "singleplex" proteins. Actually, multiple-location or "multiplex" proteins should not be ignored because they usually posses some unique biological functions worthy of our special notice. By introducing the "multi-labeled learning" and "accumulation-layer scale", a new predictor, called iLoc-Euk, has been developed that can be used to deal with the systems containing both singleplex and multiplex proteins. As a demonstration, the jackknife cross-validation was performed with iLoc-Euk on a benchmark dataset of eukaryotic proteins classified into the following 22 location sites: (1 acrosome, (2 cell membrane, (3 cell wall, (4 centriole, (5 chloroplast, (6 cyanelle, (7 cytoplasm, (8 cytoskeleton, (9 endoplasmic reticulum, (10 endosome, (11 extracellular, (12 Golgi apparatus, (13 hydrogenosome, (14 lysosome, (15 melanosome, (16 microsome (17 mitochondrion, (18 nucleus, (19 peroxisome, (20 spindle pole body, (21 synapse, and (22 vacuole, where none of proteins included has ≥25% pairwise sequence identity to any other in a same subset. The overall success rate thus obtained by iLoc-Euk was 79%, which is significantly higher than that by any of the existing predictors that also have the capacity to deal with such a complicated and stringent system. As a user-friendly web-server, iLoc-Euk is freely accessible to the public at the web-site http://icpr.jci.edu.cn/bioinfo/iLoc-Euk. It is anticipated that iLoc-Euk may become a useful bioinformatics tool for Molecular Cell Biology, Proteomics, System Biology, and Drug Development Also, its novel approach will further stimulate the development of

  7. Partial gigantism

    Directory of Open Access Journals (Sweden)

    М.М. Karimova

    2017-05-01

    Full Text Available A girl with partial gigantism (the increased I and II fingers of the left foot is being examined. This condition is a rare and unresolved problem, as the definite reason of its development is not determined. Wait-and-see strategy is recommended, as well as correcting operations after closing of growth zones, and forming of data pool for generalization and development of schemes of drug and radial therapeutic methods.

  8. Protein subcellular localization prediction using artificial intelligence technology.

    Science.gov (United States)

    Nair, Rajesh; Rost, Burkhard

    2008-01-01

    Proteins perform many important tasks in living organisms, such as catalysis of biochemical reactions, transport of nutrients, and recognition and transmission of signals. The plethora of aspects of the role of any particular protein is referred to as its "function." One aspect of protein function that has been the target of intensive research by computational biologists is its subcellular localization. Proteins must be localized in the same subcellular compartment to cooperate toward a common physiological function. Aberrant subcellular localization of proteins can result in several diseases, including kidney stones, cancer, and Alzheimer's disease. To date, sequence homology remains the most widely used method for inferring the function of a protein. However, the application of advanced artificial intelligence (AI)-based techniques in recent years has resulted in significant improvements in our ability to predict the subcellular localization of a protein. The prediction accuracy has risen steadily over the years, in large part due to the application of AI-based methods such as hidden Markov models (HMMs), neural networks (NNs), and support vector machines (SVMs), although the availability of larger experimental datasets has also played a role. Automatic methods that mine textual information from the biological literature and molecular biology databases have considerably sped up the process of annotation for proteins for which some information regarding function is available in the literature. State-of-the-art methods based on NNs and HMMs can predict the presence of N-terminal sorting signals extremely accurately. Ab initio methods that predict subcellular localization for any protein sequence using only the native amino acid sequence and features predicted from the native sequence have shown the most remarkable improvements. The prediction accuracy of these methods has increased by over 30% in the past decade. The accuracy of these methods is now on par with

  9. Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome.

    Science.gov (United States)

    Jadot, Michel; Boonen, Marielle; Thirion, Jaqueline; Wang, Nan; Xing, Jinchuan; Zhao, Caifeng; Tannous, Abla; Qian, Meiqian; Zheng, Haiyan; Everett, John K; Moore, Dirk F; Sleat, David E; Lobel, Peter

    2017-02-01

    Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease

  10. Imaging trace element distributions in single organelles and subcellular features

    Science.gov (United States)

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  11. Muscle glycogen and cell function - Location, location, location

    DEFF Research Database (Denmark)

    Ørtenblad, N; Nielsen, Joachim

    2015-01-01

    that the subcellular localization of glycogen has to be considered to fully understand the role of glycogen metabolism and signaling in skeletal muscle function. Here, we propose that the effect of low muscle glycogen on excitation-contraction coupling may serve as a built-in mechanism, which links the energetic state......The importance of glycogen, as a fuel during exercise, is a fundamental concept in exercise physiology. The use of electron microscopy has revealed that glycogen is not evenly distributed in skeletal muscle fibers, but rather localized in distinct pools. In this review, we present the available...... evidence regarding the subcellular localization of glycogen in skeletal muscle and discuss this from the perspective of skeletal muscle fiber function. The distribution of glycogen in the defined pools within the skeletal muscle varies depending on exercise intensity, fiber phenotype, training status...

  12. Measurement of endogenous subcellular concentration of steroids in tissue

    OpenAIRE

    Poortman, J.; Landeghem, A.A.J. van; Helmond-Agema, A.; Thussen, J.H.H.

    1984-01-01

    A reliable method for the extraction of steroid hormones from human uterine tissue and the subsequent measurement of these hormones in the subcellular compartments by radioimmunoassay is described. Extraction of radioactive steroid hormones from in vivo labelled human uterine tissue by different methods reveals that an almost quantitative extraction of steroid hormones from the nuclear fraction is obtained by sonication in ethanol-acetone. Extraction of steroid hormones with diethylether from...

  13. Functional analysis of Plasmodium vivax VIR proteins reveals different subcellular localizations and cytoadherence to the ICAM-1 endothelial receptor.

    Science.gov (United States)

    Bernabeu, M; Lopez, F J; Ferrer, M; Martin-Jaular, L; Razaname, A; Corradin, G; Maier, A G; Del Portillo, H A; Fernandez-Becerra, C

    2012-03-01

    The subcellular localization and function of variant subtelomeric multigene families in Plasmodium vivax remain vastly unknown. Among them, the vir superfamily is putatively involved in antigenic variation and in mediating adherence to endothelial receptors. In the absence of a continuous in vitro culture system for P. vivax, we have generated P. falciparum transgenic lines expressing VIR proteins to infer location and function. We chose three proteins pertaining to subfamilies A (VIR17), C (VIR14) and D (VIR10), with domains and secondary structures that predictably traffic these proteins to different subcellular compartments. Here, we showed that VIR17 remained inside the parasite and around merozoites, whereas VIR14 and VIR10 were exported to the membrane of infected red blood cells (iRBCs) in an apparent independent pathway of Maurer's clefts. Remarkably, VIR14 was exposed at the surface of iRBCs and mediated adherence to different endothelial receptors expressed in CHO cells under static conditions. Under physiological flow conditions, however, cytoadherence was only observed to ICAM-1, which was the only receptor whose adherence was specifically and significantly inhibited by antibodies against conserved motifs of VIR proteins. Immunofluorescence studies using these antibodies also showed different subcellular localizations of VIR proteins in P. vivax-infected reticulocytes from natural infections. These data suggest that VIR proteins are trafficked to different cellular compartments and functionally demonstrates that VIR proteins can specifically mediate cytoadherence to the ICAM-1 endothelial receptor. © 2011 Blackwell Publishing Ltd.

  14. Intracellular And Subcellular Partitioning Of Nickel In Aureococcus Anophagefferens

    Science.gov (United States)

    Wang, B.; Axe, L.; Wei, L.; Bagheri, S.; Michalopoulou, Z.

    2008-12-01

    Brown tides are caused by Aureococcus anophagefferens, a species of Pelagophyceae, and have been observed in NY/NJ waterways effecting ecosystems by attenuating light, changing water color, reducing eelgrass beds, decreasing shellfisheries, and further impacting the food web by reducing phytoplankton. Although the impact of macronutrients and iron on A. anophagefferens has been well studied, contaminants, and specifically trace metals have not. In long-term experiments designed to investigate the growth and toxicity, Cd, Cu, Ni, and Zn exposure was evaluated over 10-13 to 10-7 M for the free metal ion. While growth was inhibited or terminated from exposure to Cd and Cu, nickel addition ([Ni2+]: 10-11.23 to 10-10.23 M) promoted A. anophagefferens growth. Short-term experiments are being conducted to better understand mechanistically nickel speciation and distribution. Both total intracellular and subcellular metal concentrations are being assessed with radio-labeled 63Ni. Subcellular fractions are defined as metal-sensitive fractions (MSF) constituting organelles, cell debris, and heat-denatured protein [HDP] and biologically detoxified metal comprising heat-stabilized protein [HSP] and metal-rich granules [MRG]. Based on subcellular distribution, aqueous [Ni2+] concentrations, and A. anophagefferens growth rates, potential reaction pathways promoting A. anophagefferens growth can be addressed.

  15. Subcellular targeting of Salmonella virulence proteins by host-mediated S-palmitoylation.

    Science.gov (United States)

    Hicks, Stuart W; Charron, Guillaume; Hang, Howard C; Galán, Jorge E

    2011-07-21

    Several pathogenic bacteria utilize type III secretion systems (TTSS) to deliver into host cells bacterial virulence proteins with the capacity to modulate a variety of cellular pathways. Once delivered into host cells, the accurate targeting of bacterial effectors to specific locations is critical for their proper function. However, little is known about the mechanisms these virulence effectors use to reach their subcellular destination. Here we show that the Salmonella TTSS effector proteins SspH2 and SseI are localized to the plasma membrane of host cells, a process dependent on S-palmitoylation of a conserved cysteine residue within their N-terminal domains. We also show that effector protein lipidation is mediated by a specific subset of host-cell palmitoyltransferases and that lipidation is critical for effector function. This study describes a remarkable mechanism by which a pathogen exploits host-cell machinery to properly target its virulence factors. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. [Expression, subcellular localization and nuclear translocation of transcription factor up stream stimulatory factor-1 in odontoblasts].

    Science.gov (United States)

    Wu, Li-An; Wen, Ling-Ying; Yang, Fu-Sheng; Wang, Xiao-Jing; Fang, Jun

    2007-09-01

    To examine the expression and subcellular localization of transcription factor USF1 in odontoblasts and investigate whether nuclear translocation occurs under stimuli. Odontoblasts MDPC-23 were cultured on coverslips and divided into 2 groups. Group 1 received no stimuli, and group 2 was stimulated by nicotine with various concentrations respectively for 1h. Then the mountings of odontoblasts were prepared and immunocytochemical staining was performed with specific USF1 antibody via SABC method. Hela cells were used as positive control. The staining was positive in the cytoplasm of odontoblasts in group 1, but in the nuclei of Hela cells and in 100 mg/L nicotine-stimulated odontoblasts in group 2. There exists USF1 protein in odontoblasts, which locates in the cytoplasm and could translocate into nuclei under the stimulation of nicotine.

  17. Diverse subcellular localizations of the insect CMP-sialic acid synthetases.

    Science.gov (United States)

    Di, Wu; Fujita, Akiko; Hamaguchi, Kayo; Delannoy, Philippe; Sato, Chihiro; Kitajima, Ken

    2017-04-01

    The occurrence and biological importance of sialic acid (Sia) and its metabolic enzymes in insects have been studied using Drosophila melanogaster. The most prominent feature of D. melanogaster CMP-Sia synthetase (DmCSS) is its Golgi-localization, contrasted with nuclear localization of vertebrate CSSs. However, it remains unclear if the Golgi-localization is common to other insect CSSs and why it happens. To answer these questions, Aedes aegypti (mosquito) CSS (AaCSS) and Tribolium castaneum (beetle) CSS (TcCSS) were cloned and characterized for their activity and subcellular localization. Our new findings show: (1) AaCSS and TcCSS share a common overall structure with DmCSS in terms of evolutionarily conserved motifs and the absence of the C-terminal domain typical to vertebrate CSSs; (2) when expressed in mammalian and insect cells, AaCSS and TcCSS showed in vivo and in vitro CSS activities, similar to DmCSS. In contrast, when expressed in bacteria, they lacked CSS activity because the N-terminal hydrophobic region appeared to induce protein aggregation; (3) when expressed in Drosophila S2 cells, AaCSS and TcCSS were predominantly localized in the ER, but not in the Golgi. Surprisingly, DmCSS was mainly secreted into the culture medium, although partially detected in Golgi. Consistent with these results, the N-terminal hydrophobic regions of AaCSS and TcCSS functioned as a signal peptide to render them soluble in the ER, while the N-terminus of DmCSS functioned as a membrane-spanning region of type II transmembrane proteins whose cytosolic KLK sequence functioned as an ER export signal. Accordingly, the differential subcellular localization of insect CSSs are distinctively more diverse than previously recognized. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  18. Partial gravity habitat study

    Science.gov (United States)

    Capps, Stephen; Lorandos, Jason; Akhidime, Eval; Bunch, Michael; Lund, Denise; Moore, Nathan; Murakawa, Kiosuke

    1989-01-01

    The purpose of this study is to investigate comprehensive design requirements associated with designing habitats for humans in a partial gravity environment, then to apply them to a lunar base design. Other potential sites for application include planetary surfaces such as Mars, variable-gravity research facilities, and a rotating spacecraft. Design requirements for partial gravity environments include locomotion changes in less than normal earth gravity; facility design issues, such as interior configuration, module diameter, and geometry; and volumetric requirements based on the previous as well as psychological issues involved in prolonged isolation. For application to a lunar base, it is necessary to study the exterior architecture and configuration to insure optimum circulation patterns while providing dual egress; radiation protection issues are addressed to provide a safe and healthy environment for the crew; and finally, the overall site is studied to locate all associated facilities in context with the habitat. Mission planning is not the purpose of this study; therefore, a Lockheed scenario is used as an outline for the lunar base application, which is then modified to meet the project needs. The goal of this report is to formulate facts on human reactions to partial gravity environments, derive design requirements based on these facts, and apply the requirements to a partial gravity situation which, for this study, was a lunar base.

  19. pLoc-mGneg: Predict subcellular localization of Gram-negative bacterial proteins by deep gene ontology learning via general PseAAC.

    Science.gov (United States)

    Cheng, Xiang; Xiao, Xuan; Chou, Kuo-Chen

    2017-10-06

    Information of the proteins' subcellular localization is crucially important for revealing their biological functions in a cell, the basic unit of life. With the avalanche of protein sequences generated in the postgenomic age, it is highly desired to develop computational tools for timely identifying their subcellular locations based on the sequence information alone. The current study is focused on the Gram-negative bacterial proteins. Although considerable efforts have been made in protein subcellular prediction, the problem is far from being solved yet. This is because mounting evidences have indicated that many Gram-negative bacterial proteins exist in two or more location sites. Unfortunately, most existing methods can be used to deal with single-location proteins only. Actually, proteins with multi-locations may have some special biological functions important for both basic research and drug design. In this study, by using the multi-label theory, we developed a new predictor called "pLoc-mGneg" for predicting the subcellular localization of Gram-negative bacterial proteins with both single and multiple locations. Rigorous cross-validation on a high quality benchmark dataset indicated that the proposed predictor is remarkably superior to "iLoc-Gneg", the state-of-the-art predictor for the same purpose. For the convenience of most experimental scientists, a user-friendly web-server for the novel predictor has been established at http://www.jci-bioinfo.cn/pLoc-mGneg/, by which users can easily get their desired results without the need to go through the complicated mathematics involved. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Solid-state nuclear magnetic resonance measurements of HIV fusion peptide 13CO to lipid 31P proximities support similar partially inserted membrane locations of the α helical and β sheet peptide structures.

    Science.gov (United States)

    Gabrys, Charles M; Qiang, Wei; Sun, Yan; Xie, Li; Schmick, Scott D; Weliky, David P

    2013-10-03

    Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the ∼25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of "HFP", i.e., a ∼25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was (13)CO backbone labeled. Samples were then prepared that each contained a singly (13)CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP (13)CO nuclei and (31)P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct (13)CO shifts for the α helical and β sheet structures so that the proximities to (31)P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the (13)CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. "HFPmn" was a linear peptide that contained the 23 N-terminal residues of gp41. "HFPmn_V2E" contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and

  1. Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide 13CO to Lipid 31P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

    Science.gov (United States)

    Gabrys, Charles M.; Qiang, Wei; Sun, Yan; Xie, Li; Schmick, Scott D.; Weliky, David P.

    2013-10-01

    Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the -25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of -HFP-, i.e., a -25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was 13CO backbone labeled. Samples were then prepared that each contained a singly 13CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric - sheet structure. Proximity between the HFP 13CO nuclei and 31P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct 13CO shifts for the α helical and - sheet structures so that the proximities to 31P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the 13CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. -HFPmn- was a linear peptide that contained the 23 N-terminal residues of gp41. -HFPmn_V2E- contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and infection. The

  2. Exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors.

    Science.gov (United States)

    Hicks, Stuart W; Galán, Jorge E

    2013-05-01

    Several bacterial species have evolved specialized secretion systems to deliver bacterial effector proteins into eukaryotic cells. These effectors have the capacity to modulate host cell pathways in order to promote bacterial survival and replication. The spatial and temporal context in which the effectors exert their biochemical activities is crucial for their function. To fully understand effector function in the context of infection, we need to understand the mechanisms that lead to the precise subcellular localization of effectors following their delivery into host cells. Recent studies have shown that bacterial effectors exploit host cell machinery to accurately target their biochemical activities within the host cell.

  3. Subcellular Localization of Carotenoid Biosynthesis in Synechocystis sp. PCC 6803.

    Directory of Open Access Journals (Sweden)

    Lifang Zhang

    Full Text Available The biosynthesis pathway of carotenoids in cyanobacteria is partly described. However, the subcellular localization of individual steps is so far unknown. Carotenoid analysis of different membrane subfractions in Synechocystis sp. PCC6803 shows that "light" plasma membranes have a high carotenoid/protein ratio, when compared to "heavier" plasma membranes or thylakoids. The localization of CrtQ and CrtO, two well-defined carotenoid synthesis pathway enzymes in Synechocystis, was studied by epitope tagging and western blots. Both enzymes are locally more abundant in plasma membranes than in thylakoids, implying that the plasma membrane has higher synthesis rates of β-carotene precursor molecules and echinenone.

  4. Validating subcellular localization prediction tools with mycobacterial proteins

    Directory of Open Access Journals (Sweden)

    Niño Luis F

    2009-05-01

    Full Text Available Abstract Background The computational prediction of mycobacterial proteins' subcellular localization is of key importance for proteome annotation and for the identification of new drug targets and vaccine candidates. Several subcellular localization classifiers have been developed over the past few years, which have comprised both general localization and feature-based classifiers. Here, we have validated the ability of different bioinformatics approaches, through the use of SignalP 2.0, TatP 1.0, LipoP 1.0, Phobius, PA-SUB 2.5, PSORTb v.2.0.4 and Gpos-PLoc, to predict secreted bacterial proteins. These computational tools were compared in terms of sensitivity, specificity and Matthew's correlation coefficient (MCC using a set of mycobacterial proteins having less than 40% identity, none of which are included in the training data sets of the validated tools and whose subcellular localization have been experimentally confirmed. These proteins belong to the TBpred training data set, a computational tool specifically designed to predict mycobacterial proteins. Results A final validation set of 272 mycobacterial proteins was obtained from the initial set of 852 mycobacterial proteins. According to the results of the validation metrics, all tools presented specificity above 0.90, while dispersion sensitivity and MCC values were above 0.22. PA-SUB 2.5 presented the highest values; however, these results might be biased due to the methodology used by this tool. PSORTb v.2.0.4 left 56 proteins out of the classification, while Gpos-PLoc left just one protein out. Conclusion Both subcellular localization approaches had high predictive specificity and high recognition of true negatives for the tested data set. Among those tools whose predictions are not based on homology searches against SWISS-PROT, Gpos-PLoc was the general localization tool with the best predictive performance, while SignalP 2.0 was the best tool among the ones using a feature

  5. Validating subcellular localization prediction tools with mycobacterial proteins

    Science.gov (United States)

    Restrepo-Montoya, Daniel; Vizcaíno, Carolina; Niño, Luis F; Ocampo, Marisol; Patarroyo, Manuel E; Patarroyo, Manuel A

    2009-01-01

    Background The computational prediction of mycobacterial proteins' subcellular localization is of key importance for proteome annotation and for the identification of new drug targets and vaccine candidates. Several subcellular localization classifiers have been developed over the past few years, which have comprised both general localization and feature-based classifiers. Here, we have validated the ability of different bioinformatics approaches, through the use of SignalP 2.0, TatP 1.0, LipoP 1.0, Phobius, PA-SUB 2.5, PSORTb v.2.0.4 and Gpos-PLoc, to predict secreted bacterial proteins. These computational tools were compared in terms of sensitivity, specificity and Matthew's correlation coefficient (MCC) using a set of mycobacterial proteins having less than 40% identity, none of which are included in the training data sets of the validated tools and whose subcellular localization have been experimentally confirmed. These proteins belong to the TBpred training data set, a computational tool specifically designed to predict mycobacterial proteins. Results A final validation set of 272 mycobacterial proteins was obtained from the initial set of 852 mycobacterial proteins. According to the results of the validation metrics, all tools presented specificity above 0.90, while dispersion sensitivity and MCC values were above 0.22. PA-SUB 2.5 presented the highest values; however, these results might be biased due to the methodology used by this tool. PSORTb v.2.0.4 left 56 proteins out of the classification, while Gpos-PLoc left just one protein out. Conclusion Both subcellular localization approaches had high predictive specificity and high recognition of true negatives for the tested data set. Among those tools whose predictions are not based on homology searches against SWISS-PROT, Gpos-PLoc was the general localization tool with the best predictive performance, while SignalP 2.0 was the best tool among the ones using a feature-based approach. Even though PA-SUB 2

  6. Precise Photodynamic Therapy of Cancer via Subcellular Dynamic Tracing of Dual-loaded Upconversion Nanophotosensitizers

    National Research Council Canada - National Science Library

    Yulei Chang; Xiaodan Li; Li Zhang; Lu Xia; Xiaomin Liu; Cuixia Li; Youlin Zhang; Langping Tu; Bin Xue; Huiying Zhao; Hong Zhang; Xianggui Kong

    2017-01-01

    ...) light have led to substantial progress in improving photodynamic therapy (PDT) of cancer. For a successful PDT, subcellular organelles are promising therapeutic targets for reaching a satisfactory efficacy...

  7. Genetically targeted fluorogenic macromolecules for subcellular imaging and cellular perturbation.

    Science.gov (United States)

    Magenau, Andrew J D; Saurabh, Saumya; Andreko, Susan K; Telmer, Cheryl A; Schmidt, Brigitte F; Waggoner, Alan S; Bruchez, Marcel P

    2015-10-01

    The alteration of cellular functions by anchoring macromolecules to specified organelles may reveal a new area of therapeutic potential and clinical treatment. In this work, a unique phenotype was evoked by influencing cellular behavior through the modification of subcellular structures with genetically targetable macromolecules. These fluorogen-functionalized polymers, prepared via controlled radical polymerization, were capable of exclusively decorating actin, cytoplasmic, or nuclear compartments of living cells expressing localized fluorgen-activating proteins. The macromolecular fluorogens were optimized by establishing critical polymer architecture-biophysical property relationships which impacted binding rates, binding affinities, and the level of internalization. Specific labeling of subcellular structures was realized at nanomolar concentrations of polymer, in the absence of membrane permeabilization or transduction domains, and fluorogen-modified polymers were found to bind to protein intact after delivery to the cytosol. Cellular motility was found to be dependent on binding of macromolecular fluorogens to actin structures causing rapid cellular ruffling without migration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Studies on the subcellular localization of the porphycene CPO.

    Science.gov (United States)

    Kessel, David; Conley, Mary; Vicente, M Graça H; Reiners, John J

    2005-01-01

    This study was designed to provide more detailed information on the subcellular sites of binding of the porphycene, termed 9-capronyloxytetrakis (methoxyethyl) porphycene (CPO), with a fluorescence resonance energy transfer (FRET) technique. The proximity of CPO to two fluorescent probes was determined: nonyl acridine orange (NAO), a dye with specific affinity for the mitochondrial lipid cardiolipin, and dihexa-oxacarbocyanine iodide (DiOC6), an agent that labels the endoplasmic reticulum (ER). FRET spectra indicated energy transfer between DiOC6 and CPO but no significant transfer between NAO and CPO. These results confirm data obtained by fluorescence microscopy, suggesting a similar pattern of subcellular localization by CPO and DiOC6 but not by CPO and NAO. However, when cells containing CPO were irradiated and then loaded with NAO, FRET between the two fluorophores was observed. Hence, a relocalization of CPO can occur during irradiation. These data provide an explanation for recent studies on CPO-catalyzed photodamage to both ER and mitochondrial Bcl-2.

  9. Studies on the Subcellular Localization of the Porphycene CPO¶

    Science.gov (United States)

    Kessel, David; Conley, Mary; Vicente, M. Graça H.; Reiners, John J.

    2010-01-01

    This study was designed to provide more detailed information on the subcellular sites of binding of the porphycene, termed 9-capronyloxytetrakis (methoxyethyl) porphycene (CPO), with a fluorescence resonance energy transfer (FRET) technique. The proximity of CPO to two fluorescent probes was determined: nonyl acridine orange (NAO), a dye with specific affinity for the mitochondrial lipid cardiolipin, and dihexaoxacarbocyanine iodide (DiOC6), an agent that labels the endoplasmic reticulum (ER). FRET spectra indicated energy transfer between DiOC6 and CPO but no significant transfer between NAO and CPO. These results confirm data obtained by fluorescence microscopy, suggesting a similar pattern of subcellular localization by CPO and DiOC6 but not by CPO and NAO. However, when cells containing CPO were irradiated and then loaded with NAO, FRET between the two fluorophores was observed. Hence, a relocalization of CPO can occur during irradiation. These data provide an explanation for recent studies on CPO-catalyzed photodamage to both ER and mitochondrial Bcl-2. PMID:15745423

  10. Subcellular Distribution of Glutathione Precursors in Arabidopsis thaliana

    Science.gov (United States)

    Koffler, Barbara Eva; Maier, Romana; Zechmann, Bernd

    2011-01-01

    Abstract Glutathione is an important antioxidant and has many important functions in plant development, growth and defense. Glutathione synthesis and degradation is highly compartment-specific and relies on the subcellular availability of its precursors, cysteine, glutamate, glycine and γ-glutamylcysteine especially in plastids and the cytosol which are considered as the main centers for glutathione synthesis. The availability of glutathione precursors within these cell compartments is therefore of great importance for successful plant development and defense. The aim of this study was to investigate the compartment-specific importance of glutathione precursors in Arabidopsis thaliana. The subcellular distribution was compared between wild type plants (Col-0), plants with impaired glutathione synthesis (glutathione deficient pad2-1 mutant, wild type plants treated with buthionine sulfoximine), and one complemented line (OE3) with restored glutathione synthesis. Immunocytohistochemistry revealed that the inhibition of glutathione synthesis induced the accumulation of the glutathione precursors cysteine, glutamate and glycine in most cell compartments including plastids and the cytosol. A strong decrease could be observed in γ-glutamylcysteine (γ-EC) contents in these cell compartments. These experiments demonstrated that the inhibition of γ-glutamylcysteine synthetase (GSH1) – the first enzyme of glutathione synthesis – causes a reduction of γ-EC levels and an accumulation of all other glutathione precursors within the cells. PMID:22050910

  11. Gene ontology based transfer learning for protein subcellular localization

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    Zhou Shuigeng

    2011-02-01

    Full Text Available Abstract Background Prediction of protein subcellular localization generally involves many complex factors, and using only one or two aspects of data information may not tell the true story. For this reason, some recent predictive models are deliberately designed to integrate multiple heterogeneous data sources for exploiting multi-aspect protein feature information. Gene ontology, hereinafter referred to as GO, uses a controlled vocabulary to depict biological molecules or gene products in terms of biological process, molecular function and cellular component. With the rapid expansion of annotated protein sequences, gene ontology has become a general protein feature that can be used to construct predictive models in computational biology. Existing models generally either concatenated the GO terms into a flat binary vector or applied majority-vote based ensemble learning for protein subcellular localization, both of which can not estimate the individual discriminative abilities of the three aspects of gene ontology. Results In this paper, we propose a Gene Ontology Based Transfer Learning Model (GO-TLM for large-scale protein subcellular localization. The model transfers the signature-based homologous GO terms to the target proteins, and further constructs a reliable learning system to reduce the adverse affect of the potential false GO terms that are resulted from evolutionary divergence. We derive three GO kernels from the three aspects of gene ontology to measure the GO similarity of two proteins, and derive two other spectrum kernels to measure the similarity of two protein sequences. We use simple non-parametric cross validation to explicitly weigh the discriminative abilities of the five kernels, such that the time & space computational complexities are greatly reduced when compared to the complicated semi-definite programming and semi-indefinite linear programming. The five kernels are then linearly merged into one single kernel for

  12. Prequels to Synthetic Biology: From Candidate Gene Identification and Validation to Enzyme Subcellular Localization in Plant and Yeast Cells.

    Science.gov (United States)

    Foureau, E; Carqueijeiro, I; Dugé de Bernonville, T; Melin, C; Lafontaine, F; Besseau, S; Lanoue, A; Papon, N; Oudin, A; Glévarec, G; Clastre, M; St-Pierre, B; Giglioli-Guivarc'h, N; Courdavault, V

    2016-01-01

    Natural compounds extracted from microorganisms or plants constitute an inexhaustible source of valuable molecules whose supply can be potentially challenged by limitations in biological sourcing. The recent progress in synthetic biology combined to the increasing access to extensive transcriptomics and genomics data now provide new alternatives to produce these molecules by transferring their whole biosynthetic pathway in heterologous production platforms such as yeasts or bacteria. While the generation of high titer producing strains remains per se an arduous field of investigation, elucidation of the biosynthetic pathways as well as characterization of their complex subcellular organization are essential prequels to the efficient development of such bioengineering approaches. Using examples from plants and yeasts as a framework, we describe potent methods to rationalize the study of partially characterized pathways, including the basics of computational applications to identify candidate genes in transcriptomics data and the validation of their function by an improved procedure of virus-induced gene silencing mediated by direct DNA transfer to get around possible resistance to Agrobacterium-delivery of viral vectors. To identify potential alterations of biosynthetic fluxes resulting from enzyme mislocalizations in reconstituted pathways, we also detail protocols aiming at characterizing subcellular localizations of protein in plant cells by expression of fluorescent protein fusions through biolistic-mediated transient transformation, and localization of transferred enzymes in yeast using similar fluorescence procedures. Albeit initially developed for the Madagascar periwinkle, these methods may be applied to other plant species or organisms in order to establish synthetic biology platform. © 2016 Elsevier Inc. All rights reserved.

  13. The in vitro sub-cellular localization and in vivo efficacy of novel chitosan/GMO nanostructures containing paclitaxel.

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    Trickler, W J; Nagvekar, A A; Dash, A K

    2009-08-01

    To determine the in vitro sub-cellular localization and in vivo efficacy of chitosan/GMO nanostructures containing paclitaxel (PTX) compared to a conventional PTX treatment (Taxol). The sub-cellular localization of coumarin-6 labeled chitosan/GMO nanostructures was determined by confocal microscopy in MDA-MB-231 cells. The antitumor efficacy was evaluated in two separate studies using FOX-Chase (CB17) SCID Female-Mice MDA-MB-231 xenograph model. Treatments consisted of intravenous Taxol or chitosan/GMO nanostructures with or without PTX, local intra-tumor bolus of Taxol or chitosan/GMO nanostructures with or without PTX. The tumor diameter and animal weight was monitored at various intervals. Histopathological changes were evaluated in end-point tumors. The tumor diameter increased at a constant rate for all the groups between days 7-14. After a single intratumoral bolus dose of chitosan/GMO containing PTX showed significant reduction in tumor diameter on day 15 when compared to control, placebo and intravenous PTX administration. The tumor diameter reached a maximal decrease (4-fold) by day 18, and the difference was reduced to approximately 2-fold by day 21. Qualitatively similar results were observed in a separate study containing PTX when administered intravenously. Chitosan/GMO nanostructures containing PTX are safe and effective administered locally or intravenously. Partially supported by DOD Award BC045664.

  14. Distribution and Characterization of Antigens Found in Subcellular Fractions of African Trypanosomes.

    Science.gov (United States)

    1979-08-01

    Islation of bodies % containing the cyanide insensitive glycerophosphate oxidase of Trypanosoma - equiperdum . Comp. Biochem. Physiol. 30 1049-1054. S...commenced on a project aimed at characterizing the subcellular distribution and nature of antigens found in the African trypanosome, Trypanosoma rhodesiense...current project concerning the subcellular distribution and characterization of antigens in the African trypanosome, Trypanosoma rhodesiense. During

  15. Protein-protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery

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    Lynn eRichardson

    2011-06-01

    Full Text Available The Endosomal Sorting Complex Required for Transport (ESCRT consists of several multi-protein subcomplexes which assemble sequentially at the endosomal surface and function in multivesicular body (MVB biogenesis. While ESCRT has been relatively well characterized in yeasts and mammals, comparably little is known about ESCRT in plants. Here we explored the yeast two-hybrid protein interaction network and subcellular localization of the Arabidopsis thaliana ESCRT machinery. We show that Arabidopsis ESCRT interactome possess a number of protein-protein interactions that are either conserved in yeasts and mammals or distinct to plants. We show also that most of the Arabidopsis ESCRT proteins examined at least partially localize to MVBs in plant cells when ectopically expressed on their own or co-expressed with other interacting ESCRT proteins, and some also induce abnormal MVB phenotypes, consistent with their proposed functional roles in MVB biogenesis. Overall, our results help define the plant ESCRT machinery by highlighting both conserved and unique features when compared to ESCRT in other evolutionarily diverse organisms, providing a foundation for further exploration of ESCRT in plants.

  16. Dynamic subcellular localization of a respiratory complex controls bacterial respiration.

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    Alberge, François; Espinosa, Leon; Seduk, Farida; Sylvi, Léa; Toci, René; Walburger, Anne; Magalon, Axel

    2015-06-16

    Respiration, an essential process for most organisms, has to optimally respond to changes in the metabolic demand or the environmental conditions. The branched character of their respiratory chains allows bacteria to do so by providing a great metabolic and regulatory flexibility. Here, we show that the native localization of the nitrate reductase, a major respiratory complex under anaerobiosis in Escherichia coli, is submitted to tight spatiotemporal regulation in response to metabolic conditions via a mechanism using the transmembrane proton gradient as a cue for polar localization. These dynamics are critical for controlling the activity of nitrate reductase, as the formation of polar assemblies potentiates the electron flux through the complex. Thus, dynamic subcellular localization emerges as a critical factor in the control of respiration in bacteria.

  17. Vacuoles in mammals: a subcellular structure indispensable for early embryogenesis.

    Science.gov (United States)

    Wada, Yoh

    2013-01-01

    A vacuole is a membrane-bound subcellular structure involved in intracellular digestion. Instead of the large "vacuolar" organelles that are found in plants and fungi, animal cells possess lysosomes that are smaller in size and are enriched with hydrolytic enzymes similar to those found in the vacuoles. Large vacuolar structures are often observed in highly differentiated mammalian tissues such as embryonic visceral endoderm and absorbing epithelium. Vacuoles/lysosomes share a conserved mechanism of biogenesis, and they are at the terminal of the endocytic pathways, Recent genetic studies of the mammalian orthologs of Vam/Vps genes, which have essential functions for vacuole assembly, revealed that the dynamics of vacuoles/lysosomes are important for tissue differentiation and patterning through regulation of various molecular signaling events in mammals.

  18. Enhanced Glycogen Storage of a Subcellular Hot Spot in Human Skeletal Muscle during Early Recovery from Eccentric Contractions.

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    Joachim Nielsen

    Full Text Available Unaccustomed eccentric exercise is accompanied by muscle damage and impaired glucose uptake and glycogen synthesis during subsequent recovery. Recently, it was shown that the role and regulation of glycogen in skeletal muscle are dependent on its subcellular localization, and that glycogen synthesis, as described by the product of glycogen particle size and number, is dependent on the time course of recovery after exercise and carbohydrate availability. In the present study, we investigated the subcellular distribution of glycogen in fibers with high (type I and low (type II mitochondrial content during post-exercise recovery from eccentric contractions. Analysis was completed on five male subjects performing an exercise bout consisting of 15 x 10 maximal eccentric contractions. Carbohydrate-rich drinks were subsequently ingested throughout a 48 h recovery period and muscle biopsies for analysis included time points 3, 24 and 48 h post exercise from the exercising leg, whereas biopsies corresponding to prior to and at 48 h after the exercise bout were collected from the non-exercising, control leg. Quantitative imaging by transmission electron microscopy revealed an early (post 3 and 24 h enhanced storage of intramyofibrillar glycogen (defined as glycogen particles located within the myofibrils of type I fibers, which was associated with an increase in the number of particles. In contrast, late in recovery (post 48 h, intermyofibrillar, intramyofibrillar and subsarcolemmal glycogen in both type I and II fibers were lower in the exercise leg compared with the control leg, and this was associated with a smaller size of the glycogen particles. We conclude that in the carbohydrate-supplemented state, the effect of eccentric contractions on glycogen metabolism depends on the subcellular localization, muscle fiber's oxidative capacity, and the time course of recovery. The early enhanced storage of intramyofibrillar glycogen after the eccentric

  19. Cellular and subcellular localization of Marlin-1 in the brain

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    Luján Rafael

    2009-04-01

    Full Text Available Abstract Background Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Results Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Conclusion Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking.

  20. Cellular and subcellular localization of Marlin-1 in the brain.

    Science.gov (United States)

    Vidal, René L; Valenzuela, José I; Luján, Rafael; Couve, Andrés

    2009-04-22

    Marlin-1 is a microtubule binding protein that associates specifically with the GABAB1 subunit in neurons and with members of the Janus kinase family in lymphoid cells. In addition, it binds the molecular motor kinesin-I and nucleic acids, preferentially single stranded RNA. Marlin-1 is expressed mainly in the central nervous system but little is known regarding its cellular and subcellular distribution in the brain. Here we have studied the localization of Marlin-1 in the rodent brain and cultured neurons combining immunohistochemistry, immunofluorescence and pre-embedding electron microscopy. We demonstrate that Marlin-1 is enriched in restricted areas of the brain including olfactory bulb, cerebral cortex, hippocampus and cerebellum. Marlin-1 is abundant in dendrites and axons of GABAergic and non-GABAergic hippocampal neurons. At the ultrastructural level, Marlin-1 is present in the cytoplasm and the nucleus of CA1 neurons in the hippocampus. In the cytoplasm it associates to microtubules in the dendritic shaft and occasionally with the Golgi apparatus, the endoplasmic reticulum (ER) and dendritic spines. In the nucleus, clusters of Marlin-1 associate to euchromatin. Our results demonstrate that Marlin-1 is expressed in discrete areas of the brain. They also confirm the microtubule association at the ultrastructural level in neurons. Together with the abundance of the protein in dendrites and axons they are consistent with the emerging role of Marlin-1 as an intracellular protein linking the cytoskeleton and transport. Our study constitutes the first detailed description of the cellular and subcellular distribution of Marlin-1 in the brain. As such, it will set the basis for future studies on the functional implications of Marlin-1 in protein trafficking.

  1. Subcellular distribution and chemical forms of antimony in Ficus tikoua.

    Science.gov (United States)

    Wang, Yong; Chai, Liyuan; Yang, Zhihui; Mubarak, Hussani; Xiao, Ruiyang; Tang, Chongjian

    2017-02-01

    Ficus tikoua (F. tikoua) was a potential species for antimony (Sb) phytoremediation due to its wide growth in the mining area. However, little was known about its tolerance mechanisms toward Sb. The determination of the distribution and chemical speciation of Sb in F. tikoua is essential for understanding the mechanisms involved in Sb accumulation, transportation, and detoxification. The present study investigated the subcellular distribution and chemical forms of Sb in F. tikoua. The plant was exposed to different Sb concentrations (0, 30, 90, and 180 μmol/L) for 30 days. The results showed that F. tikoua possessed a marked ability to tolerate and accumulate Sb. The proportional Sb increased with increasing Sb concentration in the solution, and the highest Sb concentration occurred in roots (1274.5-1580.9 mg/kg), followed by stems (133.5-498.9 mg/kg) and leaves (4.1-15.7 mg/kg). In the subcellular sequestration of Sb in F. tikoua, the largest accumulation of Sb occurred in cell walls (72.4-87.5%) followed by cytoplasmic organelles (8.2-18.6%) and cytoplasmic supernatant. The results suggested that cell walls act as important protective barriers against Sb toxicity in F. tikoua. Although Sb in all plant tissues found primarily in the fractions extracted by ethanol and distilled water, the current study found that the Sb amounts in the HAc-extractable fraction, HCl-extractable fraction, and residue fraction increased at the highest Sb level (180 μmol/L) compared to that under lower Sb levels. These results indicate that excessive Sb accumulated in F. tikoua under Sb stress is bound to non-dissolved or low-bioavailable compounds, a biochemical mechanism that benefits F. tikoua because it helps alleviate Sb toxicity.

  2. Laserspritzer: a simple method for optogenetic investigation with subcellular resolutions.

    Science.gov (United States)

    Sun, Qian-Quan; Wang, Xinjun; Yang, Weiguo

    2014-01-01

    To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2) is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites). We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.

  3. Laserspritzer: a simple method for optogenetic investigation with subcellular resolutions.

    Directory of Open Access Journals (Sweden)

    Qian-Quan Sun

    Full Text Available To build a detailed circuit diagram in the brain, one needs to measure functional synaptic connections between specific types of neurons. A high-resolution circuit diagram should provide detailed information at subcellular levels such as soma, distal and basal dendrites. However, a limitation lies in the difficulty of studying long-range connections between brain areas separated by millimeters. Brain slice preparations have been widely used to help understand circuit wiring within specific brain regions. The challenge exists because long-range connections are likely to be cut in a brain slice. The optogenetic approach overcomes these limitations, as channelrhodopsin 2 (ChR2 is efficiently transported to axon terminals that can be stimulated in brain slices. Here, we developed a novel fiber optic based simple method of optogenetic stimulation: the laserspritzer approach. This method facilitates the study of both long-range and local circuits within brain slice preparations. This is a convenient and low cost approach that can be easily integrated with a slice electrophysiology setup, and repeatedly used upon initial validation. Our data with direct ChR2 mediated-current recordings demonstrates that the spatial resolution of the laserspritzer is correlated with the size of the laserspritzer, and the resolution lies within the 30 µm range for the 5 micrometer laserspritzer. Using olfactory cortical slices, we demonstrated that the laserspritzer approach can be applied to selectively activate monosynaptic perisomatic GABAergic basket synapses, or long-range intracortical glutamatergic inputs formed on different subcellular domains within the same cell (e.g. distal and proximal dendrites. We discuss significant advantages of the laserspritzer approach over the widely used collimated LED whole-field illumination method in brain slice electrophysiological research.

  4. Thyroid states regulate subcellular glucose phosphorylation activity in male mice

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    Flavia Letícia Martins Peçanha

    2017-07-01

    Full Text Available The thyroid hormones (THs, triiodothyronine (T3 and thyroxine (T4, are very important in organism metabolism and regulate glucose utilization. Hexokinase (HK is responsible for the first step of glycolysis, catalyzing the conversion of glucose to glucose 6-phosphate. HK has been found in different cellular compartments, and new functions have been attributed to this enzyme. The effects of hyperthyroidism on subcellular glucose phosphorylation in mouse tissues were examined. Tissues were removed, subcellular fractions were isolated from eu- and hyperthyroid (T3, 0.25 μg/g, i.p. during 21 days mice and HK activity was assayed. Glucose phosphorylation was increased in the particulate fraction in soleus (312.4% ± 67.1, n = 10, gastrocnemius (369.2% ± 112.4, n = 10 and heart (142.2% ± 13.6, n = 10 muscle in the hyperthyroid group compared to the control group. Hexokinase activity was not affected in brain or liver. No relevant changes were observed in HK activity in the soluble fraction for all tissues investigated. Acute T3 administration (single dose of T3, 1.25 μg/g, i.p. did not modulate HK activity. Interestingly, HK mRNA levels remained unchanged and HK bound to mitochondria was increased by T3 treatment, suggesting a posttranscriptional mechanism. Analysis of the AKT pathway showed a 2.5-fold increase in AKT and GSK3B phosphorylation in the gastrocnemius muscle in the hyperthyroid group compared to the euthyroid group. Taken together, we show for the first time that THs modulate HK activity specifically in particulate fractions and that this action seems to be under the control of the AKT and GSK3B pathways.

  5. Subcellular distribution of glutathione and cysteine in cyanobacteria

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    Tomašić, Ana; Horvat, Lucija; Fulgosi, Hrvoje

    2010-01-01

    Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria. PMID:20349253

  6. Subcellular distribution of nitroblue tetrazolium reductase (NBT-R) in human polymorphonuclear leukocytes (PMN).

    Science.gov (United States)

    Baehner, R L

    1975-11-01

    Subcellular distribution study of cytoplasmic organelles was performed on human polymorphonuclear leukocytes after homogenization in 0.34 molar sucrose by differential centrifugation and sucrose density gradient centrifugation of the homogenate. The whole homogenate and each fraction was assayed for nitroblue tetrazolium (NBT)-reductase with and without 1 mM potassium cyanide, and the distribution of this enzyme was compared to the distribution of lysozyme, peroxidase, beta-glucuronidase, and acid and alkaline phosphatase. Enzyme recovery was 97 per cent and ranged between 74 and 124 per cent. Latent activity of all enzymes except NBT-reductase, acid, and alkaline phosphatase was demonstrated by observing a four- to sixfold increase in activity after the addition of Triton-X 100. Maximal relative specific activity using either DPNH or without cyanide for NBT-reductase was found in the 100,000 x g differential centrifugation fraction and was concentrated in the less dense top fraction of the sucrose density gradient. The distribution pattern was similar to acid and alkaline phosphatase. In contrast, the maximal concentration of beta-glucuronidase and peroxidase was found in the heavier 7,200 x g granule fraction and in the more dense bottom fractions of the sucrose density gradient. Maximal lysozyme activity was concentrated in the 30,000 x g granule fraction and in the fractions located between the heaviest and lightest fractions of the sucrose density gradient. The lack of latent activity and the similarity of subcellular distribution of NBT-reductase to acid and alkaline phosphatase, two enzymes associated with microsomes and plasmalemal membranes in human polymorphonuclear leukocytes (PMN), indicates that NBT-reductase is also a nonlysosomal enzyme located in microsomes or in plasmalemal membranes. These findings support the previously described histochemical observations that initial reduction of NBT to formazan occurs on the PMN plasmalemal surface membrane at

  7. A functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity.

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    Gil, Anabel; Rodríguez-Escudero, Isabel; Stumpf, Miriam; Molina, María; Cid, Víctor J; Pulido, Rafael

    2015-01-01

    Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.

  8. Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania amazonensis promastigotes

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    José Andrés Morgado-Díaz

    2005-07-01

    Full Text Available Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME as substrate, phenylmethylsulphone fluoride (PMSF and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate, but also in a crude plasma membrane fraction (2.0-fold. Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP, with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

  9. Adenovirus E1A proteins direct subcellular redistribution of Nek9, a NimA-related kinase.

    Science.gov (United States)

    Pelka, Peter; Scimè, Anthony; Mandalfino, Christina; Joch, Monica; Abdulla, Parween; Whyte, Peter

    2007-07-01

    A monoclonal antibody raised against adenovirus E1A-associated cellular proteins recognized Nek9, a NimA-related protein kinase. Subcellular fractionation and immunofluorescence indicated that Nek9 was primarily cytoplasmic with a small portion located in the nucleus whereas E1A was primarily nuclear. Although co-immunoprecipitation experiments indicated that nuclear Nek9 interacted, directly or indirectly, with E1A, the major effect of E1A was to diminish the amount of Nek9 in the nucleus suggesting that E1A alters the subcellular distribution of Nek9 and that the interaction is transient. A Nek9 deletion mutant lacking a central RCC1-like domain interacted stably with E1A and accumulated in the nucleus in the presence of E1A, possibly representing an intermediate stage of the normally transient Nek9/E1A interaction. The interaction of Nek9 with E1A was dependent on the N-terminal sequences of E1A. Attempts to stably overexpress either Nek9 or the kinase-inactive mutant in various cell lines were unsuccessful; however, the presence of E1A allowed stable overexpression of both proteins. These results suggest that E1A disrupts a nuclear function of Nek9.

  10. iLoc-Animal: a multi-label learning classifier for predicting subcellular localization of animal proteins.

    Science.gov (United States)

    Lin, Wei-Zhong; Fang, Jian-An; Xiao, Xuan; Chou, Kuo-Chen

    2013-04-05

    Predicting protein subcellular localization is a challenging problem, particularly when query proteins have multi-label features meaning that they may simultaneously exist at, or move between, two or more different subcellular location sites. Most of the existing methods can only be used to deal with the single-label proteins. Actually, multi-label proteins should not be ignored because they usually bear some special function worthy of in-depth studies. By introducing the "multi-label learning" approach, a new predictor, called iLoc-Animal, has been developed that can be used to deal with the systems containing both single- and multi-label animal (metazoan except human) proteins. Meanwhile, to measure the prediction quality of a multi-label system in a rigorous way, five indices were introduced; they are "Absolute-True", "Absolute-False" (or Hamming-Loss"), "Accuracy", "Precision", and "Recall". As a demonstration, the jackknife cross-validation was performed with iLoc-Animal on a benchmark dataset of animal proteins classified into the following 20 location sites: (1) acrosome, (2) cell membrane, (3) centriole, (4) centrosome, (5) cell cortex, (6) cytoplasm, (7) cytoskeleton, (8) endoplasmic reticulum, (9) endosome, (10) extracellular, (11) Golgi apparatus, (12) lysosome, (13) mitochondrion, (14) melanosome, (15) microsome, (16) nucleus, (17) peroxisome, (18) plasma membrane, (19) spindle, and (20) synapse, where many proteins belong to two or more locations. For such a complicated system, the outcomes achieved by iLoc-Animal for all the aforementioned five indices were quite encouraging, indicating that the predictor may become a useful tool in this area. It has not escaped our notice that the multi-label approach and the rigorous measurement metrics can also be used to investigate many other multi-label problems in molecular biology. As a user-friendly web-server, iLoc-Animal is freely accessible to the public at the web-site .

  11. State and location dependence of action potential metabolic cost in cortical pyramidal neurons

    NARCIS (Netherlands)

    Hallermann, Stefan; de Kock, Christiaan P. J.; Stuart, Greg J.; Kole, Maarten H. P.

    2012-01-01

    Action potential generation and conduction requires large quantities of energy to restore Na+ and K+ ion gradients. We investigated the subcellular location and voltage dependence of this metabolic cost in rat neocortical pyramidal neurons. Using Na+/K+ charge overlap as a measure of action

  12. State and location dependence of action potential metabolic cost in cortical pyramidal neurons

    NARCIS (Netherlands)

    Hallermann, S.; de Kock, C.P.J.; Stuart, G.J.; Kole, M.H.

    2012-01-01

    Action potential generation and conduction requires large quantities of energy to restore Na + and K + ion gradients. We investigated the subcellular location and voltage dependence of this metabolic cost in rat neocortical pyramidal neurons. Using Na +K + charge overlap as a measure of action

  13. Carotenoids located in human lymphocyte subpopulations and Natural Killer cells by Raman microspectroscopy

    NARCIS (Netherlands)

    Puppels, G.J.; Puppels, G.J.; Garritsen, H.S.P.; Garritsen, H.S.P.; Kummer, J.A.; Greve, Jan

    1993-01-01

    The presence and subcellular location of carotenoids in human lymphocyte sub-populations (CD4+, CD8+, T-cell receptor-γδ+, and CD19+ ) and natural killer cells (CD16+ ) were studied by means of Raman microspectroscopy. In CD4+ lymphocytes a high concentration (10-3M) of carotenoids was found in the

  14. Subcellular targeting domains of sphingomyelin synthase 1 and 2.

    Science.gov (United States)

    Yeang, Calvin; Ding, Tingbo; Chirico, William J; Jiang, Xian-Cheng

    2011-12-14

    Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. Furthermore, its product SM has been implicated in atherogenic processes such as retention of lipoproteins in the blood vessel intima. There are two mammalian sphingomyelin synthases: SMS1 and SMS2. SMS1 is found exclusively in the Golgi at steady state, whereas SMS2 exists in the Golgi and plasma membrane. Conventional motifs responsible for protein targeting to the plasma membrane or Golgi are either not present in, or unique to, SMS1 and SMS2. In this study, we examined how SMS1 and SMS2 achieve their respective subcellular localization patterns. Brefeldin A treatment prevented SMS1 and SMS2 from exiting the ER, demonstrating that they transit through the classical secretory pathway. We created truncations and chimeras of SMS1 and SMS2 to define their targeting signals. We found that SMS1 contains a C-terminal Golgi targeting signal and that SMS2 contains a C-terminal plasma membrane targeting signal.

  15. Intracellular delivery of nanocarriers and targeting to subcellular organelles.

    Science.gov (United States)

    Jhaveri, Aditi; Torchilin, Vladimir

    2016-01-01

    Recent trends in drug delivery indicate a steady increase in the use of targeted therapeutics to enhance the specific delivery of biologically active payloads to diseased tissues while avoiding their off-target effects. However, in most cases, the distribution of therapeutics inside cells and their targeting to intracellular targets still presents a formidable challenge. The main barrier to intracellular delivery is the translocation of therapeutic molecules across the cell membrane, and ultimately through the membrane of their intracellular target organelles. Another prerequisite for an efficient intracellular localization of active molecules is their escape from the endocytic pathway. Pharmaceutical nanocarriers have demonstrated substantial advantages for the delivery of therapeutics and offer elegant platforms for intracellular delivery. They can be engineered with both intracellular and organelle-specific targeting moieties to deliver encapsulated or conjugated cargoes to specific sub-cellular targets. In this review, we discuss important aspects of intracellular drug targeting and delivery with a focus on nanocarriers modified with various ligands to specifically target intracellular organelles. Intracellular delivery affords selective localization of molecules to their target site, thus maximizing their efficacy and safety. The advent of novel nanocarriers and targeting ligands as well as exploration of alternate routes for the intracellular delivery and targeting has prompted extensive research, and promises an exciting future for this field.

  16. Subcellular localization of calcium deposits during zebrafish (Danio rerio) oogenesis.

    Science.gov (United States)

    Golpour, Amin; Pšenička, Martin; Niksirat, Hamid

    2016-01-01

    Calcium plays prominent roles in regulating a broad range of physiological events in reproduction. The aim of this study was to describe the subcellular distribution of calcium deposits during stages of oogenesis in zebrafish using a combined oxalate-pyroantimonate technique. The oocyte development of zebrafish was categorized into four stages: primary growth, cortical-alveolus, vitellogenic, and maturation, based on morphological criteria. Calcium deposits in the primary growth stage were detected in the cytoplasm, mitochondria, nucleus, and follicular cells. At the cortical-alveolus stage, calcium particles were transported from follicular cells and deposited in the cortical alveoli. In the vitellogenic stage, some cortical alveoli were compacted and transformed from flocculent electron-lucent to electron-dense objects with the progression of the stage. Calcium deposits were transformed from larger to smaller particles, coinciding with compaction of cortical alveoli. In the maturation stage, calcium deposits in all oocyte compartments decreased, with the exception of those in mitochondria. The proportion of area covered by calcium deposits in the mitochondria and cortical alveoli of oocytes at different stages of development was significantly different (poogenesis may contribute to better understanding of its role in oogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Spatiotemporal visualization of subcellular dynamics of carbon nanotubes

    KAUST Repository

    Serag, Maged F.

    2012-12-12

    To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of postuptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards. © 2012 American Chemical Society.

  18. A neural network classifier capable of recognizing the patterns of all major subcellular structures in fluorescence microscope images of HeLa cells.

    Science.gov (United States)

    Boland, M V; Murphy, R F

    2001-12-01

    Assessment of protein subcellular location is crucial to proteomics efforts since localization information provides a context for a protein's sequence, structure, and function. The work described below is the first to address the subcellular localization of proteins in a quantitative, comprehensive manner. Images for ten different subcellular patterns (including all major organelles) were collected using fluorescence microscopy. The patterns were described using a variety of numeric features, including Zernike moments, Haralick texture features, and a set of new features developed specifically for this purpose. To test the usefulness of these features, they were used to train a neural network classifier. The classifier was able to correctly recognize an average of 83% of previously unseen cells showing one of the ten patterns. The same classifier was then used to recognize previously unseen sets of homogeneously prepared cells with 98% accuracy. Algorithms were implemented using the commercial products Matlab, S-Plus, and SAS, as well as some functions written in C. The scripts and source code generated for this work are available at http://murphylab.web.cmu.edu/software. murphy@cmu.edu

  19. Predicting the subcellular localization of viral proteins within a mammalian host cell

    Directory of Open Access Journals (Sweden)

    Thomas DY

    2006-04-01

    Full Text Available Abstract Background The bioinformatic prediction of protein subcellular localization has been extensively studied for prokaryotic and eukaryotic organisms. However, this is not the case for viruses whose proteins are often involved in extensive interactions at various subcellular localizations with host proteins. Results Here, we investigate the extent of utilization of human cellular localization mechanisms by viral proteins and we demonstrate that appropriate eukaryotic subcellular localization predictors can be used to predict viral protein localization within the host cell. Conclusion Such predictions provide a method to rapidly annotate viral proteomes with subcellular localization information. They are likely to have widespread applications both in the study of the functions of viral proteins in the host cell and in the design of antiviral drugs.

  20. F-box protein specificity for g1 cyclins is dictated by subcellular localization.

    Directory of Open Access Journals (Sweden)

    Benjamin D Landry

    Full Text Available Levels of G1 cyclins fluctuate in response to environmental cues and couple mitotic signaling to cell cycle entry. The G1 cyclin Cln3 is a key regulator of cell size and cell cycle entry in budding yeast. Cln3 degradation is essential for proper cell cycle control; however, the mechanisms that control Cln3 degradation are largely unknown. Here we show that two SCF ubiquitin ligases, SCF(Cdc4 and SCF(Grr1, redundantly target Cln3 for degradation. While the F-box proteins (FBPs Cdc4 and Grr1 were previously thought to target non-overlapping sets of substrates, we find that Cdc4 and Grr1 each bind to all 3 G1 cyclins in cell extracts, yet only Cln3 is redundantly targeted in vivo, due in part to its nuclear localization. The related cyclin Cln2 is cytoplasmic and exclusively targeted by Grr1. However, Cdc4 can interact with Cdk-phosphorylated Cln2 and target it for degradation when cytoplasmic Cdc4 localization is forced in vivo. These findings suggest that Cdc4 and Grr1 may share additional redundant targets and, consistent with this possibility, grr1Δ cdc4-1 cells demonstrate a CLN3-independent synergistic growth defect. Our findings demonstrate that structurally distinct FBPs are capable of interacting with some of the same substrates; however, in vivo specificity is achieved in part by subcellular localization. Additionally, the FBPs Cdc4 and Grr1 are partially redundant for proliferation and viability, likely sharing additional redundant substrates whose degradation is important for cell cycle progression.

  1. Imbalanced multi-modal multi-label learning for subcellular localization prediction of human proteins with both single and multiple sites.

    Directory of Open Access Journals (Sweden)

    Jianjun He

    Full Text Available It is well known that an important step toward understanding the functions of a protein is to determine its subcellular location. Although numerous prediction algorithms have been developed, most of them typically focused on the proteins with only one location. In recent years, researchers have begun to pay attention to the subcellular localization prediction of the proteins with multiple sites. However, almost all the existing approaches have failed to take into account the correlations among the locations caused by the proteins with multiple sites, which may be the important information for improving the prediction accuracy of the proteins with multiple sites. In this paper, a new algorithm which can effectively exploit the correlations among the locations is proposed by using gaussian process model. Besides, the algorithm also can realize optimal linear combination of various feature extraction technologies and could be robust to the imbalanced data set. Experimental results on a human protein data set show that the proposed algorithm is valid and can achieve better performance than the existing approaches.

  2. The Subcellular Distribution of Small Molecules: from Pharmacokinetics to Synthetic Biology

    Science.gov (United States)

    Zheng, Nan; Tsai, Hobart Ng; Zhang, Xinyuan; Rosania, Gus R.

    2011-01-01

    The systemic pharmacokinetics and pharmacodynamics of small molecules are determined by subcellular transport phenomena. Although approaches used to study the subcellular distribution of small molecules have gradually evolved over the past several decades, experimental analysis and prediction of cellular pharmacokinetics remains a challenge. In this article, we surveyed the progress of subcellular distribution research since the 1960s, with a focus on the advantages, disadvantages and limitations of the various experimental techniques. Critical review of the existing body of knowledge pointed to many opportunities to advance the rational design of organelle-targeted chemical agents. These opportunities include: 1) development of quantitative, nonfluorescence-based, whole cell methods and techniques to measure the subcellular distribution of chemical agents in multiple compartments; 2) exploratory experimentation with nonspecific transport probes that have not been enriched with putative, organelle-targeting features; 3) elaboration of hypothesis-driven, mechanistic and modeling-based approaches to guide experiments aimed at elucidating subcellular distribution and transport; and 4) introduction of revolutionary conceptual approaches borrowed from the field of synthetic biology combined with cutting edge experimental strategies. In our laboratory, state-of-the-art subcellular transport studies are now being aimed at understanding the formation of new intracellular membrane structures in response to drug therapy, exploring the function of drug-membrane complexes as intracellular drug depots, and synthesizing new organelles with extraordinary physical and chemical properties. PMID:21805990

  3. Linking Subcellular Disturbance to Physiological Behavior and Toxicity Induced by Quantum Dots in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Qin; Zhou, Yanfeng; Song, Bin; Zhong, Yiling; Wu, Sicong; Cui, Rongrong; Cong, Haixia; Su, Yuanyuan; Zhang, Huimin; He, Yao

    2016-06-01

    The wide-ranging applications of fluorescent semiconductor quantum dots (QDs) have triggered increasing concerns about their biosafety. Most QD-related toxicity studies focus on the subcellular processes in cultured cells or global physiological effects on whole animals. However, it is unclear how QDs affect subcellular processes in living organisms, or how the subcellular disturbance contributes to the overall toxicity. Here the behavior and toxicity of QDs of three different sizes in Caenorhabditis elegans (C. elegans) are systematically investigated at both the systemic and the subcellular level. Specifically, clear size-dependent distribution and toxicity of the QDs in the digestive tract are observed. Short-term exposure of QDs leads to acute toxicity on C. elegans, yet incurring no lasting, irreversible damage. In contrast, chronic exposure of QDs severely inhibits development and shortens lifespan. Subcellular analysis reveals that endocytosis and nutrition storage are disrupted by QDs, which likely accounts for the severe deterioration in growth and longevity. This work reveals that QDs invasion disrupts key subcellular processes in living organisms, and may cause permanent damage to the tissues and organs over long-term retention. The findings provide invaluable information for safety evaluations of QD-based applications and offer new opportunities for design of novel nontoxic nanoprobes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. A sub-cellular viscoelastic model for cell population mechanics.

    Directory of Open Access Journals (Sweden)

    Yousef Jamali

    Full Text Available Understanding the biomechanical properties and the effect of biomechanical force on epithelial cells is key to understanding how epithelial cells form uniquely shaped structures in two or three-dimensional space. Nevertheless, with the limitations and challenges posed by biological experiments at this scale, it becomes advantageous to use mathematical and 'in silico' (computational models as an alternate solution. This paper introduces a single-cell-based model representing the cross section of a typical tissue. Each cell in this model is an individual unit containing several sub-cellular elements, such as the elastic plasma membrane, enclosed viscoelastic elements that play the role of cytoskeleton, and the viscoelastic elements of the cell nucleus. The cell membrane is divided into segments where each segment (or point incorporates the cell's interaction and communication with other cells and its environment. The model is capable of simulating how cells cooperate and contribute to the overall structure and function of a particular tissue; it mimics many aspects of cellular behavior such as cell growth, division, apoptosis and polarization. The model allows for investigation of the biomechanical properties of cells, cell-cell interactions, effect of environment on cellular clusters, and how individual cells work together and contribute to the structure and function of a particular tissue. To evaluate the current approach in modeling different topologies of growing tissues in distinct biochemical conditions of the surrounding media, we model several key cellular phenomena, namely monolayer cell culture, effects of adhesion intensity, growth of epithelial cell through interaction with extra-cellular matrix (ECM, effects of a gap in the ECM, tensegrity and tissue morphogenesis and formation of hollow epithelial acini. The proposed computational model enables one to isolate the effects of biomechanical properties of individual cells and the

  5. Sub-cellular distribution and translocation of TRP channels.

    Science.gov (United States)

    Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

    2011-01-01

    Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

  6. Residential Location, Job Location, and Wages: Theory and Empirics

    DEFF Research Database (Denmark)

    Vejlin, Rune Majlund

    I develop a stylized partial on-the-job equilibrium search model which incorporate a spatial dimension. Workers reside on a circle and can move at a cost. Each point on the circle has a wage distribution. Implications about wages and job mobility are drawn from the model and tested on Danish...... matched employer-employee data. The model predictions hold true. I find that workers working farther away from their residence earn higher wages. When a worker is making a job-to-job transition where he changes workplace location he experiences a higher wage change than a worker making a job-to-job...... transition without changing workplace location. However, workers making a job-to-job transition which makes the workplace location closer to the residence experiences a wage drop. Furthermore, low wage workers and workers with high transportation costs are more likely to make job-to-job transitions, but also...

  7. Pressure Transducer Locations

    Data.gov (United States)

    National Aeronautics and Space Administration — Files are located here, defining the locations of the pressure transducers on the HIRENASD model. These locations also correspond to the locations that analysts...

  8. A new method for predicting the subcellular localization of eukaryotic proteins with both single and multiple sites: Euk-mPLoc 2.0.

    Directory of Open Access Journals (Sweden)

    Kuo-Chen Chou

    Full Text Available Information of subcellular locations of proteins is important for in-depth studies of cell biology. It is very useful for proteomics, system biology and drug development as well. However, most existing methods for predicting protein subcellular location can only cover 5 to 12 location sites. Also, they are limited to deal with single-location proteins and hence failed to work for multiplex proteins, which can simultaneously exist at, or move between, two or more location sites. Actually, multiplex proteins of this kind usually posses some important biological functions worthy of our special notice. A new predictor called "Euk-mPLoc 2.0" is developed by hybridizing the gene ontology information, functional domain information, and sequential evolutionary information through three different modes of pseudo amino acid composition. It can be used to identify eukaryotic proteins among the following 22 locations: (1 acrosome, (2 cell wall, (3 centriole, (4 chloroplast, (5 cyanelle, (6 cytoplasm, (7 cytoskeleton, (8 endoplasmic reticulum, (9 endosome, (10 extracell, (11 Golgi apparatus, (12 hydrogenosome, (13 lysosome, (14 melanosome, (15 microsome (16 mitochondria, (17 nucleus, (18 peroxisome, (19 plasma membrane, (20 plastid, (21 spindle pole body, and (22 vacuole. Compared with the existing methods for predicting eukaryotic protein subcellular localization, the new predictor is much more powerful and flexible, particularly in dealing with proteins with multiple locations and proteins without available accession numbers. For a newly-constructed stringent benchmark dataset which contains both single- and multiple-location proteins and in which none of proteins has pairwise sequence identity to any other in a same location, the overall jackknife success rate achieved by Euk-mPLoc 2.0 is more than 24% higher than those by any of the existing predictors. As a user-friendly web-server, Euk-mPLoc 2.0 is freely accessible at http

  9. Partial tooth gear bearings

    Science.gov (United States)

    Vranish, John M. (Inventor)

    2010-01-01

    A partial gear bearing including an upper half, comprising peak partial teeth, and a lower, or bottom, half, comprising valley partial teeth. The upper half also has an integrated roller section between each of the peak partial teeth with a radius equal to the gear pitch radius of the radially outwardly extending peak partial teeth. Conversely, the lower half has an integrated roller section between each of the valley half teeth with a radius also equal to the gear pitch radius of the peak partial teeth. The valley partial teeth extend radially inwardly from its roller section. The peak and valley partial teeth are exactly out of phase with each other, as are the roller sections of the upper and lower halves. Essentially, the end roller bearing of the typical gear bearing has been integrated into the normal gear tooth pattern.

  10. Subcellular localization and displacement by diuretics of the peripheral benzodiazepine binding site (PBS) from rat kidney

    Energy Technology Data Exchange (ETDEWEB)

    Lukeman, S.; Fanestil, D.

    1986-03-05

    Although the PBS has been identified in many organs, its function and cellular location are speculative. Using rapid filtration, binding of (/sup 3/H)RO 5-4864 (*RO) (.75 nM) was assessed in four subcellular fractions (.3 mg/ml) derived from depapillated rat kidney by differential centrifugation: N (450g x 2 min), O (13,000 x 10), P (105,000 x 30), and S. The binding distribution was: N-18%, O-74%, P-6%, and S-2%. Marker enzyme analysis revealed that O was enriched in mitochondria (M), lysosomes (L), peroxisomes (P), and endoplasmic reticulum (ER), but not plasma membrane, and that N contained small amounts (10-15%) of markers for the above. Repeated washing of O removed ER enzymes but preserved *RO binding. O was further fractionated with centrifugation (57,000g x 4 hr) on a linear sucrose gradient (18-65%); *RO binding then comigrated with M but not P and L markers. Centrifugation of isolated M (5500 x 10 min) on another linear sucrose gradient (37-65%) gave low and high density bands, which contained 65% and 35% of *RO binding activity, resp. *RO binding in O was specific, saturable, reversible, and inhibited by diuretics. Inhibitors with the highest potency were indacrinone (K/sub d/ = 35 ..mu..M), hydrochlorothiazide (100 ..mu..M), and ethacrynic acid (325 ..mu..M). Low potency inhibitors (K/sub d/ greater than or equal to 1 mM) included amiloride, triamterene, furosemide, bumetanide, and ozolinone.

  11. PA-GFP: a window into the subcellular adventures of the individual mitochondrion.

    Science.gov (United States)

    Haigh, Sarah E; Twig, Gilad; Molina, Anthony A J; Wikstrom, Jakob D; Deutsch, Motti; Shirihai, Orian S

    2007-01-01

    Mitochondrial connectivity is characterized by matrix lumen continuity and by dynamic rewiring through fusion and fission events. While these mechanisms homogenize the mitochondrial population, a number of studies looking at mitochondrial membrane potential have demonstrated that mitochondria exist as a heterogeneous population within individual cells. To address the relationship between mitochondrial dynamics and heterogeneity, we tagged and tracked individual mitochondria over time while monitoring their mitochondrial membrane potential (deltapsi(m)). By utilizing photoactivatible-GFP (PA-GFP), targeted to the mitochondrial matrix, we determined the boundaries of the individual mitochondrion. A single mitochondrion is defined by the continuity of its matrix lumen. The boundaries set by luminal continuity matched those set by electrical coupling, indicating that the individual mitochondrion is equipotential throughout the entire organelle. Similar results were obtained with PA-GFP targeted to the inner membrane indicating that matrix continuity parallels inner membrane continuity. Sequential photoconversion of matrix PA-GFP in multiple locations within the mitochondrial web reveals that each ramified mitochondrial structure is composed of juxtaposed but discontinuous units. Moreover, as many as half of the events in which mitochondria come into contact, do not result in fusion. While all fission events generated two electrically uncoupled discontinuous matrices, the two daughter mitochondria frequently remained juxtaposed, keeping the tubular appearance unchanged. These morphologically invisible fission events illustrate the difference between mitochondrial fission and fragmentation; the latter representing the movement and separation of disconnected units. Simultaneous monitoring of deltapsi(m) of up to four individual mitochondria within the same cell revealed that subcellular heterogeneity in deltapsi(m) does not represent multiple unstable mitochondria that

  12. Palmitoylation of stathmin family proteins domain A controls Golgi versus mitochondrial subcellular targeting.

    Science.gov (United States)

    Chauvin, Stéphanie; Poulain, Fabienne E; Ozon, Sylvie; Sobel, André

    2008-10-01

    Precise localization of proteins to specialized subcellular domains is fundamental for proper neuronal development and function. The neural microtubule-regulatory phosphoproteins of the stathmin family are such proteins whose specific functions are controlled by subcellular localization. Whereas stathmin is cytosolic, SCG10, SCLIP and RB3/RB3'/RB3'' are localized to the Golgi and vesicle-like structures along neurites and at growth cones. We examined the molecular determinants involved in the regulation of this specific subcellular localization in hippocampal neurons in culture. We show that their conserved N-terminal domain A carrying two palmitoylation sites is dominant over the others for Golgi and vesicle-like localization. Using palmitoylation-deficient GFP (green fluorescent protein) fusion mutants, we demonstrate that domains A of stathmin proteins have the particular ability to control protein targeting to either Golgi or mitochondria, depending on their palmitoylation. This regulation involves the co-operation of two subdomains within domain A, and seems also to be under the control of its SLD (stathmin-like domain) extension. Our results unravel that, in specific biological conditions, palmitoylation of stathmin proteins might be able to control their targeting to express their functional activities at appropriate subcellular sites. They, more generally, open new perspectives regarding the role of palmitoylation as a signalling mechanism orienting proteins to their functional subcellular compartments.

  13. Sub-cellular force microscopy in single normal and cancer cells.

    Science.gov (United States)

    Babahosseini, H; Carmichael, B; Strobl, J S; Mahmoodi, S N; Agah, M

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Boosting accuracy of automated classification of fluorescence microscope images for location proteomics

    Directory of Open Access Journals (Sweden)

    Huang Kai

    2004-06-01

    Full Text Available Abstract Background Detailed knowledge of the subcellular location of each expressed protein is critical to a full understanding of its function. Fluorescence microscopy, in combination with methods for fluorescent tagging, is the most suitable current method for proteome-wide determination of subcellular location. Previous work has shown that neural network classifiers can distinguish all major protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Building on these results, we evaluate here new classifiers and features to improve the recognition of protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Results We report here a thorough comparison of the performance on this problem of eight different state-of-the-art classification methods, including neural networks, support vector machines with linear, polynomial, radial basis, and exponential radial basis kernel functions, and ensemble methods such as AdaBoost, Bagging, and Mixtures-of-Experts. Ten-fold cross validation was used to evaluate each classifier with various parameters on different Subcellular Location Feature sets representing both 2D and 3D fluorescence microscope images, including new feature sets incorporating features derived from Gabor and Daubechies wavelet transforms. After optimal parameters were chosen for each of the eight classifiers, optimal majority-voting ensemble classifiers were formed for each feature set. Comparison of results for each image for all eight classifiers permits estimation of the lower bound classification error rate for each subcellular pattern, which we interpret to reflect the fraction of cells whose patterns are distorted by mitosis, cell death or acquisition errors. Overall, we obtained statistically significant improvements in classification accuracy over the best previously published results, with the overall error rate being reduced by one-third to one-half and with the average

  15. Boosting accuracy of automated classification of fluorescence microscope images for location proteomics.

    Science.gov (United States)

    Huang, Kai; Murphy, Robert F

    2004-06-18

    Detailed knowledge of the subcellular location of each expressed protein is critical to a full understanding of its function. Fluorescence microscopy, in combination with methods for fluorescent tagging, is the most suitable current method for proteome-wide determination of subcellular location. Previous work has shown that neural network classifiers can distinguish all major protein subcellular location patterns in both 2D and 3D fluorescence microscope images. Building on these results, we evaluate here new classifiers and features to improve the recognition of protein subcellular location patterns in both 2D and 3D fluorescence microscope images. We report here a thorough comparison of the performance on this problem of eight different state-of-the-art classification methods, including neural networks, support vector machines with linear, polynomial, radial basis, and exponential radial basis kernel functions, and ensemble methods such as AdaBoost, Bagging, and Mixtures-of-Experts. Ten-fold cross validation was used to evaluate each classifier with various parameters on different Subcellular Location Feature sets representing both 2D and 3D fluorescence microscope images, including new feature sets incorporating features derived from Gabor and Daubechies wavelet transforms. After optimal parameters were chosen for each of the eight classifiers, optimal majority-voting ensemble classifiers were formed for each feature set. Comparison of results for each image for all eight classifiers permits estimation of the lower bound classification error rate for each subcellular pattern, which we interpret to reflect the fraction of cells whose patterns are distorted by mitosis, cell death or acquisition errors. Overall, we obtained statistically significant improvements in classification accuracy over the best previously published results, with the overall error rate being reduced by one-third to one-half and with the average accuracy for single 2D images being higher than

  16. Comparison of expressed human and mouse sodium/iodide sym-porters reveals differences in transport properties and subcellular localization

    Energy Technology Data Exchange (ETDEWEB)

    Dayem, M.; Basquin, C.; Navarro, V.; Carrier, P.; Marsault, R.; Lindenthal, S.; Pourcher, T. [Univ Nice Sophia Antipolis, Sch Med, CEA, DSV, iBEB, SBTN, TIRO, F-06107 Nice (France); Chang, P. [CNRS, UPMC Biol Dev, UMR 7009, F-06230 Villefranche Sur Mer (France); Huc, S.; Darrouzet, E. [CEA Valrho, DSV, iBEB, SBTN, F-30207 Bagnols Sur Ceze (France)

    2008-07-01

    The active transport of iodide from the blood stream into thyroid follicular cells is mediated by the Na{sup +}/I{sup -} sym-porter (NIS). We studied mouse NIS (mNIS) and found that it catalyzes iodide transport into transfected cells more efficiently than human NIS (hNIS). To further characterize this difference,we compared {sup 125}I, uptake in the transiently transfected human embryonic kidney (HEK) 293 cells. We found that the Vmax for mNIS was four times higher than that for hNIS, and that the iodide transport constant (Km) was 2-5-fold lower for hNIS than mNIS. We also performed immuno-cyto-localization studies and observed that the subcellular distribution of the two ortho-logs differed. While the mouse protein was predominantly found at the plasma membrane, its human ortho-log was intracellular in {approx} 40% of the expressing cells. Using cell surface protein-labeling assays, we found that the plasma membrane localization frequency of the mouse protein was only 2-5-fold higher than that of the human protein, and therefore cannot alone account for,x values. We reasoned that the difference in the obtained Vmax the observed difference could also be caused by a higher turnover number for iodide transport in the mouse protein. We then expressed and analyzed chimeric proteins. The data obtained with these constructs suggest that the iodide recognition site could be located in the region extending from the N-terminus to transmembrane domain 8, and that the region between transmembrane domain 5 and the C-terminus could play a role in the subcellular localization of the protein. (authors)

  17. A Location Privacy Aware Friend Locator

    DEFF Research Database (Denmark)

    Siksnys, Laurynas; Thomsen, Jeppe Rishede; Saltenis, Simonas

    2009-01-01

    to trade their location privacy for quality of service, limiting the attractiveness of the services. The challenge is to develop a communication-efficient solution such that (i) it detects proximity between a user and the user’s friends, (ii) any other party is not allowed to infer the location of the user...

  18. Global targeting of subcellular heat shock protein-90 networks for therapy of glioblastoma.

    Science.gov (United States)

    Siegelin, Markus D; Plescia, Janet; Raskett, Christopher M; Gilbert, Candace A; Ross, Alonzo H; Altieri, Dario C

    2010-06-01

    Drug discovery for complex and heterogeneous tumors now aims at dismantling global networks of disease maintenance, but the subcellular requirements of this approach are not understood. Here, we simultaneously targeted the multiple subcellular compartments of the molecular chaperone heat shock protein-90 (Hsp90) in a model of glioblastoma, a highly lethal human malignancy in urgent need of fresh therapeutic strategies. Treatment of cultured or patient-derived glioblastoma cells with Shepherdin, a dual peptidomimetic inhibitor of mitochondrial and cytosolic Hsp90, caused irreversible collapse of mitochondria, degradation of Hsp90 client proteins in the cytosol, and tumor cell killing by apoptosis and autophagy. Stereotactic or systemic delivery of Shepherdin was well tolerated and suppressed intracranial glioma growth via inhibition of cell proliferation, induction of apoptosis, and reduction of angiogenesis in vivo. These data show that disabling Hsp90 cancer networks in their multiple subcellular compartments improves strategies for drug discovery and may provide novel molecular therapy for highly recalcitrant human tumors.

  19. Cystic partially differentiated nephroblastoma.

    Science.gov (United States)

    Puvaneswary, M; Macintosh, J; Cassey, J

    2006-06-01

    Cystic partially differentiated nephroblastoma is a relatively rare tumour of the kidney usually affecting infants. Cystic Wilms' tumour and multilocular cystic nephroma should be distinguished from cystic partially differentiated nephroblastoma. Multilocular cystic nephroma is a benign tumour whereas cystic Wilms' tumour is at the malignant end of the range of classification of such tumours. Cystic partially differentiated nephroblastoma may undergo local recurrence but there is no report of metastasis.

  20. Comparative study of human mitochondrial proteome reveals extensive protein subcellular relocalization after gene duplications

    Directory of Open Access Journals (Sweden)

    Huang Yong

    2009-11-01

    Full Text Available Abstract Background Gene and genome duplication is the principle creative force in evolution. Recently, protein subcellular relocalization, or neolocalization was proposed as one of the mechanisms responsible for the retention of duplicated genes. This hypothesis received support from the analysis of yeast genomes, but has not been tested thoroughly on animal genomes. In order to evaluate the importance of subcellular relocalizations for retention of duplicated genes in animal genomes, we systematically analyzed nuclear encoded mitochondrial proteins in the human genome by reconstructing phylogenies of mitochondrial multigene families. Results The 456 human mitochondrial proteins selected for this study were clustered into 305 gene families including 92 multigene families. Among the multigene families, 59 (64% consisted of both mitochondrial and cytosolic (non-mitochondrial proteins (mt-cy families while the remaining 33 (36% were composed of mitochondrial proteins (mt-mt families. Phylogenetic analyses of mt-cy families revealed three different scenarios of their neolocalization following gene duplication: 1 relocalization from mitochondria to cytosol, 2 from cytosol to mitochondria and 3 multiple subcellular relocalizations. The neolocalizations were most commonly enabled by the gain or loss of N-terminal mitochondrial targeting signals. The majority of detected subcellular relocalization events occurred early in animal evolution, preceding the evolution of tetrapods. Mt-mt protein families showed a somewhat different pattern, where gene duplication occurred more evenly in time. However, for both types of protein families, most duplication events appear to roughly coincide with two rounds of genome duplications early in vertebrate evolution. Finally, we evaluated the effects of inaccurate and incomplete annotation of mitochondrial proteins and found that our conclusion of the importance of subcellular relocalization after gene duplication on

  1. Subcellular partitioning of metals in Aporrectodea caliginosa along a gradient of metal exposure in 31 field-contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Beaumelle, Léa [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France); Gimbert, Frédéric [Laboratoire Chrono-Environnement, UMR 6249 University of Franche-Comté/CNRS Usc INRA, 16 route de Gray, 25030 Besançon Cedex (France); Hedde, Mickaël [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France); Guérin, Annie [INRA, US 0010 LAS Laboratoire d' analyses des sols, 273 rue de Cambrai, 62000 Arras (France); Lamy, Isabelle, E-mail: lamy@versailles.inra.fr [INRA, UR 251 PESSAC, 78026 Versailles Cedex (France)

    2015-07-01

    Subcellular fractionation of metals in organisms was proposed as a better way to characterize metal bioaccumulation. Here we report the impact of a laboratory exposure to a wide range of field-metal contaminated soils on the subcellular partitioning of metals in the earthworm Aporrectodea caliginosa. Soils moderately contaminated were chosen to create a gradient of soil metal availability; covering ranges of both soil metal contents and of several soil parameters. Following exposure, Cd, Pb and Zn concentrations were determined both in total earthworm body and in three subcellular compartments: cytosolic, granular and debris fractions. Three distinct proxies of soil metal availability were investigated: CaCl{sub 2}-extractable content dissolved content predicted by a semi-mechanistic model and free ion concentration predicted by a geochemical speciation model. Subcellular partitionings of Cd and Pb were modified along the gradient of metal exposure, while stable Zn partitioning reflected regulation processes. Cd subcellular distribution responded more strongly to increasing soil Cd concentration than the total internal content, when Pb subcellular distribution and total internal content were similarly affected. Free ion concentrations were better descriptors of Cd and Pb subcellular distribution than CaCl{sub 2} extractable and dissolved metal concentrations. However, free ion concentrations and soil total metal contents were equivalent descriptors of the subcellular partitioning of Cd and Pb because they were highly correlated. Considering lowly contaminated soils, our results raise the question of the added value of three proxies of metal availability compared to soil total metal content in the assessment of metal bioavailability to earthworm. - Highlights: • Earthworms were exposed to a wide panel of historically contaminated soils • Subcellular partitioning of Cd, Pb and Zn was investigated in earthworms • Three proxies of soil metal availability were

  2. Neonatal anoxia in rats: hippocampal cellular and subcellular changes related to cell death and spatial memory.

    Science.gov (United States)

    Takada, S H; dos Santos Haemmerle, C A; Motta-Teixeira, L C; Machado-Nils, A V; Lee, V Y; Takase, L F; Cruz-Rizzolo, R J; Kihara, A H; Xavier, G F; Watanabe, I-S; Nogueira, M I

    2015-01-22

    Neonatal anoxia in rodents has been used to understand brain changes and cognitive dysfunction following asphyxia. This study investigated the time-course of cellular and subcellular changes and hippocampal cell death in a non-invasive model of anoxia in neonatal rats, using Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) to reveal DNA fragmentation, Fluoro-Jade® B (FJB) to show degenerating neurons, cleaved caspase-3 immunohistochemistry (IHC) to detect cells undergoing apoptosis, and transmission electron microscopy (TEM) to reveal fine ultrastructural changes related to cell death. Anoxia was induced by exposing postnatal day 1 (P1) pups to a flow of 100% gaseous nitrogen for 25 min in a chamber maintained at 37 °C. Control rats were similarly exposed to this chamber but with air flow instead of nitrogen. Brain changes following anoxia were evaluated at postnatal days 2, 14, 21 and 60 (P2, P14, P21 and P60). In addition, spatial reference memory following anoxia and control treatments was evaluated in the Morris water maze, starting at P60. Compared to their respective controls, P2 anoxic rats exhibited (1) higher TUNEL labeling in cornus ammonis (CA) 1 and the dentate gyrus (DG), (2) higher FJB-positive cells in the CA2-3, and (3) somato-dendritic swelling, mitochondrial injury and chromatin condensation in irregular bodies, as well as other subcellular features indicating apoptosis, necrosis, autophagy and excitotoxicity in the CA1, CA2-3 and DG, as revealed by TEM. At P14, P21 and P60, both groups showed small numbers of TUNEL-positive and FJB-positive cells. Stereological analysis at P2, P14, P21 and P60 revealed a lack of significant differences in cleaved caspase-3 IHC between anoxic and control subjects. These results suggest that the type of hippocampal cell death following neonatal anoxia is likely independent of caspase-3 activation. Neonatal anoxia induced deficits in acquisition and performance of spatial reference

  3. Subcellular Redox Targeting: Bridging in Vitro and in Vivo Chemical Biology.

    Science.gov (United States)

    Long, Marcus J C; Poganik, Jesse R; Ghosh, Souradyuti; Aye, Yimon

    2017-03-17

    Networks of redox sensor proteins within discrete microdomains regulate the flow of redox signaling. Yet, the inherent reactivity of redox signals complicates the study of specific redox events and pathways by traditional methods. Herein, we review designer chemistries capable of measuring flux and/or mimicking subcellular redox signaling at the cellular and organismal level. Such efforts have begun to decipher the logic underlying organelle-, site-, and target-specific redox signaling in vitro and in vivo. These data highlight chemical biology as a perfect gateway to interrogate how nature choreographs subcellular redox chemistry to drive precision redox biology.

  4. Calculation of the relative metastabilities of proteins in subcellular compartments of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Dick Jeffrey M

    2009-07-01

    Full Text Available Abstract Background Protein subcellular localization and differences in oxidation state between subcellular compartments are two well-studied features of the the cellular organization of S. cerevisiae (yeast. Theories about the origin of subcellular organization are assisted by computational models that can integrate data from observations of compositional and chemical properties of the system. Presentation and implications of the hypothesis I adopt the hypothesis that the state of yeast subcellular organization is in a local energy minimum. This hypothesis implies that equilibrium thermodynamic models can yield predictions about the interdependence between populations of proteins and their subcellular chemical environments. Testing the hypothesis Three types of tests are proposed. First, there should be correlations between modeled and observed oxidation states for different compartments. Second, there should be a correspondence between the energy requirements of protein formation and the order the appearance of organelles during cellular development. Third, there should be correlations between the predicted and observed relative abundances of interacting proteins within compartments. Results The relative metastability fields of subcellular homologs of glutaredoxin and thioredoxin indicate a trend from less to more oxidizing as mitochondrion – cytoplasm – nucleus. Representing the overall amino acid compositions of proteins in 23 different compartments each with a single reference model protein suggests that the formation reactions for proteins in the vacuole (in relatively oxidizing conditions, ER and early Golgi (in relatively reducing conditions are relatively highly favored, while that for the microtubule is the most costly. The relative abundances of model proteins for each compartment inferred from experimental data were found in some cases to correlate with the predicted abundances, and both positive and negative correlations were

  5. Subcellular Localization of Galloylated Catechins in Tea Plants (Camellia sinensis (L. O. Kuntze Assessed via Immunohistochemistry

    Directory of Open Access Journals (Sweden)

    Huanhuan eXu

    2016-05-01

    Full Text Available Galloylated catechins, as the main secondary metabolites in the tea plant, including (--epigallocatechin-3-gallate and (--epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physiological functions. Classical histochemical localization staining reagents can not specifically detect galloylated catechins; thus, their subcellular localization remains controversial. In the present study, we generated a monoclonal antibody (mAb against galloylated catechins, which can be used for the subcellular localization of galloylated catechins in the tea plant by immunohistochemistry. Direct ELISA and ForteBio Octet Red 96 System assay indicated the mAb could recognize the galloylated catechins with high specificities and affinities. In addition, tea bud was ascertained as the optimal tissue for freezing microtomic sections for immunohistochemistry. What’s more, the high quality mAbs which exhibited excellent binding capability to galloylated catechins were utilised for the visualization of them via immunohistochemistry. Our findings demonstrated that vacuoles were the primary sites of localization of galloylated catechins at the subcellular level.

  6. Halides tuning the subcellular-targeting in two-photon emissive complexes via different uptake mechanisms.

    Science.gov (United States)

    Tian, Xiaohe; Zhu, Yingzhong; Zhang, Qiong; Zhang, Ruilong; Wu, Jieying; Tian, Yupeng

    2017-07-11

    We reported a simple and universal strategy by tuning halides (Cl, Br and I) in terpyridine-Zn(ii) complexes to achieve different subcellular organelle targeting (nucleolus, nucleus and intracellular membrane systems, respectively) via different cellular uptake mechanisms, resulting from halide triggering different polymorphs of these complexes.

  7. Multivalency Effect of TAT-Peptide-Functionalized Nanoparticle in Cellular Endocytosis and Subcellular Trafficking.

    Science.gov (United States)

    Dalal, Chumki; Jana, Nikhil R

    2017-04-13

    Although trans-activating transcription (TAT) peptide-functionalized nanoparticle/polymer/liposome is widely used for cellular transfection applications, the multivalency (number of attached peptide per particle) effect on cell uptake mechanism and subcellular targeting performance is largely unexplored. Here we show that multivalency of nanoparticle controls the cellular interaction, cellular entry/exit mechanism, and subcellular targeting performance. We have synthesized TAT-peptide functionalized quantum dot (QD) of 30-35 nm hydrodynamic diameter with varied multivalency from 10 to 75 (e.g., QD(TAT)10, QD(TAT)20, QD(TAT)40, QD(TAT)75) and studied the role of multivalency in endocytosis and subcellular trafficking. We found that both low and high multivalent nanoparticles enter into cell predominantly via lipid-raft mediated endocytosis but the higher multivalency of 40 and 75 induces vesicular trapping followed by exocytosis within 12 h. In contrast, lower multivalency of 10 and 20 offers efficient trafficking toward perinuclear region and Golgi apparatus. This work shows the functional role of nanoparticle multivalency in cellular uptake mechanism and importance of lower multivalency for efficient subcellular targeting.

  8. Parasites modify sub-cellular partitioning of metals in the gut of fish

    NARCIS (Netherlands)

    Oyoo-Okoth, E.; Admiraal, W.; Osano, O.; Kraak, M.H.S.; Gichuki, J.; Ogwai, C.

    2012-01-01

    Infestation of fish by parasites may influence metal accumulation patterns in the host. However, the subcellular mechanisms of these processes have rarely been studied. Therefore, this study determined how a cyprinid fish (Rastrineobola argentea) partitioned four metals (Cd, Cr, Zn and Cu) in the

  9. Exploring Nanostructure Arrays for Single-Cell and Subcellular Manipulation and Detection

    DEFF Research Database (Denmark)

    Buch-Månson, Nina

    these, arrays of vertical nanostructures (NSs) with submicron diametersand microscale lengths are particularly promising and are currently being established as bothhighly sensitive protein arrays and as platforms for manipulations and investigations at thesingle-cell or even subcellular level.To date...

  10. Subcellular localization and mechanism of secretion of vascular endothelial growth factor in human skeletal muscle

    DEFF Research Database (Denmark)

    Høier, Birgitte; Prats Gavalda, Clara; Qvortrup, Klaus

    2013-01-01

    The subcellular distribution and secretion of vascular endothelial growth factor (VEGF) was examined in skeletal muscle of healthy humans. Skeletal muscle biopsies were obtained from m.v. lateralis before and after a 2 h bout of cycling exercise. VEGF localization was conducted on preparations...

  11. Development of a Charged Particle Microbeam for Targeted and Single Particle Subcellular Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Yanch, Jacquelyn C.

    2004-03-12

    The development of a charged particle microbeam for single particle, subcellular irradiations at the Massachusetts Institute of Technology Laboratory for Accelerator Beam Applications (MIT LABA) was initiated under this NEER aeard. The Microbeam apparatus makes use of a pre-existing electrostatic accelerator with a horizontal beam tube.

  12. DeepLoc: prediction of protein subcellular localization using deep learning.

    Science.gov (United States)

    Almagro Armenteros, José Juan; Sønderby, Casper Kaae; Sønderby, Søren Kaae; Nielsen, Henrik; Winther, Ole

    2017-11-01

    The prediction of eukaryotic protein subcellular localization is a well-studied topic in bioinformatics due to its relevance in proteomics research. Many machine learning methods have been successfully applied in this task, but in most of them, predictions rely on annotation of homologues from knowledge databases. For novel proteins where no annotated homologues exist, and for predicting the effects of sequence variants, it is desirable to have methods for predicting protein properties from sequence information only. Here, we present a prediction algorithm using deep neural networks to predict protein subcellular localization relying only on sequence information. At its core, the prediction model uses a recurrent neural network that processes the entire protein sequence and an attention mechanism identifying protein regions important for the subcellular localization. The model was trained and tested on a protein dataset extracted from one of the latest UniProt releases, in which experimentally annotated proteins follow more stringent criteria than previously. We demonstrate that our model achieves a good accuracy (78% for 10 categories; 92% for membrane-bound or soluble), outperforming current state-of-the-art algorithms, including those relying on homology information. The method is available as a web server at http://www.cbs.dtu.dk/services/DeepLoc. Example code is available at https://github.com/JJAlmagro/subcellular_localization. The dataset is available at http://www.cbs.dtu.dk/services/DeepLoc/data.php. jjalma@dtu.dk.

  13. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns

    DEFF Research Database (Denmark)

    Lundby, Alicia; Hansen, Kasper Lage; Weinert, Brian Tate

    2012-01-01

    that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle...

  14. Subcellular localization of Bombyx mori ribosomal protein S3a and ...

    African Journals Online (AJOL)

    Subcellular localization of Bombyx mori ribosomal protein S3a and effect of its over-expression on BmNPV infection. Z Wu-song, B Xian-xun, X Jia-ping, Y Zheng-ying, Y Ying, W Hui-ling, W Wen-bing ...

  15. MultiLoc2: integrating phylogeny and Gene Ontology terms improves subcellular protein localization prediction

    Directory of Open Access Journals (Sweden)

    Kohlbacher Oliver

    2009-09-01

    Full Text Available Abstract Background Knowledge of subcellular localization of proteins is crucial to proteomics, drug target discovery and systems biology since localization and biological function are highly correlated. In recent years, numerous computational prediction methods have been developed. Nevertheless, there is still a need for prediction methods that show more robustness and higher accuracy. Results We extended our previous MultiLoc predictor by incorporating phylogenetic profiles and Gene Ontology terms. Two different datasets were used for training the system, resulting in two versions of this high-accuracy prediction method. One version is specialized for globular proteins and predicts up to five localizations, whereas a second version covers all eleven main eukaryotic subcellular localizations. In a benchmark study with five localizations, MultiLoc2 performs considerably better than other methods for animal and plant proteins and comparably for fungal proteins. Furthermore, MultiLoc2 performs clearly better when using a second dataset that extends the benchmark study to all eleven main eukaryotic subcellular localizations. Conclusion MultiLoc2 is an extensive high-performance subcellular protein localization prediction system. By incorporating phylogenetic profiles and Gene Ontology terms MultiLoc2 yields higher accuracies compared to its previous version. Moreover, it outperforms other prediction systems in two benchmarks studies. MultiLoc2 is available as user-friendly and free web-service, available at: http://www-bs.informatik.uni-tuebingen.de/Services/MultiLoc2.

  16. Chagas' disease: humoral response to subcellular fraction of Trypanosoma cruzi in symptomatic and asymptomatic patients.

    Science.gov (United States)

    de Titto, E H; Moreno, M; Braun, M; Segura, E L

    1987-09-01

    The capacity of antibodies in serum from individuals with chronic Chagas' disease to react with antigens in different subcellular fractions of Trypanosoma cruzi varied according to the clinical status of the patients. Antibodies in serum of asymptomatic patients were directed mostly against antigens in the citosol of the parasite, whereas in overtly cardiopathic patients antibodies were directed mostly against antigens in the microsomal fractions.

  17. Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa

    Directory of Open Access Journals (Sweden)

    Yasuhide Osaki

    2017-09-01

    Full Text Available Particle bombardment is a powerful and relatively easy method for transient expression of genes of interest in plant cells, especially those that are recalcitrant to other transformation methods. This method has facilitated numerous analyses of subcellular localization of fluorescent fusion protein constructs. Particle bombardment delivers genes to the first layer of plant tissue. In leaves of higher plants, epidermal cells are the first cell layer. Many studies have used the epidermal cell layer of onion bulb (Allium cepa as the experimental tissue, because these cells are relatively large. However, onion epidermal cells lack developed plastids (i.e., chloroplasts, thereby precluding subcellular localization analysis of chloroplastic proteins. In this study, we developed a protocol for particle bombardment of the aquatic plant Egeria densa, and showed that it is a useful system for subcellular localization analysis of higher plant proteins. E. densa leaflets contain only two cell layers, and cells in the adaxial layer are sufficiently large for observation. The cells in both layers contain well-developed chloroplasts. We fused fluorescent proteins to conventional plant localization signals for the nucleus, cytosol, mitochondria, peroxisome, and chloroplast, and used particle bombardment to transiently express these fusion constructs in E. densa leaves. The plant subcellular localization signals functioned normally and displayed the expected distributions in transiently transformed E. densa cells, and even chloroplastic structures could be clearly visualized.

  18. Organelle-targeting surface-enhanced Raman scattering (SERS) nanosensors for subcellular pH sensing.

    Science.gov (United States)

    Shen, Yanting; Liang, Lijia; Zhang, Shuqin; Huang, Dianshuai; Zhang, Jing; Xu, Shuping; Liang, Chongyang; Xu, Weiqing

    2018-01-25

    The pH value of subcellular organelles in living cells is a significant parameter in the physiological activities of cells. Its abnormal fluctuations are commonly believed to be associated with cancers and other diseases. Herein, a series of surface-enhanced Raman scattering (SERS) nanosensors with high sensitivity and targeting function was prepared for the quantification and monitoring of pH values in mitochondria, nucleus, and lysosome. The nanosensors were composed of gold nanorods (AuNRs) functionalized with a pH-responsive molecule (4-mercaptopyridine, MPy) and peptides that could specifically deliver the AuNRs to the targeting subcellular organelles. The localization of our prepared nanoprobes in specific organelles was confirmed by super-high resolution fluorescence imaging and bio-transmission electron microscopy (TEM) methods. By the targeting ability, the pH values of the specific organelles can be determined by monitoring the vibrational spectral changes of MPy with different pH values. Compared to the cases of reported lysosome and cytoplasm SERS pH sensors, more accurate pH values of mitochondria and nucleus, which could be two additional intracellular tracers for subcellular microenvironments, were disclosed by this SERS approach, further improving the accuracy of discrimination of related diseases. Our sensitive SERS strategy can also be employed to explore crucial physiological and biological processes that are related to subcellular pH fluctuations.

  19. Subcellular interactions of dietary cadmium, copper and zinc in rainbow trout (Oncorhynchus mykiss)

    Energy Technology Data Exchange (ETDEWEB)

    Kamunde, Collins, E-mail: ckamunde@upei.ca [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3 (Canada); MacPhail, Ruth [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, C1A 4P3 (Canada)

    2011-10-15

    Highlights: Interactions of Cu, Cd and Zn were studied at the subcellular level in rainbow trout. Metals accumulated in the liver were predominantly metabolically active. Cd, Cu and Zn exhibited both competitive and cooperative interactions. The metal-metal interactions altered subcellular metals partitioning. - Abstract: Interactions of Cu, Cd and Zn were studied at the subcellular level in juvenile rainbow trout (Oncorhynchus mykiss) fed diets containing ({mu}g/g) 500 Cu, 1000 Zn and 500 Cd singly and as a ternary mixture for 28 days. Livers were harvested and submitted to differential centrifugation to isolate components of metabolically active metal pool (MAP: heat-denaturable proteins (HDP), organelles, nuclei) and metabolically detoxified metal pool (MDP: heat stable proteins (HSP), NaOH-resistant granules). Results indicated that Cd accumulation was enhanced in all the subcellular compartments, albeit at different time points, in fish exposed to the metals mixture relative to those exposed to Cd alone, whereas Cu alone exposure increased Cd partitioning. Exposure to the metals mixture reduced (HDP) and enhanced (HSP, nuclei and granules) Cu accumulation while exposure to Zn alone enhanced Cu concentration in all the fractions analyzed without altering proportional distribution in MAP and MDP. Although subcellular Zn accumulation was less pronounced than that of either Cu or Cd, concentrations of Zn were enhanced in HDP, nuclei and granules from fish exposed to the metals mixture relative to those exposed to Zn alone. Cadmium alone exposure mobilized Zn and Cu from the nuclei and increased Zn accumulation in organelles and Cu in granules, while Cu alone exposure stimulated Zn accumulation in HSP, HDP and organelles. Interestingly, Cd alone exposure increased the partitioning of the three metals in MDP indicative of enhanced detoxification. Generally the accumulated metals were predominantly metabolically active: Cd, 67-83%; Cu, 68-79% and Zn, 60-76%. Taken

  20. Hyperbolic partial differential equations

    CERN Document Server

    Witten, Matthew

    1986-01-01

    Hyperbolic Partial Differential Equations III is a refereed journal issue that explores the applications, theory, and/or applied methods related to hyperbolic partial differential equations, or problems arising out of hyperbolic partial differential equations, in any area of research. This journal issue is interested in all types of articles in terms of review, mini-monograph, standard study, or short communication. Some studies presented in this journal include discretization of ideal fluid dynamics in the Eulerian representation; a Riemann problem in gas dynamics with bifurcation; periodic M

  1. Beginning partial differential equations

    CERN Document Server

    O'Neil, Peter V

    2011-01-01

    A rigorous, yet accessible, introduction to partial differential equations-updated in a valuable new edition Beginning Partial Differential Equations, Second Edition provides a comprehensive introduction to partial differential equations (PDEs) with a special focus on the significance of characteristics, solutions by Fourier series, integrals and transforms, properties and physical interpretations of solutions, and a transition to the modern function space approach to PDEs. With its breadth of coverage, this new edition continues to present a broad introduction to the field, while also addres

  2. Segmentation and Location Computation of Bin Objects

    Directory of Open Access Journals (Sweden)

    C.R. Hema

    2008-11-01

    Full Text Available In this paper we present a stereo vision based system for segmentation and location computation of partially occluded objects in bin picking environments. Algorithms to segment partially occluded objects and to find the object location [midpoint,x, y and z coordinates] with respect to the bin area are proposed. The z co ordinate is computed using stereo images and neural networks. The proposed algorithms is tested using two neural network architectures namely the Radial Basis Function nets and Simple Feedforward nets. The training results fo feedforward nets are found to be more suitable for the current application.The proposed stereo vision system is interfaced with an Adept SCARA Robot to perform bin picking operations. The vision system is found to be effective for partially occluded objects, in the absence of albedo effects. The results are validated through real time bin picking experiments on the Adept Robot.

  3. Locating Educational Facilities. An Annotated Reference List.

    Science.gov (United States)

    Wakefield, Howard E.

    An annotated reference list of documents received and processed by the ERIC Clearinghouse on Educational Facilities. These documents are concerned wholly or partially with school and facilities location and site selection. All levels of education are covered and each document i s indexed and abstracted. (NI)

  4. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Directory of Open Access Journals (Sweden)

    Pei-Chi Yang

    2016-07-01

    Full Text Available Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and

  5. Sub-cellular force microscopy in single normal and cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Babahosseini, H. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Carmichael, B. [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Strobl, J.S. [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States); Mahmoodi, S.N., E-mail: nmahmoodi@eng.ua.edu [Nonlinear Intelligent Structures Laboratory, Department of Mechanical Engineering, University of Alabama, Tuscaloosa, AL 35487-0276 (United States); Agah, M., E-mail: agah@vt.edu [VT MEMS Laboratory, The Bradley Department of Electrical and Computer Engineering, Blacksburg, VA 24061 (United States)

    2015-08-07

    This work investigates the biomechanical properties of sub-cellular structures of breast cells using atomic force microscopy (AFM). The cells are modeled as a triple-layered structure where the Generalized Maxwell model is applied to experimental data from AFM stress-relaxation tests to extract the elastic modulus, the apparent viscosity, and the relaxation time of sub-cellular structures. The triple-layered modeling results allow for determination and comparison of the biomechanical properties of the three major sub-cellular structures between normal and cancerous cells: the up plasma membrane/actin cortex, the mid cytoplasm/nucleus, and the low nuclear/integrin sub-domains. The results reveal that the sub-domains become stiffer and significantly more viscous with depth, regardless of cell type. In addition, there is a decreasing trend in the average elastic modulus and apparent viscosity of the all corresponding sub-cellular structures from normal to cancerous cells, which becomes most remarkable in the deeper sub-domain. The presented modeling in this work constitutes a unique AFM-based experimental framework to study the biomechanics of sub-cellular structures. - Highlights: • The cells are modeled as a triple-layered structure using Generalized Maxwell model. • The sub-domains include membrane/cortex, cytoplasm/nucleus, and nuclear/integrin. • Biomechanics of corresponding sub-domains are compared among normal and cancer cells. • Viscoelasticity of sub-domains show a decreasing trend from normal to cancer cells. • The decreasing trend becomes most significant in the deeper sub-domain.

  6. Law Enforcement Locations

    Data.gov (United States)

    Kansas Data Access and Support Center — Law Enforcement Locations in Kansas Any location where sworn officers of a law enforcement agency are regularly based or stationed. Law enforcement agencies "are...

  7. Shapley Facility Location Games

    OpenAIRE

    Ben-Porat, Omer; Tennenholtz, Moshe

    2017-01-01

    Facility location games have been a topic of major interest in economics, operations research and computer science, starting from the seminal work by Hotelling. Spatial facility location models have successfully predicted the outcome of competition in a variety of scenarios. In a typical facility location game, users/customers/voters are mapped to a metric space representing their preferences, and each player picks a point (facility) in that space. In most facility location games considered i...

  8. Partial discharges in ellipsoidal and spheroidal voids

    DEFF Research Database (Denmark)

    Crichton, George C; Karlsson, P. W.; Pedersen, Aage

    1989-01-01

    Transients associated with partial discharges in voids can be described in terms of the charges induced on the terminal electrodes of the system. The relationship between the induced charge and the properties which are usually measured is discussed. The method is illustrated by applying it to a s...... it to a spheroidal void located in a simple disk-type spacer...

  9. Location, Location, Location: Where Do Location-Based Services Fit into Your Institution's Social Media Mix?

    Science.gov (United States)

    Nekritz, Tim

    2011-01-01

    Foursquare is a location-based social networking service that allows users to share their location with friends. Some college administrators have been thinking about whether and how to take the leap into location-based services, which are also known as geosocial networking services. These platforms, which often incorporate gaming elements like…

  10. Partial knee replacement - slideshow

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/presentations/100225.htm Partial knee replacement - series—Normal anatomy To use the sharing ... A.M. Editorial team. Related MedlinePlus Health Topics Knee Replacement A.D.A.M., Inc. is accredited ...

  11. Point mutations in GLI3 lead to misregulation of its subcellular localization.

    Directory of Open Access Journals (Sweden)

    Sybille Krauss

    Full Text Available BACKGROUND: Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS, or Pallister-Hall-syndrome (PHS. Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A regulates the nuclear localization and transcriptional activity a of GLI3 function. PRINCIPAL FINDINGS: We have shown recently that protein phosphatase 2A (PP2A and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-alpha4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. CONCLUSIONS: The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and

  12. Location, location, location: the BRMS1 protein and melanoma progression

    Directory of Open Access Journals (Sweden)

    Riker Adam I

    2012-02-01

    Full Text Available Abstract The metastasis suppressor, BRMS1, has been demonstrated to cause dramatic regression of metastatic lesions without blocking orthotopic tumor growth. The role of BRMS1 is well-documented for several non-melanoma malignancies, such as breast cancer, ovarian cancer and non-small-cell lung cancer. However, its role in melanoma is just beginning to be understood, with a recent article by Slipicevic et al. highlighting the levels of expression of BRMS1 in benign nevi, primary and metastatic melanoma samples. Their findings emphasize that the intracellular location of BRMS1 protein (cytoplasmic or nuclear, appears to have a significant impact upon the metastatic capacity of melanoma cells. Interestingly, this selective localization translates into a statistically significant decrease in the relapse-free period in melanoma patients, further associated with a thicker Breslow's depth of primary melanomas. However, and more importantly, this study begins to define a clearer role for BRMS1 in melanoma that is strictly dependent upon its cellular location, with nuclear expression associated with invasive and metastatic capacity and cytoplasmic expression resulting in repressive effects upon progression and metastasis. Please see related article: http://www.biomedcentral.com/1471-2407/12/73

  13. Beginning partial differential equations

    CERN Document Server

    O'Neil, Peter V

    2014-01-01

    A broad introduction to PDEs with an emphasis on specialized topics and applications occurring in a variety of fields Featuring a thoroughly revised presentation of topics, Beginning Partial Differential Equations, Third Edition provides a challenging, yet accessible,combination of techniques, applications, and introductory theory on the subjectof partial differential equations. The new edition offers nonstandard coverageon material including Burger's equation, the telegraph equation, damped wavemotion, and the use of characteristics to solve nonhomogeneous problems. The Third Edition is or

  14. Smartphones as locative media

    CERN Document Server

    Frith, Jordan

    2015-01-01

    Smartphone adoption has surpassed 50% of the population in more than 15 countries, and there are now more than one million mobile applications people can download to their phones. Many of these applications take advantage of smartphones as locative media, which is what allows smartphones to be located in physical space. Applications that take advantage of people's location are called location-based services, and they are the focus of this book. Smartphones as locative media raise important questions about how we understand the complicated relationship between the Internet and physical space

  15. Noise Source Location Optimization

    Directory of Open Access Journals (Sweden)

    Ed O’Keefe

    1994-01-01

    Full Text Available This article describes a method to determine locations of noise sources that minimize modal coupling in complex acoustic volumes. Using the acoustic source scattering capabilities of the boundary element method, predictions are made of mode shape and pressure levels due to various source locations. Combining knowledge of the pressure field with a multivariable function minimization technique, the source location generating minimum pressure levels can be determined. The analysis also allows for an objective comparison of “best/worst” locations. The technique was implemented on a personal computer for the U.S. Space Station, predicting 5–10 dB noise reduction using optimum source locations.

  16. Algebraic partial Boolean algebras

    CERN Document Server

    Smith, D

    2003-01-01

    Partial Boolean algebras, first studied by Kochen and Specker in the 1960s, provide the structure for Bell-Kochen-Specker theorems which deny the existence of non-contextual hidden variable theories. In this paper, we study partial Boolean algebras which are 'algebraic' in the sense that their elements have coordinates in an algebraic number field. Several of these algebras have been discussed recently in a debate on the validity of Bell-Kochen-Specker theorems in the context of finite precision measurements. The main result of this paper is that every algebraic finitely-generated partial Boolean algebra B(T) is finite when the underlying space H is three-dimensional, answering a question of Kochen and showing that Conway and Kochen's infinite algebraic partial Boolean algebra has minimum dimension. This result contrasts the existence of an infinite (non-algebraic) B(T) generated by eight elements in an abstract orthomodular lattice of height 3. We then initiate a study of higher-dimensional algebraic partial...

  17. Protein targeting to subcellular organelles via MRNA localization.

    Science.gov (United States)

    Weis, Benjamin L; Schleiff, Enrico; Zerges, William

    2013-02-01

    Cells have complex membranous organelles for the compartmentalization and the regulation of most intracellular processes. Organelle biogenesis and maintenance requires newly synthesized proteins, each of which needs to go from the ribosome translating its mRNA to the correct membrane for insertion or transclocation to an a organellar subcompartment. Decades of research have revealed how proteins are targeted to the correct organelle and translocated across one or more organelle membranes ro the compartment where they function. The paradigm examples involve interactions between a peptide sequence in the protein, localization factors, and various membrane embedded translocation machineries. Membrane translocation is either cotranslational or posttranslational depending on the protein and target organelle. Meanwhile research in embryos, neurons and yeast revealed an alternative targeting mechanism in which the mRNA is localized and only then translated to synthesize the protein in the correct location. In these cases, the targeting information is coded by the cis-acting sequences in the mRNA ("Zipcodes") that interact with localization factors and, in many cases, are transported by the molecular motors on the cytoskeletal filaments. Recently, evidence has been found for this "mRNA based" mechanism in organelle protein targeting to endoplasmic reticulum, mitochondria, and the photosynthetic membranes within chloroplasts. Here we review known and potential roles of mRNA localization in protein targeting to and within organelles. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  18. Automated Learning of Subcellular Variation among Punctate Protein Patterns and a Generative Model of Their Relation to Microtubules.

    Directory of Open Access Journals (Sweden)

    Gregory R Johnson

    2015-12-01

    Full Text Available Characterizing the spatial distribution of proteins directly from microscopy images is a difficult problem with numerous applications in cell biology (e.g. identifying motor-related proteins and clinical research (e.g. identification of cancer biomarkers. Here we describe the design of a system that provides automated analysis of punctate protein patterns in microscope images, including quantification of their relationships to microtubules. We constructed the system using confocal immunofluorescence microscopy images from the Human Protein Atlas project for 11 punctate proteins in three cultured cell lines. These proteins have previously been characterized as being primarily located in punctate structures, but their images had all been annotated by visual examination as being simply "vesicular". We were able to show that these patterns could be distinguished from each other with high accuracy, and we were able to assign to one of these subclasses hundreds of proteins whose subcellular localization had not previously been well defined. In addition to providing these novel annotations, we built a generative approach to modeling of punctate distributions that captures the essential characteristics of the distinct patterns. Such models are expected to be valuable for representing and summarizing each pattern and for constructing systems biology simulations of cell behaviors.

  19. Molecular cloning, subcellular localization and characterization of two adenylate kinases from cassava, Manihot esculenta Crantz cv. KU50.

    Science.gov (United States)

    Boonrueng, Channarong; Tangpranomkorn, Surachat; Yazhisai, Uthaman; Sirikantaramas, Supaart

    2016-10-01

    Adenylate kinase (ADK) is a phosphotransferase that plays an important role in cellular energy homeostasis. Many isozymes located in different subcellular compartments have been reported. In this study, we focus on the characterization of cassava (Manihot esculenta) ADKs. We found 15 ADKs that are publicly available in the African cassava genome database. We cloned two ADKs, namely MeADK1 and MeADK2, which are phylogenetically grouped together with the plastidial ADK in potato. Both MeADK1 and MeADK2 showed 66% identity in the amino acid sequences with plastidial ADK in potato. However, we demonstrated that they are localized to mitochondria using GFP fusions of MeADK1 and MeADK2. The Escherichia coli-produced recombinant MeADK1 and MeADK2 preferred forward reactions that produce ATP. They exhibited similar specific activities. The semi-quantitative RT-PCR analysis showed that MeADK1 and MeADK2 in 2-month-old leaves have similar expression patterns under a diurnal light-dark cycle. However, MeADK2 transcripts were expressed at much higher levels than MeADK1 in 5-month-old leaves and roots. Thus, we conclude that MeADK2 might play a vital role in energy homeostasis in cassava mitochondria. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Robustness in facility location

    OpenAIRE

    Van Lokven, Sander W.M.

    2009-01-01

    Facility location concerns the placement of facilities, for various objectives, by use of mathematical models and solution procedures. Almost all facility location models that can be found in literature are based on minimizing costs or maximizing cover, to cover as much demand as possible. These models are quite efficient for finding an optimal location for a new facility for a particular data set, which is considered to be constant and known in advance. In a real world situation, input da...

  1. Lost in Location

    DEFF Research Database (Denmark)

    Hansen, Lone Koefoed

    2009-01-01

    performance, the article examines how the growing locative media industry can learn from the location-aware performative strategies employed by artists who create situated and urban performances for the curious participant. The academic frames employed in the analysis draw on psychogeography, site......-specific performance, social and cultural geography, and aspects of phenomenology....... traversed. While becoming destination aware, the individual loses her location awareness. The article proposes that the reason people get lost when using sat-nav is due to a wrong location-performative paradigm. As an alternative, the article introduces and analyzes two performance-related examples...

  2. Smart Location Database - Download

    Data.gov (United States)

    U.S. Environmental Protection Agency — The Smart Location Database (SLD) summarizes over 80 demographic, built environment, transit service, and destination accessibility attributes for every census block...

  3. Smart Location Database - Service

    Data.gov (United States)

    U.S. Environmental Protection Agency — The Smart Location Database (SLD) summarizes over 80 demographic, built environment, transit service, and destination accessibility attributes for every census block...

  4. Generic Network Location Service

    Directory of Open Access Journals (Sweden)

    Laban Mwansa

    2010-11-01

    Full Text Available This work presents the Generic Network Location Service based on the Chord implementation utilizing data structures called distributed hash tables (DHT or structured overlay networks, which are used to build scalable self-managing distributed systems. The provided algorithms guarantee resilience in the presence of dynamism: they guarantee consistent lookup results in the presence of nodes failing and leaving. Generic Network Location Service provides a Location Service system based on DHT technology, which is storing device location records in nodes within a Chord DHT. Location records are consisting of network device identification keys as attributes, which are used to create replicas of additional location records through established Chord hashing mechanisms. Storing device location records, in places address-able (using the DHT lookup by individual location record keys provides a simple way of implementing transla¬tion functions similar to well¬ known network services (e.g. ARP, DNS, ENUM. The generic network location ser¬vice presented in the paper is not supposed to be a substitu¬tion of the existing translation techniques (e.g. ARP, DNS, ENUM, but it is considered as an overlay service that uses data available in existing systems and provides some translations currently unavailable.

  5. Methanol partial oxidation reformer

    Science.gov (United States)

    Ahmed, Shabbir; Kumar, Romesh; Krumpelt, Michael

    1999-01-01

    A partial oxidation reformer comprising a longitudinally extending chamber having a methanol, water and an air inlet and an outlet. An igniter mechanism is near the inlets for igniting a mixture of methanol and air, while a partial oxidation catalyst in the chamber is spaced from the inlets and converts methanol and oxygen to carbon dioxide and hydrogen. Controlling the oxygen to methanol mole ratio provides continuous slightly exothermic partial oxidation reactions of methanol and air producing hydrogen gas. The liquid is preferably injected in droplets having diameters less than 100 micrometers. The reformer is useful in a propulsion system for a vehicle which supplies a hydrogen-containing gas to the negative electrode of a fuel cell.

  6. Partial differential equations

    CERN Document Server

    Evans, Lawrence C

    2010-01-01

    This text gives a comprehensive survey of modern techniques in the theoretical study of partial differential equations (PDEs) with particular emphasis on nonlinear equations. The exposition is divided into three parts: representation formulas for solutions; theory for linear partial differential equations; and theory for nonlinear partial differential equations. Included are complete treatments of the method of characteristics; energy methods within Sobolev spaces; regularity for second-order elliptic, parabolic, and hyperbolic equations; maximum principles; the multidimensional calculus of variations; viscosity solutions of Hamilton-Jacobi equations; shock waves and entropy criteria for conservation laws; and, much more.The author summarizes the relevant mathematics required to understand current research in PDEs, especially nonlinear PDEs. While he has reworked and simplified much of the classical theory (particularly the method of characteristics), he primarily emphasizes the modern interplay between funct...

  7. Methanol partial oxidation reformer

    Science.gov (United States)

    Ahmed, S.; Kumar, R.; Krumpelt, M.

    1999-08-24

    A partial oxidation reformer is described comprising a longitudinally extending chamber having a methanol, water and an air inlet and an outlet. An igniter mechanism is near the inlets for igniting a mixture of methanol and air, while a partial oxidation catalyst in the chamber is spaced from the inlets and converts methanol and oxygen to carbon dioxide and hydrogen. Controlling the oxygen to methanol mole ratio provides continuous slightly exothermic partial oxidation reactions of methanol and air producing hydrogen gas. The liquid is preferably injected in droplets having diameters less than 100 micrometers. The reformer is useful in a propulsion system for a vehicle which supplies a hydrogen-containing gas to the negative electrode of a fuel cell. 7 figs.

  8. Dynamics of partial control.

    Science.gov (United States)

    Sabuco, Juan; Sanjuán, Miguel A F; Yorke, James A

    2012-12-01

    Safe sets are a basic ingredient in the strategy of partial control of chaotic systems. Recently we have found an algorithm, the sculpting algorithm, which allows us to construct them, when they exist. Here we define another type of set, an asymptotic safe set, to which trajectories are attracted asymptotically when the partial control strategy is applied. We apply all these ideas to a specific example of a Duffing oscillator showing the geometry of these sets in phase space. The software for creating all the figures appearing in this paper is available as supplementary material.

  9. [Effects of pentacin on subcellular distribution of Pu-239 nitrate in the lungs of rats].

    Science.gov (United States)

    Kabirova, N R; Sokhranich, A L

    1991-01-01

    The method of differential centrifugation has shown that 26% of 239Pu-nitrate injected intratracheally, is detected in cells in 3 h (24.4% of Pu is bound with subcellular organellae). In 24 h the nuclear fraction acquires a paramount importance in Pu binding. In the hyaloplasm 239Pu is distributed equally between low molecular (32.9-42.9%) and high molecular (54.1-55.2%) proteins. Pentacine decreases the level of Pu in the lungs, in the sum of subcellular fractions and in the sum of organellae, but produces no noticeable action on the level of the radionuclide in hyaloplasmic proteins though promotes certain redistribution of the radionuclide between low- and high molecular proteins.

  10. Fast Fourier transform-based support vector machine for subcellular localization prediction using different substitution models.

    Science.gov (United States)

    Wang, Zhimeng; Jiang, Lin; Li, Menglong; Sun, Lina; Lin, Rongying

    2007-09-01

    There are approximately 10(9) proteins in a cell. A hotspot in bioinformatics is how to identify a protein subcellular localization, if its sequence is known. In this paper, a method using fast Fourier transform-based support vector machine is developed to predict the subcellular localization of proteins from their physicochemical properties and structural parameters. The prediction accuracies reached 83% in prokaryotic organisms and 84% in eukaryotic organisms with the substitution model of the c-p-v matrix (c, composition; p, polarity; and v, molecular volume). The overall prediction accuracy was also evaluated using the "leave-one-out" jackknife procedure. The influence of the substitution model on prediction accuracy has also been discussed in the work. The source code of the new program is available on request from the authors.

  11. Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling.

    Science.gov (United States)

    Atwood, James A; Minning, Todd; Ludolf, Fernanda; Nuccio, Arthur; Weatherly, Daniel B; Alvarez-Manilla, Gerardo; Tarleton, Rick; Orlando, Ron

    2006-12-01

    Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans.

  12. Differential subcellular distribution of ion channels and the diversity of neuronal function.

    Science.gov (United States)

    Nusser, Zoltan

    2012-06-01

    Following the astonishing molecular diversity of voltage-gated ion channels that was revealed in the past few decades, the ion channel repertoire expressed by neurons has been implicated as the major factor governing their functional heterogeneity. Although the molecular structure of ion channels is a key determinant of their biophysical properties, their subcellular distribution and densities on the surface of nerve cells are just as important for fulfilling functional requirements. Recent results obtained with high resolution quantitative localization techniques revealed complex, subcellular compartment-specific distribution patterns of distinct ion channels. Here I suggest that within a given neuron type every ion channel has a unique cell surface distribution pattern, with the functional consequence that this dramatically increases the computational power of nerve cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Industrial location and competitiveness

    NARCIS (Netherlands)

    S. Brakman (Steven); J.H. Garretsen (Harry); J.G.M. van Marrewijk (Charles)

    2006-01-01

    textabstractThe interaction between the extent of location advantages and the intensity of firm competition relative to the size of the market jointly determines the location of industrial activity. Technology, factor endowments, geography, and scale economies are influential for determining

  14. Subcellular Localization of Galloylated Catechins in Tea Plants [Camellia sinensis (L.) O. Kuntze] Assessed via Immunohistochemistry

    OpenAIRE

    Xu, Huanhuan; Wang, Ya; Chen, Yana; Zhang, Pan; Zhao, Yi; Huang, Yewei; Wang, Xuanjun; Sheng, Jun

    2016-01-01

    Galloylated catechins, as the main secondary metabolites in the tea plant, including (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, comprise approximately three-quarters of all the tea plant catechins and have stronger effects than non-galloylated catechins, both on the product quality in tea processing and the pharmacological efficacy to human beings. The subcellular localization of galloylated catechins has been the primary focus of studies that assess biosynthesis and physio...

  15. Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting

    Directory of Open Access Journals (Sweden)

    Salih Maysoon

    2009-06-01

    Full Text Available Abstract Background Tail anchored (TA membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP possesses two alternatively spliced tail anchors (TA1 or TA2 but their specificity of subcellular targeting remains unknown. Results TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER, whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. Conclusion Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

  16. Alternative splicing and differential subcellular localization of the rat FGF antisense gene product

    Directory of Open Access Journals (Sweden)

    Casson Alan G

    2008-01-01

    Full Text Available Abstract Background GFG/NUDT is a nudix hydrolase originally identified as the product of the fibroblast growth factor-2 antisense (FGF-AS gene. While the FGF-AS RNA has been implicated as an antisense regulator of FGF-2 expression, the expression and function of the encoded GFG protein is largely unknown. Alternative splicing of the primary FGF-AS mRNA transcript predicts multiple GFG isoforms in many species including rat. In the present study we focused on elucidating the expression and subcellular distribution of alternatively spliced rat GFG isoforms. Results RT-PCR and immunohistochemistry revealed tissue-specific GFG mRNA isoform expression and subcellular distribution of GFG immunoreactivity in cytoplasm and nuclei of a wide range of normal rat tissues. FGF-2 and GFG immunoreactivity were co-localized in some, but not all, tissues examined. Computational analysis identified a mitochondrial targeting sequence (MTS in the N-terminus of three previously described rGFG isoforms. Confocal laser scanning microscopy and subcellular fractionation analysis revealed that all rGFG isoforms bearing the MTS were specifically targeted to mitochondria whereas isoforms and deletion mutants lacking the MTS were localized in the cytoplasm and nucleus. Mutation and deletion analysis confirmed that the predicted MTS was necessary and sufficient for mitochondrial compartmentalization. Conclusion Previous findings strongly support a role for the FGF antisense RNA as a regulator of FGF2 expression. The present study demonstrates that the antisense RNA itself is translated, and that protein isoforms resulting form alternative RNA splicing are sorted to different subcellular compartments. FGF-2 and its antisense protein are co-expressed in many tissues and in some cases in the same cells. The strong conservation of sequence and genomic organization across animal species suggests important functional significance to the physical association of these transcript

  17. Subcellular targeting and interactions among the Potato virus X TGB proteins.

    Science.gov (United States)

    Samuels, Timmy D; Ju, Ho-Jong; Ye, Chang-Ming; Motes, Christy M; Blancaflor, Elison B; Verchot-Lubicz, Jeanmarie

    2007-10-25

    Potato virus X (PVX) encodes three proteins named TGBp1, TGBp2, and TGBp3 which are required for virus cell-to-cell movement. To determine whether PVX TGB proteins interact during virus cell-cell movement, GFP was fused to each TGB coding sequence within the viral genome. Confocal microscopy was used to study subcellular accumulation of each protein in virus-infected plants and protoplasts. GFP:TGBp2 and TGBp3:GFP were both seen in the ER, ER-associated granular vesicles, and perinuclear X-bodies suggesting that these proteins interact in the same subdomains of the endomembrane network. When plasmids expressing CFP:TGBp2 and TGBp3:GFP were co-delivered to tobacco leaf epidermal cells, the fluorescent signals overlapped in ER-associated granular vesicles indicating that these proteins colocalize in this subcellular compartment. GFP:TGBp1 was seen in the nucleus, cytoplasm, rod-like inclusion bodies, and in punctate sites embedded in the cell wall. The puncta were reminiscent of previous reports showing viral proteins in plasmodesmata. Experiments using CFP:TGBp1 and YFP:TGBp2 or TGBp3:GFP showed CFP:TGBp1 remained in the cytoplasm surrounding the endomembrane network. There was no evidence that the granular vesicles contained TGBp1. Yeast two hybrid experiments showed TGBp1 self associates but failed to detect interactions between TGBp1 and TGBp2 or TGBp3. These experiments indicate that the PVX TGB proteins have complex subcellular accumulation patterns and likely cooperate across subcellular compartments to promote virus infection.

  18. Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting.

    Science.gov (United States)

    Byers, Joseph T; Guzzo, Rosa M; Salih, Maysoon; Tuana, Balwant S

    2009-06-19

    Tail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown. TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1. Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.

  19. Capillary electrophoretic analysis reveals subcellular binding between individual mitochondria and cytoskeleton

    Science.gov (United States)

    Kostal, Vratislav; Arriaga, Edgar A.

    2011-01-01

    Interactions between the cytoskeleton and mitochondria are essential for normal cellular function. An assessment of such interactions is commonly based on bulk analysis of mitochondrial and cytoskeletal markers present in a given sample, which assumes complete binding between these two organelle types. Such measurements are biased because they rarely account for non-bound ‘free’ subcellular species. Here we report on the use of capillary electrophoresis with dual laser induced fluorescence detection (CE-LIF) to identify, classify, count and quantify properties of individual binding events of mitochondria and cytoskeleton. Mitochondria were fluorescently labeled with DsRed2 while F-actin, a major cytoskeletal component, was fluorescently labeled with Alexa488-phalloidin. In a typical subcellular fraction of L6 myoblasts, 79% of mitochondrial events did not have detectable levels of F-actin, while the rest had on average ~2 zeptomole F-actin, which theoretically represents a ~ 2.5-μm long network of actin filaments per event. Trypsin treatment of L6 subcellular fractions prior to analysis decreased the fraction of mitochondrial events with detectable levels of F-actin, which is expected from digestion of cytoskeletal proteins on the surface of mitochondria. The electrophoretic mobility distributions of the individual events were also used to further distinguish between cytoskeleton-bound from cytoskeleton-free mitochondrial events. The CE-LIF approach described here could be further developed to explore cytoskeleton interactions with other subcellular structures, the effects of cytoskeleton destabilizing drugs, and the progression of viral infections. PMID:21309532

  20. Nanopipette-Based SERS Aptasensor for Subcellular Localization of Cancer Biomarker in Single Cells.

    Science.gov (United States)

    Hanif, Sumaira; Liu, Hai-Ling; Ahmed, Saud Asif; Yang, Jin-Mei; Zhou, Yue; Pang, Jie; Ji, Li-Na; Xia, Xing-Hua; Wang, Kang

    2017-09-19

    Single cell analysis is essential for understanding the heterogeneity, behaviors of cells, and diversity of target analyte in different subcellular regions. Nucleolin (NCL) is a multifunctional protein that is markedly overexpressed in most of the cancer cells. The variant expression levels of NCL in subcellular regions have a marked influence on cancer proliferation and treatments. However, the specificity of available methods to identify the cancer biomarkers is limited because of the high level of subcellular matrix effect. Herein, we proposed a novel technique to increase both the molecular and spectral specificity of cancer diagnosis by using aptamers affinity based portable nanopipette with distinctive surface-enhanced Raman scattering (SERS) activities. The aptamers-functionalized gold-coated nanopipette was used to capture target, while p-mercaptobenzonitrile (MBN) and complementary DNA modified Ag nanoparticles (AgNPs) worked as Raman reporter to produce SERS signal. The SERS signal of Raman nanotag was lost upon NCL capturing via modified DNA aptamers on nanoprobe, which further helped to verify the specificity of nanoprobe. For proof of concept, NCL protein was specifically extracted from different cell lines by aptamers modified SERS active nanoprobe. The nanoprobes manifested specifically good affinity for NCL with a dissociation constant Kd of 36 nM and provided a 1000-fold higher specificity against other competing proteins. Furthermore, the Raman reporter moiety has a vibrational frequency in the spectroscopically silent region (1800-2300 cm-1) with a negligible matrix effect from cell analysis. The subcellular localization and spatial distribution of NCL were successfully achieved in various types of cells, including MCF-7A, HeLa, and MCF-10A cells. This type of probing technique for single cell analysis could lead to the development of a new perspective in cancer diagnosis and treatment at the cellular level.

  1. Subcellular Localization of APE1/Ref-1 in Human Hepatocellular Carcinoma: Possible Prognostic Significance

    OpenAIRE

    Di Maso, Vittorio; Avellini, Claudio; Crocè, Lory Saveria; Rosso, Natalia; Quadrifoglio, Franco; Cesaratto, Laura; Codarin, Erika; Bedogni, Giorgio; Beltrami, Carlo Alberto; Tell, Gianluca; Tiribelli, Claudio

    2007-01-01

    APE1/Ref-1, normally localized in the nucleus, is a regulator of the cellular response to oxidative stress. Cytoplasmic localization has been observed in several tumors and correlates with a poor prognosis. Because no data are available on liver tumors, we investigated APE1/Ref-1 subcellular localization and its correlation with survival in 47 consecutive patients undergoing hepatocellular carcinoma (HCC) resection. APE1/Ref-1 expression was determined by immunohistochemistry in HCC and surro...

  2. Pendrin gene ablation alters ENaC subcellular distribution and open probability.

    Science.gov (United States)

    Pech, Vladimir; Wall, Susan M; Nanami, Masayoshi; Bao, Hui-Fang; Kim, Young Hee; Lazo-Fernandez, Yoskaly; Yue, Qiang; Pham, Truyen D; Eaton, Douglas C; Verlander, Jill W

    2015-07-15

    The present study explored whether the intercalated cell Cl(-)/HCO3(-) exchanger pendrin modulates epithelial Na(+) channel (ENaC) function by changing channel open probability and/or channel density. To do so, we measured ENaC subunit subcellular distribution by immunohistochemistry, single channel recordings in split open cortical collecting ducts (CCDs), as well as transepithelial voltage and Na(+) absorption in CCDs from aldosterone-treated wild-type and pendrin-null mice. Because pendrin gene ablation reduced 70-kDa more than 85-kDa γ-ENaC band density, we asked if pendrin gene ablation interferes with ENaC cleavage. We observed that ENaC-cleaving protease application (trypsin) increased the lumen-negative transepithelial voltage in pendrin-null mice but not in wild-type mice, which raised the possibility that pendrin gene ablation blunts ENaC cleavage, thereby reducing open probability. In mice harboring wild-type ENaC, pendrin gene ablation reduced ENaC-mediated Na(+) absorption by reducing channel open probability as well as by reducing channel density through changes in subunit total protein abundance and subcellular distribution. Further experiments used mice with blunted ENaC endocytosis and degradation (Liddle's syndrome) to explore the significance of pendrin-dependent changes in ENaC open probability. In mouse models of Liddle's syndrome, pendrin gene ablation did not change ENaC subunit total protein abundance, subcellular distribution, or channel density, but markedly reduced channel open probability. We conclude that in mice harboring wild-type ENaC, pendrin modulates ENaC function through changes in subunit abundance, subcellular distribution, and channel open probability. In a mouse model of Liddle's syndrome, however, pendrin gene ablation reduces channel activity mainly through changes in open probability. Copyright © 2015 the American Physiological Society.

  3. Cdc2/cyclin B1 regulates centrosomal Nlp proteolysis and subcellular localization.

    Science.gov (United States)

    Zhao, Xuelian; Jin, Shunqian; Song, Yongmei; Zhan, Qimin

    2010-11-01

    The formation of proper mitotic spindles is required for appropriate chromosome segregation during cell division. Aberrant spindle formation often causes aneuploidy and results in tumorigenesis. However, the underlying mechanism of regulating spindle formation and chromosome separation remains to be further defined. Centrosomal Nlp (ninein-like protein) is a recently characterized BRCA1-regulated centrosomal protein and plays an important role in centrosome maturation and spindle formation. In this study, we show that Nlp can be phosphorylated by cell cycle protein kinase Cdc2/cyclin B1. The phosphorylation sites of Nlp are mapped at Ser185 and Ser589. Interestingly, the Cdc2/cyclin B1 phosphorylation site Ser185 of Nlp is required for its recognition by PLK1, which enable Nlp depart from centrosomes to allow the establishment of a mitotic scaffold at the onset of mitosis . PLK1 fails to dissociate the Nlp mutant lacking Ser185 from centrosome, suggesting that Cdc2/cyclin B1 might serve as a primary kinase of PLK1 in regulating Nlp subcellular localization. However, the phosphorylation at the site Ser589 by Cdc2/cyclin B1 plays an important role in Nlp protein stability probably due to its effect on protein degradation. Furthermore, we show that deregulated expression or subcellular localization of Nlp lead to multinuclei in cells, indicating that scheduled levels of Nlp and proper subcellular localization of Nlp are critical for successful completion of normal cell mitosis, These findings demonstrate that Cdc2/cyclin B1 is a key regulator in maintaining appropriate degradation and subcellular localization of Nlp, providing novel insights into understanding on the role of Cdc2/cyclin B1 in mitotic progression.

  4. PlantLoc: an accurate web server for predicting plant protein subcellular localization by substantiality motif

    OpenAIRE

    Tang, Shengnan; Li, Tonghua; Cong, Peisheng; Xiong, Wenwei; Wang, Zhiheng; Sun, Jiangming

    2013-01-01

    Knowledge of subcellular localizations (SCLs) of plant proteins relates to their functions and aids in understanding the regulation of biological processes at the cellular level. We present PlantLoc, a highly accurate and fast webserver for predicting the multi-label SCLs of plant proteins. The PlantLoc server has two innovative characters: building localization motif libraries by a recursive method without alignment and Gene Ontology information; and establishing simple architecture for rapi...

  5. High-Resolution Secondary Ion Mass Spectrometry Reveals the Contrasting Subcellular Distribution of Arsenic and Silicon in Rice Roots

    National Research Council Canada - National Science Library

    Katie L. Moore; Markus Schröder; Zhongchang Wu; Barry G.H. Martin; Chris R. Hawes; Steve P. McGrath; Malcolm J. Hawkesford; Jian Feng Ma; Fang-Jie Zhao; Chris R.M. Grovenor

    2011-01-01

    .... In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy...

  6. Probing the subcellular localization of hopanoid lipids in bacteria using NanoSIMS.

    Directory of Open Access Journals (Sweden)

    David M Doughty

    Full Text Available The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary (2H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes.

  7. Internalization and subcellular fate of aptamer and peptide dual-functioned nanoparticles.

    Science.gov (United States)

    Gao, Huile; Yang, Zhi; Zhang, Shuang; Pang, Zhiqing; Jiang, Xinguo

    2014-06-01

    To evaluate the internalization and subcellular fate of AS1411 aptamer (for glioma targeting) and TGN peptide (for blood-brain barrier targeting)-modified nanoparticles (AsTNPs), which was important for optimizing targeted delivery systems and realizing the potential toxicity to cells. Organelles were labelled with specific markers. Several uptake inhibitors were used to determine the endocytosis pathways. Transmission electron microscopy (TEM) was utilized to directly observe the endocytosis procedure and subcellular fate of AsTNPs. Subcellular localization demonstrated that endosomes and mitochondria were involved in the uptake of AsTNPs by both C6 and bEnd.3 cells, however, lysosomes and Golgi apparatus were only involved in the internalization by C6 cells rather than bEnd.3 cells. Uptake mechanism study demonstrated the clathrin- and caveolae-mediated endocytosis were the main pathways in the uptake of AsTNPs by C6 and bEnd.3 cells. However, other pathways, including clathrin- and caveolae-independent endocytosis and macropinocytosis are also involved in the uptake by C6 cells and not by bEnd.3 cells. TEM directly demonstrated the involvement of these pathways. Particles could be found mostly in endosomes. Compared to unmodified nanoparticles, AsTNPs displayed different internalization pathways involved in several cell organelles.

  8. Efficacy Dependence of Photodynamic Therapy Mediated by Upconversion Nanoparticles: Subcellular Positioning and Irradiation Productivity.

    Science.gov (United States)

    Chen, Dexin; Tao, Ran; Tao, Ke; Chen, Biqiong; Choi, Seok Ki; Tian, Qing; Xu, Yawen; Zhou, Guangdong; Sun, Kang

    2017-04-01

    Singlet oxygen (1 O2 ), as an important kind of reactive oxygen species (ROS) and main therapeutic agent in photodynamic therapy (PDT), only have a half-life of 40 ns and an effective radius of 20 nm, which cause significant obstacles for improving PDT efficacy. In this work, novel upconversion nanoparticle (UCN)-based nanoplatforms are developed with a minimized distance between UCNs and a photosensitizer, protoporphyrin IX (PpIX), and a controllable payload of PpIX, to enhance and control ROS production. The ability of the nanoplatform to target different subcellular organelles such as cell membrane and mitochondria is demonstrated via surface modification of the nanoplatform with different targeting ligands. The results show that the mitochondria-targeting nanoplatforms result in significantly increased capability of both tumor cell killing and inhibition of tumor growth. Subcellular targeting of nanoparticles leads to the death of cancer cells in different manners. However, the efficiency of ROS generation almost have no influence on the tumor cell viability during the period of evaluation. These findings suggest that specific subcellular targeting of the nanoplatforms enhances the PDT efficacy more effectively than the increase of ROS production, and may shed light on future novel designs of effective and controllable PDT nanoplatforms. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. KCC2-dependent subcellular ECl difference of ON-OFF retinal ganglion cells in larval zebrafish

    Directory of Open Access Journals (Sweden)

    Rongwei eZhang

    2013-05-01

    Full Text Available Subcellular difference in the reversal potential of Cl- (ECl has been found in many types of neurons. As local ECl largely determines the action of nearby GABAergic/glycinergic synapses, subcellular ECl difference can effectively regulate neuronal computation. The ON-OFF retinal ganglion cell (RGC processes both ON and OFF visual signals via its ON and OFF dendrites, respectively. It is thus interesting to investigate whether the ON and OFF dendrites of single RGCs exhibit different local ECl. Here, using in vivo gramicidin-perforated patch recording in larval zebrafish ON-OFF RGCs, we examine local ECl at the ON and OFF dendrites, and soma through measuring light-evoked ON and OFF inhibitory responses, and GABA-induced response at the soma, respectively. We find there are subcellular ECl differences between the soma and dendrite, as well as between the ON and OFF dendrites of single RGCs. These somato-dendritic and inter-dendritic ECl differences are dependent on the Cl- extruder, K+/Cl- co-transporter (KCC2, because they are largely diminished by down-regulating kcc2 expression with morpholino oligonucleotides or by blocking KCC2 function with furosemide. Thus, our findings indicate that there exists KCC2-dependent ECl difference between the ON and OFF dendrites of individual ON-OFF RGCs that may differentially affect visual processing in the ON and OFF pathways.

  10. Inducible control of subcellular RNA localization using a synthetic protein-RNA aptamer interaction.

    Directory of Open Access Journals (Sweden)

    Brian J Belmont

    Full Text Available Evidence is accumulating in support of the functional importance of subcellular RNA localization in diverse biological contexts. In different cell types, distinct RNA localization patterns are frequently observed, and the available data indicate that this is achieved through a series of highly coordinated events. Classically, cis-elements within the RNA to be localized are recognized by RNA-binding proteins (RBPs, which then direct specific localization of a target RNA. Until now, the precise control of the spatiotemporal parameters inherent to regulating RNA localization has not been experimentally possible. Here, we demonstrate the development and use of a chemically-inducible RNA-protein interaction to regulate subcellular RNA localization. Our system is composed primarily of two parts: (i the Tet Repressor protein (TetR genetically fused to proteins natively involved in localizing endogenous transcripts; and (ii a target transcript containing genetically encoded TetR-binding RNA aptamers. TetR-fusion protein binding to the target RNA and subsequent localization of the latter are directly regulated by doxycycline. Using this platform, we demonstrate that enhanced and controlled subcellular localization of engineered transcripts are achievable. We also analyze rules for forward engineering this RNA localization system in an effort to facilitate its straightforward application to studying RNA localization more generally.

  11. Differential subcellular distribution of four phospholipase C isoforms and secretion of GPI-PLC activity.

    Science.gov (United States)

    Staudt, Emanuel; Ramasamy, Pathmanaban; Plattner, Helmut; Simon, Martin

    2016-12-01

    Phospholipase C (PLC) is an important enzyme of signal transduction pathways by generation of second messengers from membrane lipids. PLCs are also indicated to cleave glycosylphosphatidylinositol (GPI)-anchors of surface proteins thus releasing these into the environment. However, it remains unknown whether this enzymatic activity on the surface is due to distinct PLC isoforms in higher eukaryotes. Ciliates have, in contrast to other unicellular eukaryotes, multiple PLC isoforms as mammals do. Thus, Paramecium represents a perfect model to study subcellular distribution and potential surface activity of PLC isoforms. We have identified distinct subcellular localizations of four PLC isoforms indicating functional specialization. The association with different calcium release channels (CRCs) argues for distinct subcellular functions. They may serve as PI-PLCs in microdomains for local second messenger responses rather than free floating IP3. In addition, all isoforms can be found on the cell surface and they are found together with GPI-cleaved surface proteins in salt/ethanol washes of cells. We can moreover show them in medium supernatants of living cells where they have access to GPI-anchored surface proteins. Among the isoforms we cannot assign GPI-PLC activity to specific PLC isoforms; rather each PLC is potentially responsible for the release of GPI-anchored proteins from the surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Analysis of the subcellular localization of the human histone methyltransferase SETDB1

    Energy Technology Data Exchange (ETDEWEB)

    Tachibana, Keisuke, E-mail: nya@phs.osaka-u.ac.jp [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Gotoh, Eiko; Kawamata, Natsuko [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Ishimoto, Kenji [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Uchihara, Yoshie [Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan); Iwanari, Hiroko [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sugiyama, Akira; Kawamura, Takeshi [Radioisotope Center, The University of Tokyo, 2-11-16 Yayoi, Bunkyo, Tokyo 113-0032 (Japan); Mochizuki, Yasuhiro [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Tanaka, Toshiya [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Sakai, Juro [Division of Metabolic Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Hamakubo, Takao [Department of Quantitative Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); Kodama, Tatsuhiko [Laboratory for System Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro, Tokyo 153-8904 (Japan); and others

    2015-10-02

    SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol. - Highlights: • Endogenous human SETDB1 was localized mainly in the cytoplasm. • Combined treatment with LMB and MG132 led to accumulation of human SETDB1 in the nucleus. • HeLa cells expressing EFGP-hSETDB1 are useful for subcellular localization analyses.

  13. Subcellular localization and function study of a secreted phospholipase C from Nocardia seriolae.

    Science.gov (United States)

    Xia, Liqun; Liang, Haiying; Xu, Liang; Chen, Jianlin; Bekaert, Michaël; Zhang, Honglian; Lu, Yishan

    2017-09-15

    Fish nocardiosis is a chronic systemic granulomatous disease, and Nocardia seriolae is the main pathogen that causes it. The pathogenesis and virulence factors of N. seriolae are not fully understood. A phospholipase C (PLC), which is likely to be a secreted protein targeting host cell mitochondria, was found by a bioinformatics analysis of the whole genome sequence of N. seriolae. In order to determine the subcellular localization and study the preliminary function of PLC from N. seriolae (NsPLC), in this study gene cloning, secreted protein identification, subcellular localization in host cells and apoptosis detection of NsPLC were carried out. Mass spectrometry analysis of extracellular products from N. seriolae showed that NsPLC was a secreted protein. Subcellular localization of NsPLC-GFP fusion protein in fathead minnow (FHM) cells revealed that the green fluorescence exhibited a punctate distribution near the nucleus and did not co-localize with mitochondria. In addition, an apoptosis assay suggested that apoptosis was induced in FHM cells by the overexpression of NsPLC. This study may lay the foundations for further studies on the function of NsPLC and promote the understanding of the virulence factors and pathogenic mechanism of N. seriolae. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. A workflow for mathematical modeling of subcellular metabolic pathways in leaf metabolism of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Thomas eNägele

    2013-12-01

    Full Text Available During the last decade genome sequencing has experienced a rapid technological development resulting in numerous sequencing projects and applications in life science. In plant molecular biology, the availability of sequence data on whole genomes has enabled the reconstruction of metabolic networks. Enzymatic reactions are predicted by the sequence information. Pathways arise due to the participation of chemical compounds as substrates and products in these reactions. Although several of these comprehensive networks have been reconstructed for the genetic model plant Arabidopsis thaliana, the integration of experimental data is still challenging. Particularly the analysis of subcellular organization of plant cells limits the understanding of regulatory instances in these metabolic networks in vivo. In this study, we develop an approach for the functional integration of experimental high-throughput data into such large-scale networks. We present a subcellular metabolic network model comprising 524 metabolic intermediates and 548 metabolic interactions derived from a total of 2769 reactions. We demonstrate how to link the metabolite covariance matrix of different Arabidopsis thaliana accessions with the subcellular metabolic network model for the inverse calculation of the biochemical Jacobian, finally resulting in the calculation of a matrix which satisfies a Lyaponov equation involving a covariance matrix. In this way, differential strategies of metabolite compartmentation and involved reactions were identified in the accessions when exposed to low temperature.

  15. LOCnet and LOCtarget: sub-cellular localization for structural genomics targets

    Science.gov (United States)

    Nair, Rajesh; Rost, Burkhard

    2004-01-01

    LOCtarget is a web server and database that predicts and annotates sub-cellular localization for structural genomics targets; LOCnet is one of the methods used in LOCtarget that can predict sub-cellular localization for all eukaryotic and prokaryotic proteins. Targets are taken from the central registration database for structural genomics, namely, TargetDB. LOCtarget predicts localization through a combination of four different methods: known nuclear localization signals (PredictNLS), homology-based transfer of experimental annotations (LOChom), inference through automatic text analysis of SWISS-PROT keywords (LOCkey) and de novo prediction through a system of neural networks (LOCnet). Additionally, we report predictions from SignalP. The final prediction is based on the method with the highest confidence. The web server can be used to predict sub-cellular localization of proteins from their amino acid sequence. The LOCtarget database currently contains localization predictions for all eukaryotic proteins from TargetDB and is updated every week. The server is available at http://www.rostlab.org/services/LOCtarget/. PMID:15215440

  16. Subcellular distribution of non-muscle myosin IIb is controlled by FILIP through Hsc70.

    Directory of Open Access Journals (Sweden)

    Hideshi Yagi

    Full Text Available The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP. However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology.

  17. Optically-controlled platforms for transfection and single- and sub-cellular surgery

    DEFF Research Database (Denmark)

    Villangca, Mark Jayson; Casey, Duncan; Glückstad, Jesper

    2015-01-01

    Improving the resolution of biological research to the single- or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual, unpredic......Improving the resolution of biological research to the single- or sub-cellular level is of critical importance in a wide variety of processes and disease conditions. Most obvious are those linked to aging and cancer, many of which are dependent upon stochastic processes where individual...... and specificity of optical trapping in conjunction with other modalities to perform single and sub-cellular surgery. These tools form highly tuneable platforms for the delivery or removal of material from cells of interest, but can simultaneously excite fluorescent probes for imaging purposes or plasmonic...... structures for very local heating. We discuss both the history and recent applications of the field, highlighting the key findings and developments over the last 40 years of biophotonics research....

  18. Fundamental partial compositeness

    DEFF Research Database (Denmark)

    Sannino, Francesco; Strumia, Alessandro; Tesi, Andrea

    2016-01-01

    We construct renormalizable Standard Model extensions, valid up to the Planck scale, that give a composite Higgs from a new fundamental strong force acting on fermions and scalars. Yukawa interactions of these particles with Standard Model fermions realize the partial compositeness scenario. Unde...

  19. Partially ordered models

    NARCIS (Netherlands)

    Fernandez, R.; Deveaux, V.

    2010-01-01

    We provide a formal definition and study the basic properties of partially ordered chains (POC). These systems were proposed to model textures in image processing and to represent independence relations between random variables in statistics (in the later case they are known as Bayesian networks).

  20. Partial Biotinidase Deficiency

    OpenAIRE

    J Gordon Millichap

    1990-01-01

    The symptoms, biochemical features and inheritance pattern of partial biotinidase deficiency have been studied at the Departments of Human Genetics and Pediatrics, Medical College of Virginia, Richmond, VA; the State Laboratory Institute, Massachusetts Department of Public Health; Massachusetts General Hospital, Boston; the Lincoln Clinic, NB; and the Division of Human Genetics, university of Maryland School of Medicine, Baltimore.

  1. Partially Hidden Markov Models

    DEFF Research Database (Denmark)

    Forchhammer, Søren Otto; Rissanen, Jorma

    1996-01-01

    Partially Hidden Markov Models (PHMM) are introduced. They differ from the ordinary HMM's in that both the transition probabilities of the hidden states and the output probabilities are conditioned on past observations. As an illustration they are applied to black and white image compression wher...

  2. Honesty in partial logic

    NARCIS (Netherlands)

    W. van der Hoek (Wiebe); J.O.M. Jaspars; E. Thijsse

    1995-01-01

    textabstractWe propose an epistemic logic in which knowledge is fully introspective and implies truth, although truth need not imply epistemic possibility. The logic is presented in sequential format and is interpreted in a natural class of partial models, called balloon models. We examine the

  3. Partial Remission Definition

    DEFF Research Database (Denmark)

    Andersen, Marie Louise Max; Hougaard, Philip; Pörksen, Sven

    2014-01-01

    OBJECTIVE: To validate the partial remission (PR) definition based on insulin dose-adjusted HbA1c (IDAA1c). SUBJECTS AND METHODS: The IDAA1c was developed using data in 251 children from the European Hvidoere cohort. For validation, 129 children from a Danish cohort were followed from the onset...

  4. CELLO2GO: a web server for protein subCELlular LOcalization prediction with functional gene ontology annotation.

    Directory of Open Access Journals (Sweden)

    Chin-Sheng Yu

    Full Text Available CELLO2GO (http://cello.life.nctu.edu.tw/cello2go/ is a publicly available, web-based system for screening various properties of a targeted protein and its subcellular localization. Herein, we describe how this platform is used to obtain a brief or detailed gene ontology (GO-type categories, including subcellular localization(s, for the queried proteins by combining the CELLO localization-predicting and BLAST homology-searching approaches. Given a query protein sequence, CELLO2GO uses BLAST to search for homologous sequences that are GO annotated in an in-house database derived from the UniProt KnowledgeBase database. At the same time, CELLO attempts predict at least one subcellular localization on the basis of the species in which the protein is found. When homologs for the query sequence have been identified, the number of terms found for each of their GO categories, i.e., cellular compartment, molecular function, and biological process, are summed and presented as pie charts representing possible functional annotations for the queried protein. Although the experimental subcellular localization of a protein may not be known, and thus not annotated, CELLO can confidentially suggest a subcellular localization. CELLO2GO should be a useful tool for research involving complex subcellular systems because it combines CELLO and BLAST into one platform and its output is easily manipulated such that the user-specific questions may be readily addressed.

  5. Global Volcano Locations Database

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — NGDC maintains a database of over 1,500 volcano locations obtained from the Smithsonian Institution Global Volcanism Program, Volcanoes of the World publication. The...

  6. Uranium Location Database

    Data.gov (United States)

    U.S. Environmental Protection Agency — A GIS compiled locational database in Microsoft Access of ~15,000 mines with uranium occurrence or production, primarily in the western United States. The metadata...

  7. SGA Project Locations

    Data.gov (United States)

    Vermont Center for Geographic Information — The stream geomorphic assessment is a physical assessment competed by geomorphologists to determine the condition and sensitivity of a stream. The SGA locations...

  8. Waste Recovery Locations

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — Locations where City residents are encouraged to drop off and dispose or recycle of unwanted materials. Information provided is subject to change. Please call ahead...

  9. USAID Activity Locations

    Data.gov (United States)

    US Agency for International Development — The USAID Activities dataset is a snapshot of activities supported by USAID including their geographical locations within countries at the time of the snapshot. The...

  10. Smart Location Mapping

    Science.gov (United States)

    The Smart Location Database, Access to Jobs and Workers via Transit, and National Walkability Index tools can help assess indicators related to the built environment, transit accessibility, and walkability.

  11. Allegheny County Dam Locations

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — This dataset shows the point locations of dams in Allegheny County. If viewing this description on the Western Pennsylvania Regional Data Center’s open data portal...

  12. VT Hospital Site Locations

    Data.gov (United States)

    Vermont Center for Geographic Information — (Link to Metadata) This data layer contains point locations of all major community, regional, comprehensive health, and healthcare provider hospitals in the state of...

  13. Location and logistics

    OpenAIRE

    Alumura, Sibel A.; Karab, Bahar Y.; Melo, M. Teresa

    2013-01-01

    Facility location decisions play a critical role in designing logistics networks. This article provides some guidelines on how location decisions and logistics functions can be integrated into a single mathematical model to optimize the configuration of a logistics network. This will be illustrated by two generic models, one supporting the design of a forward logistics network and the other addressing the specific requirements of a reverse logistics network. Several special cases and extensio...

  14. Partially composite Higgs models

    DEFF Research Database (Denmark)

    Alanne, Tommi; Buarque Franzosi, Diogo; Frandsen, Mads T.

    2018-01-01

    constraints. A small part of parameter space around the classically conformal limit is stable up to the Planck scale. This is however already strongly disfavored by LHC results. in different limits, the models realize both (partially) composite-Higgs and (bosonic) technicolor models and a dynamical extension......We study the phenomenology of partially composite-Higgs models where electroweak symmetry breaking is dynamically induced, and the Higgs is a mixture of a composite and an elementary state. The models considered have explicit realizations in terms of gauge-Yukawa theories with new strongly...... interacting fermions coupled to elementary scalars and allow for a very SM-like Higgs state. We study constraints on their parameter spaces from vacuum stability and perturbativity as well as from LHC results and find that requiring vacuum stability up to the compositeness scale already imposes relevant...

  15. Arthroscopic partial medial meniscectomy

    Directory of Open Access Journals (Sweden)

    Dašić Žarko

    2011-01-01

    Full Text Available Background/Aim. Meniscal injuries are common in professional or recreational sports as well as in daily activities. If meniscal lesions lead to physical impairment they usually require surgical treatment. Arthroscopic treatment of meniscal injuries is one of the most often performed orthopedic operative procedures. Methods. The study analyzed the results of arthroscopic partial medial meniscectomy in 213 patients in a 24-month period, from 2006, to 2008. Results. In our series of arthroscopically treated medial meniscus tears we noted 78 (36.62% vertical complete bucket handle lesions, 19 (8.92% vertical incomplete lesions, 18 (8.45% longitudinal tears, 35 (16.43% oblique tears, 18 (8.45% complex degenerative lesions, 17 (7.98% radial lesions and 28 (13.14% horisontal lesions. Mean preoperative International Knee Documentation Committee (IKDC score was 49.81%, 1 month after the arthroscopic partial medial meniscectomy the mean IKDC score was 84.08%, and 6 months after mean IKDC score was 90.36%. Six months after the procedure 197 (92.49% of patients had good or excellent subjective postoperative clinical outcomes, while 14 (6.57% patients subjectively did not notice a significant improvement after the intervention, and 2 (0.93% patients had no subjective improvement after the partial medial meniscectomy at all. Conclusion. Arthroscopic partial medial meniscetomy is minimally invasive diagnostic and therapeutic procedure and in well selected cases is a method of choice for treatment of medial meniscus injuries when repair techniques are not a viable option. It has small rate of complications, low morbidity and fast rehabilitation.

  16. Mergers by Partial Acquisition

    OpenAIRE

    Lindqvist, Tobias

    2004-01-01

    This paper evaluates partial acquisition strategies. The model allows for buying a share of a firm before the actual acquisition takes place. Holding a share in a competing firm before the acquisition of another firm, outsider-toehold, eliminates the insiders' dilemma, i.e. profitable mergers do not occur. This strategy may thus be more profitable for a buyer than acquiring entire firms at once. Furthermore, the insiders' dilemma arises from the assumption of a positive externality on the out...

  17. Optimal Location of an Oil Storage Facility

    Directory of Open Access Journals (Sweden)

    Giovanni Quiel

    2010-01-01

    Full Text Available Given three oil drilling sites, we devise a method to determine the optimal location for a storage facility such that the total length of pipeline required to connect each site to the facility is minimized. First we represent the total distance of piping as the sum of the individual distances between the storage facility and each oil well. From this, both the optimal overall pipeline length and the location of the storage facility which realizes the minimal length can be determined through the first and second partial derivative tests.

  18. Partial Pole Placement in LMI Region

    Directory of Open Access Journals (Sweden)

    Liuli Ou

    2014-01-01

    Full Text Available A new approach for pole placement of single-input system is proposed in this paper. Noncritical closed loop poles can be placed arbitrarily in a specified convex region when dominant poles are fixed in anticipant locations. The convex region is expressed in the form of linear matrix inequality (LMI, with which the partial pole placement problem can be solved via convex optimization tools. The validity and applicability of this approach are illustrated by two examples.

  19. Geostar - Navigation location system

    Science.gov (United States)

    Keyser, Donald A.

    The author describes the Radiodetermination Satellite Service (RDSS). The initial phase of the RDSS provides for a unique service enabling central offices and headquarters to obtain position-location information and receive short digital messages from mobile user terminals throughout the contiguous United States, southern Canada, and northern Mexico. The system employs a spread-spectrum, CDMA modulation technique allowing multiple customers to use the system simultaneously, without preassigned coordination with fellow users. Position location is currently determined by employing an existing radio determination receiver, such as Loran-C, GPS, or Transit, in the mobile user terminal. In the early 1990s position location will be determined at a central earth station by time-differential ranging of the user terminals via two or more geostationary satellites. A brief overview of the RDSS system architecture is presented with emphasis on the user terminal and its diverse applications.

  20. Robust facility location

    OpenAIRE

    Carrizosa Priego, Emilio José; Nickel, Stefan

    2003-01-01

    Let A be a nonempty finite subset of the plane representing the geographical coordinates of a set of demand points (towns, …), to be served by a facility, whose location within a given region S is sought. Assuming that the unit cost for a∈A if the facility is located at x∈S is proportional to dist(x,a) — the distance from x to a — and that demand of point a is given by ωa, minimizing the total transportation cost TC(ω,x) amounts to solving the Weber problem. In practice, it may be the case, h...

  1. Location-based games

    DEFF Research Database (Denmark)

    Ejsing-Duun, Stine

    to create meaningful experiences; and of the prerequisites of a meaningful meeting between players and locations. The dissertation contributes to the field of LBG research by offering an enhanced understanding of LBGs, and LBG player experiences, as well as providing an expanded vocabulary describing LBG...... elements. In addition, the dissertation provides design knowledge concerning creating LBGs that uses certain emergent opportunities when combining location-aware technologies with game mechanics to make use of the six dimensions of LBGs and to involve the player’s body – i.e. make a meaningful meeting...

  2. Partial model checking

    DEFF Research Database (Denmark)

    Andersen, Henrik Reif

    1995-01-01

    A major obstacle in applying finite-state model checking to the verification of large systems is the combinatorial explosion of the state space arising when many loosely coupled parallel processes are considered. The problem also known as the state-explosion problem has been attacked from various...... sides. This paper presents a new approach based on partial model checking where parts of the concurrent system are gradually removed while transforming the specification accordingly. When the intermediate specifications constructed in this manner can be kept small, the state-explosion problem is avoided...

  3. Partial differential equations

    CERN Document Server

    Agranovich, M S

    2002-01-01

    Mark Vishik's Partial Differential Equations seminar held at Moscow State University was one of the world's leading seminars in PDEs for over 40 years. This book celebrates Vishik's eightieth birthday. It comprises new results and survey papers written by many renowned specialists who actively participated over the years in Vishik's seminars. Contributions include original developments and methods in PDEs and related fields, such as mathematical physics, tomography, and symplectic geometry. Papers discuss linear and nonlinear equations, particularly linear elliptic problems in angles and gener

  4. Partially ordered algebraic systems

    CERN Document Server

    Fuchs, Laszlo

    2011-01-01

    Originally published in an important series of books on pure and applied mathematics, this monograph by a distinguished mathematician explores a high-level area in algebra. It constitutes the first systematic summary of research concerning partially ordered groups, semigroups, rings, and fields. The self-contained treatment features numerous problems, complete proofs, a detailed bibliography, and indexes. It presumes some knowledge of abstract algebra, providing necessary background and references where appropriate. This inexpensive edition of a hard-to-find systematic survey will fill a gap i

  5. Generalized Partial Volume

    DEFF Research Database (Denmark)

    Darkner, Sune; Sporring, Jon

    2011-01-01

    Mutual Information (MI) and normalized mutual information (NMI) are popular choices as similarity measure for multimodal image registration. Presently, one of two approaches is often used for estimating these measures: The Parzen Window (PW) and the Generalized Partial Volume (GPV). Their theoret...... of view as well as w.r.t. computational complexity. Finally, we present algorithms for both approaches for NMI which is comparable in speed to Sum of Squared Differences (SSD), and we illustrate the differences between PW and GPV on a number of registration examples....

  6. Partial differential equations

    CERN Document Server

    Levine, Harold

    1997-01-01

    The subject matter, partial differential equations (PDEs), has a long history (dating from the 18th century) and an active contemporary phase. An early phase (with a separate focus on taut string vibrations and heat flow through solid bodies) stimulated developments of great importance for mathematical analysis, such as a wider concept of functions and integration and the existence of trigonometric or Fourier series representations. The direct relevance of PDEs to all manner of mathematical, physical and technical problems continues. This book presents a reasonably broad introductory account of the subject, with due regard for analytical detail, applications and historical matters.

  7. Elliptic partial differential equations

    CERN Document Server

    Han, Qing

    2011-01-01

    Elliptic Partial Differential Equations by Qing Han and FangHua Lin is one of the best textbooks I know. It is the perfect introduction to PDE. In 150 pages or so it covers an amazing amount of wonderful and extraordinary useful material. I have used it as a textbook at both graduate and undergraduate levels which is possible since it only requires very little background material yet it covers an enormous amount of material. In my opinion it is a must read for all interested in analysis and geometry, and for all of my own PhD students it is indeed just that. I cannot say enough good things abo

  8. Partial differential equations

    CERN Document Server

    Sloan, D; Süli, E

    2001-01-01

    /homepage/sac/cam/na2000/index.html7-Volume Set now available at special set price ! Over the second half of the 20th century the subject area loosely referred to as numerical analysis of partial differential equations (PDEs) has undergone unprecedented development. At its practical end, the vigorous growth and steady diversification of the field were stimulated by the demand for accurate and reliable tools for computational modelling in physical sciences and engineering, and by the rapid development of computer hardware and architecture. At the more theoretical end, the analytical insight in

  9. Comparison of subcellular distribution and chemical forms of cadmium among four soybean cultivars at young seedlings.

    Science.gov (United States)

    Wang, Peng; Deng, Xiaojuan; Huang, Yian; Fang, Xiaolong; Zhang, Jie; Wan, Haibo; Yang, Cunyi

    2015-12-01

    The hydroponic experiment was carried out to investigate the Cd subcellular distribution and chemical forms in roots and shoots among four soybean seedling cultivars with two Cd treatments. HX3 and GC8, two tolerant and low-grain-Cd-accumulating cultivars, had the lowest Cd concentration in roots and high Cd concentration in shoots, while BX10 and ZH24, two sensitive and high-grain-Cd-accumulating cultivars, had the highest Cd concentration in roots and the lowest Cd concentration in shoots at young seedling stage. Furthermore, the sequence of Cd subcellular distribution in roots at two Cd levels was cell wall (53.4-75.5 %) > soluble fraction (15.8-40.4 %) > organelle fraction (2.0-14.7 %), but in shoots, was soluble fraction (39.3-74.8 %) > cell wall (16.0-52.0 %) > organelle (4.8-19.5 %). BX10 and ZH24 had higher Cd concentration in all subcellular fractions in roots, but HX3 and GC8 had higher Cd concentration of soluble fraction in shoots. The sequence of Cd chemical forms in roots was FNacl (64.1-79.5 %) > FHAC (3.4-21.5 %) > Fd-H2O (3.6-13.0 %) > Fethanol (1.4-21.8) > FHCl (0.3-1.6 %) > Fother (0.2-1.4 %) at two Cd levels but, in shoots, was FNacl (19.7-51.4 %) ≥ FHAC (10.2-31.4 %) ≥ Fd-H2O (8.8-28.2 %) ≥ Fethanol (8.9-38.6 %) > FHCl (0.2-9.6 %) > Fother (2.5-11.2 %). BX10 and ZH24 had higher Cd concentrations in each extracted solutions from roots, but from shoots for GC8 and HX3. Taken together, the results uncover that root cell walls and leaf vacuoles might play important roles in Cd detoxification and limiting the symplastic movement of Cd.

  10. Molecular basis for the dual subcellular distribution of microsomal glutathione transferase 1.

    Science.gov (United States)

    Shimoji, Miyuki; Figueroa, Ricardo A; Neve, Etienne; Maksel, Danuta; Imreh, Gabriela; Morgenstern, Ralf; Hallberg, Einar

    2017-02-01

    Microsomal glutathione transferase 1 (MGST1) is a membrane bound enzyme involved in the detoxification of reactive electrophiles and protection of membranes from oxidative stress. The enzyme displays an unusual and broad subcellular distribution with especially high levels in the endoplasmic reticulum (ER) and outer mitochondrial membrane (OMM). Here we examined the molecular basis for this dual distribution. We hypothesized that the amphipathic properties of the first transmembrane segment (TMS), that contains a positively charged lysine (K25), is a central feature guiding dual targeting. The lysine-25 was substituted to alanine by site directed mutagenesis. We also increased the amphipathic character of the helix by inserting an additional lysine either one turn above or below K25. Expressing these constructs in simian COS cells, and analyzing subcellular distribution by immunocytochemistry, we observed an increased ER targeting of K25A-MGST1. In contrast I22K-MGST1 and F28K-MGST1 displayed pronounced mitochondrial targeting. By using in vitro transcription-translation we examined whether insertion of WT-MGST1 into ER is co- or post-translational and provide evidence for the former. In the same experimental set-up, mitochondrial insertion was shown to depend on the positive charge. Together these results show that removing the positive charge of lysine-25 promotes ER incorporation, but counteracts mitochondrial insertion. In contrast, introducing an extra lysine in the first TMS of MGST1 had opposite effects. The amphipathic character of the first TMS thus constitutes a molecular determinant for the dual targeting of MGST1. Broad subcellular distribution is consistent with a physiological role in protection from reactive intermediates and oxidative stress. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Imaging cellular and subcellular structure of human brain tissue using micro computed tomography

    Science.gov (United States)

    Khimchenko, Anna; Bikis, Christos; Schweighauser, Gabriel; Hench, Jürgen; Joita-Pacureanu, Alexandra-Teodora; Thalmann, Peter; Deyhle, Hans; Osmani, Bekim; Chicherova, Natalia; Hieber, Simone E.; Cloetens, Peter; Müller-Gerbl, Magdalena; Schulz, Georg; Müller, Bert

    2017-09-01

    Brain tissues have been an attractive subject for investigations in neuropathology, neuroscience, and neurobiol- ogy. Nevertheless, existing imaging methodologies have intrinsic limitations in three-dimensional (3D) label-free visualisation of extended tissue samples down to (sub)cellular level. For a long time, these morphological features were visualised by electron or light microscopies. In addition to being time-consuming, microscopic investigation includes specimen fixation, embedding, sectioning, staining, and imaging with the associated artefacts. More- over, optical microscopy remains hampered by a fundamental limit in the spatial resolution that is imposed by the diffraction of visible light wavefront. In contrast, various tomography approaches do not require a complex specimen preparation and can now reach a true (sub)cellular resolution. Even laboratory-based micro computed tomography in the absorption-contrast mode of formalin-fixed paraffin-embedded (FFPE) human cerebellum yields an image contrast comparable to conventional histological sections. Data of a superior image quality was obtained by means of synchrotron radiation-based single-distance X-ray phase-contrast tomography enabling the visualisation of non-stained Purkinje cells down to the subcellular level and automated cell counting. The question arises, whether the data quality of the hard X-ray tomography can be superior to optical microscopy. Herein, we discuss the label-free investigation of the human brain ultramorphology be means of synchrotron radiation-based hard X-ray magnified phase-contrast in-line tomography at the nano-imaging beamline ID16A (ESRF, Grenoble, France). As an example, we present images of FFPE human cerebellum block. Hard X-ray tomography can provide detailed information on human tissues in health and disease with a spatial resolution below the optical limit, improving understanding of the neuro-degenerative diseases.

  12. Evolution and comparative genomics of subcellular specializations: EST sequencing of Torpedo electric organ.

    Science.gov (United States)

    Nazarian, Javad; Berry, Deborah L; Sanjari, Salar; Razvi, Mohammed; Brown, Kristy; Hathout, Yetrib; Vertes, Akos; Dadgar, Sherry; Hoffman, Eric P

    2011-03-01

    Uncharacterized open reading frames (ORFs) in human genomic sequence often show a high degree of evolutionary conservation, yet have little or no tissue EST or protein data suggestive of protein product function. The encoded proteins may have highly restricted expression in specialized cells, subcellular specializations, and/or narrow windows during development. One such highly specialized and minute subcellular compartment is the neuromuscular junction (NMJ), where motorneurons contact muscle fibers. The electric Torpedo ray has evolved to expand the NMJ structure to the size of a large organ (electroplax organ), and we hypothesized that Torpedo electroplax proteins would be candidates for human ESTs expressed at the human NMJ. A total of 9719 primary electroplax cDNA clones were sequenced. We identified 44 human ORFs showing high (>63%) amino acid identity to Torpedo electroplax transcripts with enrichment for mRNA splicing motifs (SH2 and pre-mRNA splicing domains), an observation potentially important for the strict nuclear domains maintained by myonuclei underlying the NMJ. We generated antibodies against two uncharacterized human genes (C19orf29 [Drosophila cactin] and C15orf24) and showed that these were indeed expressed at the murine NMJ. Cactin, a member of the Rel transcription factor family in Drosophila, localized to the postsynaptic cytosol of the NMJ and nuclear membrane. C15orf24 protein localized to the murine postsynaptic sarcolemma. We show a novel approach towards identifying proteins expressed at a subcellular specialization using evolutionary diversity of organ function and cross-species mapping. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Effect heat stress on subcellular localization of Ca2+ in tomato fruits

    Directory of Open Access Journals (Sweden)

    Grażyna Garbaczewska

    2014-01-01

    Full Text Available The aim of this paper was to compare the fruit cell ultrastructure and subcellular localization of Ca2+ after heat stress with the use of the potassium antimonate method (Slocum and Roux 1982, Tretyn et al. 1992. The tomato plants Robin cv., relatively tolerant to heat stress, were grown under uncontrolled greenhouse conditions to the stage of fruiting. The plants were placed for 20h in two temperature regimes: 23oC (optimal temperature or 40oC (heat stress in darkness, under water vapour saturated atmosphere. Immediately after heat stress the fruits were harvested to estimate water soluble and insoluble calcium contents and subcellular localization of Ca2+. After heating the concentration of calcium in tomato fruits increased about twice. In both temperature treatments the water soluble fractions were lower than insoluble ones at smaller differences between insoluble and soluble fractions after heat stress. The shapes and localization of Ca2+ detected with the use of potassium antimonate method show that in fruits of control plants the precipitates were numerous, small and of oval shape. They were dispersed in cytosol or adjoined to endoplasmic reticulum or to external membrane of chloroplast. In the fruit of heated plants the precipitates were irregular in shape, amorphous and singly dispersed in the cytosol. We observed also some cytological changes in the structure of membranes and organelles of the plants of both experimental treatments. The heat induced increase of calcium content and the changes in subcellular localization of Ca2+ under heat stress suggest that calcium ions may be involved in avoiding heat injury. The problem requires more detailed further investigations.

  14. Subcellular distribution of molybdenum, ultrastructural and antioxidative responses in soybean seedlings under excess molybdenum stress.

    Science.gov (United States)

    Xu, Shoujun; Hu, Chengxiao; Tan, Qiling; Qin, Shiyu; Sun, Xuecheng

    2017-12-05

    Some studies have shown that excess molybdenum (Mo) could produce toxic effects on plants. However, little is known about the subcellular distribution of Mo and cell ultrastructure within plants under excess Mo stress. Here, we comprehensively analyzed the changes of Mo distribution in subcellular fractions, cell ultrastructure and antioxidant enzymes in leaves and roots of soybean seedlings in response to excess Mo stress. The results showed that roots exhibited higher Mo accumulation than leaves at the 100 mg L-1 Mo level, about 38.58-, 171.48- and 52.99-fold higher in cell walls, cell organelles and soluble fractions, respectively. Subcellular fractionations of Mo-containing tissues indicated that approximately 90% of Mo was accumulated in the soluble fractions and cell walls of the roots and leaves, and soluble fractions (accumulated 66.3-72.2% Mo) might serve as an effective storage site for excess Mo. Furthermore, excess Mo caused ultrastructural alterations in roots and leaves of soybean seedlings, leading to structural abnormality of chloroplast in leaf cells, plasmolysis, cellular deformity, vacuole enlargement and the swelling of cell wall and cytoplasm in root cells. Meanwhile, under excess Mo stress, the activity of POD, CAT and APX enzymes in roots was 1.43, 2.35 and 1.23 times that under standard Mo condition, while that of SOD and CAT enzymes in leaves was 1.23 and 1.94 times, respectively. This study provided novel insights into the mechanisms of excess Mo toxicity in soybean seedlings. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  15. Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities

    Directory of Open Access Journals (Sweden)

    Bo Fu

    2015-12-01

    Full Text Available The in vitro maturation (IVM efficiency of porcine embryos is still low because of poor oocyte quality. Although brilliant cresyl blue positive (BCB+ oocytes with low glucose-6-phosphate dehydrogenase (G6PDH activity have shown superior quality than BCB negative (− oocytes with high G6PDH activity, the use of a BCB staining test before IVM is still controversial. This study aimed to shed more light on the subcellular characteristics of porcine oocytes after selection using BCB staining. We assessed germinal vesicle chromatin configuration, cortical granule (CG migration, mitochondrial distribution, the levels of acetylated lysine 9 of histone H3 (AcH3K9 and nuclear apoptosis features to investigate the correlation between G6PDH activity and these developmentally related features. A pattern of chromatin surrounding the nucleoli was seen in 53.0% of BCB+ oocytes and 77.6% of BCB+ oocytes showed peripherally distributed CGs. After IVM, 48.7% of BCB+ oocytes had a diffused mitochondrial distribution pattern. However, there were no significant differences in the levels of AcH3K9 in the nuclei of blastocysts derived from BCB+ and BCB− oocytes; at the same time, we observed a similar incidence of apoptosis in the BCB+ and control groups. Although this study indicated that G6PDH activity in porcine oocytes was correlated with several subcellular characteristics such as germinal vesicle chromatin configuration, CG migration and mitochondrial distribution, other features such as AcH3K9 level and nuclear apoptotic features were not associated with G6PDH activity and did not validate the BCB staining test. In using this test for selecting porcine oocytes, subcellular characteristics such as the AcH3K9 level and apoptotic nuclear features should also be considered. Adding histone deacetylase inhibitors or apoptosis inhibitors into the culture medium used might improve the efficiency of IVM of BCB+ oocytes.

  16. Crime Location Choice

    NARCIS (Netherlands)

    Bernasco, Wim; Ruiter, Stijn

    2014-01-01

    Most behavior of interest to social scientists is choice behavior: actions people commit while they could also have done something else. In geographical and environmental criminology, a new framework has emerged for analyzing individual crime location choice. It is based on the principle of random

  17. The Czech Locative Chameleon

    Directory of Open Access Journals (Sweden)

    Tarald Taraldsen

    2007-12-01

    Full Text Available We show that under certain circumstances, the Czech locative prepositions (LOC show up as directional prepositions (DIR and vice versa, (under different circumstances the Czech DIR PPs show up as LOC. We argue that such a chameleon life of the PPs is structurally dependent.

  18. Tracking, say, SKYPE Locations

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. Tracking, say, SKYPE Locations. Real Time Communication: Peer-to-Peer (P2P). Datagram flows between the two conversing partners; Exposes the IP addresses of all the participants to one another. If A knows B's VoIP ID, she can establish a call with Bob & obtain his current ...

  19. General minisum circle location

    DEFF Research Database (Denmark)

    Körner, Mark; Brimberg, Jack; Juel, Henrik

    2009-01-01

    In our paper we approximate a set of given points by a general circle. More precisely, we consider the problem of locating and scaling the unit ball of some given norm k1 with respect to xed points on the plane such that the sum of weighted distances between the circle and the xed points...

  20. ATL: ASA Tutor Locator

    NARCIS (Netherlands)

    Brouwers, Maurice; Brouns, Francis; Van Rosmalen, Peter; Sloep, Peter; Koper, Rob

    2006-01-01

    ASA Tutor Locator creates ad hoc transient communities consisting of peer tutors, seeded with document fragments found in the learning network, to answer content questions of students. The sources are available from http://sourceforge.net/projects/asa-atl. Available under the three clause BSD

  1. ATL: A Tutor Locator

    NARCIS (Netherlands)

    De Jong, André; Brouwers, Maurice; Brouns, Francis; Van Rosmalen, Peter; Kester, Liesbeth; Sloep, Peter

    2007-01-01

    A Tutor Locator creates ad hoc transient communities consisting of peer tutors, seeded with document fragments found in the learning network, to answer content questions of students. The sources are available from http://sourceforge.net/projects/asa-atl.Available under the three clause BSD licence,

  2. Immigrants' location preferences

    DEFF Research Database (Denmark)

    Damm, Anna Piil

    This paper exploits a spatial dispersal policy for refugee immigrants to estimate the importance of local and regional factors for refugees' location preferences. The main results of a mixed proportional hazard competing risks model are that placed refugees react to high regional unemployment...

  3. RFID Location Algorithm

    Directory of Open Access Journals (Sweden)

    Wang Zi Min

    2016-01-01

    Full Text Available With the development of social services, people’s living standards improve further requirements, there is an urgent need for a way to adapt to the complex situation of the new positioning technology. In recent years, RFID technology have a wide range of applications in all aspects of life and production, such as logistics tracking, car alarm, security and other items. The use of RFID technology to locate, it is a new direction in the eyes of the various research institutions and scholars. RFID positioning technology system stability, the error is small and low-cost advantages of its location algorithm is the focus of this study.This article analyzes the layers of RFID technology targeting methods and algorithms. First, RFID common several basic methods are introduced; Secondly, higher accuracy to political network location method; Finally, LANDMARC algorithm will be described. Through this it can be seen that advanced and efficient algorithms play an important role in increasing RFID positioning accuracy aspects.Finally, the algorithm of RFID location technology are summarized, pointing out the deficiencies in the algorithm, and put forward a follow-up study of the requirements, the vision of a better future RFID positioning technology.

  4. Optimal Facility-Location.

    Science.gov (United States)

    Goldman, A J

    2006-01-01

    Dr. Christoph Witzgall, the honoree of this Symposium, can count among his many contributions to applied mathematics and mathematical operations research a body of widely-recognized work on the optimal location of facilities. The present paper offers to non-specialists a sketch of that field and its evolution, with emphasis on areas most closely related to Witzgall's research at NBS/NIST.

  5. Spatiotemporal AED Location Optimization

    NARCIS (Netherlands)

    Chan, Timothy C.Y.; Sun, Christopher L.F.; Demirtas, Derya; Morrison, Laurie J.; Brooks, Steven C.

    2015-01-01

    Background: Mathematical optimization can be used to plan future AED placement to maximize out-of-hospital cardiac arrest (OHCA) coverage. Many public access AEDs are placed in locations without 24/7 access. AED coverage can be overestimated unless temporal availability is considered. Objective: To

  6. Experts' Understanding of Partial Derivatives Using the Partial Derivative Machine

    Science.gov (United States)

    Roundy, David; Weber, Eric; Dray, Tevian; Bajracharya, Rabindra R.; Dorko, Allison; Smith, Emily M.; Manogue, Corinne A.

    2015-01-01

    Partial derivatives are used in a variety of different ways within physics. Thermodynamics, in particular, uses partial derivatives in ways that students often find especially confusing. We are at the beginning of a study of the teaching of partial derivatives, with a goal of better aligning the teaching of multivariable calculus with the needs of…

  7. Subcellular Targeting of Proteins Involved in Modification of Plant N- and O-Glycosylation.

    Science.gov (United States)

    Dicker, Martina; Schoberer, Jennifer; Vavra, Ulrike; Strasser, Richard

    2015-01-01

    Plants are attractive expression hosts for the production of recombinant glycoprotein therapeutics. The quality and efficiency of these biopharmaceuticals are very often influenced by the glycosylation profile. Consequently, approaches are needed that enable the production of recombinant glycoproteins with customized and homogenous N- and O-glycan structures. Here, we describe convenient tools that allow targeting and retention of glycan-modifying enzymes in the early secretory pathway of plants. These protocols can be used to fine-tune the subcellular localization of glycosyltransferases and glycosidases in plants and consequently to increase the homogeneity of glycosylation on recombinant glycoproteins.

  8. Subcellular localization of the five members of the human steroid 5α-reductase family

    Directory of Open Access Journals (Sweden)

    Antonella Scaglione

    2017-06-01

    We report the cloning and transient expression in HeLa cells of the five members of the human steroid 5α-reductase family as both N- and C-terminus green fluorescent protein tagged protein constructs. Following the intrinsic fluorescence of the tag, we have determined that the subcellular localization of these enzymes is in the endoplasmic reticulum, upon expression in HeLa cells. The presence of the tag at either end of the polypeptide chain can affect protein expression and, in the case of trans enoyl-CoA reductase, it induces the formation of protein aggregates.

  9. Subcellular localization of YKL-40 in normal and malignant epithelial cells of the breast

    DEFF Research Database (Denmark)

    Roslind, A.; Balslev, E.; Kruse, H.

    2008-01-01

    . YKL-40 protein expression was redistributed in carcinoma versus normal glandular tissue of the breast. A reduced expression of YKL-40 in relation to intermediate filaments and desmosomes was found in tumor cells. Changes in YKL-40 expression suggest that the function of YKL-40 in cells of epithelial......YKL-40 is a new prognostic biomarker in cancer. The biological function is only poorly understood. This study aimed at determining the subcellular localization of YKL-40, using immunogold labeling, in normal epithelial cells and in malignant tumor cells of the breast by immunoelectron microscopy...

  10. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    Science.gov (United States)

    Chandra, Subhash

    2008-12-01

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O 2+) revealed local secondary ion signal enhancements correlated with the water image signals of 19(H 3O) +. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K + and Na + in physiologically relevant concentrations. The high K-low Na signature in individual cells

  11. Challenges of biological sample preparation for SIMS imaging of elements and molecules at subcellular resolution

    Energy Technology Data Exchange (ETDEWEB)

    Chandra, Subhash [Cornell SIMS Laboratory, Department of Earth and Atmospheric Sciences, Snee Hall, Cornell University, Ithaca, NY 14853 (United States)], E-mail: sc40@cornell.edu

    2008-12-15

    Secondary ion mass spectrometry (SIMS) based imaging techniques capable of subcellular resolution characterization of elements and molecules are becoming valuable tools in many areas of biology and medicine. Due to high vacuum requirements of SIMS, the live cells cannot be analyzed directly in the instrument. The sample preparation, therefore, plays a critical role in preserving the native chemical composition for SIMS analysis. This work focuses on the evaluation of frozen-hydrated and frozen freeze-dried sample preparations for SIMS studies of cultured cells with a CAMECA IMS-3f dynamic SIMS ion microscope instrument capable of producing SIMS images with a spatial resolution of 500 nm. The sandwich freeze-fracture method was used for fracturing the cells. The complimentary fracture planes in the plasma membrane were characterized by field-emission secondary electron microscopy (FESEM) in the frozen-hydrated state. The cells fractured at the dorsal surface were used for SIMS analysis. The frozen-hydrated SIMS analysis of individual cells under dynamic primary ion beam (O{sub 2}{sup +}) revealed local secondary ion signal enhancements correlated with the water image signals of {sup 19}(H{sub 3}O){sup +}. A preferential removal of water from the frozen cell matrix in the Z-axis was also observed. These complications render the frozen-hydrated sample type less desirable for subcellular dynamic SIMS studies. The freeze-drying of frozen-hydrated cells, either inside the instrument or externally in a freeze-drier, allowed SIMS imaging of subcellular chemical composition. Morphological evaluations of fractured freeze-dried cells with SEM and confocal laser scanning microscopy (CLSM) revealed well-preserved mitochondria, Golgi apparatus, and stress fibers. SIMS analysis of fractured freeze-dried cells revealed well-preserved chemical composition of even the most highly diffusible ions like K{sup +} and Na{sup +} in physiologically relevant concentrations. The high K

  12. DeepLoc: prediction of protein subcellular localization using deep learning

    DEFF Research Database (Denmark)

    Almagro Armenteros, Jose Juan; Sønderby, Casper Kaae; Sønderby, Søren Kaae

    2017-01-01

    current state-of-the-art algorithms, including those relying on homology information. The method is available as a web server at http://www.cbs.dtu.dk/services/DeepLoc . Example code is available at https://github.com/JJAlmagro/subcellular_localization . The dataset is available at http://www.cbs.dtu.dk/services/Deep...... knowledge databases. For novel proteins where no annotated homologues exist, and for predicting the effects of sequence variants, it is desirable to have methods for predicting protein properties from sequence information only. Here, we present a prediction algorithm using deep neural networks to predict...

  13. A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain.

    Science.gov (United States)

    Garrone, Nicholas F; Blazer-Yost, Bonnie L; Weiss, Robert B; Lalouel, Jean-Marc; Rohrwasser, Andreas

    2009-04-13

    Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence beta-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase activity. This observation suggests a new mechanism for the

  14. A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

    Directory of Open Access Journals (Sweden)

    Lalouel Jean-Marc

    2009-04-01

    Full Text Available Abstract Background Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. Results To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. Conclusion NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase

  15. A novel BAT3 sequence generated by alternative RNA splicing of exon 11B displays cell type-specific expression and impacts on subcellular localization.

    Directory of Open Access Journals (Sweden)

    Nadine Kämper

    Full Text Available The human lymphocyte antigen (HLA encoded BAT3/BAG6 recently attracted interest as a regulator of protein targeting and degradation, a function that could be exerted in the cytosol and in the nucleus. The BAT3 gene was described to consist of 25 exons. Diversity of transcripts can be generated by alternative RNA splicing, which may control subcellular distribution of BAT3.By cDNA sequencing we identified a novel alternatively spliced sequence of the BAT3 gene located between exons 11 and 12, which was designated as exon 11B. Using PCR and colony hybridization we identified six cDNA variants, which were produced by RNA splicing of BAT3 exons 5, 11B and 24. In four examined cell types the content of BAT3 splice variants was examined. Most of the cDNA clones from monocyte-derived dendritic cells contain exon 11B, whereas this sequence was almost absent in the B lymphoma Raji. Exon 5 was detected in most and exon 24 in approximately half of the cDNA clones. The subcellular distribution of endogenous BAT3 largely correlates with a cell type specific splicing pattern. In cells transfected with BAT3 variants, full-length and Δ24 BAT3 displayed nearly exclusive nuclear staining, whereas variants deleted of exon 11B showed substantial cytosolic expression. We show here that BAT3 is mainly expressed in the cytosol of Raji cells, while other cell types displayed both cytosolic and nuclear staining. Export of BAT3 from the nucleus to the cytosol is inhibited by treatment with leptomycin B, indicating that the Crm1 pathway is involved. Nuclear expression of BAT3 containing exon 11B suggests that this sequence plays a role for nuclear retention of the protein.Cell type-specific subcellular expression of BAT3 suggests distinct functions in the cytosol and in the nucleus. Differential expression of BAT3 variants may reconcile the multiple roles described for BAT3.

  16. Furnace brazing under partial vacuum

    Science.gov (United States)

    Mckown, R. D.

    1979-01-01

    Brazing furnace utilizing partial-vacuum technique reduces tooling requirements and produces better bond. Benefit in that partial vacuum helps to dissociate metal oxides that inhibit metal flow and eliminates heavy tooling required to hold parts together during brazing.

  17. Distribution, isomerization and enantiomer selectivity of hexabromocyclododecane (HBCD) diastereoisomers in different tissue and subcellular fractions of earthworms.

    Science.gov (United States)

    Li, Bing; Chen, Hao; Sun, Hongwen; Lan, Zhonghui

    2017-05-01

    In this study, earthworms Eisenia fetida (E. fetida) were exposed to a soil artificially contaminated with individual hexabromocyclododecane (HBCD) diastereoisomers (α-, β- and γ-HBCDs) to investigate the distribution, isomerization and enantiomer selectivity of HBCDs at tissue and subcellular levels. At the tissue level, the concentrations of HBCDs all followed the order of gut>bodyfluid>body wall, which suggested that earthworms accumulated HBCDs mainly via ingesting soil particles. At the subcellular level, the concentrations of HBCDs in an extracellular fraction consisting of granules, tissue fragment, cell membrane and intact cells (fraction A) were higher than those in an intracellular fractions consisting of the microsomal and cytosol (fraction B+C). This confirmed the passive diffusion during the distribution of HBCDs into the intracellular compartment. The distribution proportions of HBCDs varied among different tissue and subcellular fractions, and all changed over time within 14 days. The variable distributions of HBCDs in different fractions were a result of the comprehensive effects of dynamics and thermodynamics processes. The β- and γ-HBCDs were isomerized to α-HBCD in all tissue and subcellular fractions except for fraction C, and the isomerization ratios varied a lot, which seemed to be related to HBCDs residence time. The selective enrichment of (-) α-, (-) β and (-) γ-HBCDs was found in all fractions and this is consistent with that in the whole earthworm. Besides, the extents of enantio-selectivity did not change significantly among different tissue and subcellular fractions. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Predicting Locations of Pollution Sources using Convolutional Neural Networks

    OpenAIRE

    Chi, Yiheng; Winovich, Nickolas D.; Lin, Guang

    2017-01-01

    Pollution is a severe problem today, and the main challenge in water and air pollution controls and eliminations is detecting and locating pollution sources. This research project aims to predict the locations of pollution sources given diffusion information of pollution in the form of array or image data. These predictions are done using machine learning. The relations between time, location, and pollution concentration are first formulated as pollution diffusion equations, which are partial...

  19. In vivo subcellular localization of Mal de Rio Cuarto virus (MRCV) non-structural proteins in insect cells reveals their putative functions

    Energy Technology Data Exchange (ETDEWEB)

    Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar [Instituto de Biotecnologia, CICVyA, Instituto Nacional de Tecnologia Agropecuaria (IB-INTA), Las cabanas y Los Reseros s/n. Hurlingham Cp 1686, Buenos Aires (Argentina); Vas, Mariana del, E-mail: mdelvas@cnia.inta.gov.ar [Instituto de Biotecnologia, CICVyA, Instituto Nacional de Tecnologia Agropecuaria (IB-INTA), Las cabanas y Los Reseros s/n. Hurlingham Cp 1686, Buenos Aires (Argentina)

    2012-09-01

    The in vivo subcellular localization of Mal de Rio Cuarto virus (MRCV, Fijivirus, Reoviridae) non-structural proteins fused to GFP was analyzed by confocal microscopy. P5-1 showed a cytoplasmic vesicular-like distribution that was lost upon deleting its PDZ binding TKF motif, suggesting that P5-1 interacts with cellular PDZ proteins. P5-2 located at the nucleus and its nuclear import was affected by the deletion of its basic C-termini. P7-1 and P7-2 also entered the nucleus and therefore, along with P5-2, could function as regulators of host gene expression. P6 located in the cytoplasm and in perinuclear cloud-like inclusions, was driven to P9-1 viroplasm-like structures and co-localized with P7-2, P10 and {alpha}-tubulin, suggesting its involvement in viroplasm formation and viral intracellular movement. Finally, P9-2 was N-glycosylated and located at the plasma membrane in association with filopodia-like protrusions containing actin, suggesting a possible role in virus cell-to-cell movement and spread.

  20. Locating and Tracking Everything Digital

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. Telcos need actual locations for highly localized and targeted ads, location based services. Telcos need actual locations for highly localized and targeted ads, location based services. Governments need to know the location of the buyers for tax – state tax etc. Locating friends ...

  1. Tutorial on Online Partial Evaluation

    Directory of Open Access Journals (Sweden)

    William R. Cook

    2011-09-01

    Full Text Available This paper is a short tutorial introduction to online partial evaluation. We show how to write a simple online partial evaluator for a simple, pure, first-order, functional programming language. In particular, we show that the partial evaluator can be derived as a variation on a compositionally defined interpreter. We demonstrate the use of the resulting partial evaluator for program optimization in the context of model-driven development.

  2. Type-Directed Partial Evaluation

    DEFF Research Database (Denmark)

    Danvy, Olivier

    1998-01-01

    Type-directed partial evaluation uses a normalization function to achieve partial evaluation. These lecture notes review its background, foundations, practice, and applications. Of specific interest is the modular technique of offline and online type-directed partial evaluation in Standard ML of ...

  3. Type-Directed Partial Evaluation

    DEFF Research Database (Denmark)

    Danvy, Olivier

    1998-01-01

    Type-directed partial evaluation uses a normalization function to achieve partial evaluation. These lecture notes review its background, foundations, practice, and applications. Of specific interest is the modular technique of offline and online type-directed partial evaluation in Standard ML...

  4. Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines.

    Directory of Open Access Journals (Sweden)

    Michael I Koukourakis

    Full Text Available LC3s (MAP1-LC3A, B and C are structural proteins of autophagosomal membranes, widely used as biomarkers of autophagy. Whether these three LC3 proteins have a similar biological role in autophagy remains obscure. We examine in parallel the subcellular expression patterns of the three LC3 proteins in a panel of human cancer cell lines, as well as in normal MRC5 fibroblasts and HUVEC, using confocal microscopy and western blot analysis of cell fractions. In the cytoplasm, there was a minimal co-localization between LC3A, B and C staining, suggesting that the relevant autophagosomes are formed by only one out of the three LC3 proteins. LC3A showed a perinuclear and nuclear localization, while LC3B was equally distributed throughout the cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli, where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies.

  5. Electric current locator

    Science.gov (United States)

    King, Paul E [Corvallis, OR; Woodside, Charles Rigel [Corvallis, OR

    2012-02-07

    The disclosure herein provides an apparatus for location of a quantity of current vectors in an electrical device, where the current vector has a known direction and a known relative magnitude to an input current supplied to the electrical device. Mathematical constants used in Biot-Savart superposition equations are determined for the electrical device, the orientation of the apparatus, and relative magnitude of the current vector and the input current, and the apparatus utilizes magnetic field sensors oriented to a sensing plane to provide current vector location based on the solution of the Biot-Savart superposition equations. Description of required orientations between the apparatus and the electrical device are disclosed and various methods of determining the mathematical constants are presented.

  6. Aquatic Nuisance Species Locator

    Science.gov (United States)

    Data in this map has been collected by the United States Geological Survey's Nonindigenous Aquatic Species program located in Gainesville, Florida (http://nas.er.usgs.gov/default.aspx). This dataset may have some inaccuracies and is only current to June 15, 2012. The species identified in this dataset are not inclusive of all aquatic nuisance species, but rather a subset identified to be at risk for transport by recreational activities such as boating and angling. Additionally, the locations where organisims have been identified are also not inclusive and should be treated as a guide. Organisms are limited to the following: American bullfrog, Asian clam, Asian shore crab, Asian tunicate, Australian spotted jellyfish, Chinese mitten crab, New Zealand mudsnail, Colonial sea squirt, Alewife, Bighead carp, Black carp, Flathead catfish, Grass carp, Green crab, Lionfish, Northern snakehead, Quagga mussel, Round Goby, Ruffe, Rusty crayfish, Sea lamprey, Silver carp, Spiny water flea, Veined rapa whelk, Zebra mussel

  7. Production Subcontracting and Location

    OpenAIRE

    Holl, Adelheid

    2007-01-01

    Using data from a panel of Spanish manufacturing firms, I examine factors that explain firms’ production subcontracting decisions and test whether there is any evidence that production subcontracting is facilitated in areas typically associated with higher agglomeration economies. The results show that location matters. Firms in industry agglomerations are more likely to subcontract production activities. While in general, larger and older firms as well as high wage firms show a greater proba...

  8. Choosing locations of landfills

    OpenAIRE

    Špacapan, Matjaž

    2008-01-01

    In my Graduation thesis we deal with the problem of choosing the most appropriate location of landfills and the criteria used in this process. The problem of waste management is one of the most critical areas of environmental protection in Slovenia. In these matters, the existing legislation and law directives in the Republic of Slovenia are followed. After this we have to pay attention to the composition of the waste and the technologies of waste management. Before we place a new facility fo...

  9. Optimal Facility-Location

    OpenAIRE

    Goldman, A. J.

    2006-01-01

    Dr. Christoph Witzgall, the honoree of this Symposium, can count among his many contributions to applied mathematics and mathematical operations research a body of widely-recognized work on the optimal location of facilities. The present paper offers to non-specialists a sketch of that field and its evolution, with emphasis on areas most closely related to Witzgall?s research at NBS/NIST.

  10. Fundamental partial compositeness

    CERN Document Server

    Sannino, Francesco

    2016-01-01

    We construct renormalizable Standard Model extensions, valid up to the Planck scale, that give a composite Higgs from a new fundamental strong force acting on fermions and scalars. Yukawa interactions of these particles with Standard Model fermions realize the partial compositeness scenario. Successful models exist because gauge quantum numbers of Standard Model fermions admit a minimal enough 'square root'. Furthermore, right-handed SM fermions have an SU(2)$_R$-like structure, yielding a custodially-protected composite Higgs. Baryon and lepton numbers arise accidentally. Standard Model fermions acquire mass at tree level, while the Higgs potential and flavor violations are generated by quantum corrections. We further discuss accidental symmetries and other dynamical features stemming from the new strongly interacting scalars. If the same phenomenology can be obtained from models without our elementary scalars, they would reappear as composite states.

  11. Applied partial differential equations

    CERN Document Server

    Logan, J David

    2004-01-01

    This primer on elementary partial differential equations presents the standard material usually covered in a one-semester, undergraduate course on boundary value problems and PDEs. What makes this book unique is that it is a brief treatment, yet it covers all the major ideas: the wave equation, the diffusion equation, the Laplace equation, and the advection equation on bounded and unbounded domains. Methods include eigenfunction expansions, integral transforms, and characteristics. Mathematical ideas are motivated from physical problems, and the exposition is presented in a concise style accessible to science and engineering students; emphasis is on motivation, concepts, methods, and interpretation, rather than formal theory. This second edition contains new and additional exercises, and it includes a new chapter on the applications of PDEs to biology: age structured models, pattern formation; epidemic wave fronts, and advection-diffusion processes. The student who reads through this book and solves many of t...

  12. Fundamental partial compositeness

    Energy Technology Data Exchange (ETDEWEB)

    Sannino, Francesco [CP-Origins and Danish IAS, University of Southern Denmark,Campusvej 55 (Denmark); Strumia, Alessandro [Dipartimento di Fisica dell’Università di Pisa and INFN,Pisa (Italy); Theory Division, CERN,Geneva (Switzerland); Tesi, Andrea [Department of Physics, Enrico Fermi Institute, University of Chicago,Chicago, IL 60637 (United States); Vigiani, Elena [Dipartimento di Fisica dell’Università di Pisa and INFN,Pisa (Italy)

    2016-11-07

    We construct renormalizable Standard Model extensions, valid up to the Planck scale, that give a composite Higgs from a new fundamental strong force acting on fermions and scalars. Yukawa interactions of these particles with Standard Model fermions realize the partial compositeness scenario. Under certain assumptions on the dynamics of the scalars, successful models exist because gauge quantum numbers of Standard Model fermions admit a minimal enough ‘square root’. Furthermore, right-handed SM fermions have an SU(2){sub R}-like structure, yielding a custodially-protected composite Higgs. Baryon and lepton numbers arise accidentally. Standard Model fermions acquire mass at tree level, while the Higgs potential and flavor violations are generated by quantum corrections. We further discuss accidental symmetries and other dynamical features stemming from the new strongly interacting scalars. If the same phenomenology can be obtained from models without our elementary scalars, they would reappear as composite states.

  13. Elliptic partial differential equations

    CERN Document Server

    Volpert, Vitaly

    If we had to formulate in one sentence what this book is about it might be "How partial differential equations can help to understand heat explosion, tumor growth or evolution of biological species". These and many other applications are described by reaction-diffusion equations. The theory of reaction-diffusion equations appeared in the first half of the last century. In the present time, it is widely used in population dynamics, chemical physics, biomedical modelling. The purpose of this book is to present the mathematical theory of reaction-diffusion equations in the context of their numerous applications. We will go from the general mathematical theory to specific equations and then to their applications. Mathematical anaylsis of reaction-diffusion equations will be based on the theory of Fredholm operators presented in the first volume. Existence, stability and bifurcations of solutions will be studied for bounded domains and in the case of travelling waves. The classical theory of reaction-diffusion equ...

  14. Partial pressure gauges

    CERN Document Server

    Peter, G J

    2007-01-01

    The determination of partial pressures in vacuum systems is usually performed by mass spectrometers. The most common type is the QMS. Quadrupole mass spectrometers were developed decades ago and have been used by vacuum specialists as a diagnostic tool since then. In the first part of the paper the principles of these mass spectrometers are briefly reviewed together with the key features of the instruments. This is necessary to operate these instruments. In the second part the boundary conditions which arise from the application as residual gas analyser in UHV/XHV plants are described. These lead to special versions of mass spectrometers. Results obtained with these instruments and typical artefacts in mass spectra obtained in the UHV are discussed.

  15. Prediction of essential proteins based on subcellular localization and gene expression correlation.

    Science.gov (United States)

    Fan, Yetian; Tang, Xiwei; Hu, Xiaohua; Wu, Wei; Ping, Qing

    2017-12-01

    Essential proteins are indispensable to the survival and development process of living organisms. To understand the functional mechanisms of essential proteins, which can be applied to the analysis of disease and design of drugs, it is important to identify essential proteins from a set of proteins first. As traditional experimental methods designed to test out essential proteins are usually expensive and laborious, computational methods, which utilize biological and topological features of proteins, have attracted more attention in recent years. Protein-protein interaction networks, together with other biological data, have been explored to improve the performance of essential protein prediction. The proposed method SCP is evaluated on Saccharomyces cerevisiae datasets and compared with five other methods. The results show that our method SCP outperforms the other five methods in terms of accuracy of essential protein prediction. In this paper, we propose a novel algorithm named SCP, which combines the ranking by a modified PageRank algorithm based on subcellular compartments information, with the ranking by Pearson correlation coefficient (PCC) calculated from gene expression data. Experiments show that subcellular localization information is promising in boosting essential protein prediction.

  16. An improved procedure for subcellular spatial alignment during live-cell CLEM.

    Directory of Open Access Journals (Sweden)

    Benjamin S Padman

    Full Text Available Live-cell correlative light and electron microscopy (CLEM offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope.

  17. The Salmonella effector SteA binds phosphatidylinositol 4-phosphate for subcellular targeting within host cells.

    Science.gov (United States)

    Domingues, Lia; Ismail, Ahmad; Charro, Nuno; Rodríguez-Escudero, Isabel; Holden, David W; Molina, María; Cid, Víctor J; Mota, Luís Jaime

    2016-07-01

    Many bacterial pathogens use specialized secretion systems to deliver virulence effector proteins into eukaryotic host cells. The function of these effectors depends on their localization within infected cells, but the mechanisms determining subcellular targeting of each effector are mostly elusive. Here, we show that the Salmonella type III secretion effector SteA binds specifically to phosphatidylinositol 4-phosphate [PI(4)P]. Ectopically expressed SteA localized at the plasma membrane (PM) of eukaryotic cells. However, SteA was displaced from the PM of Saccharomyces cerevisiae in mutants unable to synthesize the local pool of PI(4)P and from the PM of HeLa cells after localized depletion of PI(4)P. Moreover, in infected cells, bacterially translocated or ectopically expressed SteA localized at the membrane of the Salmonella-containing vacuole (SCV) and to Salmonella-induced tubules; using the PI(4)P-binding domain of the Legionella type IV secretion effector SidC as probe, we found PI(4)P at the SCV membrane and associated tubules throughout Salmonella infection of HeLa cells. Both binding of SteA to PI(4)P and the subcellular localization of ectopically expressed or bacterially translocated SteA were dependent on a lysine residue near the N-terminus of the protein. Overall, this indicates that binding of SteA to PI(4)P is necessary for its localization within host cells. © 2015 John Wiley & Sons Ltd.

  18. Subcellular targeting and dynamic regulation of PTEN: Implications for neuronal cells and neurological disorders

    Directory of Open Access Journals (Sweden)

    Patricia eKreis

    2014-04-01

    Full Text Available PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum, the mitochondria or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.

  19. Improving drug potency and efficacy by nanocarrier-mediated subcellular targeting.

    Science.gov (United States)

    Murakami, Mami; Cabral, Horacio; Matsumoto, Yu; Wu, Shourong; Kano, Mitsunobu R; Yamori, Takao; Nishiyama, Nobuhiro; Kataoka, Kazunori

    2011-01-05

    Nanocarrier-mediated drug targeting is an emerging strategy for cancer therapy and is being used, for example, with chemotherapeutic agents for ovarian cancer. Nanocarriers are selectively accumulated in tumors as a result of their enhanced permeability and retention of macromolecules, thereby enhancing the antitumor activity of the nanocarrier-associated drugs. We investigated the real-time subcellular fate of polymeric micelles incorporating (1,2-diaminocyclohexane) platinum(II) (DACHPt/m), the parent complex of oxaliplatin, in tumor tissues by fluorescence-based assessment of their kinetic stability. These observations revealed that DACHPt/m was extravasated from blood vessels to the tumor tissue and dissociated inside each cell. Furthermore, DACHPt/m selectively dissociated within late endosomes, enhancing drug delivery to the nearby nucleus relative to free oxaliplatin, likely by circumvention of the cytoplasmic detoxification systems such as metallothionein and methionine synthase. Thus, these drug-loaded micelles exhibited higher antitumor activity than did oxaliplatin alone, even against oxaliplatin-resistant tumors. These findings suggest that nanocarriers targeting subcellular compartments may have considerable benefits in clinical applications.

  20. Engineering an effective Mn-binding MRI reporter protein by subcellular targeting.

    Science.gov (United States)

    Bartelle, Benjamin B; Mana, Miyeko D; Suero-Abreu, Giselle A; Rodriguez, Joe J; Turnbull, Daniel H

    2015-12-01

    Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely used for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as a genetic reporter for MEMRI. The bacterial Mn-binding protein, MntR was identified as a candidate reporter protein. MntR was engineered for expression in mammalian cells, and targeted to different subcellular organelles, including the Golgi Apparatus where cellular Mn is enriched. Transfected HEK293 cells and B16 melanoma cells were tested in vitro and in vivo, using immunocytochemistry, MR imaging and relaxometry. Subcellular targeting of MntR to the cytosol, endoplasmic reticulum and Golgi apparatus was verified with immunocytochemistry. After targeting to the Golgi, MntR expression produced robust R1 changes and T1 contrast in cells, in vitro and in vivo. Co-expression with the divalent metal transporter DMT1, a previously described Mn-based reporter, further enhanced contrast in B16 cells in culture, but in the in vivo B16 tumor model tested was not significantly better than MntR alone. This second-generation reporter system both expands the capabilities of genetically encoded reporters for imaging with MEMRI and provides important insights into the mechanisms of Mn biology which create endogenous MEMRI contrast. © 2014 Wiley Periodicals, Inc.

  1. rAAV-mediated subcellular targeting of optogenetic tools in retinal ganglion cells in vivo.

    Science.gov (United States)

    Wu, Chaowen; Ivanova, Elena; Zhang, Yi; Pan, Zhuo-Hua

    2013-01-01

    Expression of optogenetic tools in surviving inner retinal neurons to impart retinal light sensitivity has been a new strategy for restoring vision after photoreceptor degeneration. One potential approach for restoring retinal light sensitivity after photoreceptor degeneration is to express optogenetic tools in retinal ganglion cells (RGCs). For this approach, restoration of ON and OFF center-surround receptive fields in RGCs, a key feature of visual information processing, may be important. A possible solution is to differentially express depolarizing and hyperpolarizing optogenetic tools, such as channelrhodopsin-2 and halorhodopsin, to the center and peripheral regions of the RGC dendritic field by using protein targeting motifs. Recombinant adeno-associated virus (rAAV) vectors have proven to be a powerful vehicle for in vitro and in vivo gene delivery, including in the retina. Therefore, the search for protein targeting motifs that can achieve rAAV-mediated subcellular targeted expression would be particularly valuable for developing therapeutic applications. In this study, we identified two protein motifs that are suitable for rAAV-mediated subcellular targeting for generating center-surround receptive fields while reducing the axonal expression in RGCs. Resulting morphological dendritic field and physiological response field by center-targeting were significantly smaller than those produced by surround-targeting. rAAV motif-mediated protein targeting could also be a valuable tool for studying physiological function and clinical applications in other areas of the central nervous system.

  2. Subcellular targeting and dynamic regulation of PTEN: implications for neuronal cells and neurological disorders.

    Science.gov (United States)

    Kreis, Patricia; Leondaritis, George; Lieberam, Ivo; Eickholt, Britta J

    2014-01-01

    PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5)P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum (ER), the mitochondria, or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions, or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i) recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii) current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.

  3. Characterization of Subcellular Responses Induced by Exposure of Microbubbles to Astrocytes.

    Science.gov (United States)

    Sun, Shan; Kanagaraj, Johnwesly; Cho, Lindsey; Kang, Dongkoo; Xiao, Shu; Cho, Michael

    2015-10-01

    Blast traumatic brain injury (bTBI) has now been identified to associate with adverse health consequences among combat veterans. Post-traumatic stress disorder linked with explosive blasts, for example, may result from such brain injury. The fundamental questions about the nature, diagnosis, and long-term consequences of bTBI and causative relationship to post-traumatic stress disorder remain elusive, however. A better understanding of brain tissue injury requires elucidation of potential mechanisms. One such mechanism may be generation of microcavitation bubbles in the brain after an explosive blast and their subsequent interaction with brain cells. Using a controlled electrical discharge system, we have successfully generated shock waves (∼10 MPa) and microbubbles (20-30 μm) in the cell culture of mouse astrocytes. Detachment of astrocytes from the substrate after exposure to microbubbles was observed, and it depended on repetitive exposures. Of the cells that survived the initial assault, several subcellular changes were monitored and determined using fluorescent microscopy, including cell viability, cytoskeletal reorganization, changes in focal adhesion, membrane permeability, and potential onset of apoptosis. While the astrocytes impacted by the shock wave only demonstrated essentially unaltered cellular behavior, the astrocytes exposed to microbubbles exhibited significantly different responses, including production of reactive oxygen species by collapse of microbubbles. In the present study, we characterized and report for the first time the altered biophysical and subcellular properties in astrocytes in response to exposure to the combination of shock waves and microbubbles.

  4. Subcellular localization of leptin and leptin receptor in breast cancer detected in an electron microscopic study.

    Science.gov (United States)

    Al-Shibli, Saad M; Amjad, Nasser M; Al-Kubaisi, Muna K; Mizan, Shaikh

    2017-01-22

    Leptin (LEP) and leptin receptor (LEPR) have long been found associated with breast cancer. So far no high-resolution method such as electron microscopy has been used to investigate the subcellular localization of leptin and leptin receptor in breast cancer. We collected cancer and non-cancer breast tissues from 51 women with invasive ductal breast cancer. Leptin and leptin receptor in the tissues were estimated using immunohistochemistry (IHC). LEP and LEPR were localized at subcellular level by immunocytochemistry (ICC) using ultra-fine gold particle conjugated antibody, and visualized with transmission electron microscopy (TEM). IHC showed high presence of LEP and LEPR in 65% and 67% respectively of the breast cancer samples, 100% and 0% respectively of the adipose tissue samples, and no high presence in the non-cancer breast tissue samples. On TEM views both LEP and LEPR were found highly concentrated within the nucleus of the cancer cells, indicating that nucleus is the principal seat of action. However, presence of high concentration of LEP does not necessarily prove its over-expression, as often concluded, because LEP could be internalized from outside by LEPR in the cells. In contrast, LEPR is definitely over-expressed in the ductal breast cancer cells. Therefore, we hypothesize that over-expression of LEPR, rather than that of LEP has a fundamental role in breast carcinogenesis in particular, and probably for LEP-LEPR associated tumors in general. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. STUDY OF SUBCELLULAR DISTRIBUTION OF CRYSTALLINE MESO-TETRA(3-PYRIDYLBACTERIOCHLORIN NANOPARTICLES

    Directory of Open Access Journals (Sweden)

    Yu. S. Maklygina

    2016-01-01

    Full Text Available The results of the study of subcellular distribution of molecular meso-tetra(3-pyridylbacteriochlorin nanocrystals proposed as therapeutic agents for photodynamic therapy are represented in the article. Investigations and measurement of spectroscopic properties of molecular crystals of near-infrared photosensitizer were conducted using special device complex based on fiber-optic spectrometer. Investigation and analysis of the pattern of subcellular accumulation of meso-tetra(3-pyridylbacteriochlorin in molecular (dimethyl sulfoxide (DMSO as solvent and nanocrystalline forms on different cell lines: human monocytes (THP-1, human cervical cancer cells (HeLa and mouse malignant brain tumor cells (glioma C6. The dynamics of subcellylar accumulation of the agent at concentration of 5 and 10 mg/l was assessed with laser microscope-spectrum analyzer and by confocal microscopy. The study showed that in the course of interaction with cell lines molecular nanocrystals of the agent developed ability to fluorescence. Hence, in the cellular environment meso-tetra(3-pyridyl bacteriochlorin nanoparticles became phototoxic giving opportunities for their use for fluorescence diagnosis and photodynamic therapy. Specific role of meso-tetra(3-pyridylbacteriochlorin in the range of photosensitizers is determined by its spectral characteristics, i.e. absorption and fluorescence in near-infrared band, which allows measuring and affecting on deeper layers of biotissue. Thus, the use of meso-tetra(3-pyridylbacteriochlorin nanoparticles as nanophotosensitizers may improve the efficacy of diagnosis and treatment of deep-seated tumors.

  6. Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis

    Directory of Open Access Journals (Sweden)

    Cheevadhanarak Supapon

    2009-09-01

    Full Text Available Abstract The present study examined the changes in protein expression in Spirulina platensis upon exposure to high temperature, with the changes in expression analyzed at the subcellular level. In addition, the transcriptional expression level of some differentially expressed proteins, the expression pattern clustering, and the protein-protein interaction network were analyzed. The results obtained from differential expression analysis revealed up-regulation of proteins involved in two-component response systems, DNA damage and repair systems, molecular chaperones, known stress-related proteins, and proteins involved in other biological processes, such as capsule formation and unsaturated fatty acid biosynthesis. The clustering of all differentially expressed proteins in the three cellular compartments showed: (i the majority of the proteins in all fractions were sustained tolerance proteins, suggesting the roles of these proteins in the tolerance to high temperature stress, (ii the level of resistance proteins in the photosynthetic membrane was 2-fold higher than the level in two other fractions, correlating with the rapid inactivation of the photosynthetic system in response to high temperature. Subcellular communication among the three cellular compartments via protein-protein interactions was clearly shown by the PPI network analysis. Furthermore, this analysis also showed a connection between temperature stress and nitrogen and ammonia assimilation.

  7. An Improved Procedure for Subcellular Spatial Alignment during Live-Cell CLEM

    Science.gov (United States)

    Padman, Benjamin S.; Bach, Markus; Ramm, Georg

    2014-01-01

    Live-cell correlative light and electron microscopy (CLEM) offers unique insights into the ultrastructure of dynamic cellular processes. A critical and technically challenging part of CLEM is the 3-dimensional relocation of the intracellular region of interest during sample processing. We have developed a simple CLEM procedure that uses toner particles from a laser printer as orientation marks. This facilitates easy tracking of a region of interest even by eye throughout the whole procedure. Combined with subcellular fluorescence markers for the plasma membrane and nucleus, the toner particles allow for precise subcellular spatial alignment of the optical and electron microscopy data sets. The toner-based reference grid is printed and transferred onto a polymer film using a standard office printer and laminator. We have also designed a polymer film holder that is compatible with most inverted microscopes, and have validated our strategy by following the ultrastructure of mitochondria that were selectively photo-irradiated during live-cell microscopy. In summary, our inexpensive and robust CLEM procedure simplifies optical imaging, without limiting the choice of optical microscope. PMID:24755651

  8. Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization

    DEFF Research Database (Denmark)

    Petersen, B O; Lukas, J; Sørensen, Claus Storgaard

    1999-01-01

    Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated...... by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo...... phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear...

  9. Subcellular Localization of HIV-1 gag-pol mRNAs Regulates Sites of Virion Assembly.

    Science.gov (United States)

    Becker, Jordan T; Sherer, Nathan M

    2017-03-15

    Full-length unspliced human immunodeficiency virus type 1 (HIV-1) RNAs serve dual roles in the cytoplasm as mRNAs encoding the Gag and Gag-Pol capsid proteins as well as genomic RNAs (gRNAs) packaged by Gag into virions undergoing assembly at the plasma membrane (PM). Because Gag is sufficient to drive the assembly of virus-like particles even in the absence of gRNA binding, whether viral RNA trafficking plays an active role in the native assembly pathway is unknown. In this study, we tested the effects of modulating the cytoplasmic abundance or distribution of full-length viral RNAs on Gag trafficking and assembly in the context of single cells. Increasing full-length viral RNA abundance or distribution had little-to-no net effect on Gag assembly competency when provided in trans In contrast, artificially tethering full-length viral RNAs or surrogate gag-pol mRNAs competent for Gag synthesis to non-PM membranes or the actin cytoskeleton severely reduced net virus particle production. These effects were explained, in large part, by RNA-directed changes to Gag's distribution in the cytoplasm, yielding aberrant subcellular sites of virion assembly. Interestingly, RNA-dependent disruption of Gag trafficking required either of two cis-acting RNA regulatory elements: the 5' packaging signal (Psi) bound by Gag during genome encapsidation or, unexpectedly, the Rev response element (RRE), which regulates the nuclear export of gRNAs and other intron-retaining viral RNAs. Taken together, these data support a model for native infection wherein structural features of the gag-pol mRNA actively compartmentalize Gag to preferred sites within the cytoplasm and/or PM.IMPORTANCE The spatial distribution of viral mRNAs within the cytoplasm can be a crucial determinant of efficient translation and successful virion production. Here we provide direct evidence that mRNA subcellular trafficking plays an important role in regulating the assembly of human immunodeficiency virus type 1 (HIV

  10. Robot-assisted partial nephrectomy: Superiority over laparoscopic partial nephrectomy.

    Science.gov (United States)

    Shiroki, Ryoichi; Fukami, Naohiko; Fukaya, Kosuke; Kusaka, Mamoru; Natsume, Takahiro; Ichihara, Takashi; Toyama, Hiroshi

    2016-02-01

    Nephron-sparing surgery has been proven to positively impact the postoperative quality of life for the treatment of small renal tumors, possibly leading to functional improvements. Laparoscopic partial nephrectomy is still one of the most demanding procedures in urological surgery. Laparoscopic partial nephrectomy sometimes results in extended warm ischemic time and severe complications, such as open conversion, postoperative hemorrhage and urine leakage. Robot-assisted partial nephrectomy exploits the advantages offered by the da Vinci Surgical System to laparoscopic partial nephrectomy, equipped with 3-D vision and a better degree in the freedom of surgical instruments. The introduction of the da Vinci Surgical System made nephron-sparing surgery, specifically robot-assisted partial nephrectomy, safe with promising results, leading to the shortening of warm ischemic time and a reduction in perioperative complications. Even for complex and challenging tumors, robotic assistance is expected to provide the benefit of minimally-invasive surgery with safe and satisfactory renal function. Warm ischemic time is the modifiable factor during robot-assisted partial nephrectomy to affect postoperative kidney function. We analyzed the predictive factors for extended warm ischemic time from our robot-assisted partial nephrectomy series. The surface area of the tumor attached to the kidney parenchyma was shown to significantly affect the extended warm ischemic time during robot-assisted partial nephrectomy. In cases with tumor-attached surface area more than 15 cm(2) , we should consider switching robot-assisted partial nephrectomy to open partial nephrectomy under cold ischemia if it is imperative. In Japan, a nationwide prospective study has been carried out to show the superiority of robot-assisted partial nephrectomy to laparoscopic partial nephrectomy in improving warm ischemic time and complications. By facilitating robotic technology, robot-assisted partial nephrectomy

  11. Location of Planet X

    Energy Technology Data Exchange (ETDEWEB)

    Harrington, R.S.

    1988-10-01

    Observed positions of Uranus and Neptune along with residuals in right ascension and declination are used to constrain the location of a postulated tenth planet. The residuals are converted into residuals in ecliptic longitude and latitude. The results are then combined into seasonal normal points, producing average geocentric residuals spaced slightly more than a year apart that are assumed to represent the equivalent heliocentric average residuals for the observed oppositions. Such a planet is found to most likely reside in the region of Scorpius, with considerably less likelihood that it is in Taurus. 8 references.

  12. Locating Nordic Noir

    DEFF Research Database (Denmark)

    Hansen, Kim Toft; Waade, Anne Marit

    such as Beck, The Killing, Trapped and The Bridge as well as a range of other important Nordic Noir cases. The book positions the development of Nordic Noir on the global market for popular television drama and places the international attention towards Nordic crime dramas within regional development of drama......This book is a comprehensive study of Nordic Noir television drama from the 1990’s until today. The authors introduce the history of contemporary Nordic Noir from the perspective of place, production and location studies. The chapters include readings of well-known television crime dramas...

  13. The LHCb Vertex Locator

    CERN Document Server

    Eklund, L

    2004-01-01

    LHCb is an experiment at the Large Hadron Collider (LHC), dedicated to the study of b-hadron physics. The Vertex Locator (VELO) is a sub-detector which reconstructs primary and secondary vertices, which is of importance for the processes under study. It is a silicon micro-strip detector operated in a harsh radiation environment with demands of high read-out speed and the use of minimal material. In this paper, the LHCb VELO is introduced and the technology choices of the detector are motivated. We report on the present status of the project and one possible upgrade technology is presented.

  14. Locating Nordic Noir

    DEFF Research Database (Denmark)

    Hansen, Kim Toft; Waade, Anne Marit

    This book is a comprehensive study of Nordic Noir television drama from the 1990’s until today. The authors introduce the history of contemporary Nordic Noir from the perspective of place, production and location studies. The chapters include readings of well-known television crime dramas...... production in Sweden, Denmark, Norway and Iceland. Consequently, Nordic Noir is read as both a transnational financial and creative phenomenon and as a local possibility for community building. The book is aimed at readers with an interest in crime drama as well as television scholars and students...

  15. Competitive facility location models

    Science.gov (United States)

    Kononov, A. V.; Kochetov, Yu. A.; Plyasunov, A. V.

    2009-06-01

    Two classes of competitive facility location models are considered, in which several persons (players) sequentially or simultaneously open facilities for serving clients. The first class consists of discrete two-level programming models. The second class consists of game models with several independent players pursuing selfish goals. For the first class, its relationship with pseudo-Boolean functions is established and a novel method for constructing a family of upper and lower bounds on the optimum is proposed. For the second class, the tight PLS-completeness of the problem of finding Nash equilibriums is proved.

  16. Flexible thermoplastic denture base materials for aesthetical removable partial denture framework.

    Science.gov (United States)

    Singh, Kunwarjeet; Aeran, Himanshu; Kumar, Narender; Gupta, Nidhi

    2013-10-01

    Conventional fixed partial dentures, implant supported Fixed Partial Dentures (FDPs) and removable partial dentures are the most common treatment modalities for the aesthetic and functional rehabilitation of partially edentulous patients. Although implants and FDP have certain advantages over removable partial dentures, in some cases, removable partial dentures may be the only choice which is available. Removable cast partial dentures are used as definitive removable prostheses when indicated, but location of clasps may affect aesthetics. So, when patient is concerned about aesthetics, flexible partial dentures which is aesthetically superior to flipper and cast partial dentures, may be considered. But for the success of flexible removable partial denture, proper diagnosis, treatment planning and insertion technique of this prosthesis is very important, which have been thoroughly described in this article.

  17. Partially supervised speaker clustering.

    Science.gov (United States)

    Tang, Hao; Chu, Stephen Mingyu; Hasegawa-Johnson, Mark; Huang, Thomas S

    2012-05-01

    Content-based multimedia indexing, retrieval, and processing as well as multimedia databases demand the structuring of the media content (image, audio, video, text, etc.), one significant goal being to associate the identity of the content to the individual segments of the signals. In this paper, we specifically address the problem of speaker clustering, the task of assigning every speech utterance in an audio stream to its speaker. We offer a complete treatment to the idea of partially supervised speaker clustering, which refers to the use of our prior knowledge of speakers in general to assist the unsupervised speaker clustering process. By means of an independent training data set, we encode the prior knowledge at the various stages of the speaker clustering pipeline via 1) learning a speaker-discriminative acoustic feature transformation, 2) learning a universal speaker prior model, and 3) learning a discriminative speaker subspace, or equivalently, a speaker-discriminative distance metric. We study the directional scattering property of the Gaussian mixture model (GMM) mean supervector representation of utterances in the high-dimensional space, and advocate exploiting this property by using the cosine distance metric instead of the euclidean distance metric for speaker clustering in the GMM mean supervector space. We propose to perform discriminant analysis based on the cosine distance metric, which leads to a novel distance metric learning algorithm—linear spherical discriminant analysis (LSDA). We show that the proposed LSDA formulation can be systematically solved within the elegant graph embedding general dimensionality reduction framework. Our speaker clustering experiments on the GALE database clearly indicate that 1) our speaker clustering methods based on the GMM mean supervector representation and vector-based distance metrics outperform traditional speaker clustering methods based on the “bag of acoustic features” representation and statistical

  18. Indoor Location Technologies

    CERN Document Server

    Goswami, Subrata

    2013-01-01

    Focusing on the special challenges posed by accurately pinpointing a location indoors, this volume reflects the distance we have come in the handful of decades since the germination of GPS technology. Not only can we locate a signal to within a meter’s accuracy, but we now have this technology in the most basic mobile phone. Tracing recent practical developments in positioning technology and in the market it supplies, the author examines the contributions of the varied research—in silicon, signal and image processing, radio communications and software—to a fast-evolving field. The book looks forward to a time when, in addition to directing your road journey, positioning systems can peer indoors and guide you to an available photocopier in your office building. Featuring standalone chapters each dealing with a specific aspect of the subject, including treatments of systems such as Zebra, Awarepoint, Aeroscout, IEEE 802.11, etc. This study has all the detail needed to get up to speed on a key modern techn...

  19. [Post traumatic partial seizures].

    Science.gov (United States)

    Carvajal, P; Almárcegui, C; Pablo, M J; Peralta, P; Bernal, M; Valdizán, J R

    Post traumatic epilepsy represents 4% of the prevalence of the disorder and is one of the sequelas which is most difficult to prevent. Risk factors have been described to predict the appearance of seizures. A seven year old boy with a severe head injury was admitted to the Intensive Care Unit. On neuroimaging studies there were multiple foci of contusion, mainly in the left hemisphere, and blood in the III and IV ventricles and frontal horn of the left lateral ventricle. The patient had severe sequelae of head injury with a right spastic hemiplegia and hemiparesia with hypertony of the left side, together with complete blindness of both eyes due to bilateral atrophy of the optic nerve. Serial EEG were done, in which a recording showed alternating periods of hypervoltage grapho elements superimposed on a trace of very low voltage, with continuous activity of low voltage and low frequency. There were no grapho elements with acute morphology. However, the patient had a first partial seizure a year and a half after his head injury. On the EEG an epileptogenic focus was identified in the left hemisphere. Within two years of his head injury he had seven seizures. He had not received prophylactic antiepileptic treatment after the head injury. We report a case of epilepsy secondary to a head injury, in which the first seizure occurred one and a half years after injury. In view of the risk factors, we discuss whether prophylactic anti epileptic treatment might have been beneficial.

  20. Absorption Kinetics and Subcellular Fractionation of Zinc in Winter Wheat in Response to Nitrogen Supply

    Directory of Open Access Journals (Sweden)

    Zhaojun Nie

    2017-08-01

    Full Text Available Nitrogen (N is critical for zinc (Zn absorption into plant roots; this in turn allows for Zn accumulation and biofortification of grain in winter wheat (Triticum aestivum L., an important food crop. However, little is known about root morphology and subcellular Zn distribution in response to N treatment at different levels of Zn supply. In this study, two nutrient solution culture experiments were conducted to examine Zn accumulation, Zn absorption kinetics, root morphology, and Zn subcellular distribution in wheat seedlings pre-cultured with different N concentrations. The results showed positive correlations between N and Zn concentrations, and N and Zn accumulation, respectively. The findings suggested that an increase in N supply enhanced root absorption and the root-to-shoot transport of Zn. Nitrogen combined with the high Zn (Zn10 treatment increased the Zn concentration and consequently its accumulation in both shoots and roots. The maximum influx rate (Vmax, root length, surface area, and volume of 14-d-old seedlings, and root growth from 7 to 14 d in the medium N (N7.5 treatment were higher, but the Michaelis constant (Km and minimum equilibrium concentrations (Cmin in this treatment were lower than those in the low (N0.05 and high (N15 N treatments, when Zn was supplied at a high level (Zn10. Meanwhile, there were no pronounced differences in the above root traits between the N0.05Zn0 and N7.5Zn10 treatments. An increase in N supply decreased Zn in cell walls and cell organelles, while it increased Zn in the root soluble fraction. In leaves, an increase in N supply significantly decreased Zn in cell walls and the soluble fraction, while it increased Zn in cell organelles under Zn deficiency, but increased Zn distribution in the soluble fraction under medium and high Zn treatments. Therefore, a combination of medium N and high Zn treatments enhanced Zn absorption, apparently by enhancing Zn membrane transport and stimulating root

  1. Metabolism of the environmental toxicant benzo(a)pyrene by subcellular fractions of human ovary

    Science.gov (United States)

    Rekhadevi, PV; Diggs, DL; Huderson, AC; Harris, KL; Archibong, AE; Ramesh, A

    2014-01-01

    Knowledge of the ability of the female reproductive system to metabolize environmental chemicals is critical not only from the standpoint of toxicity but also from infertility risk assessment. Benzo(a)pyrene (BaP) is a toxicant that is released into the environment from automobile exhausts, cigarette smoke, burning of refuse, industrial emissions, and hazardous waste sites. In exposed animals, BaP becomes activated to reactive meta-bolites that interfere with target organ function and as a consequence cause toxicity. Studies on animal models conducted in our laboratories and those of others have shown that BaP possess endocrine disrupting properties. Thus, this chemical has the potential to cause infertility and cancers in the female genital tract. An understanding of BaP metabolism in the female reproductive system will be of importance in the diagnosis and management of female fertility as well as cancers in the reproductive tissues. Therefore, the objective of our study was to examine the metabolism of BaP by human ovarian subcellular fractions. Human ovary samples (eight individuals) were obtained from postoperative tissue removed from subjects with uterine tumors. Sub-cellular fractions (nuclear, cytosolic, mitochondrial, and microsomal) were prepared by differential centrifugation. BaP (1 μM and 3 μM) was individually incubated with individual subcellular fractions for 15 min and the products were analyzed by high-performance liquid chromatography. Among the different fractions tested, microsomal BaP metabolism was higher than the rest of the fractions. The BaP metabolites identified were as follows: BaP-9,10-diol, BaP-4,5-diol, BaP-7,8-diol, 9(OH) BaP, 3(OH) BaP, BaP-1,6-dione, BaP-3,6-dione, and BaP-6,12-dione. Of interest was the presence of DNA-reactive metabolites such as BaP-3,6-dione, BaP-6,12-dione, and BaP 7,8-diol, which have been implicated in the causation of infertility and cancer. Our results indicate that women who are exposed to BaP via

  2. Locating Nordic Noir

    DEFF Research Database (Denmark)

    Hansen, Kim Toft; Waade, Anne Marit

    This book is a comprehensive study of Nordic Noir television drama from the 1990’s until today. The authors introduce the history of contemporary Nordic Noir from the perspective of place, production and location studies. The chapters include readings of well-known television crime dramas...... such as Beck, The Killing, Trapped and The Bridge as well as a range of other important Nordic Noir cases. The book positions the development of Nordic Noir on the global market for popular television drama and places the international attention towards Nordic crime dramas within regional development of drama...... production in Sweden, Denmark, Norway and Iceland. Consequently, Nordic Noir is read as both a transnational financial and creative phenomenon and as a local possibility for community building. The book is aimed at readers with an interest in crime drama as well as television scholars and students...

  3. Locating Nordic Noir

    DEFF Research Database (Denmark)

    Hansen, Kim Toft; Waade, Anne Marit

    This book is a comprehensive study of Nordic Noir television drama from the 1990’s until today. The authors introduce the history of contemporary Nordic Noir from the perspective of place, production and location studies. The chapters include readings of well-known television crime dramas...... production in Sweden, Denmark, Norway and Iceland. Consequently, Nordic Noir is read as both a transnational financial and creative phenomenon and as a local possibility for community building. The book is aimed at readers with an interest in crime drama as well as television scholars and students...... of television drama, and it offers a comprehensible, scholarly and methodologically original approach to the popularity of Nordic television crime dramas....

  4. Demographics and remote locations

    Energy Technology Data Exchange (ETDEWEB)

    Coppus, G. [Dynawise Inc., Calgary, AB (Canada)

    2005-07-01

    Within the next decade, the number of people leaving the workforce will exceed the number of new entrants. The demand for workers is expected to increase in Alberta due to oil sands industry growth. Sixty-four per cent of all responding federal, provincial, and municipal government organizations have reported shortages in their work environments, and almost 8 in 10 organizations predict they will continue to be understaffed in the next 3 to 5 years. Tightness in the general labour market will have an impact on employers in remote locations, and planning is essential to avoid the creation of a mercenary culture and a reliance on compensation. Challenges found in remote locations include high turnover rates among recent hires; critical gaps in service length; aggressive retirement; and the fact that the local population is often seen as unqualified for many industrial jobs. This Power Point presentation suggested that although decisions to join or quit a company are often based on considerations such as the organizational environment and amount of compensation, the relationship between employees and management is the most important attribute in many career decisions. Rigorous quantitative analysis of current demographics combined with forecasting analysis may help to forestall recruitment difficulties for many companies. Productivity analyses, and the effects of work overload on absenteeism, attrition and quality of workmanship must also be examined. Many companies are now focusing on retention and development strategies on mid-career staff to address depletion, as well as redesigning procedures to operate with less skilled staff. It was concluded that extra efforts are now being made to attract non-traditional employees, and non-traditional employment models are being considered by some companies. refs., tabs., figs.

  5. Four Novel NR5A1 Mutations in 46,XY Gonadal Dysgenesis Patients Including Frameshift Mutations with Altered Subcellular SF-1 Localization.

    Science.gov (United States)

    Rehkämper, Jan; Tewes, Ann-Christin; Horvath, Judit; Scherer, Gerd; Wieacker, Peter; Ledig, Susanne

    2017-12-01

    46,XY gonadal dysgenesis (46,XY GD) is a disorder of sexual development caused by mutations in genes involved in early gonadal development (bipotential gonads) and testis differentiation. In 46,XY GD individuals, mutations of the SRY gene are detected most frequently, followed by mutations in the NR5A1 (SF-1) gene, but in a lot of cases, the underlying molecular mechanism remains elusive. In this study, we retrospectively performed sequence analyses of the NR5A1 (SF-1) gene in 84 patients with complete, partial, and syndromic forms of 46,XY GD. In total, 7 heterozygous mutations were found in 6 of 84 patients (7.1%). Among these, we identified 4 mutations that, to the best of our knowledge, have not been reported before (c.268G>T, c.369del, c.871-1G>C, and c.893T>C). Transfection of different mutations revealed altered subcellular localization of the mutant SF-1 protein in the case of the frameshift mutations, indicating an impaired protein function. In conclusion, we present 4 novel mutations of the NR5A1 gene associated with 46,XY GD together with in vitro data pointing towards a possible functional impairment of the mutant SF-1 proteins. © 2017 S. Karger AG, Basel.

  6. Ultrasonic partial discharge monitoring method on instrument transformers

    Directory of Open Access Journals (Sweden)

    Kartalović Nenad

    2012-01-01

    Full Text Available Sonic and ultrasonic partial discharge monitoring have been applied since the early days of these phenomena monitoring. Modern measurement and partial discharge acoustic (ultrasonic and sonic monitoring method has been rapidly evolving as a result of new electronic component design, information technology and updated software solutions as well as the development of knowledge in the partial discharge diagnosis. Electrical discharges in the insulation system generate voltage-current pulses in the network and ultrasonic waves that propagate through the insulation system and structure. Amplitude-phase-frequency analysis of these signals reveals information about the intensity, type and location of partial discharges. The paper discusses the possibility of ultrasonic method selectivity improvement and the increase of diagnosis reliability in the field. Measurements were performed in the laboratory and in the field while a number of transformers were analysed for dissolved gases in the oil. A comparative review of methods for the partial discharge detection is also presented in this paper.

  7. Locating Minimal Fault Interaction in Combinatorial Testing

    Directory of Open Access Journals (Sweden)

    Wei Zheng

    2016-01-01

    Full Text Available Combinatorial testing (CT technique could significantly reduce testing cost and increase software system quality. By using the test suite generated by CT as input to conduct black-box testing towards a system, we are able to detect interactions that trigger the system’s faults. Given a test case, there may be only part of all its parameters relevant to the defects in system and the interaction constructed by those partial parameters is key factor of triggering fault. If we can locate those parameters accurately, this will facilitate the software diagnosing and testing process. This paper proposes a novel algorithm named complete Fault Interaction Location (comFIL to locate those interactions that cause system’s failures and meanwhile obtains the minimal set of target interactions in test suite produced by CT. By applying this method, testers can analyze and locate the factors relevant to defects of system more precisely, thus making the process of software testing and debugging easier and more efficient. The results of our empirical study indicate that comFIL performs better compared with known fault location techniques in combinatorial testing because of its improved effectiveness and precision.

  8. Laboratory Experimental System for Examination of Acoustic Emission Generated by Partial Discharges

    Directory of Open Access Journals (Sweden)

    I. M. Salom

    2013-11-01

    Full Text Available One of the major causes of transformer failures is dielectric breakdown. Partial discharges cause gradual insulation degradation thus partial discharge activity monitoring provides transformer state insight. This paper gives an overview of common methods for partial discharges detection and source location in transformers, with a special reference to the acoustic method as an noninvasive and interference resistant method suitable for application. For laboratory testing a laboratory experimental system for partial discharge diagnostics using acoustic emission measurement was developed.

  9. Experts’ understanding of partial derivatives using the partial derivative machine

    Directory of Open Access Journals (Sweden)

    David Roundy

    2015-09-01

    Full Text Available [This paper is part of the Focused Collection on Upper Division Physics Courses.] Partial derivatives are used in a variety of different ways within physics. Thermodynamics, in particular, uses partial derivatives in ways that students often find especially confusing. We are at the beginning of a study of the teaching of partial derivatives, with a goal of better aligning the teaching of multivariable calculus with the needs of students in STEM disciplines. In this paper, we report on an initial study of expert understanding of partial derivatives across three disciplines: physics, engineering, and mathematics. We report on the central research question of how disciplinary experts understand partial derivatives, and how their concept images of partial derivatives differ, with a focus on experimentally measured quantities. Using the partial derivative machine (PDM, we probed expert understanding of partial derivatives in an experimental context without a known functional form. In particular, we investigated which representations were cued by the experts’ interactions with the PDM. Whereas the physicists and engineers were quick to use measurements to find a numeric approximation for a derivative, the mathematicians repeatedly returned to speculation as to the functional form; although they were comfortable drawing qualitative conclusions about the system from measurements, they were reluctant to approximate the derivative through measurement. On a theoretical front, we found ways in which existing frameworks for the concept of derivative could be expanded to include numerical approximation.

  10. Triple subcellular targeting of isopentenyl diphosphate isomerases encoded by a single gene.

    Science.gov (United States)

    Guirimand, Grégory; Guihur, Anthony; Phillips, Michael A; Oudin, Audrey; Glévarec, Gaëlle; Mahroug, Samira; Melin, Céline; Papon, Nicolas; Clastre, Marc; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Rodríguez-Concepción, Manuel; Burlat, Vincent; Courdavault, Vincent

    2012-11-01

    Isopentenyl diphosphate isomerase (IDI) is a key enzyme of the isoprenoid pathway, catalyzing the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, the universal precursors of all isoprenoids. In plants, several subcellular compartments, including cytosol/ER, peroxisomes, mitochondria and plastids, are involved in isoprenoid biosynthesis. Here, we report on the unique triple targeting of two Catharanthus roseus IDI isoforms encoded by a single gene (CrIDI1). The triple localization of CrIDI1 in mitochondria, plastids and peroxisomes is explained by alternative transcription initiation of CrIDI1, by the specificity of a bifunctional N-terminal mitochondria/plastid transit peptide and by the presence of a C-terminal peroxisomal targeting signal. Moreover, bimolecular fluorescence complementation assays revealed self-interactions suggesting that the IDI likely acts as a multimer in vivo.

  11. In vivo imaging of specific drug-target binding at subcellular resolution

    Science.gov (United States)

    Dubach, J. M.; Vinegoni, C.; Mazitschek, R.; Fumene Feruglio, P.; Cameron, L. A.; Weissleder, R.

    2014-05-01

    The possibility of measuring binding of small-molecule drugs to desired targets in live cells could provide a better understanding of drug action. However, current approaches mostly yield static data, require lysis or rely on indirect assays and thus often provide an incomplete understanding of drug action. Here, we present a multiphoton fluorescence anisotropy microscopy live cell imaging technique to measure and map drug-target interaction in real time at subcellular resolution. This approach is generally applicable using any fluorescently labelled drug and enables high-resolution spatial and temporal mapping of bound and unbound drug distribution. To illustrate our approach we measure intracellular target engagement of the chemotherapeutic Olaparib, a poly(ADP-ribose) polymerase inhibitor, in live cells and within a tumour in vivo. These results are the first generalizable approach to directly measure drug-target binding in vivo and present a promising tool to enhance understanding of drug activity.

  12. Analytical model of ionization and energy deposition by proton beams in subcellular compartments

    Science.gov (United States)

    de Vera, Pablo; Surdutovich, Eugene; Abril, Isabel; Garcia-Molina, Rafael; Solov'yov, Andrey V.

    2014-04-01

    We present an analytical model to evaluate in a fast, simple and effective manner the energy delivered by proton beams moving through a cell model made of nucleus and cytoplasm, taking into account the energy carried by the secondary electrons generated along the proton tracks. The electronic excitation spectra of these subcellular compartments have been modelled by means of an empirical parameterization of their dielectric properties. The energy loss rate and target ionization probability induced by swift protons are evaluated by means of the dielectric formalism. With the present model we have quantified the energy delivered, the specific energy, and the number of ionizations produced per incoming ion in a typical human cell by a typical hadrontherapy proton beam having energies usually reached around the Bragg peak (below 20 MeV). We find that the specific energy per incoming ion delivered in the nucleus and in the cytoplasm are rather similar for all the proton energy range analyzed.

  13. Accurate Classification of Protein Subcellular Localization from High-Throughput Microscopy Images Using Deep Learning

    Directory of Open Access Journals (Sweden)

    Tanel Pärnamaa

    2017-05-01

    Full Text Available High-throughput microscopy of many single cells generates high-dimensional data that are far from straightforward to analyze. One important problem is automatically detecting the cellular compartment where a fluorescently-tagged protein resides, a task relatively simple for an experienced human, but difficult to automate on a computer. Here, we train an 11-layer neural network on data from mapping thousands of yeast proteins, achieving per cell localization classification accuracy of 91%, and per protein accuracy of 99% on held-out images. We confirm that low-level network features correspond to basic image characteristics, while deeper layers separate localization classes. Using this network as a feature calculator, we train standard classifiers that assign proteins to previously unseen compartments after observing only a small number of training examples. Our results are the most accurate subcellular localization classifications to date, and demonstrate the usefulness of deep learning for high-throughput microscopy.

  14. MECHANISMS OF DAMAGING EFFECT OF MANGENESE IN TOXIC CONCENTRATIONS ON CELLULAR AND SUBCELLULAR LEVELS

    Directory of Open Access Journals (Sweden)

    Goncharenko A. V.

    2012-11-01

    Full Text Available Influence of subtoxic concentration of manganese chloride in dose equal to LD 50 on condition of plasmatic membranes (model: erythrocytes and functional activity of cell power (model: the isolated liver mitochondrion of rats was studied. It was established that manganese chloride in fixed concentration caused authentic augmentation of sorption capacity of erythrocytes towards alcian blue, influenced increasing of their spontaneous haemolysis and activation of peroxide oxidation of lipids. In experiment on the isolated mitochondrion it was proved that manganese chloride caused dissociation of an oxidizing phosphorusling and complete inhibition of respiration in concentrations of 3 and 4,5mM. These dependences testify that subtoxic concentration of manganese can damage the cell energy. Thus, this pilot research indicated damaging effect of manganese on cellular (erythrocytes and subcellular (mitochondrion levels which are realized through external functioning of membrane structures and deprived them from restoration.

  15. Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds 1

    Science.gov (United States)

    Swain, Elisabeth; Li, Chun Ping; Poulton, Jonathan E.

    1992-01-01

    In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two β-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. Images Figure 5 Figure 1 Figure 2 Figure 3 Figure 4 Figure 6 PMID:16652960

  16. Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds.

    Science.gov (United States)

    Swain, E; Li, C P; Poulton, J E

    1992-09-01

    In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two beta-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.

  17. Lipidomics: analysis of the lipid composition of cells and subcellular organelles by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Brügger, Britta

    2014-01-01

    Lipidomics aims to quantitatively define lipid classes, including their molecular species, in biological systems. Lipidomics has experienced rapid progress, mainly because of continuous technical advances in instrumentation that are now enabling quantitative lipid analyses with an unprecedented level of sensitivity and precision. The still-growing category of lipids includes a broad diversity of chemical structures with a wide range of physicochemical properties. Reflecting this diversity, different methods and strategies are being applied to the quantification of lipids. Here, I review state-of-the-art electrospray ionization tandem mass spectrometric approaches and direct infusion to quantitatively assess lipid compositions of cells and subcellular fractions. Finally, I discuss a few examples of the power of mass spectrometry-based lipidomics in addressing cell biological questions.

  18. Phosphatidic acid phosphatase activity in subcellular fractions of normal and dystrophic human muscle.

    Science.gov (United States)

    Kunze, D; Rüstow, B; Olthoff, D; Jung, K

    1985-03-15

    Biopsy samples from normal and dystrophic human muscle (Duchenne type) were fractionated by differential centrifugation and microsomes, mitochondria and cytosol were assayed for phosphatidic acid phosphatase (EC 3.1.3.4) and marker enzymes of mitochondria and cytosol. The activity of phosphatidic acid phosphatase was significantly lower in microsomes and higher in cytosol and mitochondria of dystrophic muscle than in the corresponding subcellular fractions of normal muscle. The results support an explanation of earlier findings that there is reduced G3P incorporation into diglycerides and phosphatidylcholine and a qualitative and quantitative change in the amount of phosphatidylcholine in dystrophic microsomes. The possible reasons for the reduction in the activity of only microsomal PA-P-ase were discussed.

  19. In situ spatiotemporal mapping of flow fields around seeded stem cells at the subcellular length scale.

    Directory of Open Access Journals (Sweden)

    Min Jae Song

    2010-09-01

    Full Text Available A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms.

  20. mEosFP-based green-to-red photoconvertible subcellular probes for plants.

    Science.gov (United States)

    Mathur, Jaideep; Radhamony, Resmi; Sinclair, Alison M; Donoso, Ana; Dunn, Natalie; Roach, Elyse; Radford, Devon; Mohaghegh, P S Mohammad; Logan, David C; Kokolic, Ksenija; Mathur, Neeta

    2010-12-01

    Photoconvertible fluorescent proteins (FPs) are recent additions to the biologists' toolbox for understanding the living cell. Like green fluorescent protein (GFP), monomeric EosFP is bright green in color but is efficiently photoconverted into a red fluorescent form using a mild violet-blue excitation. Here, we report mEosFP-based probes that localize to the cytosol, plasma membrane invaginations, endosomes, prevacuolar vesicles, vacuoles, the endoplasmic reticulum, Golgi bodies, mitochondria, peroxisomes, and the two major cytoskeletal elements, filamentous actin and cortical microtubules. The mEosFP fusion proteins are smaller than GFP/red fluorescent protein-based probes and, as demonstrated here, provide several significant advantages for imaging of living plant cells. These include an ability to differentially color label a single cell or a group of cells in a developing organ, selectively highlight a region of a cell or a subpopulation of organelles and vesicles within a cell for tracking them, and understanding spatiotemporal aspects of interactions between similar as well as different organelles. In addition, mEosFP probes introduce a milder alternative to fluorescence recovery after photobleaching, whereby instead of photobleaching, photoconversion followed by recovery of green fluorescence can be used for estimating subcellular dynamics. Most importantly, the two fluorescent forms of mEosFP furnish bright internal controls during imaging experiments and are fully compatible with cyan fluorescent protein, GFP, yellow fluorescent protein, and red fluorescent protein fluorochromes for use in simultaneous, multicolor labeling schemes. Photoconvertible mEosFP-based subcellular probes promise to usher in a much higher degree of precision to live imaging of plant cells than has been possible so far using single-colored FPs.

  1. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion.

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    Gonzalo P Solis

    Full Text Available Analyses of cultured cells and transgenic mice expressing prion protein (PrP deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1 the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2 the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP's essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP's ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and

  2. Prolactin-induced Subcellular Targeting of GLUT1 Glucose Transporter in Living Mammary Epithelial Cells.

    Science.gov (United States)

    Riskin, Arieh; Mond, Yehudit

    2015-10-26

    Studying the biological pathways involved in mammalian milk production during lactation could have many clinical implications. The mammary gland is unique in its requirement for transport of free glucose into the cell for the synthesis of lactose, the primary carbohydrate in milk. To study GLUT1 trafficking and subcellular targeting in living mammary epithelial cells (MEC) in culture. Immunocytochemistry was used to study GLUT1 hormonally regulated subcellular targeting in human MEC (HMEC). To study GLUT1 targeting and recycling in living mouse MEC (MMEC) in culture, we constructed fusion proteins of GLUT1 and green fluorescent protein (GFP) and expressed them in CIT3 MMEC. Cells were maintained in growth medium (GM), or exposed to secretion medium (SM), containing prolactin. GLUT1 in HMEC localized primarily to the plasma membrane in GM. After exposure to prolactin for 4 days, GLUT1 was targeted intracellularly and demonstrated a perinuclear distribution, co-localizing with lactose synthetase. The dynamic trafficking of GFP-GLUT1 fusion proteins in CIT3 MMEC suggested a basal constitutive GLUT1 recycling pathway between an intracellular pool and the cell surface that targets most GLUT1 to the plasma membrane in GM. Upon exposure to prolactin in SM, GLUT1 was specifically targeted intracellularly within 90-110 minutes. Our studies suggest intracellular targeting of GLUT1 to the central vesicular transport system upon exposure to prolactin. The existence of a dynamic prolactin-induced sorting machinery for GLUT1 could be important for transport of free glucose into the Golgi for lactose synthesis during lactation.

  3. Expression and subcellular targeting of human complement factor C5a in Nicotiana species.

    Science.gov (United States)

    Nausch, Henrik; Mikschofsky, Heike; Mischofsky, Heike; Koslowski, Roswitha; Meyer, Udo; Broer, Inge; Huckauf, Jana

    2012-01-01

    We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a). C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T(0) transgenic plants were low in terms of the proportion of total soluble protein (TSP) when targeted to the apoplast (0.0002% TSP) or endoplasmic reticulum (0.0003% TSP) but was one order of magnitude higher when targeted to the vacuole (0.001% TSP). The yields could be increased by conventional breeding (up to 0.014% TSP in the T₂ generation). C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.

  4. Analysis of the subcellular targeting of the smaller replicase protein of Pelargonium flower break virus.

    Science.gov (United States)

    Martínez-Turiño, Sandra; Hernández, Carmen

    2012-02-01

    Replication of all positive RNA viruses occurs in association with intracellular membranes. In many cases, the mechanism of membrane targeting is unknown and there appears to be no correlation between virus phylogeny and the membrane systems recruited for replication. Pelargonium flower break virus (PFBV, genus Carmovirus, family Tombusviridae) encodes two proteins, p27 and its read-through product p86 (the viral RNA dependent-RNA polymerase), that are essential for replication. Recent reports with other members of the family Tombusviridae have shown that the smaller replicase protein is targeted to specific intracellular membranes and it is assumed to determine the subcellular localization of the replication complex. Using in vivo expression of green fluorescent protein (GFP) fusions in plant and yeast cells, we show here that PFBV p27 localizes in mitochondria. The same localization pattern was found for p86 that contains the p27 sequence at its N-terminus. Cellular fractionation of p27GFP-expressing cells confirmed the confocal microscopy observations and biochemical treatments suggested a tight association of the protein to membranes. Analysis of deletion mutants allowed identification of two regions required for targeting of p27 to mitochondria. These regions mapped toward the N- and C-terminus of the protein, respectively, and could function independently though with distinct efficiency. In an attempt to search for putative cellular factors involved in p27 localization, the subcellular distribution of the protein was checked in a selected series of knockout yeast strains and the outcome of this approach is discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Effect of chilling on porcine germinal vesicle stage oocytes at the subcellular level.

    Science.gov (United States)

    Gerelchimeg, Bou; Li-Qing, Liu; Zhong, Zheng; Jiang-Tian, Tian; Qing-Ran, Kong; Jun, Song; Xue-Dong, Wang; Zhong-Hua, Liu

    2009-08-01

    The potential subcellular consequence of chilling on porcine germinal vesicle (GV) stage oocytes was examined. Prior to in vitro maturation (IVM), Cumulus-oocyte complexes (COCs) freshly collected from antral follicles (3-6mm in diameter) were evenly divided into four groups and immediately incubated in PVA-TL-HEPES medium at the temperature of 39 degrees C (control group), 23 degrees C (room temperature), 15 degrees C and 10 degrees C for 10min, respectively. Following 42h of IVM at 39 degrees C, the survival rates were examined. There was no significant difference between the survival rate of 23 degrees C chilled group and control group (77.92 and 91.89%), but the survival rate of 15 and 10 degrees C chilled group were significantly decreased (46.34 and 4.81%, Peffects of chilling on oocytes at the subcellular level, the control and 15 degrees C chilled group COCs fixed at different time points of the IVM cultures (2, 2.5, 3, 3.5 and 4h of IVM) were prepared for transmission electron microscope (TEM) observation. As the result, compared with the control group, there were two significant changes in the ultrastructural morphology of 15 degrees C treatment group: (1) dramatic reduction of heterogeneous lipid, (2) disorganized mitochondria-endoplasmic reticulum-lipid vesicles (M-E-L) combination. These results indicate that 15 degrees C is a critical chilling temperature for porcine GV stage oocyte and the alteration of cellular chemical composition and the destruction of M-E-L combination maybe responsible for chilling injury of porcine oocyte at this stage.

  6. In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

    Science.gov (United States)

    Song, Min Jae; Dean, David; Knothe Tate, Melissa L.

    2010-01-01

    A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms. PMID:20862249

  7. Subcellular localisation of radionuclides by transmission electronic microscopy in aquatic and terrestrial organisms

    Energy Technology Data Exchange (ETDEWEB)

    Floriani, M.; Grasset, G.; Simon, O.; Morlon, H.; Laroche, L. [CEA Cadarache (DEI/SECRE/LRE), Laboratory of Radioecology and Ecotoxicology, Institute for Radioprotection and Nuclear Safety, 13 - Saint-Paul-lez-Durance (France)

    2004-07-01

    The global framework of this study is to go further in the understanding of the involved mechanisms of uranium and selenium internalisation at the subcellular level and of their toxicity towards several aquatic and terrestrial organisms. In this context, the applications and performances of a Scanning Transmission Electron Microscope (TEM/STEM) equipped with CCD camera and Energy-Dispersive- X-Ray (EDAX) analysis are reported. The principal merit of this equipment is the clear expression of element distribution with nanometer resolution. The sample for TEM analysis were prepared in ultrathin sections of 70-140 nm (thickness) and those for EDAX in sections of 200-500 nm. This method offers the possibility of a direct correlation between histological image and distribution map of trace elements. For each sample, following TEM analysis, EDAX spectra or EDAX mapping were also recorded to confirm the identity of the electron dense material in the scanned sections. Demonstration of the usefulness of this method to understand the bioaccumulation mechanisms and to study the effect of the pollutant uptake at the subcellular level was performed for target organs of a metal (U) and a metalloid (Se) in various biological models: a higher rooted plant (Phaseolus vulgaris)) and a freshwater invertebrate (Orconectes Limosus) and a unicellular green alga (Chlamydomonas reinhardtii)). TEM-EDAX analysis revealed the presence of U-deposits in gills and digestive gland in crayfish, and in vacuoles or in the cytoplasm of different rooted cells bean. In the alga, the accumulation of Se was found in electron-dense granules within cytoplasm associated with ultrastructural changes and starch accumulation. (author)

  8. Integration of Cadmium Accumulation, Subcellular Distribution, and Physiological Responses to Understand Cadmium Tolerance in Apple Rootstocks

    Directory of Open Access Journals (Sweden)

    Jiangtao Zhou

    2017-06-01

    Full Text Available Cadmium (Cd is a nonessential and highly toxic element causing agricultural problems. However, little information is available about the variation in Cd tolerance among apple rootstocks and its underlying physiological regulation mechanisms. This study investigated Cd accumulation, subcellular distribution, and chemical forms as well as physiological changes among four apple rootstocks exposed to either 0 or 300 μM CdCl2. The results showed that variations in Cd tolerance existed among these rootstocks. Cd exposure caused decline in photosynthesis, chlorophyll and biomass in four apple rootstocks, which was less pronounced in M. baccata, indicating its higher Cd tolerance. This finding was corroborated with higher Cd tolerance indexes (TIs of the whole plant in M. baccata than those in the other three apple rootstocks. Among the four apple rootstocks, M. baccata displayed the lowest Cd concentrations in roots, wood, and leaves, the smallest total Cd amounts as well as the lowest BCF. In apple rootstocks, it was found that to immobilize Cd in cell wall and soluble fraction (most likely in vacuole and to convert it into pectate- or protein- integrated forms and undissolved Cd phosphate forms may be the primary strategies to reduce Cd mobility and toxicity. The physiological changes including ROS, carbohydrates and antioxidants were in line with the variations of Cd tolerance among four apple rootstocks. In comparison with the other three apple rootstocks, M. baccata had lower concentrations of ROS in roots and bark, H2O2 in roots and leaves and MDA in roots, wood and bark, but higher concentrations of soluble sugars in bark and starch in roots and leaves, and enhanced antioxidants. These results indicate that M. baccata are more tolerant to Cd stress than the other three apple rootstocks under the current experiment conditions, which is probably related to Cd accumulation, subcellular partitioning and chemical forms of Cd and well

  9. Expression and subcellular targeting of human complement factor C5a in Nicotiana species.

    Directory of Open Access Journals (Sweden)

    Henrik Nausch

    Full Text Available We evaluated transgenic tobacco plants as an alternative to Escherichia coli for the production of recombinant human complement factor 5a (C5a. C5a has not been expressed in plants before and is highly unstable in vivo in its native form, so it was necessary to establish the most suitable subcellular targeting strategy. We used the strong and constitutive CaMV 35S promoter to drive transgene expression and compared three different subcellular compartments. The yields of C5a in the T(0 transgenic plants were low in terms of the proportion of total soluble protein (TSP when targeted to the apoplast (0.0002% TSP or endoplasmic reticulum (0.0003% TSP but was one order of magnitude higher when targeted to the vacuole (0.001% TSP. The yields could be increased by conventional breeding (up to 0.014% TSP in the T₂ generation. C5a accumulated to the same level in seeds and leaves when targeted to the apoplast but was up to 1.7-fold more abundant in the seeds when targeted to the ER or vacuole, although this difference was less striking in the better-performing lines. When yields were calculated as an amount per gram fresh weight of transgenic plant tissue, the vacuole targeting strategy was clearly more efficient in seeds, reaching 35.8 µg C5a per gram of fresh seed weight compared to 10.62 µg C5a per gram fresh weight of leaves. Transient expression of C5aER and C5aVac in N. benthamiana, using MagnICON vectors, reached up to 0.2% and 0.7% of TSP, respectively, but was accompanied by cytotoxic effects and induced leaf senescence. Western blot of the plant extracts revealed a band matching the corresponding glycosylated native protein and the bioassay demonstrated that recombinant C5a was biologically active.

  10. ESLpred2: improved method for predicting subcellular localization of eukaryotic proteins

    Directory of Open Access Journals (Sweden)

    Raghava Gajendra PS

    2008-11-01

    Full Text Available Abstract Background The expansion of raw protein sequence databases in the post genomic era and availability of fresh annotated sequences for major localizations particularly motivated us to introduce a new improved version of our previously forged eukaryotic subcellular localizations prediction method namely "ESLpred". Since, subcellular localization of a protein offers essential clues about its functioning, hence, availability of localization predictor would definitely aid and expedite the protein deciphering studies. However, robustness of a predictor is highly dependent on the superiority of dataset and extracted protein attributes; hence, it becomes imperative to improve the performance of presently available method using latest dataset and crucial input features. Results Here, we describe augmentation in the prediction performance obtained for our most popular ESLpred method using new crucial features as an input to Support Vector Machine (SVM. In addition, recently available, highly non-redundant dataset encompassing three kingdoms specific protein sequence sets; 1198 fungi sequences, 2597 from animal and 491 plant sequences were also included in the present study. First, using the evolutionary information in the form of profile composition along with whole and N-terminal sequence composition as an input feature vector of 440 dimensions, overall accuracies of 72.7, 75.8 and 74.5% were achieved respectively after five-fold cross-validation. Further, enhancement in performance was observed when similarity search based results were coupled with whole and N-terminal sequence composition along with profile composition by yielding overall accuracies of 75.9, 80.8, 76.6% respectively; best accuracies reported till date on the same datasets. Conclusion These results provide confidence about the reliability and accurate prediction of SVM modules generated in the present study using sequence and profile compositions along with similarity search

  11. Ligand-binding properties and subcellular localization of maize cytokinin receptors

    Science.gov (United States)

    Lomin, Sergey N.; Yonekura-Sakakibara, Keiko; Romanov, Georgy A.; Sakakibara, Hitoshi

    2011-01-01

    The ligand-binding properties of the maize (Zea mays L.) cytokinin receptors ZmHK1, ZmHK2, and ZmHK3a have been characterized using cytokinin binding assays with living cells or membrane fractions. According to affinity measurements, ZmHK1 preferred N6-(Δ2-isopentenyl)adenine (iP) and had nearly equal affinities to trans-zeatin (tZ) and cis-zeatin (cZ). ZmHK2 preferred tZ and iP to cZ, while ZmHK3a preferred iP. Only ZmHK2 had a high affinity to dihydrozeatin (DZ). Analysis of subcellular fractions from leaves and roots of maize seedlings revealed specific binding of tZ in the microsome fraction but not in chloroplasts or mitochondria. In competitive binding assays with microsomes, tZ and iP were potent competitors of [3H]tZ while cZ demonstrated significantly lower affinity; adenine was almost ineffective. The binding specificities of microsomes from leaf and root cells for cytokinins were consistent with the expression pattern of the ZmHKs and our results on individual receptor properties. Aqueous two-phase partitioning and sucrose density-gradient centrifugation followed by immunological detection with monoclonal antibody showed that ZmHK1 was associated with the endoplasmic reticulum (ER). This was corroborated by observations of the subcellular localization of ZmHK1 fusions with green fluorescent protein in maize protoplasts. All these data strongly suggest that at least a part of cytokinin perception occurs in the ER. PMID:21778179

  12. Osmotic stress changes the expression and subcellular localization of the Batten disease protein CLN3.

    Directory of Open Access Journals (Sweden)

    Amanda Getty

    Full Text Available Juvenile CLN3 disease (formerly known as juvenile neuronal ceroid lipofuscinosis is a fatal childhood neurodegenerative disorder caused by mutations in the CLN3 gene. CLN3 encodes a putative lysosomal transmembrane protein with unknown function. Previous cell culture studies using CLN3-overexpressing vectors and/or anti-CLN3 antibodies with questionable specificity have also localized CLN3 in cellular structures other than lysosomes. Osmoregulation of the mouse Cln3 mRNA level in kidney cells was recently reported. To clarify the subcellular localization of the CLN3 protein and to investigate if human CLN3 expression and localization is affected by osmotic changes we generated a stably transfected BHK (baby hamster kidney cell line that expresses a moderate level of myc-tagged human CLN3 under the control of the human ubiquitin C promoter. Hyperosmolarity (800 mOsm, achieved by either NaCl/urea or sucrose, dramatically increased the mRNA and protein levels of CLN3 as determined by quantitative real-time PCR and Western blotting. Under isotonic conditions (300 mOsm, human CLN3 was found in a punctate vesicular pattern surrounding the nucleus with prominent Golgi and lysosomal localizations. CLN3-positive early endosomes, late endosomes and cholesterol/sphingolipid-enriched plasma membrane microdomain caveolae were also observed. Increasing the osmolarity of the culture medium to 800 mOsm extended CLN3 distribution away from the perinuclear region and enhanced the lysosomal localization of CLN3. Our results reveal that CLN3 has multiple subcellular localizations within the cell, which, together with its expression, prominently change following osmotic stress. These data suggest that CLN3 is involved in the response and adaptation to cellular stress.

  13. Identification of an intrinsic determinant critical for maspin subcellular localization and function.

    Directory of Open Access Journals (Sweden)

    Sijana H Dzinic

    Full Text Available Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1. Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that maspin reactive center loop (RCL and its neighboring sequence are critical for maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate(346 (D(346 to Glutamate (E(346, maspin(D346E, was predominantly nuclear, whereas wild type maspin (maspin(WT was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC showed that the increased nuclear enrichment of maspin(D346E was, at least in part, due to its increased affinity to HDAC1. Maspin(D346E was also more potent than maspin(WT as an HDAC inhibitor. Taken together, our evidence demonstrates that D(346 is a critical cis-element in maspin sequence that determines the molecular context and subcellular localization of maspin. A mechanistic model derived from our evidence suggests a new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.

  14. Evaluation on subcellular partitioning and biodynamics of pulse copper toxicity in tilapia reveals impacts of a major environmental disturbance.

    Science.gov (United States)

    Ju, Yun-Ru; Yang, Ying-Fei; Tsai, Jeng-Wei; Cheng, Yi-Hsien; Chen, Wei-Yu; Liao, Chung-Min

    2017-07-01

    Fluctuation exposure of trace metal copper (Cu) is ubiquitous in aquatic environments. The purpose of this study was to investigate the impacts of chronically pulsed exposure on biodynamics and subcellular partitioning of Cu in freshwater tilapia (Oreochromis mossambicus). Long-term 28-day pulsed Cu exposure experiments were performed to explore subcellular partitioning and toxicokinetics/toxicodynamics of Cu in tilapia. Subcellular partitioning linking with a metal influx scheme was used to estimate detoxification and elimination rates. A biotic ligand model-based damage assessment model was used to take into account environmental effects and biological mechanisms of Cu toxicity. We demonstrated that the probability causing 50% of susceptibility risk in response to pulse Cu exposure in generic Taiwan aquaculture ponds was ~33% of Cu in adverse physiologically associated, metabolically active pool, implicating no significant susceptibility risk for tilapia. We suggest that our integrated ecotoxicological models linking chronic exposure measurements with subcellular partitioning can facilitate a risk assessment framework that provides a predictive tool for preventive susceptibility reduction strategies for freshwater fish exposed to pulse metal stressors.

  15. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    NARCIS (Netherlands)

    Matthews, G.D.; Gur, N.; Koopman, W.J.H.; Pines, O.; Vardimon, L.

    2010-01-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS

  16. Subcellular Targeting of VIP Boutons in Mouse Barrel Cortex is Layer-Dependent and not Restricted to Interneurons.

    Science.gov (United States)

    Zhou, Xiaojuan; Rickmann, Michael; Hafner, Georg; Staiger, Jochen F

    2017-11-01

    Neocortical vasoactive intestinal polypeptide (VIP) expressing cells are a diverse subpopulation of GABAergic interneurons issuing distinct axonal projections. They are known to inhibit other types of interneurons as well as excitatory principal neurons and possess a disinhibitory net effect in cortical circuits. In order to elucidate their targeting specificity, the output connectivity of VIP interneurons was studied at the subcellular level in barrel cortex of interneuron-specific Cre-driver mice, using pre- and postembedding electron microscopy. Systematically sampling VIP boutons across all layers, we found a substantial proportion of the innervated subcellular structures were dendrites (80%), with somata (13%), and spines (7%) being much less targeted. In layer VI, a high proportion of axosomatic synapses was found (39%). GABA-immunopositive ratio was quantified among the targets using statistically validated thresholds: only 37% of the dendrites, 7% of the spines, and 26% of the somata showed above-threshold immunogold labeling. For the main target structure "dendrite", a higher proportion of GABAergic subcellular profiles existed in deep than in superficial layers. In conclusion, VIP interneurons innervate non-GABAergic excitatory neurons and interneurons at their subcellular domains with layer-dependent specificity. This suggests a diverse output of VIP interneurons, which predicts multiple functionality in cortical circuitry beyond disinhibition. © The Author 2017. Published by Oxford University Press.

  17. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Directory of Open Access Journals (Sweden)

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  18. SPA Proteins Affect the Subcellular Localization of COP1 in the COP1/SPA Ubiquitin Ligase Complex during Photomorphogenesis.

    Science.gov (United States)

    Balcerowicz, Martin; Kerner, Konstantin; Schenkel, Christian; Hoecker, Ute

    2017-07-01

    The Arabidopsis ( Arabidopsis thaliana ) COP1/SPA ubiquitin ligase is a central repressor that suppresses light signaling in darkness by targeting positive regulators of the light response, mainly transcription factors, for degradation. Light inactivates COP1/SPA, in part by excluding COP1 from the nucleus. SPA proteins are essential cofactors of COP1, but their exact role in the COP1/SPA complex is thus far unknown. To unravel a potential role of SPA proteins in COP1 nucleocytoplasmic partitioning, we monitored the subcellular localization of COP1 in a spa1234 quadruple mutant ( spaQn ). We analyzed a YFP-COP1-expressing transgenic line and endogenous COP1 after subcellular fractionation. In dark-grown seedlings, both YFP-COP1 and endogenous COP1 accumulated in the nucleus in the absence and presence of SPA proteins, indicating that SPA proteins are not required for nuclear localization of COP1 in darkness. In contrast, in white light-grown seedlings, spaQn mutants failed to relocalize COP1 from the nucleus to the cytoplasm. Hence, SPA proteins are necessary for the light-controlled change in COP1 subcellular localization. We conclude that SPA proteins have a dual role: (1) they are required for light-responsiveness of COP1 subcellular localization, and (2) they promote COP1 activity in darkness in a fashion that is independent of the nuclear import/nuclear retention of COP1. © 2017 American Society of Plant Biologists. All Rights Reserved.

  19. Zn subcellular distribution in liver of goldfish (carassius auratus) with exposure to zinc oxide nanoparticles and mechanism of hepatic detoxification.

    Science.gov (United States)

    Fan, Wenhong; Li, Qian; Yang, Xiuping; Zhang, Li

    2013-01-01

    Zinc Oxide Nanoparticles (ZnO NPs) have attracted increasing concerns because of their widespread use and toxic potential. In this study, Zn accumulations in different tissues (gills, liver, muscle, and gut) of goldfish (Carassius auratus) after exposure to ZnO NPs were studied in comparison with bulk ZnO and Zn(2+). And the technique of subcellular partitioning was firstly used on the liver of goldfish to study the hepatic accumulation of ZnO NPs. The results showed that at sublethal Zn concentration (2 mg/L), bioaccumulation in goldfish was tissue-specific and dependent on the exposure materials. Compared with Zn(2+), the particles of bulk ZnO and the ZnO NPs appeared to aggregate in the environmentally contacted tissues (gills and gut), rather than transport to the internal tissues (liver and muscle). The subcellular distributions of liver differed for the three exposure treatments. After ZnO NPs exposure, Zn percentage in metal-rich granule (MRG) increased significantly, and after Zn(2+) exposure, it increased significantly in the organelles. Metallothionein-like proteins (MTLP) were the main target for Zn(2+), while MRG played dominant role for ZnO NPs. The different results of subcellular distributions revealed that metal detoxification mechanisms of liver for ZnO NPs, bulk ZnO, and Zn(2+) were different. Overall, subcellular partitioning provided an interesting start to better understanding of the toxicity of nano- and conventional materials.

  20. On location at CERN

    CERN Multimedia

    Joannah Caborn Wengler

    2012-01-01

    CERN continues to be a very popular candidate for film locations at the moment. Not only has it inspired a German author and a film-maker interested in the more exotic interpretations of the science being worked on at the Organization, but even the recent puppet animation film by the legendary Muppets featured some CERN scenes.   Dr Bunsen Honeydew (far left) and his friends at ATLAS. Beaker, Bunsen's assistant, has just been sucked up the vacuum tube top left... Image courtesy Walt Disney Studios. In “The Muppet Movie”, released in November 2011 in North America and world-wide in January and February this year, Kermit is reuniting his friends who have ended up in some far-flung places since they last worked together 10 years ago. CERN caught the imagination of the film-makers as the perfect place for the Muppet scientists, Dr. Honeydew Bunsen and his hapless assistant Beaker. After a brief scene filmed in front of a backdrop of the ATLAS detector, the rest of the ...

  1. Biodynamics of copper oxide nanoparticles and copper ions in an oligochaete - Part II: Subcellular distribution following sediment exposure.

    Science.gov (United States)

    Thit, Amalie; Ramskov, Tina; Croteau, Marie-Noële; Selck, Henriette

    2016-11-01

    The use and likely incidental release of metal nanoparticles (NPs) is steadily increasing. Despite the increasing amount of published literature on metal NP toxicity in the aquatic environment, very little is known about the biological fate of NPs after sediment exposures. Here, we compare the bioavailability and subcellular distribution of copper oxide (CuO) NPs and aqueous Cu (Cu-Aq) in the sediment-dwelling worm Lumbriculus variegatus. Ten days (d) sediment exposure resulted in marginal Cu bioaccumulation in L. variegatus for both forms of Cu. Bioaccumulation was detected because isotopically enriched 65Cu was used as a tracer. Neither burrowing behavior or survival was affected by the exposure. Once incorporated into tissue, Cu loss was negligible over 10 d of elimination in clean sediment (Cu elimination rate constants were not different from zero). With the exception of day 10, differences in bioaccumulation and subcellular distribution between Cu forms were either not detectable or marginal. After 10 d of exposure to Cu-Aq, the accumulated Cu was primarily partitioned in the subcellular fraction containing metallothionein-like proteins (MTLP, ≈40%) and cellular debris (CD, ≈30%). Cu concentrations in these fractions were significantly higher than in controls. For worms exposed to CuO NPs for 10 d, most of the accumulated Cu was partitioned in the CD fraction (≈40%), which was the only subcellular fraction where the Cu concentration was significantly higher than for the control group. Our results indicate that L. variegatus handle the two Cu forms differently. However, longer-term exposures are suggested in order to clearly highlight differences in the subcellular distribution of these two Cu forms. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Biodynamics of copper oxide nanoparticles and copper ions in an oligochaete - Part II: Subcellular distribution following sediment exposure

    Science.gov (United States)

    Thit, Amalie; Ramskov, Tina; Croteau, Marie-Noele; Selck, Henriette

    2016-01-01

    The use and likely incidental release of metal nanoparticles (NPs) is steadily increasing. Despite the increasing amount of published literature on metal NP toxicity in the aquatic environment, very little is known about the biological fate of NPs after sediment exposures. Here, we compare the bioavailability and subcellular distribution of copper oxide (CuO) NPs and aqueous Cu (Cu-Aq) in the sediment-dwelling worm Lumbriculus variegatus. Ten days (d) sediment exposure resulted in marginal Cu bioaccumulation in L. variegatus for both forms of Cu. Bioaccumulation was detected because isotopically enriched 65Cu was used as a tracer. Neither burrowing behavior or survival was affected by the exposure. Once incorporated into tissue, Cu loss was negligible over 10 d of elimination in clean sediment (Cu elimination rate constants were not different from zero). With the exception of day 10, differences in bioaccumulation and subcellular distribution between Cu forms were either not detectable or marginal. After 10 d of exposure to Cu-Aq, the accumulated Cu was primarily partitioned in the subcellular fraction containing metallothionein-like proteins (MTLP, ≈40%) and cellular debris (CD, ≈30%). Cu concentrations in these fractions were significantly higher than in controls. For worms exposed to CuO NPs for 10 d, most of the accumulated Cu was partitioned in the CD fraction (≈40%), which was the only subcellular fraction where the Cu concentration was significantly higher than for the control group. Our results indicate that L. variegatus handle the two Cu forms differently. However, longer-term exposures are suggested in order to clearly highlight differences in the subcellular distribution of these two Cu forms.

  3. Algorithms over partially ordered sets

    DEFF Research Database (Denmark)

    Baer, Robert M.; Østerby, Ole

    1969-01-01

    We here study some problems concerned with the computational analysis of finite partially ordered sets. We begin (in § 1) by showing that the matrix representation of a binary relationR may always be taken in triangular form ifR is a partial ordering. We consider (in § 2) the chain structure...

  4. [Acrylic resin removable partial dentures

    NARCIS (Netherlands)

    Baat, C. de; Witter, D.J.; Creugers, N.H.J.

    2011-01-01

    An acrylic resin removable partial denture is distinguished from other types of removable partial dentures by an all-acrylic resin base which is, in principle, solely supported by the edentulous regions of the tooth arch and in the maxilla also by the hard palate. When compared to the other types of

  5. Partial Epilepsy with Auditory Features

    Directory of Open Access Journals (Sweden)

    J Gordon Millichap

    2004-07-01

    Full Text Available The clinical characteristics of 53 sporadic (S cases of idiopathic partial epilepsy with auditory features (IPEAF were analyzed and compared to previously reported familial (F cases of autosomal dominant partial epilepsy with auditory features (ADPEAF in a study at the University of Bologna, Italy.

  6. Difference in subcellular distribution of vascular endothelial cadherin in cytoplasm by clathrinor caveolae-mediated endocytosis after lipopolysaccharide challenge

    Directory of Open Access Journals (Sweden)

    Ye ZHANG

    2016-03-01

    Full Text Available Objective  To investigate different subcellular distribution and the effect on cell permeability of vascular endothelial cadherin (VE-Cad in cytoplasm via clathrin-and caveolae-mediated endocytosis pathways after lipopolysaccharide (LPS challenge. Methods  The co-immunoprecipitation of VE-Cad with Rab11 (marker of the recycling endosomes and lysosome-associated membrane protein 2 (LAMP2, marker of the late endosomes/lysosomes at different time points, as well as the effects of clathrin-mediated endocytosis inhibitor chlorpromazine (CPZ and caveolae inhibitor filipin on the coimmunoprecipitation of VE-Cad with Rab11/LAMP2 and on the monolayer cell permeability after LPS(10µg/ml treatment, were observed. Results  1 The co-immunoprecipitation of VE-Cad with Rab11 was increased at 1h after LPS treatment (P<0.05, and then it decreased gradually, while the co-immunoprecipitation of VE-Cad with LAMP2 was increased with elapse of time after LPS treatment (P<0.05. 2 The increased co-immunoprecipitation of VE-Cad with Rab11 was inhibited obviously by the clathrin-mediated endocytosis inhibitor CPZ (P<0.05, while the inhibitor of caveolae filipin failed to do so. The increased coimmunoprecipitation of VE-Cad with LAMP2 was inhibited obviously by the caveolae filipin (P<0.05, while the clathrinmediated endocytosis inhibitor CPZ failed. 3 The increased monolayer cell permeability at 1h after LPS treatment was improved by the clathrin-mediated endocytosis inhibitor CPZ (P<0.05, and the increased monolayer cell hyperpermeability at 4h after LPS treatment could be significantly attenuated by the caveolae inhibitor filipin. Conclusion  VE-Cad may get into the cells and locate in recycling endosomes or lysosomes respectively through clathrin-and caveolae-mediated endocytosis after LPS treatment, thus leading to hyperpermeability in different extent of monolayer cells. DOI: 10.11855/j.issn.0577-7402.2016.01.05

  7. Cast Partial Denture versus Acrylic Partial Denture for Replacement of Missing Teeth in Partially Edentulous Patients

    Directory of Open Access Journals (Sweden)

    Pramita Suwal

    2017-03-01

    Full Text Available Aim: To compare the effects of cast partial denture with conventional all acrylic denture in respect to retention, stability, masticatory efficiency, comfort and periodontal health of abutments. Methods: 50 adult partially edentulous patient seeking for replacement of missing teeth having Kennedy class I and II arches with or without modification areas were selected for the study. Group-A was treated with cast partial denture and Group-B with acrylic partial denture. Data collected during follow-up visit of 3 months, 6 months, and 1 year by evaluating retention, stability, masticatory efficiency, comfort, periodontal health of abutment. Results: Chi-square test was applied to find out differences between the groups at 95% confidence interval where p = 0.05. One year comparison shows that cast partial denture maintained retention and stability better than acrylic partial denture (p< 0.05. The masticatory efficiency was significantly compromising from 3rd month to 1 year in all acrylic partial denture groups (p< 0.05. The comfort of patient with cast partial denture was maintained better during the observation period (p< 0.05. Periodontal health of abutment was gradually deteriorated in all acrylic denture group (p

  8. Localization and regulation of mouse pantothenate kinase 2 [The PanK2 Genes of Mouse and Human Specify Proteins with Distinct Subcellular Locations

    Energy Technology Data Exchange (ETDEWEB)

    Leonardi, Roberta [St. Jude Children' s Research Hospital, Memphis, TN (United States); Zhang, Yong-Mei [St. Jude Children' s Research Hospital, Memphis, TN (United States); Lykidis, Athanasios [DOE Joint Genome Inst., Walnut Creek, CA (United States); Rock, Charles O. [St. Jude Children' s Research Hospital, Memphis, TN (United States); Jackowski, Suzanne [St. Jude Children' s Research Hospital, Memphis, TN (United States)

    2007-09-07

    Coenzyme A (CoA) biosynthesis is initiated by pantothenatekinase (PanK) and CoA levels are controlled through differentialexpression and feedback regulation of PanK isoforms. PanK2 is amitochondrial protein in humans, but comparative genomics revealed thatacquisition of a mitochondrial targeting signal was limited to primates.Human and mouse PanK2 possessed similar biochemical properties, withinhibition by acetylCoA and activation by palmitoylcarnitine. Mouse PanK2localized in the cytosol, and the expression of PanK2 was higher in humanbrain compared to mouse brain. Differences in expression and subcellularlocalization should be considered in developing a mouse model for humanPanK2 deficiency.

  9. Haematopoietic protein tyrosine phosphatase (HePTP) phosphorylation by cAMP-dependent protein kinase in T-cells: dynamics and subcellular location.

    Science.gov (United States)

    Nika, Konstantina; Hyunh, Huong; Williams, Scott; Paul, Surojit; Bottini, Nunzio; Taskén, Kjetil; Lombroso, Paul J; Mustelin, Tomas

    2004-01-01

    The HePTP (haematopoietic protein tyrosine phosphatase) is a negative regulator of the ERK2 (extracellular signal-regulated protein kinase 2) and p38 MAP kinases (mitogen-activated protein kinases) in T-cells. This inhibitory function requires a physical association of HePTP through an N-terminal KIM (kinase-interaction motif) with ERK and p38. We previously reported that PKA (cAMP-dependent protein kinase) phosphorylates Ser-23 within the KIM of HePTP, resulting in dissociation of HePTP from ERK2. Here we follow the phosphorylation of this site in intact T-cells. We find that HePTP is phosphorylated at Ser-23 in resting T-cells and that this phosphorylation increases upon treatment of the cells with agents that elevate intracellular cAMP, such as prostaglandin E2. HePTP phosphorylation occurred at discrete regions at the cell surface. Phosphorylation was reduced by inhibitors of PKA and increased by inhibitors of protein phosphatases PP1 and PP2A, but not by inhibitors of calcineurin. In vitro, PP1 efficiently dephosphorylated HePTP at Ser-23, while PP2A was much less efficient. Activation of PP1 by treatment of the cells with ceramide suppressed Ser-23 phosphorylation, as did transfection of the catalytic subunit of PP1. Phosphorylation at Ser-23 is also increased in a transient manner upon T-cell antigen receptor ligation. In contrast, treatment of cells with phorbol ester had no effect on HePTP phosphorylation at Ser-23. We conclude from these results that HePTP is under continuous control by PKA and a serine-specific phosphatase, probably PP1, in T-cells and that this basal phosphorylation at Ser-23 can rapidly change in response to external stimuli. This, in turn, will affect the ability of HePTP to inhibit the ERK and p38 MAP kinases. PMID:14613483

  10. Indoor Space Location Model Based on Location Service

    Science.gov (United States)

    Zhou, Y.; Zeng, G.; Huang, Y.; Yang, X.

    2017-10-01

    Location is the basis for the realization of location services, the integrity of the location information and its way of representation in indoor space model directly restricts the quality of location services. The construction of the existing indoor space model is mostly for specific applications and lack of uniform representation of location information. Several geospatial standards have been developed to meet the requirement of the indoor spatial information system, among which CityGML LOD4 and IndoorGML are the most relevant ones for indoor spatial information. However, from the perspective of Location Based Service (LBS), the CityGML LOD4 is more inclined to visualize the indoor space. Although IndoorGML is mainly used for indoor space navigation and has description (such as geometry, topology, and semantics) benefiting for indoor LBS, this standard model lack explicit representation of indoor location information. In this paper, from the perspective of Location Based Service (LBS), based on the IndoorGML standard, an indoor space location model (ISLM) conforming to human cognition is proposed through integration of the geometric and topological and semantic features of the indoor spatial entity. This model has the explicit description of location information which the standard indoor space model of IndoorGML and CityGML LOD4 does not have, which can lay the theoretical foundation for indoor location service such as indoor navigation, indoor routing and location query.

  11. Partial order infinitary term rewriting

    DEFF Research Database (Denmark)

    Bahr, Patrick

    2014-01-01

    with the partial order model restricted to total terms. Hence, partial order convergence constitutes a conservative extension of metric convergence, which additionally offers a fine-grained distinction between different levels of divergence. In the second part, we focus our investigation on strong convergence...... of orthogonal systems. The main result is that the gap between the metric model and the partial order model can be bridged by extending the term rewriting system by additional rules. These extensions are the well-known Böhm extensions. Based on this result, we are able to establish that -- contrary...

  12. A GIS-Based Optimization Technique for Spatial Location of ...

    African Journals Online (AJOL)

    GIS)-based package; TransCAD v. 5.0 was used to determine the optimal locations of one to ten waste bins. This optimization technique requires less computational time and the output of ten computer runs showed that partial service coverage ...

  13. ICE Online Detainee Locator System

    Data.gov (United States)

    Department of Homeland Security — The Online Detainee Locator datasets provide the location of a detainee who is currently in ICE custody, or who was release from ICE custody for any reason with the...

  14. VT Transportation Project Point Locations

    Data.gov (United States)

    Vermont Center for Geographic Information — Project information for project that are located off system or on system at a discrete location. The projects in this data set represent the active projects in the...

  15. Duality in constrained location problems

    DEFF Research Database (Denmark)

    Juel, Henrik; Love, Robert F.

    1987-01-01

    The dual of a facility location problem with general norms, distance constraints, and linear constraints is formulated.......The dual of a facility location problem with general norms, distance constraints, and linear constraints is formulated....

  16. Mathematical modeling of sub-cellular asymmetry of fat-dachsous heterodimer for generation of planar cell polarity.

    Directory of Open Access Journals (Sweden)

    Mohit Kumar Jolly

    Full Text Available Planar Cell Polarity (PCP is an evolutionarily conserved characteristic of animal tissues marked by coordinated polarization of cells or structures in the plane of a tissue. In insect wing epithelium, for instance, PCP is characterized by en masse orientation of hairs orthogonal to its apical-basal axis and pointing along the proximal-distal axis of the organ. Directional cue for PCP has been proposed to be generated by complex sets of interactions amongst three proteins - Fat (Ft, Dachsous (Ds and Four-jointed (Fj. Ft and Ds are two atypical cadherins, which are phosphorylated by Fj, a Golgi kinase. Ft and Ds from adjacent cells bind heterophilically via their tandem cadherin repeats, and their binding affinities are regulated by Fj. Further, in the wing epithelium, sub-cellular levels of Ft-Ds heterodimers are seen to be elevated at the distal edges of individual cells, prefiguring their PCP. Mechanisms generating this sub-cellular asymmetry of Ft-Ds heterodimer in proximal and distal edges of cells, however, have not been resolved yet. Using a mathematical modeling approach, here we provide a framework for generation of this sub-cellular asymmetry of Ft-Ds heterodimer. First, we explain how the known interactions within Ft-Ds-Fj system translate into sub-cellular asymmetry of Ft-Ds heterodimer. Second, we show that this asymmetric localization of Ft-Ds heterodimer is lost when tissue-level gradient of Fj is flattened, or when phosphorylation of Ft by Fj is abolished, but not when tissue-level gradient of Ds is flattened or when phosphorylation of Ds is abrogated. Finally, we show that distal enrichment of Ds also amplifies Ft-Ds asymmetry. These observations reveal that gradient of Fj expression, phosphorylation of Ft by Fj and sub-cellular distal accumulation of Ds are three critical elements required for generating sub-cellular asymmetry of Ft-Ds heterodimer. Our model integrates the known experimental data and presents testable predictions

  17. Biodynamics of copper oxide nanoparticles and copper ions in an oligochaete - Part II: Subcellular distribution following sediment exposure

    Energy Technology Data Exchange (ETDEWEB)

    Thit, Amalie, E-mail: athitj@ruc.dk [U.S. Geological Survey, 345 Middlefield Road, Menlo Park, CA 94025 (United States); Department of Science and Environment, Roskilde University, Universitetsvej 1, Roskilde DK-4000 (Denmark); Ramskov, Tina, E-mail: tramskov@hotmail.com [U.S. Geological Survey, 345 Middlefield Road, Menlo Park, CA 94025 (United States); Department of Science and Environment, Roskilde University, Universitetsvej 1, Roskilde DK-4000 (Denmark); Croteau, Marie-Noële, E-mail: mcroteau@usgs.gov [Department of Science and Environment, Roskilde University, Universitetsvej 1, Roskilde DK-4000 (Denmark); Selck, Henriette [U.S. Geological Survey, 345 Middlefield Road, Menlo Park, CA 94025 (United States); Department of Science and Environment, Roskilde University, Universitetsvej 1, Roskilde DK-4000 (Denmark)

    2016-11-15

    Highlights: • L. variegatus was exposed to sediment spiked with either aqueous Cu or nanoparticulate CuO. • Both aqueous and nanoparticulate Cu were marginally accumulated by L. variegatus. • Elimination of Cu accumulated from both forms was limited. • The subcellular distribution of accumulated Cu varied between Cu forms. • The use of a tracer, greater exposure concentration and duration are recommended. - Abstract: The use and likely incidental release of metal nanoparticles (NPs) is steadily increasing. Despite the increasing amount of published literature on metal NP toxicity in the aquatic environment, very little is known about the biological fate of NPs after sediment exposures. Here, we compare the bioavailability and subcellular distribution of copper oxide (CuO) NPs and aqueous Cu (Cu-Aq) in the sediment-dwelling worm Lumbriculus variegatus. Ten days (d) sediment exposure resulted in marginal Cu bioaccumulation in L. variegatus for both forms of Cu. Bioaccumulation was detected because isotopically enriched {sup 65}Cu was used as a tracer. Neither burrowing behavior or survival was affected by the exposure. Once incorporated into tissue, Cu loss was negligible over 10 d of elimination in clean sediment (Cu elimination rate constants were not different from zero). With the exception of day 10, differences in bioaccumulation and subcellular distribution between Cu forms were either not detectable or marginal. After 10 d of exposure to Cu-Aq, the accumulated Cu was primarily partitioned in the subcellular fraction containing metallothionein-like proteins (MTLP, ≈40%) and cellular debris (CD, ≈30%). Cu concentrations in these fractions were significantly higher than in controls. For worms exposed to CuO NPs for 10 d, most of the accumulated Cu was partitioned in the CD fraction (≈40%), which was the only subcellular fraction where the Cu concentration was significantly higher than for the control group. Our results indicate that L. variegatus

  18. Detection and subcellular localization of dehydrin-like proteins in quinoa (Chenopodium quinoa Willd.) embryos.

    Science.gov (United States)

    Carjuzaa, P; Castellión, M; Distéfano, A J; del Vas, M; Maldonado, S

    2008-01-01

    The aim of this study was to characterize the dehydrin content in mature embryos of two quinoa cultivars, Sajama and Baer La Unión. Cultivar Sajama grows at 3600-4000 m altitude and is adapted to the very arid conditions characteristic of the salty soils of the Bolivian Altiplano, with less than 250 mm of annual rain and a minimum temperature of -1 degrees C. Cultivar Baer La Unión grows at sea-level regions of central Chile and is adapted to more humid conditions (800 to 1500 mm of annual rain), fertile soils, and temperatures above 5 degrees C. Western blot analysis of embryo tissues from plants growing under controlled greenhouse conditions clearly revealed the presence of several dehydrin bands (at molecular masses of approximately 30, 32, 50, and 55 kDa), which were common to both cultivars, although the amount of the 30 and 32 kDa bands differed. Nevertheless, when grains originated from their respective natural environments, three extra bands (at molecular masses of approximately 34, 38, and 40 kDa), which were hardly visible in Sajama, and another weak band (at a molecular mass of approximately 28 kDa) were evident in Baer La Unión. In situ immunolocalization microscopy detected dehydrin-like proteins in all axis and cotyledon tissues. At the subcellular level, dehydrins were detected in the plasma membrane, cytoplasm and nucleus. In the cytoplasm, dehydrins were found associated with mitochondria, rough endoplasmic reticulum cisternae, and proplastid membranes. The presence of dehydrins was also recognized in the matrix of protein bodies. In the nucleus, dehydrins were associated with the euchromatin. Upon examining dehydrin composition and subcellular localization in two quinoa cultivars belonging to highly contrasting environments, we conclude that most dehydrins detected here were constitutive components of the quinoa seed developmental program, but some of them (specially the 34, 38, and 40 kDa bands) may reflect quantitative molecular differences

  19. Targeted Degradation of Proteins Localized in Subcellular Compartments by Hybrid Small Molecules.

    Science.gov (United States)

    Okuhira, Keiichiro; Shoda, Takuji; Omura, Risa; Ohoka, Nobumichi; Hattori, Takayuki; Shibata, Norihito; Demizu, Yosuke; Sugihara, Ryo; Ichino, Asato; Kawahara, Haruka; Itoh, Yukihiro; Ishikawa, Minoru; Hashimoto, Yuichi; Kurihara, Masaaki; Itoh, Susumu; Saito, Hiroyuki; Naito, Mikihiko

    2017-03-01

    Development of novel small molecules that selectively degrade pathogenic proteins would provide an important advance in targeted therapy. Recently, we have devised a series of hybrid small molecules named SNIPER (specific and nongenetic IAP-dependent protein ERaser) that induces the degradation of target proteins via the ubiquitin-proteasome system. To understand the localization of proteins that can be targeted by this protein knockdown technology, we examined whether SNIPER molecules are able to induce degradation of cellular retinoic acid binding protein II (CRABP-II) proteins localized in subcellular compartments of cells. CRABP-II is genetically fused with subcellular localization signals, and they are expressed in the cells. SNIPER(CRABP) with different IAP-ligands, SNIPER(CRABP)-4 with bestatin and SNIPER(CRABP)-11 with MV1 compound, induce the proteasomal degradation of wild-type (WT), cytosolic, nuclear, and membrane-localized CRABP-II proteins, whereas only SNIPER(CRABP)-11 displayed degradation activity toward the mitochondrial CRABP-II protein. The small interfering RNA-mediated silencing of cIAP1 expression attenuated the knockdown activity of SNIPER(CRABP) against WT and cytosolic CRABP-II proteins, indicating that cIAP1 is the E3 ligase responsible for degradation of these proteins. Against membrane-localized CRABP-II protein, cIAP1 is also a primary E3 ligase in the cells, but another E3 ligase distinct from cIAP2 and X-linked inhibitor of apoptosis protein (XIAP) could also be involved in the SNIPER(CRABP)-11-induced degradation. However, for the degradation of nuclear and mitochondrial CRABP-II proteins, E3 ligases other than cIAP1, cIAP2, and XIAP play a role in the SNIPER-mediated protein knockdown. These results indicate that SNIPER can target cytosolic, nuclear, membrane-localized, and mitochondrial proteins for degradation, but the responsible E3 ligase is different, depending on the localization of the target protein. Copyright © 2017 by

  20. Beyond co-localization: inferring spatial interactions between sub-cellular structures from microscopy images

    Directory of Open Access Journals (Sweden)

    Paul Grégory

    2010-07-01

    Full Text Available Abstract Background Sub-cellular structures interact in numerous direct and indirect ways in order to fulfill cellular functions. While direct molecular interactions crucially depend on spatial proximity, other interactions typically result in spatial correlations between the interacting structures. Such correlations are the target of microscopy-based co-localization analysis, which can provide hints of potential interactions. Two complementary approaches to co-localization analysis can be distinguished: intensity correlation methods capitalize on pattern discovery, whereas object-based methods emphasize detection power. Results We first reinvestigate the classical co-localization measure in the context of spatial point pattern analysis. This allows us to unravel the set of implicit assumptions inherent to this measure and to identify potential confounding factors commonly ignored. We generalize object-based co-localization analysis to a statistical framework involving spatial point processes. In this framework, interactions are understood as position co-dependencies in the observed localization patterns. The framework is based on a model of effective pairwise interaction potentials and the specification of a null hypothesis for the expected pattern in the absence of interaction. Inferred interaction potentials thus reflect all significant effects that are not explained by the null hypothesis. Our model enables the use of a wealth of well-known statistical methods for analyzing experimental data, as demonstrated on synthetic data and in a case study considering virus entry into live cells. We show that the classical co-localization measure typically under-exploits the information contained in our data. Conclusions We establish a connection between co-localization and spatial interaction of sub-cellular structures by formulating the object-based interaction analysis problem in a spatial statistics framework based on nearest-neighbor distance

  1. Uptake and subcellular distribution of triclosan in typical hydrophytes under hydroponic conditions.

    Science.gov (United States)

    He, Yupeng; Nie, Enguang; Li, Chengming; Ye, Qingfu; Wang, Haiyan

    2017-01-01

    The increasing discharge of pharmaceuticals and personal care products (PPCPs) into the environment has generated serious public concern. The recent awareness of the environmental impact of this emerging class of pollutants and their potential adverse effects on human health have been documented in many reports. However, information regarding uptake and intracellular distribution of PPCPs in hydrophytes under hydroponic conditions, and potential human exposure is very limited. A laboratory experiment was conducted using 14C-labeled triclosan (TCS) to investigate uptake and distribution of TCS in six aquatic plants (water spinach, purple perilla, cress, penny grass, cane shoot, and rice), and the subcellular distribution of 14C-TCS was determined in these plants. The results showed that the uptake and removal rate of TCS from nutrient solution by hydrophytes followed the order of cress (96%) > water spinach (94%) > penny grass (87%) > cane shoot (84%) > purple perilla (78%) > rice (63%) at the end of incubation period (192 h). The range of 14C-TCS content in the roots was 94.3%-99.0% of the added 14C-TCS, and the concentrations in roots were 2-3 orders of magnitude greater than those in shoots. Furthermore, the subcellular fraction-concentration factor (3.6 × 102-2.6 × 103 mL g-1), concentration (0.58-4.47 μg g-1), and percentage (30%-61%) of 14C-TCS in organelles were found predominantly greater than those in cell walls and/or cytoplasm. These results indicate that for these plants, the roots are the primary storage for TCS, and within plant cells organelles are the major domains for TCS accumulation. These findings provide a better understanding of translocation and accumulation of TCS in aquatic plants at the cellular level, which is valuable for environmental and human health assessments of TCS. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Movies of cellular and sub-cellular motion by digital holographic microscopy

    Directory of Open Access Journals (Sweden)

    Yu Lingfeng

    2006-03-01

    Full Text Available Abstract Background Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. Methods A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. Results Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable

  3. Partial domain wall partition functions

    National Research Council Canada - National Science Library

    Foda, O; Wheeler, M

    2012-01-01

    We consider six-vertex model configurations on an (n × N) lattice, n ≤ N, that satisfy a variation on domain wall boundary conditions that we define and call partial domain wall boundary conditions...

  4. Physics of partially ionized plasmas

    CERN Document Server

    Krishan, Vinod

    2016-01-01

    Plasma is one of the four fundamental states of matter; the other three being solid, liquid and gas. Several components, such as molecular clouds, diffuse interstellar gas, the solar atmosphere, the Earth's ionosphere and laboratory plasmas, including fusion plasmas, constitute the partially ionized plasmas. This book discusses different aspects of partially ionized plasmas including multi-fluid description, equilibrium and types of waves. The discussion goes on to cover the reionization phase of the universe, along with a brief description of high discharge plasmas, tokomak plasmas and laser plasmas. Various elastic and inelastic collisions amongst the three particle species are also presented. In addition, the author demonstrates the novelty of partially ionized plasmas using many examples; for instance, in partially ionized plasma the magnetic induction is subjected to the ambipolar diffusion and the Hall effect, as well as the usual resistive dissipation. Also included is an observation of kinematic dynam...

  5. Improved prediction of peroxisomal PTS1 proteins from genome sequences based on experimental subcellular targeting analyses as exemplified for protein kinases from Arabidopsis.

    Science.gov (United States)

    Ma, Changle; Reumann, Sigrun

    2008-01-01

    Due to current experimental limitations in peroxisome proteome research, the identification of low-abundance regulatory proteins such as protein kinases largely relies on computational protein prediction. To test and improve the identification of regulatory proteins by such a prediction-based approach, the Arabidopsis genome was screened for genes that encode protein kinases with predicted type 1 or type 2 peroxisome targeting signals (PTS1 or PTS2). Upon transient expression in onion epidermal cells, the predicted PTS1 domains of four of the seven protein kinases re-directed the reporter protein, enhanced yellow green fluorescent (EYFP), to peroxisomes and were thus verified as functional PTS1 domains. The full-length fusions, however, remained cytosolic, suggesting that PTS1 exposure is induced by specific signals. To investigate why peroxisome targeting of three other kinases was incorrectly predicted and ultimately to improve the prediction algorithms, selected amino acid residues located upstream of PTS1 tripeptides were mutated and the effect on subcellular targeting of the reporter protein was analysed. Acidic residues in close proximity to major PTS1 tripeptides were demonstrated to inhibit protein targeting to plant peroxisomes even in the case of the prototypical PTS1 tripeptide SKL>, whereas basic residues function as essential auxiliary targeting elements in front of weak PTS1 tripeptides such as SHL>. The functional characterization of these inhibitory and essential enhancer-targeting elements allows their consideration in predictive algorithms to improve the prediction accuracy of PTS1 proteins from genome sequences.

  6. Hepatocyte nuclear structure and subcellular distribution of copper in zebrafish Brachydanio rerio and roach Rutilus rutilus (Teleostei, Cyprinidae) exposed to copper sulphate

    Energy Technology Data Exchange (ETDEWEB)

    Paris-Palacios, Severine [Universite de Reims Champagne-Ardenne (URCA), UFR Sciences Exactes et Naturelles, Laboratoire d' Eco-Toxicologie, Institut International de Recherche sur les Ions Metalliques, B.P. 1039-51687 Reims cedex 2 (France)]. E-mail: severine.paris@univ-reims.fr; Biagianti-Risbourg, Sylvie [Universite de Reims Champagne-Ardenne (URCA), UFR Sciences Exactes et Naturelles, Laboratoire d' Eco-Toxicologie, Institut International de Recherche sur les Ions Metalliques, B.P. 1039-51687 Reims cedex 2 (France)]. E-mail: sylvie.biagianti@univ-reims.fr

    2006-05-10

    Copper is a trace element essential to life, but also a heavy metal with toxic effect clearly demonstrated. Cu induced perturbations in fish liver are well documented but the variability of the reported results is large. In this study two cyprinids, zebrafish and roach, were exposed to copper. Reported histocytological changes are either adaptative or degenerative depending on fish species, concentration of metal, and duration of exposure. Hepatic subcellular distribution of copper was determined by X-ray microanalysis in control and Cu-exposed roach and zebrafish. Sublethal copper sulphate contamination induced the development of a particular nucleolar alteration forming a network or honeycomb like structure in liver. This perturbation is observable in almost all the hepatocytes of zebrafish and roach exposed to copper for a minimum of 4 days of exposure. It seemed to concern more precisely the pars fibrosa. X-ray microanalysis showed that the appearance of network nucleolus was in relation to a Cu accumulation. Cu deposit was well located in the network as pars granulosa and nucloplasm showed very lower metal concentrations. The origin and consequence of network structure in nucleolus was discussed.

  7. Introduction to partial differential equations

    CERN Document Server

    Greenspan, Donald

    2000-01-01

    Designed for use in a one-semester course by seniors and beginning graduate students, this rigorous presentation explores practical methods of solving differential equations, plus the unifying theory underlying the mathematical superstructure. Topics include basic concepts, Fourier series, second-order partial differential equations, wave equation, potential equation, heat equation, approximate solution of partial differential equations, and more. Exercises appear at the ends of most chapters. 1961 edition.

  8. [Acrylic resin removable partial dentures].

    Science.gov (United States)

    de Baat, C; Witter, D J; Creugers, N H J

    2011-01-01

    An acrylic resin removable partial denture is distinguished from other types of removable partial dentures by an all-acrylic resin base which is, in principle, solely supported by the edentulous regions of the tooth arch and in the maxilla also by the hard palate. When compared to the other types of removable partial dentures, the acrylic resin removable partial denture has 3 favourable aspects: the economic aspect, its aesthetic quality and the ease with which it can be extended and adjusted. Disadvantages are an increased risk of caries developing, gingivitis, periodontal disease, denture stomatitis, alveolar bone reduction, tooth migration, triggering of the gag reflex and damage to the acrylic resin base. Present-day indications are ofa temporary or palliative nature or are motivated by economic factors. Special varieties of the acrylic resin removable partial denture are the spoon denture, the flexible denture fabricated of non-rigid acrylic resin, and the two-piece sectional denture. Furthermore, acrylic resin removable partial dentures can be supplied with clasps or reinforced by fibers or metal wires.

  9. The C-terminal 18 Amino Acid Region of Dengue Virus NS5 Regulates its Subcellular Localization and Contains a Conserved Arginine Residue Essential for Infectious Virus Production.

    Directory of Open Access Journals (Sweden)

    Moon Y F Tay

    2016-09-01

    Full Text Available Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18 alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα, allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.

  10. Location theory a unified approach

    CERN Document Server

    Nickel, Stefan

    2006-01-01

    Although modern location theory is now more than 90 years old, the focus of researchers in this area has been mainly problem oriented. However, a common theory, which keeps the essential characteristics of classical location models, is still missing.This monograph addresses this issue. A flexible location problem called the Ordered Median Problem (OMP) is introduced. For all three main subareas of location theory (continuous, network and discrete location) structural properties of the OMP are presented and solution approaches provided. Numerous illustrations and examples help the reader to bec

  11. N-terminal acetylation by NatC is not a general determinant for substrate subcellular localization in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Henriette Aksnes

    Full Text Available N-terminal acetylation has been suggested to play a role in the subcellular targeting of proteins, in particular those acetylated by the N-terminal acetyltransferase complex NatC. Based on previous positional proteomics data revealing N-terminal acetylation status and the predicted NAT substrate classes, we selected 13 suitable NatC substrates for subcellular localization studies in Saccharomyces cerevisiae. Fluorescence microscopy analysis of GFP-tagged candidates in the presence or absence of the NatC catalytic subunit Naa30 (Mak3 revealed unaltered localization patterns for all 13 candidates, thus arguing against a general role for the N-terminal acetyl group as a localization determinant. Furthermore, all organelle-localized substrates indicated undisrupted structures, thus suggesting that absence of NatC acetylation does not have a vast effect on organelle morphology in yeast.

  12. Endoplasmic reticulum export, subcellular distribution, and fibril formation by Pmel17 require an intact N-terminal domain junction.

    Science.gov (United States)

    Leonhardt, Ralf M; Vigneron, Nathalie; Rahner, Christoph; Van den Eynde, Benoît J; Cresswell, Peter

    2010-05-21

    Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes.

  13. Endoplasmic Reticulum Export, Subcellular Distribution, and Fibril Formation by Pmel17 Require an Intact N-terminal Domain Junction*

    Science.gov (United States)

    Leonhardt, Ralf M.; Vigneron, Nathalie; Rahner, Christoph; Van den Eynde, Benoît J.; Cresswell, Peter

    2010-01-01

    Pmel17 is a melanocyte/melanoma-specific protein that subcellularly localizes to melanosomes, where it forms a fibrillar matrix that serves for the sequestration of potentially toxic reaction intermediates of melanin synthesis and deposition of the pigment. As a key factor in melanosomal biogenesis, understanding intracellular trafficking and processing of Pmel17 is of central importance to comprehend how these organelles are formed, how they mature, and how they function in the cell. Using a series of deletion and missense mutants of Pmel17, we are able to show that the integrity of the junction between the N-terminal region and the polycystic kidney disease-like domain is highly crucial for endoplasmic reticulum export, subcellular targeting, and fibril formation by Pmel17 and thus for establishing functional melanosomes. PMID:20231267

  14. Subcellular localization of the delayed rectifier K(+) channels KCNQ1 and ERG1 in the rat heart

    DEFF Research Database (Denmark)

    Rasmussen, Hanne Borger; Møller, Morten; Knaus, Hans-Günther

    2003-01-01

    In the heart, several K(+) channels are responsible for the repolarization of the cardiac action potential, including transient outward and delayed rectifier K(+) currents. In the present study, the cellular and subcellular localization of the two delayed rectifier K(+) channels, KCNQ1 and ether......-a-go-go-related gene-1 (ERG1), was investigated in the adult rat heart. Confocal immunofluorescence microscopy of atrial and ventricular cells revealed that whereas KCNQ1 labeling was detected in both the peripheral sarcolemma and a structure transversing the myocytes, ERG1 immunoreactivity was confined to the latter....... Immunoelectron microscopy of atrial and ventricular myocytes showed that the ERG1 channel was primarily expressed in the transverse tubular system and its entrance, whereas KCNQ1 was detected in both the peripheral sarcolemma and in the T tubules. Thus, whereas ERG1 displays a very restricted subcellular...

  15. Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

    Science.gov (United States)

    Shrestha, Bindesh; Sripadi, Prabhakar; Reschke, Brent R; Henderson, Holly D; Powell, Matthew J; Moody, Sally A; Vertes, Akos

    2014-01-01

    Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

  16. Defining the subcellular interface of nanoparticles by live-cell imaging.

    Directory of Open Access Journals (Sweden)

    Peter H Hemmerich

    Full Text Available Understanding of nanoparticle-bio-interactions within living cells requires knowledge about the dynamic behavior of nanomaterials during their cellular uptake, intracellular traffic and mutual reactions with cell organelles. Here, we introduce a protocol of combined kinetic imaging techniques that enables investigation of exemplary fluorochrome-labelled nanoparticles concerning their intracellular fate. By time-lapse confocal microscopy we observe fast, dynamin-dependent uptake of polystyrene and silica nanoparticles via the cell membrane within seconds. Fluorescence recovery after photobleaching (FRAP experiments reveal fast and complete exchange of the investigated nanoparticles at mitochondria, cytoplasmic vesicles or the nuclear envelope. Nuclear translocation is observed within minutes by free diffusion and active transport. Fluorescence correlation spectroscopy (FCS and raster image correlation spectroscopy (RICS indicate diffusion coefficients of polystyrene and silica nanoparticles in the nucleus and the cytoplasm that are consistent with particle motion in living cells based on diffusion. Determination of the apparent hydrodynamic radii by FCS and RICS shows that nanoparticles exert their cytoplasmic and nuclear effects mainly as mobile, monodisperse entities. Thus, a complete toolkit of fluorescence fluctuation microscopy is presented for the investigation of nanomaterial biophysics in subcellular microenvironments that contributes to develop a framework of intracellular nanoparticle delivery routes.

  17. Subcellular analysis of interaction between breast cancer cells and drug by digital holography

    Science.gov (United States)

    Zhao, Jie; Lin, Qiaowen; Wang, Dayong; Wang, Yunxin; Ouyang, Liting; Guo, Sha; Yao, Qian

    2017-10-01

    Digital holographic microscopy is a promising quantitative phase-contrast imaging technique, which exhibits the advantages of non-destruction, full field of view, quasi-real time, and don't need dye and external marker to the living biological sample. In this paper, the inverted off-axis image-plane digital holography with pre-magnification is built up to study the living MDA-MB-231 breast cancer cells. The lateral resolution of the proposed experimental setup is 0.87μm, which is verified by the standard USAF test target. Then the system is used to visualize the interaction between living breast cancer cells and drug. The blebbing is observed after the cells are treated by paclitaxel drug, and the distribution of the paclitaxel inside the cells is detected, which is near the cytomembrane, or in other words the end of the microtubules. It will stop the mitosis and cause the death of the cells. It is helpful to reveal the anticancer mechanism of paclitaxel in the subcellular scale.

  18. Subcellular and supracellular mechanical stress prescribes cytoskeleton behavior in Arabidopsis cotyledon pavement cells

    Science.gov (United States)

    Sampathkumar, Arun; Krupinski, Pawel; Wightman, Raymond; Milani, Pascale; Berquand, Alexandre; Boudaoud, Arezki; Hamant, Olivier; Jönsson, Henrik; Meyerowitz, Elliot M

    2014-01-01

    Although it is a central question in biology, how cell shape controls intracellular dynamics largely remains an open question. Here, we show that the shape of Arabidopsis pavement cells creates a stress pattern that controls microtubule orientation, which then guides cell wall reinforcement. Live-imaging, combined with modeling of cell mechanics, shows that microtubules align along the maximal tensile stress direction within the cells, and atomic force microscopy demonstrates that this leads to reinforcement of the cell wall parallel to the microtubules. This feedback loop is regulated: cell-shape derived stresses could be overridden by imposed tissue level stresses, showing how competition between subcellular and supracellular cues control microtubule behavior. Furthermore, at the microtubule level, we identified an amplification mechanism in which mechanical stress promotes the microtubule response to stress by increasing severing activity. These multiscale feedbacks likely contribute to the robustness of microtubule behavior in plant epidermis. DOI: http://dx.doi.org/10.7554/eLife.01967.001 PMID:24740969

  19. [Anti-tumor immunity of Newcastle disease virus HN protein is influenced by differential subcellular targeting].

    Science.gov (United States)

    Wang, Kaibing; Sui, Hong; Li, Lejing; Li, Xi; Wang, Lei

    2010-08-01

    Hemagglutinin-neuraminidase (HN) protein of newcastle disease virus is an important immunogen for oncolysis. We designed three different expression plasmids encoding the HN protein targeted to different subcellular compartments: cytoplasmic (Cy-HN), secreted (Sc-HN) and membrane-anchored (M-HN). On the basis of antitumor effect in vitro, the aim of this study is to investigate the anti-tumor immunity effect of HN protein in vivo. In the present study, we developed a mouse model in order to evaluate the anti-tumor effect of the intratumorally injected modified HN proteins and the anti-tumor immunity by lymphocyte proliferative response and CTL activity test. Although all three DNA constructs elicited an immune response, tumor-bearing mice intratumorally injected with M-HN demonstrated a significantly better anti-tumor effect than those injected with Cy-HN or Sc-HN (Day 18: P=0.022; Day 21: Psubcellular targeting. The membrane-anchored form of the HN protein appears to be an ideal candidate to improve the specific cellular immunity.

  20. Direct imaging the subcellular localization of single-walled carbon nanotubes

    Science.gov (United States)

    Zhou, Feifan; Xing, Da; Chen, Wei R.

    2011-03-01

    The development of single-walled carbon nanotubes (SWNTs) for various biomedical applications is an area of great promise. However, the contradictory data on the interaction of single-walled carbon nanotubes with cells highlight the need to study their uptake and cytotoxic effects in cells. Here, we use confocal microscopy to image the translocation of single-walled carbon nanotubes into cells and localization on the subcellular organelle. We also observe that single-walled carbon nanotubes do not affect the cellular condition and mitochondrial membrane potential. One intrinsic property of single-walled carbon nanotubes is their strong optical absorbance in the near-infrared (NIR) region. It could be used to selectively increase the thermal destructions in the target tumors. A specific type of SWNT by the CoMoCAT method has an intense absorption band at 980 nm. When irradiated with a 980-nm laser, the single-walled carbon nanotubes affect the cellular oxidation and destroy the mitochondrial membrane potential, and induce cell apoptosis. Thus, the single-walled carbon nanotubes appear to enter the cytoplasm without cytotoxic effects in cells, and can be used as effective and selective nanomaterials for cancer photothermal therapy.

  1. Systematic study of subcellular localization of Arabidopsis PPR proteins confirms a massive targeting to organelles.

    Science.gov (United States)

    Colcombet, Jean; Lopez-Obando, Mauricio; Heurtevin, Laure; Bernard, Clément; Martin, Karine; Berthomé, Richard; Lurin, Claire

    2013-01-01

    Four hundred and fifty-eight genes coding for PentatricoPeptide Repeat (PPR) proteins are annotated in the Arabidopsis thaliana genome. Over the past 10 years, numerous reports have shown that many of these proteins function in organelles to target specific transcripts and are involved in post-transcriptional regulation. Therefore, they are thought to be important players in the coordination between nuclear and organelle genome expression. Only four of these proteins have been described to be addressed outside organelles, indicating that some PPRs could function in post-transcriptional regulations of nuclear genes. In this work, we updated and improved our current knowledge on the localization of PPR proteins of Arabidopsis within the plant cell. We particularly investigated the subcellular localization of 166 PPR proteins whose targeting predictions were ambiguous, using a combination of high-throughput cloning and microscopy. Through systematic localization experiments and data integration, we confirmed that PPR proteins are largely targeted to organelles and showed that dual targeting to both the mitochondria and plastid occurs more frequently than expected. These results allow us to speculate that dual-targeted PPR proteins could be important for the fine coordination of gene expressions in both organelles.

  2. Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution

    Science.gov (United States)

    Thiery-Lavenant, Gwendoline; Zavalin, Andre I.; Caprioli, Richard M.

    2013-04-01

    Targeted multiplex imaging mass spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This article describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.

  3. Differential subcellular targeting of glutamate receptor subtypes during homeostatic synaptic plasticity.

    Science.gov (United States)

    Soares, Cary; Lee, Kevin F H; Nassrallah, Wissam; Béïque, Jean-Claude

    2013-08-14

    Homeostatic processes are believed to contribute to the stability of neuronal networks that are perpetually influenced by Hebbian forms of synaptic plasticity. Whereas the rules governing the targeting and trafficking of AMPA and NMDA subtypes of glutamate receptors during rapid Hebbian LTP have been extensively studied, those that are operant during homeostatic forms of synaptic strengthening are less well understood. Here, we used biochemical, biophysical, and pharmacological approaches to investigate glutamate receptor regulation during homeostatic synaptic plasticity. We show in rat organotypic hippocampal slices that prolonged network silencing induced a robust surface upregulation of GluA2-lacking AMPARs, not only at synapses, but also at extrasynaptic dendritic and somatic regions of CA1 pyramidal neurons. We also detected a shift in NMDAR subunit composition that, in contrast to the cell-wide surface delivery of GluA2-lacking AMPARs, occurred exclusively at synapses. The subunit composition and subcellular distribution of AMPARs and NMDARs are therefore distinctly regulated during homeostatic synaptic plasticity. Thus, because subunit composition dictates key channel properties, such as agonist affinity, gating kinetics, and calcium permeability, the homeostatic synaptic process transcends the simple modulation of synaptic strength by also regulating the signaling and integrative properties of central synapses.

  4. Diversity in subcellular targeting of the PP2A B'eta subfamily members.

    Science.gov (United States)

    Matre, Polina; Meyer, Christian; Lillo, Cathrine

    2009-10-01

    Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase comprising a catalytic subunit (C), a scaffolding subunit (A), and a regulatory subunit (B). The B subunits are believed to be responsible for substrate specificity and localization of the PP2A complex. In plants, three families of B subunits exist, i.e. B (B55), B', and B''. Here, we report differential subcellular targeting within the Arabidopsis B'eta subfamily, which consists of the close homologs B'eta, B'theta, B'gamma and B'zeta. Phenotypes of corresponding knockouts were observed, and particularly revealed delayed flowering for the B'eta knockout. The B' subunits were linked to fluorescent tags and transiently expressed in various tissues of onion, tobacco and Arabidopsis. B'eta and B'gamma targeted the cytosol and nucleus. B'zeta localized to the cytoplasm and partly co-localized with mitochondrial markers when the N-terminus was free. Provided its C-terminus was free, the B'theta subunit targeted peroxisomes. The importance of the C-terminal end for peroxisomal targeting was further confirmed by truncation of the C-terminus. The results revealed that the closely related B' subunits are targeting different organelles in plants, and exemplify the usage of the peptide serine-serine-leucine as a PTS1 peroxisomal signaling peptide.

  5. Design of a Novel Equi-Biaxial Stretcher for Live Cellular and Subcellular Imaging.

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    Jasmin Imsirovic

    Full Text Available Cells in the body experience various mechanical stimuli that are often essential to proper cell function. In order to study the effects of mechanical stretch on cell function, several devices have been built to deliver cyclic stretch to cells; however, they are generally not practical for live cell imaging. We introduce a novel device that allows for live cell imaging, using either an upright or inverted microscope, during the delivery of cyclic stretch, which can vary in amplitude and frequency. The device delivers equi-biaxial strain to cells seeded on an elastic membrane via indentation of the membrane. Membrane area strain was calibrated to indenter depth and the device showed repeatable and accurate delivery of strain at the scale of individual cells. At the whole cell level, changes in intracellular calcium were measured at different membrane area strains, and showed an amplitude-dependent response. At the subcellular level, the mitochondrial network was imaged at increasing membrane area strains to demonstrate that stretch can lead to mitochondrial fission in lung fibroblasts. The device is a useful tool for studying transient as well as long-term mechanotransduction as it allows for simultaneous stretching and imaging of live cells in the presence of various chemical stimuli.

  6. Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds

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    Miguel A. Ibeas

    2017-12-01

    Full Text Available Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

  7. Fixation protocols for subcellular imaging by synchrotron-based Fourier transform infrared microspectroscopy.

    Science.gov (United States)

    Gazi, E; Dwyer, J; Lockyer, N P; Miyan, J; Gardner, P; Hart, C; Brown, M; Clarke, N W

    2005-01-01

    Synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy is a powerful bioanalytical technique for the simultaneous analysis of lipids, proteins, carbohydrates, and a variety of phosphorylated molecules within intact cells. SR-FTIR microspectroscopy can be used in the imaging mode to generate biospectroscopic maps of the distribution and intensity profiles of subcellular biomolecular domains at diffraction-limited spatial resolution. However, the acquisition of highly spatially resolved IR images of cells is not only a function of instrumental parameters (source brightness, sampling aperture size) but also the cell preparation method employed. Additionally, for the IR data to be biochemically relevant the cells must be preserved in a life-like state without introducing artefacts. In the present study we demonstrate, for the first time, the differences in biomolecular localizations observed in SR-FTIR images of cells fixed by formalin, formalin-critical point drying (CPD), and glutaraldehyde-osmium tetroxide-CPD, using the PC-3 prostate cancer cell line. We compare these SR-FTIR images of fixed cells to unfixed cells. The influence of chemical fixatives on the IR spectrum is discussed in addition to the biological significance of the observed localizations. Our experiments reveal that formalin fixation at low concentration preserves lipid, phosphate, and protein components without significantly influencing the IR spectrum of the cell. (c) 2004 Wiley Periodicals, Inc.

  8. Arabinogalactan Glycosyltransferases: Enzyme Assay, Protein-Protein Interaction, Subcellular Localization, and Perspectives for Application

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    Naomi Geshi

    2014-01-01

    Full Text Available Arabinogalactan proteins (AGPs are abundant extracellular proteoglycans that are found in most plant species and involved in many cellular processes, such as cell proliferation and survival, pattern formation, and growth, and in plant microbe interaction. AGPs are synthesized by posttranslational O-glycosylation of proteins and attached glycan part often constitutes greater than 90% of the molecule. Subtle altered glycan structures during development have been considered to function as developmental markers on the cell surface, but little is known concerning the molecular mechanisms. My group has been working on glycosylation enzymes (glycosyltransferases of AGPs to investigate glycan function of the molecule. This review summarizes the recent findings from my group as for AtGalT31A, AtGlcAT14A-C, and AtGalT29A of Arabidopsis thaliana with a specific focus on the (i biochemical enzyme activities; (ii subcellular compartments targeted by the glycosyltransferases; and (iii protein-protein interactions. I also discuss application aspect of glycosyltransferase in improving AGP-based product used in industry, for example, gum arabic.

  9. Subcellular metabolite and lipid analysis of Xenopus laevis eggs by LAESI mass spectrometry.

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    Bindesh Shrestha

    Full Text Available Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI, an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen, were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.

  10. Subcellular Nanorheology Reveals Lysosomal Viscosity as a Reporter for Lysosomal Storage Diseases.

    Science.gov (United States)

    Devany, John; Chakraborty, Kasturi; Krishnan, Yamuna

    2018-02-14

    We describe a new method to measure viscosity within subcellular organelles of a living cell using nanorheology. We demonstrate proof of concept by measuring viscosity in lysosomes in multiple cell types and disease models. The lysosome is an organelle responsible for the breakdown of complex biomolecules. When different lysosomal proteins are defective, they are unable to break down specific biological substrates, which get stored within the lysosome, causing about 70 fatal diseases called lysosomal storage disorders (LSDs). Although the buildup of storage material is critical to the pathology of these diseases, methods to monitor cargo accumulation in the lysosome are lacking for most LSDs. Using passive particle tracking nanorheology and fluorescence recovery after photobleaching, we report that viscosity in the lysosome increases significantly during cargo accumulation in several LSD models. In a mammalian cell culture model of Niemann Pick C, lysosomal viscosity directly correlates with the levels of accumulated cholesterol. We also observed increased viscosity in diverse LSD models in Caenorhabditis elegans, revealing that lysosomal viscosity is a powerful reporter with which to monitor substrate accumulation in LSDs for new diagnostics or to assay therapeutic efficacy.

  11. Subcellular distribution of folate and folate binding protein in renal proximal tubules

    Energy Technology Data Exchange (ETDEWEB)

    Sharkey, C.; Hjelle, J.T.; Selhub, J.

    1986-03-01

    High affinity folate binding protein (FBP) found in brush border membranes derived from renal cortices is thought to be involved in the renal conservation of folate. To examine the mechanisms of folate recovery, the subcellular distribution of FBP and /sup 3/H-folate in rabbit renal proximal tubules (PT) was examined using analytical cell fractionation techniques. Tubules contain 3.41 +/- 0.32 picomoles FBP/mg protein (X +/- S.D.; n = 5). Postnuclear supernates (PNS) of PT were layered atop Percoll-sucrose gradients, centrifuged, fractions collected and assayed for various marker enzymes and FBP. Pooled fractions from such gradients were subsequently treated with digitonin and centrifuged in a stoichiometric manner with the activity of the microvillar enzyme, alanylaminopeptidase (AAP); excess FBP distributed with more buoyant particles. Infusion of /sup 3/H-folate into rabbit kidneys followed by tubule isolation and fractionation revealed a time dependent shift in distribution of radiolabel from the AAP-rich gradient fractions to a region containing more buoyant particles; radiolevel was not associated with lysosomal markers. EM-radioautography revealed grains over intracellular vesicles. These results are consistent with the hypothesis that folate is recovered by a process involving receptor-mediated endocytosis or transcytosis.

  12. Arginine methylation controls the subcellular localization and functions of the oncoprotein splicing factor SF2/ASF.

    Science.gov (United States)

    Sinha, Rahul; Allemand, Eric; Zhang, Zuo; Karni, Rotem; Myers, Michael P; Krainer, Adrian R

    2010-06-01

    Alternative splicing and posttranslational modifications (PTMs) are major sources of protein diversity in eukaryotic proteomes. The SR protein SF2/ASF is an oncoprotein that functions in pre-mRNA splicing, with additional roles in other posttranscriptional and translational events. Functional studies of SR protein PTMs have focused exclusively on the reversible phosphorylation of Ser residues in the C-terminal RS domain. We confirmed that human SF2/ASF is methylated at residues R93, R97, and R109, which were identified in a global proteomic analysis of Arg methylation, and further investigated whether these methylated residues regulate the properties of SF2/ASF. We show that the three arginines additively control the subcellular localization of SF2/ASF and that both the positive charge and the methylation state are important. Mutations that block methylation and remove the positive charge result in the cytoplasmic accumulation of SF2/ASF. The consequent decrease in nuclear SF2/ASF levels prevents it from modulating the alternative splicing of target genes, results in higher translation stimulation, and abrogates the enhancement of nonsense-mediated mRNA decay. This study addresses the mechanisms by which Arg methylation and the associated positive charge regulate the activities of SF2/ASF and emphasizes the significance of localization control for an oncoprotein with multiple functions in different cellular compartments.

  13. Cellular and subcellular aquaporin-4 distribution in the mouse neurohypophysis and the effects of osmotic stimulation.

    Science.gov (United States)

    Mesbah-Benmessaoud, Ouahiba; Benabdesselam, Roza; Hardin-Pouzet, Hélène; Dorbani-Mamine, Latifa; Grange-Messent, Valérie

    2011-01-01

    Water channel aquaporin-4 (AQP4) is the most abundant water channel in the rodent brain and is mainly expressed in cerebral areas involved in central osmoreception and osmoregulation. The neurohypophysis is the release site of hypothalamic neurohormones vasopressin and oxytocin, which are involved in the regulation of the water balance. The authors investigated the cellular and subcellular distribution of AQP4 in the mouse neurohypophysis before and after chronic osmotic stimulation, using immunofluorescence microscopy and immunoperoxidase electron microscopy. They showed that AQP4 was abundant in the mouse hypophysis, mainly in the neural lobe. AQP4 was discontinuously distributed along pituicytes plasma membranes, in the dense neurosecretory granules and microvesicles of nerve endings and fibers, and along the luminal and abluminal membranes of fenestrated capillary endothelial cells. After chronic osmotic stimulation, AQP4 immunolabeling was enhanced. Taken together, these results suggest that AQP4 could be involved in the pituicyte sensor effect during osmoregulation, the modification and/or maturation mechanism of neurosecretory granules during neurohormone release, and the blood perfusion of the hypophysis.

  14. Using fluorescence lifetime microscopy to study the subcellular localization of anthocyanins.

    Science.gov (United States)

    Chanoca, Alexandra; Burkel, Brian; Kovinich, Nik; Grotewold, Erich; Eliceiri, Kevin W; Otegui, Marisa S

    2016-12-01

    Anthocyanins are flavonoid pigments that accumulate in most seed plants. They are synthesized in the cytoplasm but accumulate inside the vacuoles. Anthocyanins are pigmented at the lower vacuolar pH, but in the cytoplasm they can be visualized based on their fluorescence properties. Thus, anthocyanins provide an ideal system for the development of new methods to investigate cytoplasmic pools and association with other molecular components. We have analyzed the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), in both in vitro and in vivo conditions, using wild-type and mutant Arabidopsis thaliana seedlings. Within plant cells, the amplitude-weighted mean fluorescence lifetime (τm ) correlated with distinct subcellular localizations of anthocyanins. The vacuolar pool of anthocyanins exhibited shorter τm than the cytoplasmic pool. Consistently, lowering the pH of anthocyanins in solution shortened their fluorescence decay. We propose that FLIM is a useful tool for understanding the trafficking of anthocyanins and, potentially, for estimating vacuolar pH inside intact plant cells. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  15. Subcellular localization of flavonol aglycone in hepatocytes visualized by confocal laser scanning fluorescence microscope.

    Science.gov (United States)

    Mukai, Rie; Shirai, Yasuhito; Saito, Naoaki; Yoshida, Ken-Ichi; Ashida, Hitoshi

    2009-04-01

    Flavonoids are widely distributed in the plant kingdom and show various biological activities. The bioavailability of flavonoids in biological samples has conventionally been quantified by high-performance liquid chromatography and mass spectrometry, but with these analytical techniques it is difficult to estimate the subcellular localization of flavonoids in intact cells. In this study, we attempted to examine the localization of flavonoids in cultured cells using a confocal laser scanning fluorescence microscope and mouse hepatoma Hepa-1c1c7 cells. Five flavonol aglycones showed autofluorescence in the cells under the conditions (Ex. 488 nm to Em. 515-535 nm), whereas three flavonol glycosides and eight compounds belonging to other flavonoid subclasses, i.e., flavones, flavanones, and catechins, did not. The autofluorescence of galangin and kaempferol appeared stronger in the nucleus than cytoplasm, suggesting that they are incorporated into the cells and accumulated in the nucleus. The proposed method provided evidence that flavonol aglycones are incorporated into, and accumulated in the nucleus of, hepatocytes.

  16. Proteomic Analysis of Lysine Acetylation Sites in Rat Tissues Reveals Organ Specificity and Subcellular Patterns

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    Alicia Lundby

    2012-08-01

    Full Text Available Lysine acetylation is a major posttranslational modification involved in a broad array of physiological functions. Here, we provide an organ-wide map of lysine acetylation sites from 16 rat tissues analyzed by high-resolution tandem mass spectrometry. We quantify 15,474 modification sites on 4,541 proteins and provide the data set as a web-based database. We demonstrate that lysine acetylation displays site-specific sequence motifs that diverge between cellular compartments, with a significant fraction of nuclear sites conforming to the consensus motifs G-AcK and AcK-P. Our data set reveals that the subcellular acetylation distribution is tissue-type dependent and that acetylation targets tissue-specific pathways involved in fundamental physiological processes. We compare lysine acetylation patterns for rat as well as human skeletal muscle biopsies and demonstrate its general involvement in muscle contraction. Furthermore, we illustrate that acetylation of fructose-bisphosphate aldolase and glycerol-3-phosphate dehydrogenase serves as a cellular mechanism to switch off enzymatic activity.

  17. Silicon modifies root anatomy, and uptake and subcellular distribution of cadmium in young maize plants.

    Science.gov (United States)

    Vaculík, Marek; Landberg, Tommy; Greger, Maria; Luxová, Miroslava; Stoláriková, Miroslava; Lux, Alexander

    2012-07-01

    Silicon (Si) has been shown to ameliorate the negative influence of cadmium (Cd) on plant growth and development. However, the mechanism of this phenomenon is not fully understood. Here we describe the effect of Si on growth, and uptake and subcellular distribution of Cd in maize plants in relation to the development of root tissues. Young maize plants (Zea mays) were cultivated for 10 d hydroponically with 5 or 50 µm Cd and/or 5 mm Si. Growth parameters and the concentrations of Cd and Si were determined in root and shoot by atomic absorption spectrometry or inductively coupled plasma mass spectroscopy. The development of apoplasmic barriers (Casparian bands and suberin lamellae) and vascular tissues in roots were analysed, and the influence of Si on apoplasmic and symplasmic distribution of (109)Cd applied at 34 nm was investigated between root and shoot. Si stimulated the growth of young maize plants exposed to Cd and influenced the development of Casparian bands and suberin lamellae as well as vascular tissues in root. Si did not affect the distribution of apoplasmic and symplasmic Cd in maize roots, but considerably decreased symplasmic and increased apoplasmic concentration of Cd in maize shoots. Differences in Cd uptake of roots and shoots are probably related to the development of apoplasmic barriers and maturation of vascular tissues in roots. Alleviation of Cd toxicity by Si might be attributed to enhanced binding of Cd to the apoplasmic fraction in maize shoots.

  18. Tissue distribution and subcellular localization of the cardiac sodium channel during mouse heart development.

    Science.gov (United States)

    Domínguez, Jorge N; de la Rosa, Angel; Navarro, Francisco; Franco, Diego; Aránega, Amelia E

    2008-04-01

    The aim of this study was to analyse the mRNA expression levels and protein distribution of the cardiac sodium channel Scn5a/Nav1.5 during mouse cardiogenesis. Scn5a mRNA levels were determined by real-time RT-PCR using embryonic hearts ranging from E9.5 to E17.5 as well as postnatal and adult hearts. In addition, Scn5a protein (Nav1.5) distribution was analysed by immunohistochemistry and confocal microscopy. Scn5a mRNA levels displayed a peak at stage E11.5, decreased during the subsequent stages and then steadily increased from E17.5 onwards, and throughout the postnatal to the adult stages. Immunohistochemistry experiments revealed comparable distribution of Nav1.5 between the different cardiac chambers at early embryonic stages. During the foetal stages, Nav1.5 showed an enhanced expression in the trabeculated myocardium and in the bundle branches. At the subcellular level, Nav1.5 and Scn1b double-immunostaining analysis is consistent with the presence of both sodium channel subunits in the T-tubule system and the intercalated discs. Our results demonstrate that the cardiac sodium channel, Nav1.5, shows a dynamic expression pattern during mouse heart development, indicating that it could play an important role in the acquisition of a mature pattern of conduction and contraction during cardiogenesis.

  19. Studying subcellular detail in fixed astrocytes: Dissociation of morphologically intact glial cells (DIMIGs

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    Julia eHaseleu

    2013-05-01

    Full Text Available Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs, since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from antigen in surrounding non-glial cells. Yet, PAPs frequently display antigens related to motility and glia-synaptic interaction. Here, we describe the dissociation of morphologically intact glial cells (DIMIGs, permitting unambiguous antigen localization using epifluorescence microscopy. Astrocytes are dissociated from juvenile (p13-15 mouse cortex by applying papain treatment and cytospin centrifugation to attach the cells to a slide. The cells and their complete processes including the PAPs is thus projected in 2D. The entire procedure takes 2½-3 hours. We show by morphometry that the diameter of DIMIGs, including the PAPs is similar to that of astrocytes in situ. In contrast to cell culture, results derived from this procedure allow for direct conclusions relating to (i the presence of an antigen in cortical astrocytes, (ii subcellular antigen distribution, in particular when localized in the PAPs. The detailed resolution is shown in an exemplary study of the organization of the astrocytic cytoskeleton components actin, ezrin, tubulin, and GFAP. The distribution of connexin 43 in relation to a single astrocyte’s process tree is also investigated.

  20. Subcellular clearance and accumulation of Huntington disease protein: A mini-review

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    Ting eZhao

    2016-04-01

    Full Text Available Huntington’s disease (HD is an autosomal dominant, progressive neurodegenerative disease caused by an expanded polyglutamine (polyQ tract in the N-terminal region of mutant huntingtin (mHtt. As a result, mHtt forms aggregates that are abundant in the nuclei and processes of neuronal cells. Although the roles of mHtt aggregates are still debated, the formation of aggregates points to deficient clearance of mHtt in brain cells. Since the accumulation of mHtt is a prerequisite for its neurotoxicity, exploring the mechanisms for mHtt accumulation and clearance would advance our understanding of HD pathogenesis and help us develop treatments for HD. We know that the ubiquitin-proteasome system and autophagy play important roles in clearing mHtt; however, how mHtt preferentially accumulates in neuronal nuclei and processes remains unclear. Studying the clearance of mHtt in neuronal cells is a challenge because neurons are morphologically and functionally polarized, which means the turnover of mHtt may be distinct in different cellular compartments. In this review, we discuss our current knowledge about the clearance and accumulation of mHtt and strategies of examining mHtt clearance and accumulation in different subcellular regions

  1. Subcellular localization of Arabidopsis arogenate dehydratases suggests novel and non-enzymatic roles

    Science.gov (United States)

    Bross, Crystal D.; Howes, Travis R.; Abolhassani Rad, Sara; Kljakic, Ornela

    2017-01-01

    Abstract Arogenate dehydratases (ADTs) catalyze the final step in phenylalanine biosynthesis in plants. The Arabidopsis thaliana genome encodes a family of six ADTs capable of decarboxylating/dehydrating arogenate into phenylalanine. Using cyan fluorescent protein (CFP)-tagged proteins, the subcellular localization patterns of all six A. thaliana ADTs were investigated in intact Nicotiana benthamiana and A. thaliana leaf cells. We show that A. thaliana ADTs localize to stroma and stromules (stroma-filled tubules) of chloroplasts. This localization pattern is consistent with the enzymatic function of ADTs as many enzymes required for amino acid biosynthesis are primarily localized to chloroplasts, and stromules are thought to increase metabolite transport from chloroplasts to other cellular compartments. Furthermore, we provide evidence that ADTs have additional, non-enzymatic roles. ADT2 localizes in a ring around the equatorial plane of chloroplasts or to a chloroplast pole, which suggests that ADT2 is a component of the chloroplast division machinery. In addition to chloroplasts, ADT5 was also found in nuclei, again suggesting a non-enzymatic role for ADT5. We also show evidence that ADT5 is transported to the nucleus via stromules. We propose that ADT2 and ADT5 are moonlighting proteins that play an enzymatic role in phenylalanine biosynthesis and a second role in chloroplast division or transcriptional regulation, respectively. PMID:28338876

  2. Sub-cellular organization of the melanin-concentrating hormone neurons in the hypothalamus.

    Science.gov (United States)

    Jancsik, Veronika; Bene, Roland; Sótonyi, Péter; Zachar, Gergely

    2018-01-01

    Melanin-concentrating hormone (MCH) is a potent orexigenic and sleep-promoting neuropeptide in mammals produced predominately by hypothalamic neurons which project to a wide variety of brain areas. Several MCH producing neurons contain MCH as the only neuropeptide, while others comprise cocaine- and amphetamine regulated transcript (CART) as well. The intrahypothalamic localization and the projection pattern of these two subpopulations are distinct. To provide structural grounding to understand the mechanism of action of MCH neurons we show here the subcellular localization of the neuropeptides in the two subpopulations within the hypothalamus of healthy young male mice by applying single and double immunofluorescence labelling.; Thick, prominent MCH immunopositive reticulation and fine discrete granules are detected within the perikarya of both CART positive and CART-free MCH neurons. Typically, one or more immunoreactive processes emanate from the perikarya. The bulk of CART immunoreactivity is also centrally positioned, surrounded by sparse immunoreactive granules within the perikarya and in the processes. In double immunopositive neurons, the two neuropeptides seem to colocalize in the heavily labelled central area, while the immunopositive granules in the cell body periphery and in the processes apparently contain either MCH or CART. This spatial arrangement suggests that MCH and CART, after being synthetized and processed in the endoplasmic reticulum/Golgi complex, are sorted into separate dense core vesicles, which then enter into the cell processes. This mechanism allows for both concerted and independent regulation of the transport and release of MCH and CART. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds.

    Science.gov (United States)

    Ibeas, Miguel A; Grant-Grant, Susana; Navarro, Nathalia; Perez, M F; Roschzttardtz, Hannetz

    2017-01-01

    Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

  4. Perspective: On the importance of hydrodynamic interactions in the subcellular dynamics of macromolecules.

    Science.gov (United States)

    Skolnick, Jeffrey

    2016-09-14

    An outstanding challenge in computational biophysics is the simulation of a living cell at molecular detail. Over the past several years, using Stokesian dynamics, progress has been made in simulating coarse grained molecular models of the cytoplasm. Since macromolecules comprise 20%-40% of the volume of a cell, one would expect that steric interactions dominate macromolecular diffusion. However, the reduction in cellular diffusion rates relative to infinite dilution is due, roughly equally, to steric and hydrodynamic interactions, HI, with nonspecific attractive interactions likely playing rather a minor role. HI not only serve to slow down long time diffusion rates but also cause a considerable reduction in the magnitude of the short time diffusion coefficient relative to that at infinite dilution. More importantly, the long range contribution of the Rotne-Prager-Yamakawa diffusion tensor results in temporal and spatial correlations that persist up to microseconds and for intermolecular distances on the order of protein radii. While HI slow down the bimolecular association rate in the early stages of lipid bilayer formation, they accelerate the rate of large scale assembly of lipid aggregates. This is suggestive of an important role for HI in the self-assembly kinetics of large macromolecular complexes such as tubulin. Since HI are important, questions as to whether continuum models of HI are adequate as well as improved simulation methodologies that will make simulations of more complex cellular processes practical need to be addressed. Nevertheless, the stage is set for the molecular simulations of ever more complex subcellular processes.

  5. Fast two-photon imaging of subcellular voltage dynamics in neuronal tissue with genetically encoded indicators.

    Science.gov (United States)

    Chamberland, Simon; Yang, Helen H; Pan, Michael M; Evans, Stephen W; Guan, Sihui; Chavarha, Mariya; Yang, Ying; Salesse, Charleen; Wu, Haodi; Wu, Joseph C; Clandinin, Thomas R; Toth, Katalin; Lin, Michael Z; St-Pierre, François

    2017-07-27

    Monitoring voltage dynamics in defined neurons deep in the brain is critical for unraveling the function of neuronal circuits but is challenging due to the limited performance of existing tools. In particular, while genetically encoded voltage indicators have shown promise for optical detection of voltage transients, many indicators exhibit low sensitivity when imaged under two-photon illumination. Previous studies thus fell short of visualizing voltage dynamics in individual neurons in single trials. Here, we report ASAP2s, a novel voltage indicator with improved sensitivity. By imaging ASAP2s using random-access multi-photon microscopy, we demonstrate robust single-trial detection of action potentials in organotypic slice cultures. We also show that ASAP2s enables two-photon imaging of graded potentials in organotypic slice cultures and in Drosophila . These results demonstrate that the combination of ASAP2s and fast two-photon imaging methods enables detection of neural electrical activity with subcellular spatial resolution and millisecond-timescale precision.

  6. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

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    S Andrea Moreno

    2015-06-01

    Full Text Available Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF of T. b. brucei: (i fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme in glycosomes, (ii enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes and a GAPDH isoenzyme in the cytosol, (iii malate dehydrogenase in cytosol and (iv glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  7. Studying subcellular detail in fixed astrocytes: dissociation of morphologically intact glial cells (DIMIGs).

    Science.gov (United States)

    Haseleu, Julia; Anlauf, Enrico; Blaess, Sandra; Endl, Elmar; Derouiche, Amin

    2013-01-01

    Studying the distribution of astrocytic antigens is particularly hard when they are localized in their fine, peripheral astrocyte processes (PAPs), since these processes often have a diameter comparable to vesicles and small organelles. The most appropriate technique is immunoelectron microscopy, which is, however, a time-consuming procedure. Even in high resolution light microscopy, antigen localization is difficult to detect due to the small dimensions of these processes, and overlay from antigen in surrounding non-glial cells. Yet, PAPs frequently display antigens related to motility and glia-synaptic interaction. Here, we describe the dissociation of morphologically intact glial cells (DIMIGs), permitting unambiguous antigen localization using epifluorescence microscopy. Astrocytes are dissociated from juvenile (p13-15) mouse cortex by applying papain treatment and cytospin centrifugation to attach the cells to a slide. The cells and their complete processes including the PAPs is thus projected in 2D. The entire procedure takes 2.5-3 h. We show by morphometry that the diameter of DIMIGs, including the PAPs is similar to that of astrocytes in situ. In contrast to cell culture, results derived from this procedure allow for direct conclusions relating to (1) the presence of an antigen in cortical astrocytes, (2) subcellular antigen distribution, in particular when localized in the PAPs. The detailed resolution is shown in an exemplary study of the organization of the astrocytic cytoskeleton components actin, ezrin, tubulin, and GFAP. The distribution of connexin 43 in relation to a single astrocyte's process tree is also investigated.

  8. Subcellular distribution of glutathione and its dynamic changes under oxidative stress in the yeast Saccharomyces cerevisiae

    Science.gov (United States)

    Zechmann, Bernd; Liou, Liang-Chun; Koffler, Barbara E; Horvat, Lucija; Tomašić, Ana; Fulgosi, Hrvoje; Zhang, Zhaojie

    2011-01-01

    Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H2O2) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress. PMID:22093747

  9. An expectation maximization based method for subcellular particle tracking using multi-angle TIRF microscopy.

    Science.gov (United States)

    Liang, Liang; Shen, Hongying; De Camilli, Pietro; Toomre, Derek K; Duncan, James S

    2011-01-01

    Multi-angle total internal reflection fluorescence microscopy (MA-TIRFM) is a new generation of TIRF microscopy to study cellular processes near dorsal cell membrane in 4 dimensions (3D+t). To perform quantitative analysis using MA-TIRFM, it is necessary to track subcellular particles in these processes. In this paper, we propose a method based on a MAP framework for automatic particle tracking and apply it to track clathrin coated pits (CCPs). The expectation maximization (EM) algorithm is employed to solve the MAP problem. To provide the initial estimations for the EM algorithm, we develop a forward filter based on the most probable trajectory (MPT) filter. Multiple linear models are used to model particle dynamics. For CCP tracking, we use two linear models to describe constrained Brownian motion and fluorophore variation according to CCP properties. The tracking method is evaluated on synthetic data and results show that it has high accuracy. The result on real data confirmed by human expert cell biologists is also presented.

  10. Immunocytochemical localization and subcellular site of lectin synthesis in developing wheat embryos

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Peumans, W.J.

    1986-04-01

    Appearance of wheat germ agglutinin (WGA) during wheat germ embryogenesis was studied using indirect immunocytochemical methods at the light and electron microscope levels. Developing embryos were labelled with (/sup 35/S) cysteine in pulse and pulse-chase experiments to study the synthesis and transport of the lectin to protein bodies (PB). The radical, and coleorhiza first accumulated WGA around 10 days post anthesis (DPA), while WGA was found in the epiblast and coleoptile 15 and 20 DPA respectively. At the subcellular level WGA can be seen first in a small developing PB which later fused with larger ones. WGA was distributed evenly throughout the PB. When tissue was pulse-labelled, fractionated on an isopycnic sucrose gradient and exposed to detergent, the incorporated radioactivity of released lectin coincided with the position of the endoplasmic reticulum (ER) marker enzyme NADH-cytochrome c reductase. Both radioactivity and enzyme activity shifted to a higher density in the presence of 2 mM Mg acetate, indicating that radioactive lectin was associated with the rough ER.

  11. Location of Urban Logistic Terminals as Hub Location Problem

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    Jasmina Pašagić Škrinjar

    2012-09-01

    Full Text Available In this paper the problems of locating urban logistic terminals are studied as hub location problems that due to a large number of potential nodes in big cities belong to hard non-polynomial problems, the so-called NP-problems. The hub location problems have found wide application in physical planning of transport and telecommunication systems, especially systems of fast delivery, networks of logistic and distribution centres and cargo traffic terminals of the big cities, etc. The paper defines single and multiple allocations and studies the numerical examples. The capacitated single allocation hub location problems have been studied, with the provision of a mathematical model of selecting the location for the hubs on the network. The paper also presents the differences in the possibilities of implementing the exact and heuristic methods to solve the actual location problems of big dimensions i.e. hub problems of the big cities.

  12. The Ibibio Locative Copular Constructions

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    Ogbonna Anyanwu

    2010-10-01

    Full Text Available The Ibibio language, a member of the Lower Cross group of languages is predominantly spoken in Akwa Ibom State, Nigeria. Ibibio has two types of copular construction. One is locative while the other is predicative. Using a purely descriptive approach/method, this paper provides a descriptive account of the copular locative constructions in Ibibio. This study is based on a database collected from adult speakers of Ibibio by the author using an elicitation list. The database includes both actual and potential words/sentences, which standard Ibibio speakers found to be in consistent with their language rules.The paper has observed that Ibibio copular locatives make use of locative copular verbs which have semantic content, but are however, with indeterminate locative precision except when they co- occur with an appropriate locative complement. It has further observed that in Ibibio, the location of a subject entity can be marked with locative elements in two ways. In the first instance, a locative copular verb which can be dórò, ‘be on’ síne ‘be in’ bà ‘be at’ obligatorily co-occurs with a deitic locative complement or a locative prepositional phrase (PP complement headed by a multipurpose preposition (P, ké. The P is semantically, interpreted as either on, in, at or under depending on the particular locative copular verb that it co-occurs with. In the second instance, in addition to the locative copular verb and the locative PP complement headed by the P ké, there is also within the PP complement (headed by the P ké , a locative relational noun modifier which, modifies the head noun, a complement to the locative P ké. Based on Ameka andOgbonna Anyanwu71Levinson’s (2007 cross-linguistic classification of positional and locative verbs, the paper concludes that Ibibio belongs to the multi-verb type of languages with inherently locative copular verbs which are used to express the spatial locative orientation of subject

  13. The subcellular fate of cadmium and zinc in the scallop Chlamys nobilis during waterborne and dietary metal exposure

    Energy Technology Data Exchange (ETDEWEB)

    Pan Ke [Department of Biology, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong); Wang Wenxiong [Department of Biology, Hong Kong University of Science and Technology (HKUST), Clear Water Bay, Kowloon (Hong Kong)], E-mail: wwang@ust.hk

    2008-12-11

    Subcellular metal distribution has received increasing attention in aquatic toxicology studies, but the relationship between metal distribution and metal biokinetics remains largely unexplored. A series of short-term experiments on different concentrations of dissolved and dietary metals and on metal elimination were conducted to investigate the dynamics of subcellular distribution of Cd and Zn in the scallop Chlamys nobilis, a bivalve species that is known to accumulate very high concentrations of Cd and Zn in its tissues. Our results showed that, in general, both Cd and Zn were sequestered in insoluble forms (organelles, metal-rich granules, and cellular debris). The main binding pool for the newly acquired metals was organelles for Cd and cellular debris for Zn. Metallothionein-like protein (MTLP) was the most important storage pool for Cd in the scallops. Storage in the non-toxic form both in organelles and MTLP instead of through exocytosis was the major detoxification strategy to control Cd and accounted for the low efflux rate of Cd from scallops. In contrast to Cd, the main binding pool for Zn was cellular debris. Significant changes were found in the scallops when they were challenged with different concentrations of metals in the aqueous and food phases. Such changes provide important information on how scallops handle metals when there is increasing metal uptake. The redistribution of Zn among each subcellular compartment was much faster than the redistribution of Cd, suggesting an effective regulation mechanism for Zn in scallops. Thus, knowing subcellular metal distribution helps in studying the toxicity of both waterborne and dietborne metals.

  14. Effects of carbohydrate supplements on exercise-induced menstrual dysfunction and ovarian subcellular structural changes in rats

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    Can Zhao

    2014-09-01

    Conclusion: Female adult rats with 9-week continuous exercise can cause menstrual dysregulation as a model for EAMD. Post-EAMD intervention with glucose and oligosaccharide intake can normalize the menstrual cycle, restore the follicular subcellular structure, and reverse the exercise-induced reduction of ovary sex hormones. It suggests a positive feedback of hypothalamus–pituitary–ovary axis might be involved in the molecular mechanisms of energy intake in treating EAMD.

  15. Changes in Subcellular Distribution of n-Octanoyl or n-Decanoyl Ghrelin in Ghrelin-Producing Cells

    OpenAIRE

    Nishi, Yoshihiro; Mifune, Hiroharu; Yabuki, Akira; Tajiri, Yuji; Hirata, Rumiko; Tanaka, Eiichiro; Hosoda, Hiroshi; Kangawa, Kenji; Kojima, Masayasu

    2013-01-01

    Background: The enzyme ghrelin O-acyltransferase (GOAT) catalyzes the acylation of ghrelin. The molecular form of GOAT, together with its reaction in vitro, has been reported previously. However, the sub-cellular processes governing the acylation of ghrelin remain to be elucidated.Methods: Double immunoelectron microscopy was used to examine changes in the relative proportions of secretory granules containing n-octanoyl ghrelin (C8-ghrelin) or n-decanoyl ghrelin (C10-ghrelin) in ghrelin-pro...

  16. Location Systems An Introduction to the Technology Behind Location Awareness

    CERN Document Server

    LaMarca, Anthony

    2008-01-01

    Advances in electronic location technology and the coming of age of mobile computing have opened the door for location-aware applications to permeate all aspects of everyday life. Location is at the core of a large number of high-value applications ranging from the life-and-death context of emergency response to serendipitous social meet-ups. For example, the market for GPS products and services alone is expected to grow to US200 billion by 2015. Unfortunately, there is no single location technology that is good for every situation and exhibits high accuracy, low cost, and universal coverage.

  17. Partial cecal necrosis treated by laparoscopic partial cecal resection.

    Science.gov (United States)

    Perko, Zdravko; Bilan, Kanito; Vilović, Katarina; Druzijanić, Nikica; Kraljević, Damir; Juriicić, Josko; Krnić, Dragan; Srsen, Darko; Pogorelić, Zenon; Tomić, Snjezana

    2006-12-01

    Acute colonic ischemia is the common cause of colitis in elderly population. However, isolated ischemic necrosis of cecum is rare entity, often associated with variety of conditions. Here we present a case of a 73-year old woman with a past history of hypertension presented with clinical symptoms of right lower quadrant abdominal pain and tenderness localized to the right lower quadrant, guarding and rebound tenderness. With diagnosis of acute appendicitis, the patient underwent laparoscopy where the cecal partial necrosis was discovered. Necrotic area of cecum was excised using two endoscopic cutters and laparoscopic appendectomy was performed. Pathologist report showed thrombosis of vessels and necrosis of entire cecal wall. The patient completely recovered without any surgical complications. This is the first case of partial cecum necrosis laparoscopicaly managed and with a partial cecal resection only.

  18. Effects of exogenous spermidine on subcellular distribution and chemical forms of cadmium in Typha latifolia L. under cadmium stress.

    Science.gov (United States)

    Tang, C F; Zhang, R Q; Wen, S Z; Li, C F; Guo, X F; Liu, Y G

    2009-01-01

    The effects of foliar spraying with spermidine (Spd) on the subcellular distribution and chemical speciation of Cd in Typha latifolia L. in hydroponic system were investigated. Increased Cd concentration in the medium caused an increase of Cd concentration in all tissues and all subcellular fractions, with the highest Cd level occurring in roots, followed by rhizomes and leaves and the greatest accumulation was in cell walls, soluble fraction and organelle containing fractions ranked next, respectively. The greatest amount of Cd was found in the extraction of 1 mol.L(-1) NaCl, followed by 2% HAC, deionized water, 80% ethanol, residues and the least in 0.6 mol.L(-1) HCl. The subcellular distribution and different chemical forms of Cd both decreased after Spd application and were more inhibited by 0.25 than 0.50 mmol.L(-1) Spd, especially in roots at 0.10 mmol.L(-1) Cd treatment. It could be suggested that the retention of Cd in roots, cell walls binding with metal, metal ion compartmentation in vacuoles and Cd forms existing in undissolved and integrated with pectates and protein play an important role in Cd detoxification and Spd could enhance the tolerance of cattail to Cd stress.

  19. Weak mitochondrial targeting sequence determines tissue-specific subcellular localization of glutamine synthetase in liver and brain cells.

    Science.gov (United States)

    Matthews, Gideon D; Gur, Noa; Koopman, Werner J H; Pines, Ophry; Vardimon, Lily

    2010-02-01

    Evolution of the uricotelic system for ammonia detoxification required a mechanism for tissue-specific subcellular localization of glutamine synthetase (GS). In uricotelic vertebrates, GS is mitochondrial in liver cells and cytoplasmic in brain. Because these species contain a single copy of the GS gene, it is not clear how tissue-specific subcellular localization is achieved. Here we show that in chicken, which utilizes the uricotelic system, the GS transcripts of liver and brain cells are identical and, consistently, there is no difference in the amino acid sequence of the protein. The N-terminus of GS, which constitutes a 'weak' mitochondrial targeting signal (MTS), is sufficient to direct a chimeric protein to the mitochondria in hepatocytes and to the cytoplasm in astrocytes. Considering that a weak MTS is dependent on a highly negative mitochondrial membrane potential (DeltaPsi) for import, we examined the magnitude of DeltaPsi in hepatocytes and astrocytes. Our results unexpectedly revealed that DeltaPsi in hepatocytes is considerably more negative than that of astrocytes and that converting the targeting signal into 'strong' MTS abolished the capability to confer tissue-specific subcellular localization. We suggest that evolutional selection of weak MTS provided a tool for differential targeting of an identical protein by taking advantage of tissue-specific differences in DeltaPsi.

  20. SLC30A3 (ZnT3 oligomerization by dityrosine bonds regulates its subcellular localization and metal transport capacity.

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    Gloria Salazar

    2009-06-01

    Full Text Available Non-covalent and covalent homo-oligomerization of membrane proteins regulates their subcellular localization and function. Here, we described a novel oligomerization mechanism affecting solute carrier family 30 member 3/zinc transporter 3 (SLC30A3/ZnT3. Oligomerization was mediated by intermolecular covalent dityrosine bonds. Using mutagenized ZnT3 expressed in PC12 cells, we identified two critical tyrosine residues necessary for dityrosine-mediated ZnT3 oligomerization. ZnT3 carrying the Y372F mutation prevented ZnT3 oligomerization, decreased ZnT3 targeting to synaptic-like microvesicles (SLMVs, and decreased resistance to zinc toxicity. Strikingly, ZnT3 harboring the Y357F mutation behaved as a "gain-of-function" mutant as it displayed increased ZnT3 oligomerization, targeting to SLMVs, and increased resistance to zinc toxicity. Single and double tyrosine ZnT3 mutants indicate that the predominant dimeric species is formed between tyrosine 357 and 372. ZnT3 tyrosine dimerization was detected under normal conditions and it was enhanced by oxidative stress. Covalent species were also detected in other SLC30A zinc transporters localized in different subcellular compartments. These results indicate that covalent tyrosine dimerization of a SLC30A family member modulates its subcellular localization and zinc transport capacity. We propose that dityrosine-dependent membrane protein oligomerization may regulate the function of diverse membrane protein in normal and disease states.