Science.gov

Sample records for subcellular structure provide

  1. Plasma effects on subcellular structures

    SciTech Connect

    Gweon, Bomi; Kim, Dan Bee; Jung, Heesoo; Choe, Wonho; Kim, Daeyeon; Shin, Jennifer H.

    2010-03-08

    Atmospheric pressure helium plasma treated human hepatocytes exhibit distinctive zones of necrotic and live cells separated by a void. We propose that plasma induced necrosis is attributed to plasma species such as oxygen radicals, charged particles, metastables and/or severe disruption of charged cytoskeletal proteins. Interestingly, uncharged cytoskeletal intermediate filaments are only minimally disturbed by plasma, elucidating the possibility of plasma induced electrostatic effects selectively destroying charged proteins. These bona fide plasma effects, which inflict alterations in specific subcellular structures leading to necrosis and cellular detachment, were not observed by application of helium flow or electric field alone.

  2. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    PubMed

    Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

    2016-04-02

    In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

  3. Geometric modeling of subcellular structures, organelles, and multiprotein complexes

    PubMed Central

    Feng, Xin; Xia, Kelin; Tong, Yiying; Wei, Guo-Wei

    2013-01-01

    SUMMARY Recently, the structure, function, stability, and dynamics of subcellular structures, organelles, and multi-protein complexes have emerged as a leading interest in structural biology. Geometric modeling not only provides visualizations of shapes for large biomolecular complexes but also fills the gap between structural information and theoretical modeling, and enables the understanding of function, stability, and dynamics. This paper introduces a suite of computational tools for volumetric data processing, information extraction, surface mesh rendering, geometric measurement, and curvature estimation of biomolecular complexes. Particular emphasis is given to the modeling of cryo-electron microscopy data. Lagrangian-triangle meshes are employed for the surface presentation. On the basis of this representation, algorithms are developed for surface area and surface-enclosed volume calculation, and curvature estimation. Methods for volumetric meshing have also been presented. Because the technological development in computer science and mathematics has led to multiple choices at each stage of the geometric modeling, we discuss the rationales in the design and selection of various algorithms. Analytical models are designed to test the computational accuracy and convergence of proposed algorithms. Finally, we select a set of six cryo-electron microscopy data representing typical subcellular complexes to demonstrate the efficacy of the proposed algorithms in handling biomolecular surfaces and explore their capability of geometric characterization of binding targets. This paper offers a comprehensive protocol for the geometric modeling of subcellular structures, organelles, and multiprotein complexes. PMID:23212797

  4. Geometric modeling of subcellular structures, organelles, and multiprotein complexes.

    PubMed

    Feng, Xin; Xia, Kelin; Tong, Yiying; Wei, Guo-Wei

    2012-12-01

    Recently, the structure, function, stability, and dynamics of subcellular structures, organelles, and multiprotein complexes have emerged as a leading interest in structural biology. Geometric modeling not only provides visualizations of shapes for large biomolecular complexes but also fills the gap between structural information and theoretical modeling, and enables the understanding of function, stability, and dynamics. This paper introduces a suite of computational tools for volumetric data processing, information extraction, surface mesh rendering, geometric measurement, and curvature estimation of biomolecular complexes. Particular emphasis is given to the modeling of cryo-electron microscopy data. Lagrangian-triangle meshes are employed for the surface presentation. On the basis of this representation, algorithms are developed for surface area and surface-enclosed volume calculation, and curvature estimation. Methods for volumetric meshing have also been presented. Because the technological development in computer science and mathematics has led to multiple choices at each stage of the geometric modeling, we discuss the rationales in the design and selection of various algorithms. Analytical models are designed to test the computational accuracy and convergence of proposed algorithms. Finally, we select a set of six cryo-electron microscopy data representing typical subcellular complexes to demonstrate the efficacy of the proposed algorithms in handling biomolecular surfaces and explore their capability of geometric characterization of binding targets. This paper offers a comprehensive protocol for the geometric modeling of subcellular structures, organelles, and multiprotein complexes. Copyright © 2012 John Wiley & Sons, Ltd.

  5. Tracking variable number of multiple subcellular structures in 3D.

    PubMed

    Wen, Quan; Gao, Jean

    2009-01-01

    With the introduction of sensitive and fast electronic imaging devices and the development of biological methods to tag proteins of interest by green fluorescent proteins (GFP), it has now become critical to develop automatic quantitative data analysis tools to study the live cell dynamics at subcellular level. In this paper, a sequential Monte Carlo (SMC) method to track variable number of multiple 3D subcellular structures is proposed. First, multiple subcellular structures are represented by a joint state. Then the distribution of the dimension changing joint state is sampled efficiently by the reverse jump Markov chain Monte Carlo (RJMCMC) method designed with update move, identity switch move, disappearing move, and appearing move. The experimental results show that the proposed method can successfully track multiple 3D subcellular structures with different motion modalities such as object appearing and disappearing.

  6. Homeostatic Plasticity of Subcellular Neuronal Structures: From Inputs to Outputs.

    PubMed

    Wefelmeyer, Winnie; Puhl, Christopher J; Burrone, Juan

    2016-10-01

    Neurons in the brain are highly plastic, allowing an organism to learn and adapt to its environment. However, this ongoing plasticity is also inherently unstable, potentially leading to aberrant levels of circuit activity. Homeostatic forms of plasticity are thought to provide a means of controlling neuronal activity by avoiding extremes and allowing network stability. Recent work has shown that many of these homeostatic modifications change the structure of subcellular neuronal compartments, ranging from changes to synaptic inputs at both excitatory and inhibitory compartments to modulation of neuronal output through changes at the axon initial segment (AIS) and presynaptic terminals. Here we review these different forms of structural plasticity in neurons and the effects they may have on network function. Copyright © 2016. Published by Elsevier Ltd.

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

    NASA Astrophysics Data System (ADS)

    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 < 0.0001) and the surrounding cytoplasm (p < 0.0001). Moreover, we demonstrate the mechanical response of cells to Latrunculin-A, a drug that reduces cell stiffness by preventing cytoskeletal assembly. Our technique can therefore generate valuable insights into cellular biomechanics and its role in pathophysiology.

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

    PubMed Central

    Antonacci, Giuseppe; Braakman, Sietse

    2016-01-01

    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 < 0.0001) and the surrounding cytoplasm (p < 0.0001). Moreover, we demonstrate the mechanical response of cells to Latrunculin-A, a drug that reduces cell stiffness by preventing cytoskeletal assembly. Our technique can therefore generate valuable insights into cellular biomechanics and its role in pathophysiology. PMID:27845411

  9. Tracking interacting subcellular structures by sequential Monte Carlo method.

    PubMed

    Wen, Quan; Gao, Jean

    2007-01-01

    With the wide application of green fluorescent protein (GFP) in the study of live cell, which leads to a better understanding of biochemical events at subcellular level, there is a surging need for the computer-aided analysis on the huge amount of image sequence data acquired by the advanced microscopy devices. One of such tasks is the motility analysis of the multiple subcellular structures. In this paper, an algorithm using sequential Monte Carlo (SMC) method for multiple interacting object tracking is proposed. We use joint state to represent all the objects together, and model the interaction between objects in the 2D plane by augmenting an extra dimension and evaluating their overlapping relationship in the 3D space. Markov chain Monte Carlo (MCMC) method with a novel height swap move is applied to sample the joint state distribution efficiently. To facilitate distinguishing between different objects, a new observation method is also proposed by matching the size and intensity profile of the object. The experimental results show that our method is promising.

  10. Visualizing Escherichia coli sub-cellular structure using sparse deconvolution Spatial Light Interference Tomography.

    PubMed

    Mir, Mustafa; Babacan, S Derin; Bednarz, Michael; Do, Minh N; Golding, Ido; Popescu, Gabriel

    2012-01-01

    Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a non-invasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM). In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIM's resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT), to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.

  11. Methods of biomedical optical imaging: from subcellular structures to tissues and organs

    NASA Astrophysics Data System (ADS)

    Turchin, I. V.

    2016-05-01

    Optical bioimaging methods have a wide range of applications in the life sciences, most notably including the molecular resolution study of subcellular structures, small animal molecular imaging, and structural and functional clinical diagnostics of tissue layers and organs. We review fluorescent microscopy, fluorescent macroscopy, optical coherence tomography, optoacoustic tomography, and optical diffuse spectroscopy and tomography from the standpoint of physical fundamentals, applications, and progress.

  12. Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography

    PubMed Central

    Chen, Huan-Yuan; Chiang, Dapi Meng-Lin; Lin, Zi-Jing; Hsieh, Chia-Chun; Yin, Gung-Chian; Weng, I.-Chun; Guttermann, Peter; Werner, Stephan; Henzler, Katja; Schneider, Gerd; Lai, Lee-Jene; Liu, Fu-Tong

    2016-01-01

    Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level. PMID:27748356

  13. Use of Xenopus cell-free extracts to study size regulation of subcellular structures

    PubMed Central

    Jevtić, Predrag; Milunović-Jevtić, ANA; Dilsaver, Matthew R.; Gatlin, Jesse C.; Levy, Daniel L.

    2016-01-01

    Striking size variations are prominent throughout biology, at the organismal, cellular, and subcellular levels. Important fundamental questions concern organelle size regulation and how organelle size is regulated relative to cell size, also known as scaling. Uncovering mechanisms of organelle size regulation will inform the functional significance of size as well as the implications of misregulated size, for instance in the case of nuclear enlargement in cancer. Xenopus egg and embryo extracts are powerful cell-free systems that have been utilized extensively for mechanistic and functional studies of various organelles and subcellular structures. The open biochemical nature of the extract permits facile manipulation of its composition, and in recent years extract approaches have illuminated mechanisms of organelle size regulation. This review largely focuses on in vitro Xenopus studies that have identified regulators of nuclear and spindle size. We also discuss potential relationships between size scaling of the nucleus and spindle, size regulation of other subcellular structures, and extract experiments that have clarified developmental timing mechanisms. We conclude by offering some future prospects, notably the integration of Xenopus extract with microfluidic technology. PMID:27759156

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

    PubMed Central

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

    2012-01-01

    Summary 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. PMID

  15. Analyzing subcellular structure with optical Fourier filtering based on Gabor filters

    NASA Astrophysics Data System (ADS)

    Boustany, Nada N.; Sierra, Heidy

    2013-02-01

    Label-free measurement of subcellular morphology can be used to track dynamically cellular function under various conditions and has important applications in cellular monitoring and in vitro cell assays. We show that optical filtering of scattered light by two-dimensional Gabor filters allows for direct and highly sensitive measurement of sample structure. The Gabor filters, which are defined by their spatial frequency, orientation and Gaussian envelope, can be used to track locally and in situ the characteristic size and orientation of structures within the sample. Our method consists of sequentially implementing a set of Gabor filters via a spatial light modulator placed in a conjugate Fourier plane during optical imaging and identifying the filters that yield maximum signal. Using this setup, we show that Gabor filtering of light forward-scattered by spheres yields an optical response which varies linearly with diameter between 100nm and 2000nm. The optical filtering sensitivity to changes in diameter is on the order of 20nm and can be achieved at low image resolution. We use numerical simulations to demonstrate that this linear response can be predicted from scatter theory and does not vary significantly with changes in refractive index ratio. By applying this Fourier filtering method in samples consisting of diatoms and cells, we generate false-color images of the object that encode at each pixel the size of the local structures within the object. The resolution of these encoded size maps in on the order of 0.36μm. The pixel histograms of these encoded images directly provide 20nm resolved "size spectra", depicting the size distribution of structures within the analyzed object. We use these size spectra to differentiate the morphology of apoptosis-competent and bax/bak null apoptosis-resistant cells during cell death. We also utilize the sensitivity of the Gabor filters to object orientation to track changes in organelle morphology, and detect mitochondrial

  16. Isotope labeling of rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments.

    USDA-ARS?s Scientific Manuscript database

    The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses considerable challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynth...

  17. Subcellular Size

    PubMed Central

    Marshall, Wallace F.

    2015-01-01

    All of the same conceptual questions about size in organisms apply equally at the level of single cells. What determines the size, not only of the whole cell, but of all of its parts? What ensures that subcellular components are properly proportioned relative to the whole cell? How does alteration in organelle size affect biochemical function? Answering such fundamental questions requires us to understand how the size of individual organelles and other cellular structures is determined. Knowledge of organelle biogenesis and dynamics has advanced rapidly in recent years. Does this knowledge give us enough information to formulate reasonable models for organelle size control, or are we still missing something? PMID:25957302

  18. Three-dimensional cellular and subcellular structures of human brain tissue determined by microtomography

    NASA Astrophysics Data System (ADS)

    Mizutani, Ryuta; Takeuchi, Akihisa; Takekoshi, Susumu; Yoshiyuki Osamura, R.; Uesugi, Kentaro; Suzuki, Yoshio

    2009-09-01

    We report here x-ray microtomographic studies of human cerebral cortex stained with high-Z elements. Brain tissues were stained with metal elements by the Golgi and Bodian impregnation methods and subjected to x-ray microtomographic analysis. Axons and dendrites arising from cell bodies were visualized as three-dimensional networks. Spherical structures of cellular nuclei were observed in the interiors of cell bodies, indicating that hard x-ray microtomography can reveal the intracellular structure. High-Z element microcontrasting in conjunction with microtomographic analysis can be applied to any soft tissues. Our results show that the metal contrasting facilitates the three-dimensional microtomographic visualization of cellular and subcellular structures of soft tissues.

  19. RNALocate: a resource for RNA subcellular localizations.

    PubMed

    Zhang, Ting; Tan, Puwen; Wang, Liqiang; Jin, Nana; Li, Yana; Zhang, Lin; Yang, Huan; Hu, Zhenyu; Zhang, Lining; Hu, Chunyu; Li, Chunhua; Qian, Kun; Zhang, Changjian; Huang, Yan; Li, Kongning; Lin, Hao; Wang, Dong

    2017-01-04

    Increasing evidence has revealed that RNA subcellular localization is a very important feature for deeply understanding RNA's biological functions after being transported into intra- or extra-cellular regions. RNALocate is a web-accessible database that aims to provide a high-quality RNA subcellular localization resource and facilitate future researches on RNA function or structure. The current version of RNALocate documents more than 37 700 manually curated RNA subcellular localization entries with experimental evidence, involving more than 21 800 RNAs with 42 subcellular localizations in 65 species, mainly including Homo sapiens, Mus musculus and Saccharomyces cerevisiae etc. Besides, RNA homology, sequence and interaction data have also been integrated into RNALocate. Users can access these data through online search, browse, blast and visualization tools. In conclusion, RNALocate will be of help in elucidating the entirety of RNA subcellular localization, and developing new prediction methods. The database is available at http://www.rna-society.org/rnalocate/.

  20. RNALocate: a resource for RNA subcellular localizations

    PubMed Central

    Zhang, Ting; Tan, Puwen; Wang, Liqiang; Jin, Nana; Li, Yana; Zhang, Lin; Yang, Huan; Hu, Zhenyu; Zhang, Lining; Hu, Chunyu; Li, Chunhua; Qian, Kun; Zhang, Changjian; Huang, Yan; Li, Kongning; Lin, Hao; Wang, Dong

    2017-01-01

    Increasing evidence has revealed that RNA subcellular localization is a very important feature for deeply understanding RNA's biological functions after being transported into intra- or extra-cellular regions. RNALocate is a web-accessible database that aims to provide a high-quality RNA subcellular localization resource and facilitate future researches on RNA function or structure. The current version of RNALocate documents more than 37 700 manually curated RNA subcellular localization entries with experimental evidence, involving more than 21 800 RNAs with 42 subcellular localizations in 65 species, mainly including Homo sapiens, Mus musculus and Saccharomyces cerevisiae etc. Besides, RNA homology, sequence and interaction data have also been integrated into RNALocate. Users can access these data through online search, browse, blast and visualization tools. In conclusion, RNALocate will be of help in elucidating the entirety of RNA subcellular localization, and developing new prediction methods. The database is available at http://www.rna-society.org/rnalocate/. PMID:27543076

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

    PubMed

    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.

  2. LiDAR: Providing structure

    USGS Publications Warehouse

    Vierling, Lee A.; Martinuzzi, Sebastián; Asner, Gregory P.; Stoker, Jason M.; Johnson, Brian R.

    2011-01-01

    Since the days of MacArthur, three-dimensional (3-D) structural information on the environment has fundamentally transformed scientific understanding of ecological phenomena (MacArthur and MacArthur 1961). Early data on ecosystem structure were painstakingly laborious to collect. However, as reviewed and reported in recent volumes of Frontiers(eg Vierling et al. 2008; Asner et al.2011), advances in light detection and ranging (LiDAR) remote-sensing technology provide quantitative and repeatable measurements of 3-D ecosystem structure that enable novel ecological insights at scales ranging from the plot, to the landscape, to the globe. Indeed, annual publication of studies using LiDAR to interpret ecological phenomena increased 17-fold during the past decade, with over 180 new studies appearing in 2010 (ISI Web of Science search conducted on 23 Mar 2011: [{lidar AND ecol*} OR {lidar AND fores*} OR {lidar AND plant*}]).

  3. Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation.

    PubMed

    Jackson, C; Dench, J E; Hall, D O; Moore, A L

    1979-07-01

    Discontinuous Percoll density gradients have been developed for the purification of mitochondria, permitting rapid separation under isosmotic and low viscosity conditions. Mitochondria from several etiolated tissues have been successfully separated from contaminating subcellular structures by this method. For potato tuber the ratio of washed to purified mitochondrial protein was 2.6, similar to the increase in specific activity of cytochrome c oxidase following separation. The purification of mitochondria from green leaf tissues on Percoll gradients has reduced chlorophyll contamination of spinach mitochondria from about 70 micrograms chlorophyll per milligram protein to approximately 8 micrograms chlorophyll per milligram protein.The ratio of protein content of the washed mitochondria compared to that in the purified preparation was 7 for spinach and respiratory activity was retained. The physiological integrity and oxidative properties of washed and gradient mitochondria are compared.

  4. Multi-scale Imaging of Cellular and Sub-cellular Structures using Scanning Probe Recognition Microscopy.

    NASA Astrophysics Data System (ADS)

    Chen, Q.; Rice, A. F.

    2005-03-01

    Scanning Probe Recognition Microscopy is a new scanning probe capability under development within our group to reliably return to and directly interact with a specific nanobiological feature of interest. In previous work, we have successfully recognized and classified tubular versus globular biological objects from experimental atomic force microscope images using a method based on normalized central moments [ref. 1]. In this paper we extend this work to include recognition schemes appropriate for cellular and sub-cellular structures. Globular cells containing tubular actin filaments are under investigation. Thus there are differences in external/internal shapes and scales. Continuous Wavelet Transform with a differential Gaussian mother wavelet is employed for multi- scale analysis. [ref. 1] Q. Chen, V. Ayres and L. Udpa, ``Biological Investigation Using Scanning Probe Recognition Microscopy,'' Proceedings 3rd IEEE Conference on Nanotechnology, vol. 2, p 863-865 (2003).

  5. Protein subcellular localization prediction based on compartment-specific features and structure conservation

    PubMed Central

    Su, Emily Chia-Yu; Chiu, Hua-Sheng; Lo, Allan; Hwang, Jenn-Kang; Sung, Ting-Yi; Hsu, Wen-Lian

    2007-01-01

    Background Protein subcellular localization is crucial for genome annotation, protein function prediction, and drug discovery. Determination of subcellular localization using experimental approaches is time-consuming; thus, computational approaches become highly desirable. Extensive studies of localization prediction have led to the development of several methods including composition-based and homology-based methods. However, their performance might be significantly degraded if homologous sequences are not detected. Moreover, methods that integrate various features could suffer from the problem of low coverage in high-throughput proteomic analyses due to the lack of information to characterize unknown proteins. Results We propose a hybrid prediction method for Gram-negative bacteria that combines a one-versus-one support vector machines (SVM) model and a structural homology approach. The SVM model comprises a number of binary classifiers, in which biological features derived from Gram-negative bacteria translocation pathways are incorporated. In the structural homology approach, we employ secondary structure alignment for structural similarity comparison and assign the known localization of the top-ranked protein as the predicted localization of a query protein. The hybrid method achieves overall accuracy of 93.7% and 93.2% using ten-fold cross-validation on the benchmark data sets. In the assessment of the evaluation data sets, our method also attains accurate prediction accuracy of 84.0%, especially when testing on sequences with a low level of homology to the training data. A three-way data split procedure is also incorporated to prevent overestimation of the predictive performance. In addition, we show that the prediction accuracy should be approximately 85% for non-redundant data sets of sequence identity less than 30%. Conclusion Our results demonstrate that biological features derived from Gram-negative bacteria translocation pathways yield a significant

  6. Direct template matching reveals a host subcellular membrane gyroid cubic structure that is associated with SARS virus.

    PubMed

    Almsherqi, Zakaria A; McLachlan, Craig S; Mossop, Peter; Knoops, Kèvin; Deng, Yuru

    2005-01-01

    Viral infection can result in alterations to the host subcellular membrane. This is often reported when using transmission electron microscopy (TEM), resulting in a description of tubuloreticular membrane subcellular ultrastructure rather than a definition based on 3-D morphology. 2-D TEM micrographs depicting subcellular membrane changes are associated with subcellular SARS virion particles [Goldsmith CS, Tatti KM, Ksiazek TG et al. Ultra-structural characterization of SARS coronavirus. Emerg Infect Dis 2004; 10: 320-326]. In the present study, we have defined the 2-D membrane pattern and shape associated with the SARS virus infection. This is by using a direct template matching method to determine what the 3-D structure of the SARS virus associated host membrane change would be. The TEM image for our purposes is defined on 2-D information, such as the membrane having undergone proliferation and from pattern recognition suggesting that the membrane-described pattern is possibly a gyroid type of membrane. Features of the membrane were used to compute and match the gyroid structure with an existing 2-D TEM micrograph, where it was revealed that the membrane structure was indeed a gyroid-based cubic membrane. The 2-D gyroid computer-simulated image that was used to match the electron micrograph of interest was derived from a mathematically well-defined 3-D structure, and it is from this 3-D derivative that allows us to make inferences about the 3-D structure of this membrane. In conclusion, we demonstrate that a 3-D structure can be defined from a 2-D membrane patterned image and that a SARS viral associated membrane change has been identified as cubic membrane morphology. Possible mechanisms for this cubic membrane change are discussed with respect to viral severity, persistence and free radical production.

  7. Model-based segmentation and quantification of subcellular structures in 2D and 3D fluorescent microscopy images

    NASA Astrophysics Data System (ADS)

    Wörz, Stefan; Heinzer, Stephan; Weiss, Matthias; Rohr, Karl

    2008-03-01

    We introduce a model-based approach for segmenting and quantifying GFP-tagged subcellular structures of the Golgi apparatus in 2D and 3D microscopy images. The approach is based on 2D and 3D intensity models, which are directly fitted to an image within 2D circular or 3D spherical regions-of-interest (ROIs). We also propose automatic approaches for the detection of candidates, for the initialization of the model parameters, and for adapting the size of the ROI used for model fitting. Based on the fitting results, we determine statistical information about the spatial distribution and the total amount of intensity (fluorescence) of the subcellular structures. We demonstrate the applicability of our new approach based on 2D and 3D microscopy images.

  8. CytoSpectre: a tool for spectral analysis of oriented structures on cellular and subcellular levels.

    PubMed

    Kartasalo, Kimmo; Pölönen, Risto-Pekka; Ojala, Marisa; Rasku, Jyrki; Lekkala, Jukka; Aalto-Setälä, Katriina; Kallio, Pasi

    2015-10-26

    Orientation and the degree of isotropy are important in many biological systems such as the sarcomeres of cardiomyocytes and other fibrillar structures of the cytoskeleton. Image based analysis of such structures is often limited to qualitative evaluation by human experts, hampering the throughput, repeatability and reliability of the analyses. Software tools are not readily available for this purpose and the existing methods typically rely at least partly on manual operation. We developed CytoSpectre, an automated tool based on spectral analysis, allowing the quantification of orientation and also size distributions of structures in microscopy images. CytoSpectre utilizes the Fourier transform to estimate the power spectrum of an image and based on the spectrum, computes parameter values describing, among others, the mean orientation, isotropy and size of target structures. The analysis can be further tuned to focus on targets of particular size at cellular or subcellular scales. The software can be operated via a graphical user interface without any programming expertise. We analyzed the performance of CytoSpectre by extensive simulations using artificial images, by benchmarking against FibrilTool and by comparisons with manual measurements performed for real images by a panel of human experts. The software was found to be tolerant against noise and blurring and superior to FibrilTool when analyzing realistic targets with degraded image quality. The analysis of real images indicated general good agreement between computational and manual results while also revealing notable expert-to-expert variation. Moreover, the experiment showed that CytoSpectre can handle images obtained of different cell types using different microscopy techniques. Finally, we studied the effect of mechanical stretching on cardiomyocytes to demonstrate the software in an actual experiment and observed changes in cellular orientation in response to stretching. CytoSpectre, a versatile, easy

  9. Role of structural and functional elements of mouse methionine-S-sulfoxide reductase in its subcellular distribution.

    PubMed

    Kim, Hwa-Young; Gladyshev, Vadim N

    2005-06-07

    Oxidized forms of methionine residues in proteins can be repaired by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). In mammals, three MsrBs are present, which are targeted to various subcellular compartments. In contrast, only a single mammalian MsrA gene is known whose products have been detected in both cytosol and mitochondria. Factors that determine the location of the protein in these compartments are not known. Here, we found that MsrA was present in cytosol, nucleus, and mitochondria in mouse cells and tissues and that the major enzyme forms detected in various compartments were generated from a single-translation product rather than by alternative translation initiation. Both cytosolic and mitochondrial forms were processed with respect to the N-terminal signal peptide, and the distribution of the protein occurred post-translationally. Deletion of amino acids 69-108, 69-83, 84-108, or 217-233, which contained elements important for MsrA structure and function, led to exclusive mitochondrial location of MsrA, whereas a region that affected substrate binding but was not part of the overall fold had no influence on the subcellular distribution. The data suggested that proper structure-function organization of MsrA played a role in subcellular distribution of this protein in mouse cells. These findings were recapitulated by expressing various forms of mouse MsrA in Saccharomyces cerevisiae, suggesting conservation of the mechanisms responsible for distribution of the mammalian enzyme among different cellular compartments.

  10. High-Content Microscopy Analysis of Subcellular Structures: Assay Development and Application to Focal Adhesion Quantification.

    PubMed

    Kroll, Torsten; Schmidt, David; Schwanitz, Georg; Ahmad, Mubashir; Hamann, Jana; Schlosser, Corinne; Lin, Yu-Chieh; Böhm, Konrad J; Tuckermann, Jan; Ploubidou, Aspasia

    2016-07-01

    High-content analysis (HCA) converts raw light microscopy images to quantitative data through the automated extraction, multiparametric analysis, and classification of the relevant information content. Combined with automated high-throughput image acquisition, HCA applied to the screening of chemicals or RNAi-reagents is termed high-content screening (HCS). Its power in quantifying cell phenotypes makes HCA applicable also to routine microscopy. However, developing effective HCA and bioinformatic analysis pipelines for acquisition of biologically meaningful data in HCS is challenging. Here, the step-by-step development of an HCA assay protocol and an HCS bioinformatics analysis pipeline are described. The protocol's power is demonstrated by application to focal adhesion (FA) detection, quantitative analysis of multiple FA features, and functional annotation of signaling pathways regulating FA size, using primary data of a published RNAi screen. The assay and the underlying strategy are aimed at researchers performing microscopy-based quantitative analysis of subcellular features, on a small scale or in large HCS experiments. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  11. Cellular and subcellular localization of CXCL12 and CXCR4 in rat nociceptive structures: physiological relevance.

    PubMed

    Reaux-Le Goazigo, Annabelle; Rivat, Cyril; Kitabgi, Patrick; Pohl, Michel; Melik Parsadaniantz, Stéphane

    2012-09-01

    Initial studies implicated the chemokine CXC motif ligand 12 (CXCL12) and its cognate CXC motif receptor 4 (CXCR4) in pain modulation. However, there has been no description of the distribution, transport and axonal sorting of CXCL12 and CXCR4 in rat nociceptive structures, and their direct participation in nociception modulation has not been demonstrated. Here, we report that acute intrathecal administration of CXCL12 induced mechanical hypersensitivity in naive rats. This effect was prevented by a CXCR4-neutralizing antibody. To determine the morphological basis of this behavioural response, we used light and electron microscopic immunohistochemistry to map CXCL12- and CXCR4-immunoreactive elements in dorsal root ganglia, lumbar spinal cord, sciatic nerve and skin. Light microscopy analysis revealed CXCL12 and CXCR4 immunoreactivity in calcitonin gene related peptide-containing peptidergic primary sensory neurons, which were both conveyed to central and peripheral sensory nerve terminals. Electron microscopy clearly demonstrated CXCL12 and CXCR4 immunoreactivity in primary sensory nerve terminals in the dorsal horn; both were sorted into small clear vesicles and large dense-core vesicles. This suggests that CXCL12 and CXCR4 are trafficked from nerve cell bodies to the dorsal horn. Double immunogold labelling for CXCL12 and calcitonin gene related peptide revealed partial vesicular colocalization in axonal terminals. We report, for the first time, that CXCR4 receptors are mainly located on the neuronal plasma membrane, where they are present at pre-synaptic and post-synaptic sites of central terminals. Receptor inactivation experiments, behavioural studies and morphological analyses provide strong evidence that the CXCL12/CXCR4 system is involved in modulation of nociceptive signalling.

  12. Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures

    NASA Astrophysics Data System (ADS)

    Li, Xuesong; Lam, Wen Jiun; Cao, Zhe; Hao, Yan; Sun, Qiqi; He, Sicong; Mak, Ho Yi; Qu, Jianan Y.

    2015-11-01

    The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.

  13. Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures.

    PubMed

    Li, Xuesong; Lam, Wen Jiun; Cao, Zhe; Hao, Yan; Sun, Qiqi; He, Sicong; Mak, Ho Yi; Qu, Jianan Y

    2015-11-01

    The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.

  14. Recent advances in imaging subcellular processes

    PubMed Central

    Myers, Kenneth A.; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology. PMID:27408708

  15. Recent advances in imaging subcellular processes.

    PubMed

    Myers, Kenneth A; Janetopoulos, Christopher

    2016-01-01

    Cell biology came about with the ability to first visualize cells. As microscopy techniques advanced, the early microscopists became the first cell biologists to observe the inner workings and subcellular structures that control life. This ability to see organelles within a cell provided scientists with the first understanding of how cells function. The visualization of the dynamic architecture of subcellular structures now often drives questions as researchers seek to understand the intricacies of the cell. With the advent of fluorescent labeling techniques, better and new optical techniques, and more sensitive and faster cameras, a whole array of questions can now be asked. There has been an explosion of new light microscopic techniques, and the race is on to build better and more powerful imaging systems so that we can further our understanding of the spatial and temporal mechanisms controlling molecular cell biology.

  16. Factors influencing subcellular localization of the human papillomavirus L2 minor structural protein

    SciTech Connect

    Kieback, Elisa; Mueller, Martin . E-mail: Martin.Mueller@dkfz.de

    2006-02-05

    Two structural proteins form the capsids of papillomaviruses. The major structural protein L1 is the structural determinant of the capsids and is present in 360 copies arranged in 72 pentamers. The minor structural protein L2 is estimated to be present in twelve copies per capsid. Possible roles for L2 in interaction with cell surface receptors and in virion uptake have been suggested. As previously reported, L2 localizes in subnuclear domains identified as nuclear domain 10 (ND10). As it was demonstrated that L2 is able to recruit viral and cellular proteins to ND10, a possible role for L2 as a mediator in viral assembly has been proposed. In this study, we determined factors influencing the localization of L2 at ND10. Under conditions of moderate L2 expression level and in the absence of heterologous viral components, we observed that, in contrast to previous reports, L2 is mainly distributed homogeneously throughout the nucleus. L2, however, is recruited to ND10 at a higher expression level or in the presence of viral components derived from vaccinia virus or from Semliki Forest virus. We observed that translocation of L2 to ND10 is not a concentration-dependent accumulation but rather seems to be triggered by yet unidentified cellular factors. In contrast to HPV 11 and 16 L2, the HPV 18 L2 protein seems to require L1 for efficient nuclear accumulation.

  17. Time-dependent subcellular structure injuries induced by nano-/micron-sized calcium oxalate monohydrate and dihydrate crystals.

    PubMed

    Sun, Xin-Yuan; Yu, Kai; Ouyang, Jian-Ming

    2017-10-01

    Comparative studies were conducted to investigate the time effect of cell injury induced by nano-sized (50nm) and micron-sized (10μm) calcium oxalate monohydrate (COM) and dihydrate (COD) crystals in African green monkey renal epithelial (Vero) cells. The effects of nano-/micron-sized COM and COD exposure on Vero cells were investigated by detecting the cell viability, cell morphology, LDH release, reactive oxygen species, mitochondrial membrane potential, cell cycle, and cell apoptosis, as well as the intracellular and extracellular crystal distribution. Nano-/micron-sized COM and COD exposure lead to subcellular organelle injury in varying degrees, but the injury sequence of various organelles differed. The time sequence of organelle injury presenting significant variation was described as follows: cell membrane injury (1h)provide insights into the damage to renal epithelial cells induced by urinary crystals and the formation mechanism of kidney stones. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Real-time Imaging and Tuning Subcellular Structures and Membrane Transport Kinetics of Single Live Cells at Nanosecond Regime

    PubMed Central

    Xu, Hongwu; Nallathamby, Prakash D.; Xu, Xiao-Hong Nancy

    2009-01-01

    We developed an electric-field exposure microchannel system with 230-nanometer thin-layer gold electrodes, and interfaced it with a single living cell imaging station and a 10-nanosecond-electric-pulse (10nsEP) generator. This design allows us to image intracellular molecules and structures, membrane transport and viability of single leukemic cells (HL60) while the cells are exposed to 10nsEPs of 0–179 kV/cm, permitting the study of subcellular responses at nanosecond regime. The electrodes confine a thin-layer section of the cells exposed to 10nsEPs, offering unprecedented high spatial resolution (230-nm at z-direction of E and imaging plane) for imaging intracellular molecules of single cells affected by 10nsEPs. We found that nucleic acids, membrane transport rates and viability of single cells depend on the number and electric-field-strength (E) of 10nsEPs, showing the cumulative effect of 10nsEPs on intracellular molecules and structures and suggesting the possibility of tuning them one-pulse-at-a-time. Using lower E (51 kV/cm) of 10nsEPs, we could manipulate nucleic acids of single living cells without disrupting their cellular membrane and viability. As E increases to 80, 124 and 179 kV/cm, membrane integrity and viability of cells exhibit higher dependence on the number of 10nsEPs in a non-linear fashion, showing that critical E and pulse number are needed to surmount cellular transport barriers and membrane integrity. PMID:19795898

  19. Longin and GAF domains: structural evolution and adaptation to the subcellular trafficking machinery.

    PubMed

    De Franceschi, Nicola; Wild, Klemens; Schlacht, Alexander; Dacks, Joel B; Sinning, Irmgard; Filippini, Francesco

    2014-01-01

    Endomembrane trafficking is one of the most prominent cytological features of eukaryotes. Given their widespread distribution and specialization, coiled-coil domains, coatomer domains, small GTPases and Longin domains are considered primordial 'building blocks' of the membrane trafficking machineries. Longin domains are conserved across eukaryotes and were likely to be present in the Last Eukaryotic Common Ancestor. The Longin fold is based on the α-β-α sandwich architecture and a unique topology, possibly accounting for the special adaptation to the eukaryotic trafficking machinery. The ancient Per ARNT Sim (PAS) and cGMP-specific phosphodiesterases, Adenylyl cyclases and FhlA (GAF) family domains show a similar architecture, and the identification of prokaryotic counterparts of GAF domains involved in trafficking provides an additional connection for the endomembrane system back into the pre-eukaryotic world. Proteome-wide, comparative bioinformatic analyses of the domains reveal three binding regions (A, B and C) mediating either specific or conserved protein-protein interactions. While the A region mediates intra- and inter-molecular interactions, the B region is involved in binding small GTPases, thus providing an evolutionary connection among major building blocks in the endomembrane system. Finally, we propose that the peculiar interaction surface of the C region of the Longin domain allowed it to extensively integrate into the endomembrane trafficking machinery in the earliest stages of building the eukaryotic cell. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Fumarate hydratase isoforms of Leishmania major: subcellular localization, structural and kinetic properties.

    PubMed

    Feliciano, Patrícia R; Gupta, Shreedhara; Dyszy, Fabio; Dias-Baruffi, Marcelo; Costa-Filho, Antonio J; Michels, Paul A M; Nonato, M Cristina

    2012-01-01

    Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs.

  1. Characterization of bud emergence 46 (BEM46) protein: Sequence, structural, phylogenetic and subcellular localization analyses

    SciTech Connect

    Kumar, Abhishek; Kollath-Leiß, Krisztina; Kempken, Frank

    2013-08-30

    Highlights: •All eukaryotes have at least a single copy of a bem46 ortholog. •The catalytic triad of BEM46 is illustrated using sequence and structural analysis. •We identified indels in the conserved domain of BEM46 protein. •Localization studies of BEM46 protein were carried out using GFP-fusion tagging. -- Abstract: The bud emergence 46 (BEM46) protein from Neurospora crassa belongs to the α/β-hydrolase superfamily. Recently, we have reported that the BEM46 protein is localized in the perinuclear ER and also forms spots close by the plasma membrane. The protein appears to be required for cell type-specific polarity formation in N. crassa. Furthermore, initial studies suggested that the BEM46 amino acid sequence is conserved in eukaryotes and is considered to be one of the widespread conserved “known unknown” eukaryotic genes. This warrants for a comprehensive phylogenetic analysis of this superfamily to unravel origin and molecular evolution of these genes in different eukaryotes. Herein, we observe that all eukaryotes have at least a single copy of a bem46 ortholog. Upon scanning of these proteins in various genomes, we find that there are expansions leading into several paralogs in vertebrates. Usingcomparative genomic analyses, we identified insertion/deletions (indels) in the conserved domain of BEM46 protein, which allow to differentiate fungal classes such as ascomycetes from basidiomycetes. We also find that exonic indels are able to differentiate BEM46 homologs of different eukaryotic lineage. Furthermore, we unravel that BEM46 protein from N. crassa possess a novel endoplasmic-retention signal (PEKK) using GFP-fusion tagging experiments. We propose that three residues namely a serine 188S, a histidine 292H and an aspartic acid 262D are most critical residues, forming a catalytic triad in BEM46 protein from N. crassa. We carried out a comprehensive study on bem46 genes from a molecular evolution perspective with combination of functional

  2. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi.

    PubMed

    Gonzalez, Soledad Natalia; Valsecchi, Wanda Mariela; Maugeri, Dante; Delfino, José María; Cazzulo, Juan José

    2017-01-01

    The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these

  3. Structure-Function Analysis of Rgs1 in Magnaporthe oryzae: Role of DEP Domains in Subcellular Targeting

    PubMed Central

    Ramanujam, Ravikrishna; Yishi, Xu; Liu, Hao; Naqvi, Naweed I.

    2012-01-01

    Background Rgs1, a prototypical Regulator of G protein Signaling, negatively modulates the cyclic AMP pathway thereby influencing various aspects of asexual development and pathogenesis in the rice-blast fungus Magnaporthe oryzae. Rgs1 possesses tandem DEP motifs (termed DEP-A and DEP-B; for Dishevelled, Egl-10, Pleckstrin) at the N-terminus, and a Gα-GTP interacting RGS catalytic core domain at the C-terminus. In this study, we focused on gaining further insights into the mechanisms of Rgs1 regulation and subcellular localization by characterizing the role(s) of the individual domains and the full-length protein during asexual development and pathogenesis in Magnaporthe. Methodology/Principal Findings Utilizing western blot analysis and specific antisera against the N- and C-terminal halves of Rgs1, we identify and report the in vivo endoproteolytic processing/cleavage of full-length Rgs1 that yields an N-terminal DEP and a RGS core domain. Independent expression of the resultant DEP-DEP half (N-Rgs1) or RGS core (C-Rgs1) fragments, failed to complement the rgs1Δ defects in colony morphology, aerial hyphal growth, surface hydrophobicity, conidiation, appressorium formation and infection. Interestingly, the full-length Rgs1-mCherry, as well as the tagged N-terminal DEP domains (individually or in conjunction) localized to distinct punctate vesicular structures in the cytosol, while the catalytic RGS core motif was predominantly vacuolar. Conclusions/Significance Based on our data from sequence alignments, immuno-blot and microscopic analysis, we propose that the post-translational proteolytic processing of Rgs1 and the vacuolar sequestration of the catalytic RGS domain represents an important means of down regulating Rgs1 function and thus forming an additional and alternative means of regulating G protein signaling in Magnaporthe. We further hypothesize the prevalence of analogous mechanisms functioning in other filamentous fungi. Furthermore, we conclusively

  4. Structure, kinetic characterization and subcellular localization of the two ribulose 5-phosphate epimerase isoenzymes from Trypanosoma cruzi

    PubMed Central

    Gonzalez, Soledad Natalia; Valsecchi, Wanda Mariela; Maugeri, Dante; Delfino, José María; Cazzulo, Juan José

    2017-01-01

    The enzyme of the pentose phosphate pathway (PPP) ribulose-5-phosphate-epimerase (RPE) is encoded by two genes present in the genome of Trypanosoma cruzi CL Brener clone: TcRPE1 and TcRPE2. Despite high sequence similarity at the amino acid residue level, the recombinant isoenzymes show a strikingly different kinetics. Whereas TcRPE2 follows a typical michaelian behavior, TcRPE1 shows a complex kinetic pattern, displaying a biphasic curve, suggesting the coexistence of -at least- two kinetically different molecular forms. Regarding the subcellular localization in epimastigotes, whereas TcRPE1 is a cytosolic enzyme, TcRPE2 is localized in glycosomes. To our knowledge, TcRPE2 is the first PPP isoenzyme that is exclusively localized in glycosomes. Over-expression of TcRPE1, but not of TcRPE2, significantly reduces the parasite doubling time in vitro, as compared with wild type epimastigotes. Both TcRPEs represent single domain proteins exhibiting the classical α/β TIM-barrel fold, as expected for enzymes with this activity. With regard to the architecture of the active site, all the important amino acid residues for catalysis -with the exception of M58- are also present in both TcRPEs models. The superimposition of the binding pocket of both isoenzyme models shows that they adopt essentially identical positions in the active site with a residue specific RMSD < 2Å, with the sole exception of S12, which displays a large deviation (residue specific RMSD: 11.07 Å). Studies on the quaternary arrangement of these isoenzymes reveal that both are present in a mixture of various oligomeric species made up of an even number of molecules, probably pointing to the dimer as their minimal functional unit. This multiplicity of oligomeric species has not been reported for any of the other RPEs studied so far and it might bear implications for the regulation of TcRPEs activity, although further investigation will be necessary to unravel the physiological significance of these

  5. Transcutical imaging with cellular and subcellular resolution.

    PubMed

    Tao, Xiaodong; Lin, Hui-Hao; Lam, Tuwin; Rodriguez, Ramiro; Wang, Jing W; Kubby, Joel

    2017-03-01

    We demonstrate transcutical structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins and calcium indicators in the living Drosophila brain with cellular and subcellular resolution.

  6. Transcutical imaging with cellular and subcellular resolution

    PubMed Central

    Tao, Xiaodong; Lin, Hui-Hao; Lam, Tuwin; Rodriguez, Ramiro; Wang, Jing W.; Kubby, Joel

    2017-01-01

    We demonstrate transcutical structural and functional imaging of neurons labeled with genetically encoded red fluorescent proteins and calcium indicators in the living Drosophila brain with cellular and subcellular resolution. PMID:28663828

  7. NAP (davunetide) provides functional and structural neuroprotection.

    PubMed

    Gozes, Illana

    2011-01-01

    NAP (davunetide) is an eight amino acid peptide (NAPVSIPQ) that has been shown to provide potent neuroprotection, in vitro and in vivo. In human clinical trials, NAP has been shown to increase memory scores in patients suffering from amnestic mild cognitive impairment, a precursor to Alzheimer's disease and to enhance functional daily behaviors in schizophrenia patients. NAP is derived from activity-dependent neuroprotective protein (ADNP) a molecule that is essential for brain formation, interacting with chromatin associated protein alpha and the chromatin remodeling complex SWI/SNF and regulating >400 genes during embryonic development. Partial loss in ADNP results in cognitive deficits and pathology of the microtubule associated protein tau (tauopathy) that is ameliorated in part by NAP replacement therapy. Recent studies increased the scope of NAP neuroprotection and provided further insights into the NAP mechanisms of action. Thus, it has been hypothesized that the presence of tau on axonal microtubules renders them notably less sensitive to the microtubule-severing protein katanin, and NAP was shown to protect microtubules from katanin disruption in the face of reduced tau expression. Parallel studies showed that NAP reduced the number of apoptotic neurons through activation of PI-3K/Akt pathway in the cortical plate or both PI-3K/Akt and MAPK/MEK1 kinases in the white matter. The interaction of these disparate yet complementary pathways is the subject of future studies toward human brain neuroprotection in the clinical scenario.

  8. Subcellular taxonomy: An ultrastructural classification system with diagnostic applications

    SciTech Connect

    McLay, A.L.C.; Toner, P.G.

    1985-01-01

    Contents of this work include: Ultrastructure, Nomenclature, and Disease; Numerical Listing: YX Cellular and Subcellular Structure; Alphabetical Listing; and Appendix: Proposed Revised Listing of M-6 Codes.

  9. Subcellular localization of mammalian type II membrane proteins.

    PubMed

    Aturaliya, Rajith N; Fink, J Lynn; Davis, Melissa J; Teasdale, Melvena S; Hanson, Kelly A; Miranda, Kevin C; Forrest, Alistair R R; Grimmond, Sean M; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Teasdale, Rohan D

    2006-05-01

    Application of a computational membrane organization prediction pipeline, MemO, identified putative type II membrane proteins as proteins predicted to encode a single alpha-helical transmembrane domain (TMD) and no signal peptides. MemO was applied to RIKEN's mouse isoform protein set to identify 1436 non-overlapping genomic regions or transcriptional units (TUs), which encode exclusively type II membrane proteins. Proteins with overlapping predicted InterPro and TMDs were reviewed to discard false positive predictions resulting in a dataset comprised of 1831 transcripts in 1408 TUs. This dataset was used to develop a systematic protocol to document subcellular localization of type II membrane proteins. This approach combines mining of published literature to identify subcellular localization data and a high-throughput, polymerase chain reaction (PCR)-based approach to experimentally characterize subcellular localization. These approaches have provided localization data for 244 and 169 proteins. Type II membrane proteins are localized to all major organelle compartments; however, some biases were observed towards the early secretory pathway and punctate structures. Collectively, this study reports the subcellular localization of 26% of the defined dataset. All reported localization data are presented in the LOCATE database (http://www.locate.imb.uq.edu.au).

  10. Biochemical characterization of USP7 reveals post-translational modification sites and structural requirements for substrate processing and subcellular localization.

    PubMed

    Fernández-Montalván, Amaury; Bouwmeester, Tewis; Joberty, Gerard; Mader, Robert; Mahnke, Marion; Pierrat, Benoit; Schlaeppi, Jean-Marc; Worpenberg, Susanne; Gerhartz, Bernd

    2007-08-01

    Ubiquitin specific protease 7 (USP7) belongs to the family of deubiquitinating enzymes. Among other functions, USP7 is involved in the regulation of stress response pathways, epigenetic silencing and the progress of infections by DNA viruses. USP7 is a 130-kDa protein with a cysteine peptidase core, N- and C-terminal domains required for protein-protein interactions. In the present study, recombinant USP7 full length, along with several variants corresponding to domain deletions, were expressed in different hosts in order to analyze post-translational modifications, oligomerization state, enzymatic properties and subcellular localization patterns of the enzyme. USP7 is phosphorylated at S18 and S963, and ubiquitinated at K869 in mammalian cells. In in vitro activity assays, N- and C-terminal truncations affected the catalytic efficiency of the enzyme different. Both the protease core alone and in combination with the N-terminal domain are over 100-fold less active than the full length enzyme, whereas a construct including the C-terminal region displays a rather small decrease in catalytic efficiency. Limited proteolysis experiments revealed that USP7 variants containing the C-terminal domain interact more tightly with ubiquitin. Besides playing an important role in substrate recognition and processing, this region might be involved in enzyme dimerization. USP7 constructs lacking the N-terminal domain failed to localize in the cell nucleus, but no nuclear localization signal could be mapped within the enzyme's first 70 amino acids. Instead, the tumor necrosis factor receptor associated factor-like region (amino acids 70-205) was sufficient to achieve the nuclear localization of the enzyme, suggesting that interaction partners might be required for USP7 nuclear import.

  11. Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.).

    PubMed

    Qin, Xiaoqiong; Coku, Ardian; Inoue, Kentaro; Tian, Li

    2011-10-01

    Carotenoids perform many critical functions in plants, animals, and humans. It is therefore important to understand carotenoid biosynthesis and its regulation in plants. Phytoene synthase (PSY) catalyzes the first committed and rate-limiting step in carotenoid biosynthesis. While PSY is present as a single copy gene in Arabidopsis, duplicated PSY genes have been identified in many economically important monocot and dicot crops. CmPSY1 was previously identified from melon (Cucumis melo L.), but was not functionally characterized. We isolated a second PSY gene, CmPSY2, from melon in this work. CmPSY2 possesses a unique intron/exon structure that has not been observed in other plant PSYs. Both CmPSY1 and CmPSY2 are functional in vitro, but exhibit distinct expression patterns in different melon tissues and during fruit development, suggesting differential regulation of the duplicated melon PSY genes. In vitro chloroplast import assays verified the plastidic localization of CmPSY1 and CmPSY2 despite the lack of an obvious plastid target peptide in CmPSY2. Promoter motif analysis of the duplicated melon and tomato PSY genes and the Arabidopsis PSY revealed distinctive cis-regulatory structures of melon PSYs and identified gibberellin-responsive motifs in all PSYs except for SlPSY1, which has not been reported previously. Overall, these data provide new insights into the evolutionary history of plant PSY genes and the regulation of PSY expression by developmental and environmental signals that may involve different regulatory networks.

  12. Identification, subcellular localization, biochemical properties, and high-resolution crystal structure of Trypanosoma brucei UDP-glucose pyrophosphorylase

    PubMed Central

    Mariño, Karina; Güther, Maria Lucia Sampaio; Wernimont, Amy K; Amani, Mernhaz; Hui, Raymond; Ferguson, Michael AJ

    2010-01-01

    The protozoan parasite Trypanosoma brucei is the causative agent of the cattle disease Nagana and human African sleeping sickness. Glycoproteins play key roles in the parasite’s survival and infectivity, and the de novo biosyntheses of the sugar nucleotides UDP-galactose (UDP-Gal), UDP-N-acetylglucosamine, and GDP-fucose have been shown to be essential for their growth. The only route to UDP-Gal in T. brucei is through the epimerization of UDP-glucose (UDP-Glc) by UDP-Glc 4′-epimerase. UDP-Glc is also the glucosyl donor for the unfolded glycoprotein glucosyltransferase (UGGT) involved in glycoprotein quality control in the endoplasmic reticulum and is the presumed donor for the synthesis of base J (β-d-glucosylhydroxymethyluracil), a rare deoxynucleotide found in telomere-proximal DNA in the bloodstream form of T. brucei. Considering that UDP-Glc plays such a central role in carbohydrate metabolism, we decided to characterize UDP-Glc biosynthesis in T. brucei. We identified and characterized the parasite UDP-glucose pyrophosphorylase (TbUGP), responsible for the formation of UDP-Glc from glucose-1-phosphate and UTP, and localized the enzyme to the peroxisome-like glycosome organelles of the parasite. Recombinant TbUGP was shown to be enzymatically active and specific for glucose-1-phosphate. The high-resolution crystal structure was also solved, providing a framework for the design of potential inhibitors against the parasite enzyme. PMID:20724435

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

    SciTech Connect

    Maroniche, Guillermo A.; Mongelli, Vanesa C.; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar

    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.

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

    PubMed

    Maroniche, Guillermo A; Mongelli, Vanesa C; Llauger, Gabriela; Alfonso, Victoria; Taboga, Oscar; del Vas, Mariana

    2012-09-01

    The in vivo subcellular localization of Mal de Río 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 α-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.

  15. Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum.

    PubMed Central

    Pologe, L G; Pavlovec, A; Shio, H; Ravetch, J V

    1987-01-01

    Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface. The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis. The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains. Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes. Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs. A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data. Images PMID:3313387

  16. Quantitative phase imaging of cellular and subcellular structures for non-invasive screening diagnostics of socially significant diseases

    NASA Astrophysics Data System (ADS)

    Vasilenko, Irina; Metelin, Vladislav; Nasyrov, Marat; Belyakov, Vladimir; Kuznetsov, Alexander; Sukhenko, Evgeniy

    2015-03-01

    The objective of the present study is to increase the quality of the early diagnosis using cytological differential-diagnostic criteria for reactive changes in the nuclear structures of the immunocompetent cells. The morphofunctional status of living cells were estimated in the real time using new technologic platform of the hardware-software complex for phase cell imaging. The level of functional activity for lymphocyte subpopulations was determined on the base of modification of nuclear structures and decreasing of nuclear phase thickness. The dynamics of nuclear parameters was used as the quantitative measuring for cell activating level and increasing of proliferative potential.

  17. A linear programming approach to reconstructing subcellular structures from confocal images for automated generation of representative 3D cellular models.

    PubMed

    Wood, Scott T; Dean, Brian C; Dean, Delphine

    2013-04-01

    This paper presents a novel computer vision algorithm to analyze 3D stacks of confocal images of fluorescently stained single cells. The goal of the algorithm is to create representative in silico model structures that can be imported into finite element analysis software for mechanical characterization. Segmentation of cell and nucleus boundaries is accomplished via standard thresholding methods. Using novel linear programming methods, a representative actin stress fiber network is generated by computing a linear superposition of fibers having minimum discrepancy compared with an experimental 3D confocal image. Qualitative validation is performed through analysis of seven 3D confocal image stacks of adherent vascular smooth muscle cells (VSMCs) grown in 2D culture. The presented method is able to automatically generate 3D geometries of the cell's boundary, nucleus, and representative F-actin network based on standard cell microscopy data. These geometries can be used for direct importation and implementation in structural finite element models for analysis of the mechanics of a single cell to potentially speed discoveries in the fields of regenerative medicine, mechanobiology, and drug discovery.

  18. A tale of the ciliate tail: investigation into the adaptive significance of this sub-cellular structure

    PubMed Central

    Gemmell, Brad J.; Jiang, Houshuo; Buskey, Edward J.

    2015-01-01

    Ciliates can form an important link between the microbial loop and higher trophic levels primarily through consumption by copepods. This high predation pressure has resulted in a number of ciliate species developing rapid escape swimming behaviour. Several species of these escaping ciliates also possess a long contractile tail for which the functionality remains unresolved. We use high-speed video, specialized optics and novel fluid visualization tools to evaluate the role of this contractile appendage in two free-swimming ciliates, Pseudotontonia sp. and Tontonia sp., and compare the performance to escape swimming behaviour of a non-tailed species, Strobilidium sp. Here, we show that ‘tailed’ species respond to hydrodynamic disturbances with extremely short response latencies (less than or equal to 0.89 ms) by rapidly contracting the tail which carries the cell body 2–4 cell diameters within a few milliseconds. This provides an advantage over non-tailed species during the critical first 10–30 ms of an escape. Two small, short-lived vortex rings are created during contraction of the tail. The flow imposed by the ciliate jumping can be described as two well-separated impulsive Stokeslets and the overall flow attenuates spatially as r−3. The high initial velocities and spatio-temporal arrangement of vortices created by tail contractions appear to provide a means for rapid escape as well as hydrodynamic ‘camouflage’ against fast striking, mechanoreceptive predators such as copepods. PMID:26180066

  19. Arabidopsis myosin XI sub-domains homologous to the yeast myo2p organelle inheritance sub-domain target subcellular structures in plant cells

    PubMed Central

    Sattarzadeh, Amirali; Schmelzer, Elmon; Hanson, Maureen R.

    2013-01-01

    Myosin XI motor proteins transport plant organelles on the actin cytoskeleton. The Arabidopsis gene family that encodes myosin XI has 13 members, 12 of which have sub-domains within the tail region that are homologous to well-characterized cargo-binding domains in the yeast myosin V myo2p. Little is presently known about the cargo-binding domains of plant myosin XIs. Prior experiments in which most or all of the tail regions of myosin XIs have been fused to yellow fluorescent protein (YFP) and transiently expressed have often not resulted in fluorescent labeling of plant organelles. We identified 42 amino-acid regions within 12 Arabidopsis myosin XIs that are homologous to the yeast myo2p tail region known to be essential for vacuole and mitochondrial inheritance. A YFP fusion of the yeast region expressed in plants did not label tonoplasts or mitochondria. We investigated whether the homologous Arabidopsis regions, termed by us the “PAL” sub-domain, could associate with subcellular structures following transient expression of fusions with YFP in Nicotiana benthamiana. Seven YFP::PAL sub-domain fusions decorated Golgi and six were localized to mitochondria. In general, the myosin XI PAL sub-domains labeled organelles whose motility had previously been observed to be affected by mutagenesis or dominant negative assays with the respective myosins. Simultaneous transient expression of the PAL sub-domains of myosin XI-H, XI-I, and XI-K resulted in inhibition of movement of mitochondria and Golgi. PMID:24187546

  20. Animal NLRs provide structural insights into plant NLR function.

    PubMed

    Bentham, Adam; Burdett, Hayden; Anderson, Peter A; Williams, Simon J; Kobe, Bostjan

    2017-03-01

    The plant immune system employs intracellular NLRs (nucleotide binding [NB], leucine-rich repeat [LRR]/nucleotide-binding oligomerization domain [NOD]-like receptors) to detect effector proteins secreted into the plant cell by potential pathogens. Activated plant NLRs trigger a range of immune responses, collectively known as the hypersensitive response (HR), which culminates in death of the infected cell. Plant NLRs show structural and functional resemblance to animal NLRs involved in inflammatory and innate immune responses. Therefore, knowledge of the activation and regulation of animal NLRs can help us understand the mechanism of action of plant NLRs, and vice versa. This review provides an overview of the innate immune pathways in plants and animals, focusing on the available structural and biochemical information available for both plant and animal NLRs. We highlight the gap in knowledge between the animal and plant systems, in particular the lack of structural information for plant NLRs, with crystal structures only available for the N-terminal domains of plant NLRs and an integrated decoy domain, in contrast to the more complete structures available for animal NLRs. We assess the similarities and differences between plant and animal NLRs, and use the structural information on the animal NLR pair NAIP/NLRC4 to derive a plausible model for plant NLR activation. Signalling by cooperative assembly formation (SCAF) appears to operate in most innate immunity pathways, including plant and animal NLRs. Our proposed model of plant NLR activation includes three key steps: (1) initially, the NLR exists in an inactive auto-inhibited state; (2) a combination of binding by activating elicitor and ATP leads to a structural rearrangement of the NLR; and (3) signalling occurs through cooperative assembly of the resistosome. Further studies, structural and biochemical in particular, will be required to provide additional evidence for the different features of this model and

  1. Critical behavior of subcellular density organization during neutrophil activation and migration.

    PubMed

    Baker-Groberg, Sandra M; Phillips, Kevin G; Healy, Laura D; Itakura, Asako; Porter, Juliana E; Newton, Paul K; Nan, Xiaolin; McCarty, Owen J T

    2015-12-01

    Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration.

  2. Critical behavior of subcellular density organization during neutrophil activation and migration

    PubMed Central

    Baker-Groberg, Sandra M.; Phillips, Kevin G.; Healy, Laura D.; Itakura, Asako; Porter, Juliana E.; Newton, Paul K.; Nan, Xiaolin; McCarty, Owen J.T.

    2015-01-01

    Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration. PMID:26640599

  3. Subcellular proteomics in neuroscience.

    PubMed

    Li, Ka Wan; Smit, August B

    2008-05-01

    The brain is the most complex and dynamically organized organ of the human body, with a high degree of computation capability enabling the execution of a wide spectrum of physiological processes and behaviors. In the past decades a large number of genomics studies have been undertaken to investigate brain function and brain disorders, but despite these efforts many of the underlying molecular mechanisms still remain largely unknown. The implementation of mass spectrometry based quantitative proteomics in recent years enabled to tap into condition-specific protein trafficking and protein interaction that are the key to organelle proteome (dys)function. The technology for neuroproteomics is still evolving; currently there are no standardized protocols. In this review we describe the most commonly used methods to prepare brain subcellular fractions suitable for proteomics analysis, and highlight the various approaches for quantitative neuroproteomics.

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

    NASA Astrophysics Data System (ADS)

    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

  5. Structure of CD84 Provides Insight into SLAM Family Function

    SciTech Connect

    Yan,Q.; Malashkevich, V.; Fedorov, A.; Fedorov, E.; Cao, E.; Lary, J.; Cole, J.; Nathenson, S.; Almo, S.

    2007-01-01

    The signaling lymphocyte activation molecule (SLAM) family includes homophilic and heterophilic receptors that modulate both adaptive and innate immune responses. These receptors share a common ectodomain organization: a membrane-proximal immunoglobulin constant domain and a membrane-distal immunoglobulin variable domain that is responsible for ligand recognition. CD84 is a homophilic family member that enhances IFN-{gamma} secretion in activated T cells. Our solution studies revealed that CD84 strongly self-associates with a K{sub d} in the submicromolar range. These data, in combination with previous reports, demonstrate that the SLAM family homophilic affinities span at least three orders of magnitude and suggest that differences in the affinities may contribute to the distinct signaling behavior exhibited by the individual family members. The 2.0 {angstrom} crystal structure of the human CD84 immunoglobulin variable domain revealed an orthogonal homophilic dimer with high similarity to the recently reported homophilic dimer of the SLAM family member NTB-A. Structural and chemical differences in the homophilic interfaces provide a mechanism to prevent the formation of undesired heterodimers among the SLAM family homophilic receptors. These structural data also suggest that, like NTB-A, all SLAM family homophilic dimers adopt a highly kinked organization spanning an end-to-end distance of {approx}140 {angstrom}. This common molecular dimension provides an opportunity for all two-domain SLAM family receptors to colocalize within the immunological synapse and bridge the T cell and antigen-presenting cell.

  6. Support vector machine approach for protein subcellular localization prediction.

    PubMed

    Hua, S; Sun, Z

    2001-08-01

    Subcellular localization is a key functional characteristic of proteins. A fully automatic and reliable prediction system for protein subcellular localization is needed, especially for the analysis of large-scale genome sequences. In this paper, Support Vector Machine has been introduced to predict the subcellular localization of proteins from their amino acid compositions. The total prediction accuracies reach 91.4% for three subcellular locations in prokaryotic organisms and 79.4% for four locations in eukaryotic organisms. Predictions by our approach are robust to errors in the protein N-terminal sequences. This new approach provides superior prediction performance compared with existing algorithms based on amino acid composition and can be a complementary method to other existing methods based on sorting signals. A web server implementing the prediction method is available at http://www.bioinfo.tsinghua.edu.cn/SubLoc/. Supplementary material is available at http://www.bioinfo.tsinghua.edu.cn/SubLoc/.

  7. Effectiveness of Structural Feedback Provided by Pathfinder Networks

    ERIC Educational Resources Information Center

    Trumpower, David L.; Sarwar, Gul Shahzad

    2010-01-01

    Within the field of education, there has been an increasing recognition of the importance of formative assessment and of structural knowledge. In an effort to fill needs in each of these areas, this article describes an innovative feedback strategy intended to improve students' structural knowledge. Twenty-four high school physics students were…

  8. Multiscale Investigation from Subcellular to Tissue Scale of Onion Epidermal Plant Cell Wall Mechanical Properties

    NASA Astrophysics Data System (ADS)

    Zamil, Mohammad Shafayet

    the best of our knowledge, the plant cell wall at subcellular scale was never characterized under tensile loading. By coupling the structure based multiscale modeling and mechanical characterizations at different length scales, an attempt was made to provide novel insights towards understanding the mechanics and architecture of cell wall. This study also suggests that a multiscale investigation is essential for garnering fundamental insights into the hierarchical deformation of biological systems.

  9. Subcellular Fractionation Enhances Proteome Coverage of Pancreatic Duct Cells

    PubMed Central

    Paulo, Joao A.; Gaun, Aleksandr; Kadiyala, Vivek; Ghoulidi, Ali; Banks, Peter A.; Conwell, Darwin L.; Steen, Hanno

    2013-01-01

    Objectives Subcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increases proteome coverage. Methods We used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared - via mass spectrometry-based analysis - the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates. Results We identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle. Conclusions Subcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease. PMID:23352835

  10. Providing structural modules with self-integrity monitoring

    NASA Technical Reports Server (NTRS)

    Walton, W. B.; Ibanez, P.; Yessaie, G.

    1988-01-01

    With the advent of complex space structures (i.e., U.S. Space Station), the need for methods for remotely detecting structural damage will become greater. Some of these structures will have hundreds of individual structural elements (i.e., strut members). Should some of them become damaged, it could be virtually impossible to detect it using visual or similar inspection techniques. The damage of only a few individual members may or may not be a serious problem. However, should a significant number of the members be damaged, a significant problem could be created. The implementation of an appropriate remote damage detection scheme would greatly reduce the likelihood of a serious problem related to structural damage ever occurring. This report presents the results of the research conducted on remote structural damage detection approaches and the related mathematical algorithms. The research was conducted for the Small Business Innovation and Research (SBIR) Phase 2 National Aeronautics and Space Administration (NASA) Contract NAS7-961.

  11. Structural disorder provides increased adaptability for vesicle trafficking pathways.

    PubMed

    Pietrosemoli, Natalia; Pancsa, Rita; Tompa, Peter

    2013-01-01

    Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (~23%) than the other two, COPI (~9%) and COPII (~8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and suggest

  12. Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

    PubMed Central

    Tompa, Peter

    2013-01-01

    Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (∼23%) than the other two, COPI (∼9%) and COPII (∼8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and

  13. VET Provider Market Structures: History, Growth and Change. Research Report

    ERIC Educational Resources Information Center

    Korbel, Patrick; Misko, Josie

    2016-01-01

    This paper tracks the development of the Australian vocational education and training (VET) provider market over the last two decades in the context of significant policy changes and generally increased competition. It provides an insight into how the sector has arrived at its current position, painting a present-day picture of great diversity.…

  14. The Emergence of Dissipative Structures within Information Provider Organizations.

    ERIC Educational Resources Information Center

    Ward, Robert; Robins, David

    1998-01-01

    Compares three system models (closed, open, dissipative) with traditional models for adoption of new technology and library operations. Argues that information-provider organizations (libraries) must be able to adapt to radical changes in their environments in order to remain viable economic entities. (Author/AEF)

  15. Primary structure and developmental expression of Bufo arenarum cellular nucleic acid-binding protein: changes in subcellular localization during early embryogenesis.

    PubMed

    Armas, P; Cabada, M O; Calcaterra, N B

    2001-02-01

    A Bufo arenarum cellular nucleic acid-binding protein (bCNBP) full-length cDNA was cloned. bCNBP is a 19.4 kDa protein containing seven CCHC zinc finger motifs, an RGG box and a Ser-rich region. Amino acid comparisons showed high values of homology in vertebrates and smaller values in insects or inferior eukaryotes. Northern blot analysis during oogenesis and early development revealed two transcripts with different expressions of pattern behavior. One of them is present in all stages analyzed, whereas the other is only detected from the beginning of zygotic transcription. Immunocytochemistry assays carried out on sections of ovary and early embryos showed that there was no specific staining of previtellogenic oocytes. In early vitellogenic oocytes, in oocytes at stages V/VI and in embryos at early blastula stage, reaction was observed inside the cytoplasm. At mid-blastula stage, CNBP was mainly detected in the epiblast. At the late gastrula stage, two layers of cells were stained in the archenteron roof, in which the internal one presented as strong staining. Nuclei in this layer were stained even stronger than the cytoplasm. Changes in mRNA expression patterns, accompanied by changes in subcellular localization, suggest that CNBP might interact with both nuclear and cytoplasmic nucleic acids.

  16. [Structural characteristics providing for high specificity of enteropeptidase].

    PubMed

    Mikhaĭlova, A G; Rumsh, L D

    1998-04-01

    The effects of structural modification upon the specificity of enteropeptidase were studied. A variation in the unique specificity of the enzyme was shown to be the result of an autolysis caused by the enzyme's loss of calcium ions. The cleavage sites of the autolysis were determined. A truncated enzyme containing the C-terminal fragment of its heavy chain (466-800 residues) and the intact light chain were shown to be the products of autolysis. The kinetic parameters of the hydrolysis of trypsinogen, a recombinant protein, and a peptide substrate with both forms of enteropeptidase were determined. Conditions were found that can help regulate the transition of the native enzyme into the truncated form. A hypothesis was proposed concerning the autoactivational character of proenteropeptidase processing.

  17. Kin structure provides no explanation for intruders in social aphids.

    PubMed

    Abbot, Patrick; Chhatre, Vikram

    2007-09-01

    Nontraditional social organisms have received increasing attention in recent years, because they present opportunities to study the convergent properties of social evolution. Some aphid species are social, occurring in dense clones with specialized morphs that attack predators and parasites. Little is known about how social aphid colonies resolve conflicts of interest when clonal barriers break down. Pemphigus obesinymphae is a North American gall-forming social aphid that produces both nymphal defenders that protect natal clones, and specialized intruders that invade other nearby clones on their host plants. We tested the hypothesis that clones are arranged on their host plants in spatial clusters of related family groups, such that intruders would be biased towards movement within kin groups. Movement within and not between kin groups would then provide insight into the nature of conflict in this social aphid. We sampled eight sites in the eastern United States and in Arizona, and used eight microsatellite markers to estimate pairwise relatedness between spatial groups. We found little evidence of deviation from random distributions of genotypes on their host plants. Evidently, Pem. obesinymphae intruders typically exploit unrelated clones, and spatial orientation provides no solution to the problem of 'polyclonality' in this species. We discuss implications of this result for our understanding of cooperation and conflict in social aphids.

  18. Subcellular compartmentation of glutathione in dicotyledonous plants

    PubMed Central

    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

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

    PubMed Central

    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. SUBCELLULAR PHARMACOKINETICS AND ITS POTENTIAL FOR LIBRARY FOCUSING (R826652)

    EPA Science Inventory

    Abstract

    Subcellular pharmacokinetics (SP) optimizes biology-related factors in the design of libraries for high throughput screening by defining comparatively narrow ranges of properties (lipophilicity, amphiphilicity, acidity, reactivity, 3D-structural features) of t...

  1. SUBCELLULAR PHARMACOKINETICS AND ITS POTENTIAL FOR LIBRARY FOCUSING (R826652)

    EPA Science Inventory

    Abstract

    Subcellular pharmacokinetics (SP) optimizes biology-related factors in the design of libraries for high throughput screening by defining comparatively narrow ranges of properties (lipophilicity, amphiphilicity, acidity, reactivity, 3D-structural features) of t...

  2. Protein subcellular localization assays using split fluorescent proteins

    DOEpatents

    Waldo, Geoffrey S.; Cabantous, Stephanie

    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).

  3. Subcellular optogenetics – controlling signaling and single-cell behavior

    PubMed Central

    Karunarathne, W. K. Ajith; O'Neill, Patrick R.; Gautam, Narasimhan

    2015-01-01

    ABSTRACT Variation in signaling activity across a cell plays a crucial role in processes such as cell migration. Signaling activity specific to organelles within a cell also likely plays a key role in regulating cellular functions. To understand how such spatially confined signaling within a cell regulates cell behavior, tools that exert experimental control over subcellular signaling activity are required. Here, we discuss the advantages of using optogenetic approaches to achieve this control. We focus on a set of optical triggers that allow subcellular control over signaling through the activation of G-protein-coupled receptors (GPCRs), receptor tyrosine kinases and downstream signaling proteins, as well as those that inhibit endogenous signaling proteins. We also discuss the specific insights with regard to signaling and cell behavior that these subcellular optogenetic approaches can provide. PMID:25433038

  4. [The contractile heart. Cellular and subcellular aspects].

    PubMed

    Larsen, T H

    1994-12-10

    The excitation-contraction coupling can be defined as the mechanisms involved when the action potential initiates a contraction response of the cardiac muscle cell. The action potential is conducted from cell to cell, resulting in a synchronized contraction of the myocardium. Both the propagation of the action potential and the myofibrillar contraction are dependent on changes in free Ca++ concentrations. Recently, the mediators and the molecular and structural components involved in the subcellular transforming of the action potential into a contraction have been characterized. The opening of voltage-dependent L-type Ca(++)-channels in the cell membrane stimulates a release of Ca++ from the sarcoplasmic reticulum. This Ca(++)-mediated Ca(++)-release appears to be a graded mechanism and is associated with the presence of structural couplings (Ca++ synapses) in the cardiac muscle cell.

  5. Subcellular localization of the yeast proteome.

    PubMed

    Kumar, Anuj; Agarwal, Seema; Heyman, John A; Matson, Sandra; Heidtman, Matthew; Piccirillo, Stacy; Umansky, Lara; Drawid, Amar; Jansen, Ronald; Liu, Yang; Cheung, Kei-Hoi; Miller, Perry; Gerstein, Mark; Roeder, G Shirleen; Snyder, Michael

    2002-03-15

    Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass approximately 5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability--a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu.

  6. Subcellular localization of the yeast proteome

    PubMed Central

    Kumar, Anuj; Agarwal, Seema; Heyman, John A.; Matson, Sandra; Heidtman, Matthew; Piccirillo, Stacy; Umansky, Lara; Drawid, Amar; Jansen, Ronald; Liu, Yang; Cheung, Kei-Hoi; Miller, Perry; Gerstein, Mark; Roeder, G. Shirleen; Snyder, Michael

    2002-01-01

    Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass ∼5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability—a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu. PMID:11914276

  7. Multiple-labelling immunoEM using different sizes of colloidal gold: alternative approaches to test for differential distribution and colocalization in subcellular structures.

    PubMed

    Mayhew, Terry M; Lucocq, John M

    2011-03-01

    Various methods for quantifying cellular immunogold labelling on transmission electron microscope thin sections are currently available. All rely on sound random sampling principles and are applicable to single immunolabelling across compartments within a given cell type or between different experimental groups of cells. Although methods are also available to test for colocalization in double/triple immunogold labelling studies, so far, these have relied on making multiple measurements of gold particle densities in defined areas or of inter-particle nearest neighbour distances. Here, we present alternative two-step approaches to codistribution and colocalization assessment that merely require raw counts of gold particles in distinct cellular compartments. For assessing codistribution over aggregate compartments, initial statistical evaluation involves combining contingency table and chi-squared analyses to provide predicted gold particle distributions. The observed and predicted distributions allow testing of the appropriate null hypothesis, namely, that there is no difference in the distribution patterns of proteins labelled by different sizes of gold particle. In short, the null hypothesis is that of colocalization. The approach for assessing colabelling recognises that, on thin sections, a compartment is made up of a set of sectional images (profiles) of cognate structures. The approach involves identifying two groups of compartmental profiles that are unlabelled and labelled for one gold marker size. The proportions in each group that are also labelled for the second gold marker size are then compared. Statistical analysis now uses a 2 × 2 contingency table combined with the Fisher exact probability test. Having identified double labelling, the profiles can be analysed further in order to identify characteristic features that might account for the double labelling. In each case, the approach is illustrated using synthetic and/or experimental datasets and can

  8. Crystal structure of human thimet oligopeptidase provides insight into substrate recognition, regulation, and localization.

    PubMed

    Ray, Kallol; Hines, Christina S; Coll-Rodriguez, Jerry; Rodgers, David W

    2004-05-07

    Thimet oligopeptidase (TOP) is a zinc metallopeptidase that metabolizes a number of bioactive peptides and degrades peptides released by the proteasome, limiting antigenic presentation by MHC class I molecules. We present the crystal structure of human TOP at 2.0-A resolution. The active site is located at the base of a deep channel that runs the length of the elongated molecule, an overall fold first seen in the closely related metallopeptidase neurolysin. Comparison of the two related structures indicates hinge-like flexibility and identifies elements near one end of the channel that adopt different conformations. Relatively few of the sequence differences between TOP and neurolysin map to the proposed substrate-binding site, and four of these variable residues may account for differences in substrate specificity. In addition, a loop segment (residues 599-611) in TOP differs in conformation and degree of order from the corresponding neurolysin loop, suggesting it may also play a role in activity differences. Cysteines thought to mediate covalent oligomerization of rat TOP, which can inactivate the enzyme, are found to be surface-accessible in the human enzyme, and additional cysteines (residues 321,350, and 644) may also mediate multimerization in the human homolog. Disorder in the N terminus of TOP indicates it may be involved in subcellular localization, but a potential nuclear import element is found to be part of a helix and, therefore, unlikely to be involved in transport. A large acidic patch on the surface could potentially mediate a protein-protein interaction, possibly through formation of a covalent linkage.

  9. An Organelle Correlation-Guided Feature Selection Approach for Classifying Multi-Label Subcellular Bio-images.

    PubMed

    Shao, Wei; Liu, Mingxia; Xu, Ying-Ying; Shen, Hong-Bin; Zhang, Daoqiang

    2017-03-03

    Nowadays, with the advances in microscopic imaging, accurate classification of bioimage-based protein subcellular location pattern has attracted as much attention as ever. One of the basic challenging problems is how to select the useful feature components among thousands of potential features to describe the images. This is not an easy task especially considering there is a high ratio of multi-location proteins. Existing feature selection methods seldom take the correlation among different cellular compartments into consideration, and thus may miss some features that will be co-important for several subcellular locations. To deal with this problem, we make use of the important structural correlation among different cellular compartments and propose an organelle structural correlation regularized feature selection method CSF (Common-Sets of Features) in this paper. We formulate the multi-label classification problem by adopting a group-sparsity regularizer to select common subsets of relevant features from different cellular compartments. In addition, we also add a cell structural correlation regularized Laplacian term, which utilizes the prior biological structural information to capture the intrinsic dependency among different cellular compartments. The CSF provides a new feature selection strategy for multi-label bio-image subcellular pattern classifications, and the experimental results also show its superiority when comparing with several existing algorithms.

  10. Subcellular localization of pituitary enzymes

    NASA Technical Reports Server (NTRS)

    Smith, R. E.

    1970-01-01

    A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

  11. Subcellular Localization of Arabidopsis 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase1

    PubMed Central

    Leivar, Pablo; González, Víctor M.; Castel, Susanna; Trelease, Richard N.; López-Iglesias, Carmen; Arró, Montserrat; Boronat, Albert; Campos, Narciso; Ferrer, Albert; Fernàndez-Busquets, Xavier

    2005-01-01

    Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-μm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR

  12. Subcellular localization of Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    Leivar, Pablo; González, Víctor M; Castel, Susanna; Trelease, Richard N; López-Iglesias, Carmen; Arró, Montserrat; Boronat, Albert; Campos, Narciso; Ferrer, Albert; Fernàndez-Busquets, Xavier

    2005-01-01

    Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-microm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of

  13. Genetically Targeted Fluorogenic Macromolecules for Subcellular Imaging and Cellular Perturbation

    PubMed Central

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

    2015-01-01

    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. PMID:26183934

  14. Experimental shielding evaluation of the radiation protection provided by the structurally significant components of residential structures.

    PubMed

    Dickson, E D; Hamby, D M

    2014-03-01

    The human health and environmental effects following a postulated accidental release of radioactive material to the environment have been a public and regulatory concern since the early development of nuclear technology. These postulated releases have been researched extensively to better understand the potential risks for accident mitigation and emergency planning purposes. The objective of this investigation is to provide an updated technical basis for contemporary building shielding factors for the US housing stock. Building shielding factors quantify the protection from ionising radiation provided by a certain building type. Much of the current data used to determine the quality of shielding around nuclear facilities and urban environments is based on simplistic point-kernel calculations for 1950s era suburbia and is no longer applicable to the densely populated urban environments realised today. To analyse a building's radiation shielding properties, the ideal approach would be to subject a variety of building types to various radioactive sources and measure the radiation levels in and around the building. While this is not entirely practicable, this research analyses the shielding effectiveness of ten structurally significant US housing-stock models (walls and roofs) important for shielding against ionising radiation. The experimental data are used to benchmark computational models to calculate the shielding effectiveness of various building configurations under investigation from two types of realistic environmental source terms. Various combinations of these ten shielding models can be used to develop full-scale computational housing-unit models for building shielding factor calculations representing 69.6 million housing units (61.3%) in the United States. Results produced in this investigation provide a comparison between theory and experiment behind building shielding factor methodology.

  15. Regulation of intracellular heme trafficking revealed by subcellular reporters

    PubMed Central

    Yuan, Xiaojing; Rietzschel, Nicole; Walter Nuno, Ana Beatriz; Hanna, David A.; Phillips, John D.; Raven, Emma L.; Reddi, Amit R.; Hamza, Iqbal

    2016-01-01

    Heme is an essential prosthetic group in proteins that reside in virtually every subcellular compartment performing diverse biological functions. Irrespective of whether heme is synthesized in the mitochondria or imported from the environment, this hydrophobic and potentially toxic metalloporphyrin has to be trafficked across membrane barriers, a concept heretofore poorly understood. Here we show, using subcellular-targeted, genetically encoded hemoprotein peroxidase reporters, that both extracellular and endogenous heme contribute to cellular labile heme and that extracellular heme can be transported and used in toto by hemoproteins in all six subcellular compartments examined. The reporters are robust, show large signal-to-background ratio, and provide sufficient range to detect changes in intracellular labile heme. Restoration of reporter activity by heme is organelle-specific, with the Golgi and endoplasmic reticulum being important sites for both exogenous and endogenous heme trafficking. Expression of peroxidase reporters in Caenorhabditis elegans shows that environmental heme influences labile heme in a tissue-dependent manner; reporter activity in the intestine shows a linear increase compared with muscle or hypodermis, with the lowest heme threshold in neurons. Our results demonstrate that the trafficking pathways for exogenous and endogenous heme are distinct, with intrinsic preference for specific subcellular compartments. We anticipate our results will serve as a heuristic paradigm for more sophisticated studies on heme trafficking in cellular and whole-animal models. PMID:27528661

  16. A Formal Ontology of Subcellular Neuroanatomy

    PubMed Central

    Larson, Stephen D.; Fong, Lisa L.; Gupta, Amarnath; Condit, Christopher; Bug, William J.; Martone, Maryann E.

    2007-01-01

    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. PMID:18974798

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

    PubMed

    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.

  18. Structural changes at the cellular and subcellular level in the cerebral cortex of mice visualized by means of trans-cranial multi photon in-vivo microscopy

    NASA Astrophysics Data System (ADS)

    Nase, Gabriele; Helm, P. Johannes; Ottersen, Ole Petter

    2006-02-01

    Neural cell structural change is associated with numerous processes in the normal and diseased brain. We report about a multi photon laser scanning microscope setup that permits visualization of such neuronal and glial structural dynamics in living mice and recent observations performed by means of this instrument. The modular system consists of a modified industrial standard CSLM and is to a large degree composed of commercially available components. From a technical point of view, two developments are essential: Firstly, a multifunctional stage adapted to both laser scanning microscopic observation and pre-observational surgery has been designed and built. Secondly, the essential component of the detection unit is a state-of-the-art photomultiplier tube installed in a Peltier cooled thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields. An electro-optical modulator is used to accurately adjust the power of the applied laser beam and to blank the beam when no data are sampled, e.g. in the dead time intervals between adjacent pixels. Photo bleaching and thermal damage are thus kept on a minimum level. In transgenic mice expressing fluorescent proteins in neuronal or glial cells, details such as spines and astrocytic endfeet can be imaged trans-cranially to at least 80 microns below the brain surface. Individual cells and cell compartments can be visualized repeatedly over extensive periods of time.

  19. Protein structure prediction provides comparable performance to crystallographic structures in docking-based virtual screening.

    PubMed

    Du, Hongying; Brender, Jeffrey R; Zhang, Jian; Zhang, Yang

    2015-01-01

    Structure based virtual screening has largely been limited to protein targets for which either an experimental structure is available or a strongly homologous template exists so that a high-resolution model can be constructed. The performance of state of the art protein structure predictions in virtual screening in systems where only weakly homologous templates are available is largely untested. Using the challenging DUD database of structural decoys, we show here that even using templates with only weak sequence homology (<30% sequence identity) structural models can be constructed by I-TASSER which achieve comparable enrichment rates to using the experimental bound crystal structure in the majority of the cases studied. For 65% of the targets, the I-TASSER models, which are constructed essentially in the apo conformations, reached 70% of the virtual screening performance of using the holo-crystal structures. A correlation was observed between the success of I-TASSER in modeling the global fold and local structures in the binding pockets of the proteins versus the relative success in virtual screening. The virtual screening performance can be further improved by the recognition of chemical features of the ligand compounds. These results suggest that the combination of structure-based docking and advanced protein structure modeling methods should be a valuable approach to the large-scale drug screening and discovery studies, especially for the proteins lacking crystallographic structures.

  20. Structural Changes in Senescing Oilseed Rape Leaves at Tissue and Subcellular Levels Monitored by Nuclear Magnetic Resonance Relaxometry through Water Status

    PubMed Central

    Musse, Maja; De Franceschi, Loriane; Cambert, Mireille; Sorin, Clément; Le Caherec, Françoise; Burel, Agnès; Bouchereau, Alain; Mariette, François; Leport, Laurent

    2013-01-01

    Nitrogen use efficiency is relatively low in oilseed rape (Brassica napus) due to weak nitrogen remobilization during leaf senescence. Monitoring the kinetics of water distribution associated with the reorganization of cell structures, therefore, would be valuable to improve the characterization of nutrient recycling in leaf tissues and the associated senescence processes. In this study, nuclear magnetic resonance (NMR) relaxometry was used to describe water distribution and status at the cellular level in different leaf ranks of well-watered plants. It was shown to be able to detect slight variations in the evolution of senescence. The NMR results were linked to physiological characterization of the leaves and to light and electron micrographs. A relationship between cell hydration and leaf senescence was revealed and associated with changes in the NMR signal. The relative intensities and the transverse relaxation times of the NMR signal components associated with vacuole water were positively correlated with senescence, describing water uptake and vacuole and cell enlargement. Moreover, the relative intensity of the NMR signal that we assigned to the chloroplast water decreased during the senescence process, in agreement with the decrease in relative chloroplast volume estimated from micrographs. The results are discussed on the basis of water flux occurring at the cellular level during senescence. One of the main applications of this study would be for plant phenotyping, especially for plants under environmental stress such as nitrogen starvation. PMID:23903438

  1. Three-dimensional preservation of cellular and subcellular structures suggests 1.6 billion-year-old crown-group red algae

    PubMed Central

    Bengtson, Stefan; Sallstedt, Therese; Belivanova, Veneta; Whitehouse, Martin

    2017-01-01

    The ~1.6 Ga Tirohan Dolomite of the Lower Vindhyan in central India contains phosphatized stromatolitic microbialites. We report from there uniquely well-preserved fossils interpreted as probable crown-group rhodophytes (red algae). The filamentous form Rafatazmia chitrakootensis n. gen, n. sp. has uniserial rows of large cells and grows through diffusely distributed septation. Each cell has a centrally suspended, conspicuous rhomboidal disk interpreted as a pyrenoid. The septa between the cells have central structures that may represent pit connections and pit plugs. Another filamentous form, Denaricion mendax n. gen., n. sp., has coin-like cells reminiscent of those in large sulfur-oxidizing bacteria but much more recalcitrant than the liquid-vacuole-filled cells of the latter. There are also resemblances with oscillatoriacean cyanobacteria, although cell volumes in the latter are much smaller. The wider affinities of Denaricion are uncertain. Ramathallus lobatus n. gen., n. sp. is a lobate sessile alga with pseudoparenchymatous thallus, “cell fountains,” and apical growth, suggesting florideophycean affinity. If these inferences are correct, Rafatazmia and Ramathallus represent crown-group multicellular rhodophytes, antedating the oldest previously accepted red alga in the fossil record by about 400 million years. PMID:28291791

  2. Expression pattern, subcellular localization and structure--function relationship of rat Tpx-1, a spermatogenic cell adhesion molecule responsible for association with Sertoli cells.

    PubMed

    Maeda, T; Nishida, J; Nakanishi, Y

    1999-12-01

    The gene for a testicular cell adhesion protein called Tpx-1, which mediates the binding of spermatogenic cells to Sertoli cells of the rat in primary culture, was previously cloned. Here the characterization of Tpx-1 is reported. Tpx-1 messenger ribonucleic acid (mRNA) became detectable in pachytene spermatocytes and continued to be present throughout development into elongated spermatids, while the amount of Tpx-1 protein seemed to increase some time after the increment of mRNA. Tpx-1 protein was also present, although less abundantly, in spermatozoa prepared from the epididymis. Tpx-1 contains a cluster of hydrophobic amino acid residues near the amino terminus and a cysteine-rich region in the carboxyl-terminal half. Tpx-1 fused with green fluorescence protein was secreted into the medium when expressed in a cultured cell line, depending on the presence of the amino-terminal hydrophobic region. Moreover, Tpx-1 was present in the medium of testicular cell primary culture. Structure-function analysis revealed that the amino-terminal 101 amino acid residues were sufficient for cell adhesion activity, whereas the carboxyl-terminal cysteine-rich region was dispensable. In conclusion, Tpx-1 is produced and secreted from spermatogenic cells at various differentiation stages, and mediates the interaction of those cells with Sertoli cells.

  3. An Overview of Sub-Cellular Mechanisms Involved in the Action of TTFields

    PubMed Central

    Tuszynski, Jack A.; Wenger, Cornelia; Friesen, Douglas E.; Preto, Jordane

    2016-01-01

    Long-standing research on electric and electromagnetic field interactions with biological cells and their subcellular structures has mainly focused on the low- and high-frequency regimes. Biological effects at intermediate frequencies between 100 and 300 kHz have been recently discovered and applied to cancer cells as a therapeutic modality called Tumor Treating Fields (TTFields). TTFields are clinically applied to disrupt cell division, primarily for the treatment of glioblastoma multiforme (GBM). In this review, we provide an assessment of possible physical interactions between 100 kHz range alternating electric fields and biological cells in general and their nano-scale subcellular structures in particular. This is intended to mechanistically elucidate the observed strong disruptive effects in cancer cells. Computational models of isolated cells subject to TTFields predict that for intermediate frequencies the intracellular electric field strength significantly increases and that peak dielectrophoretic forces develop in dividing cells. These findings are in agreement with in vitro observations of TTFields’ disruptive effects on cellular function. We conclude that the most likely candidates to provide a quantitative explanation of these effects are ionic condensation waves around microtubules as well as dielectrophoretic effects on the dipole moments of microtubules. A less likely possibility is the involvement of actin filaments or ion channels. PMID:27845746

  4. Information theory provides a comprehensive framework for the evaluation of protein structure predictions

    PubMed Central

    Swanson, Rosemarie; Vannucci, Marina; Tsai, Jerry W.

    2008-01-01

    Protein structure prediction has a number of important ad hoc similarity measures for evaluating predictions, but would benefit from a measure that is able to provide a common framework for a broad range of comparisons. Here we show that a mutual information-like measure can provide a comprehensive framework for evaluating protein structure prediction of all types. We discuss the concept of information, its application to secondary structure, and the obstacle to applying it to 3D structure. Based on insights from the secondary structure case, we present an approach to work around the 3D difficulties, and develop a method to measure the mutual information provided by a 3D structure prediction. We integrate the evaluation of all types of protein structure prediction into a single frame work, and compare the amount of information provided by various prediction methods, including secondary structure prediction. Within this broadened framework, the idea that structure is better preserved than sequence during evolution is evaluated quantitatively for the globin family. A nearly perfect sequence match in the globin family corresponds to about 300 bits of information, whereas a nearly perfect structural match for the same two proteins corresponds to about 2500 bits of information, where bits of information describes the probability of obtaining a match of similar closeness by chance. Mutual information provides both a theoretical basis for evaluating structure similarity and an explanatory surround for existing similarity measures. PMID:18704942

  5. A guided tour into subcellular colocalization analysis in light microscopy.

    PubMed

    Bolte, S; Cordelières, F P

    2006-12-01

    It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, image-analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object-based approach.

  6. Mathematical Modeling of Sub-Cellular Asymmetry of Fat-Dachsous Heterodimer for Generation of Planar Cell Polarity

    PubMed Central

    Kumar, Amit; Sinha, Pradip

    2014-01-01

    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 for future

  7. Toward a Theory of the Primo Vascular System: A Hypothetical Circulatory System at the Subcellular Level

    PubMed Central

    Lee, Byung-Cheon; Park, Sang Hyun; Yoon, Seung Zhoo

    2013-01-01

    This paper suggests a theoretical framework for the primo vascular system (PVS), a hypothetical circulatory system, in which extracellular DNA microvesicles interact to form and break down cell structures. Since Bonghan Kim reported the existence of Bonghan ducts and the SNU research team reinvestigated and named it the PVS, there has been series of studies trying to examine its structure and functions. In this paper, we hypothesize that the PVS is the network system in which extracellular DNA microvesicles circulate and interact at the subcellular level, forming and breaking down cell structures. This idea integrates A. Béchamp's idea of microzymas and Bonghan Kim's idea of sanals. A proof of this idea may complement modern medical theory, perhaps providing an essential clue for an alternative solution dealing with modern healthcare problem. PMID:23853665

  8. Toward a theory of the primo vascular system: a hypothetical circulatory system at the subcellular level.

    PubMed

    Lee, Byung-Cheon; Yoon, Ji Woong; Park, Sang Hyun; Yoon, Seung Zhoo

    2013-01-01

    This paper suggests a theoretical framework for the primo vascular system (PVS), a hypothetical circulatory system, in which extracellular DNA microvesicles interact to form and break down cell structures. Since Bonghan Kim reported the existence of Bonghan ducts and the SNU research team reinvestigated and named it the PVS, there has been series of studies trying to examine its structure and functions. In this paper, we hypothesize that the PVS is the network system in which extracellular DNA microvesicles circulate and interact at the subcellular level, forming and breaking down cell structures. This idea integrates A. Béchamp's idea of microzymas and Bonghan Kim's idea of sanals. A proof of this idea may complement modern medical theory, perhaps providing an essential clue for an alternative solution dealing with modern healthcare problem.

  9. Subcellular storage compartments of bacteriopheophorbide sensitizers

    NASA Astrophysics Data System (ADS)

    Moser, Joerg G.; Dembeck, U.; Hubert, M.; Spengler, Bernhard; Bayer, Rainer; Wagner, Birgit

    1994-03-01

    Fluorescence colocalization with the Golgi specific stain, NBD-ceramide, and the mitochondrial localizing stain, Rhodamine 123, confirmed the earlier assumption that the Golgi apparatus is one of the prominent storage compartments for bacteriopheophorbide esters in OAT 75 SCLC cells and several amelanotic melanoma cell lines (A375, Melur SP18, SkAMel 25). Furthermore, a diffuse staining of mitochondria, of non-structured cytoplasm, and an additional storage in melanine vesicles of the amelanotic melanoma cells suggests further storage compartments with quantitatively different contributions to the phototoxicity of bacteriochlorophyll-derived photosensitizers. Independent observations of early phototoxic effects on microfilamentous networks, enzymatic activities (succinate dehydrogenase, lactate dehydrogenase), and redistribution phenomena following primary uptake of the sensitizers let us assume that only a part of the 108 molecules taken up by a cell contribute directly to phototoxicity. Thus it may be asked if a proper subcellular positioning of only a few sensitizer molecules may have similar phototoxic effects as the huge amounts stored at apparently ineffective sites.

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

    SciTech Connect

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

    2016-02-25

    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. In conclusion, 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. Imaging trace element distributions in single organelles and subcellular features

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26911251

  13. Global subcellular characterization of protein degradation using quantitative proteomics.

    PubMed

    Larance, Mark; Ahmad, Yasmeen; Kirkwood, Kathryn J; Ly, Tony; Lamond, Angus I

    2013-03-01

    Protein degradation provides an important regulatory mechanism used to control cell cycle progression and many other cellular pathways. To comprehensively analyze the spatial control of protein degradation in U2OS osteosarcoma cells, we have combined drug treatment and SILAC-based quantitative mass spectrometry with subcellular and protein fractionation. The resulting data set analyzed more than 74,000 peptides, corresponding to ~5000 proteins, from nuclear, cytosolic, membrane, and cytoskeletal compartments. These data identified rapidly degraded proteasome targets, such as PRR11 and highlighted a feedback mechanism resulting in translation inhibition, induced by blocking the proteasome. We show this is mediated by activation of the unfolded protein response. We observed compartment-specific differences in protein degradation, including proteins that would not have been characterized as rapidly degraded through analysis of whole cell lysates. Bioinformatic analysis of the entire data set is presented in the Encyclopedia of Proteome Dynamics, a web-based resource, with proteins annotated for stability and subcellular distribution.

  14. Payload test philosophy. [to provide confidence in Shuttle structural math models

    NASA Technical Reports Server (NTRS)

    Mayhew, D.

    1979-01-01

    Shuttle payload test philosophy is discussed with reference to testing to provide confidence in Shuttle structural math models. Particular attention is given the Shuttle quarter-scale program and the Mated Vertical Ground Vibration Test Program.

  15. Payload test philosophy. [to provide confidence in Shuttle structural math models

    NASA Technical Reports Server (NTRS)

    Mayhew, D.

    1979-01-01

    Shuttle payload test philosophy is discussed with reference to testing to provide confidence in Shuttle structural math models. Particular attention is given the Shuttle quarter-scale program and the Mated Vertical Ground Vibration Test Program.

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

    PubMed

    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.

  17. Subcellular glucose exposure biases the spatial distribution of insulin granules in single pancreatic beta cells

    NASA Astrophysics Data System (ADS)

    Terao, Kyohei; Gel, Murat; Okonogi, Atsuhito; Fuke, Ariko; Okitsu, Teru; Tada, Takashi; Suzuki, Takaaki; Nagamatsu, Shinya; Washizu, Masao; Kotera, Hidetoshi

    2014-02-01

    In living tissues, a cell is exposed to chemical substances delivered partially to its surface. Such a heterogeneous chemical environment potentially induces cell polarity. To evaluate this effect, we developed a microfluidic device that realizes spatially confined delivery of chemical substances at subcellular resolution. Our microfluidic device allows simple setup and stable operation for over 4 h to deliver chemicals partially to a single cell. Using the device, we showed that subcellular glucose exposure triggers an intracellular [Ca2+] change in the β-cells. In addition, the imaging of a cell expressing GFP-tagged insulin showed that continuous subcellular exposure to glucose biased the spatial distribution of insulin granules toward the site where the glucose was delivered. Our approach illustrates an experimental technique that will be applicable to many biological experiments for imaging the response to subcellular chemical exposure and will also provide new insights about the development of polarity of β-cells.

  18. Sub-diffuse structured light imaging provides macroscopic maps of microscopic tissue structure (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kanick, Stephen C.

    2016-03-01

    The onset and progression of cancer introduces changes to the intra-cellular ultrastructural components and to the morphology of the extracellular matrix. While previous work has shown that localized scatter imaging is sensitive to pathology-induced differences in these aspects of tissue microstructure, wide adaptation this knowledge for surgical guidance is limited by two factors. First, the time required to image with confocal-level localization of the remission signal can be substantial. Second, localized (i.e. sub-diffuse) scatter remission intensity is influenced interchangeably by parameters that define scattering frequency and anisotropy. This similarity relationship must be carefully considered in order to obtain unique estimates of biomarkers that define either the scatter density or features that describe the distribution (e.g. shape, size, and orientation) of scatterers. This study presents a novel approach that uses structured light imaging to address both of these limitations. Monte Carlo data were used to model the reflectance intensity over a wide range of spatial frequencies, reduced scattering coefficients, absorption coefficients, and a metric of the scattering phase function that directly maps to the fractal dimension of scatter sizes. The approach is validated in tissue-simulating phantoms constructed with user-tuned scattering phase functions. The validation analysis shows that the phase function can be described in the presence of different scatter densities or background absorptions. Preliminary data from clinical tissue specimens show quantitative images of both the scatter density and the tissue fractal dimension for various tissue types and pathologies. These data represent a novel wide-field quantitative approach to mapping microscopic structural biomarkers that cannot be obtained with standard diffuse imaging. Implications for the use of this approach to assess surgical margins will be discussed.

  19. Sub-cellular proteomics of Medicago truncatula

    PubMed Central

    Lee, Jeonghoon; Lei, Zhentian; Watson, Bonnie S.; Sumner, Lloyd W.

    2013-01-01

    Medicago truncatula is a leading model species and substantial molecular, genetic, genomics, proteomics, and metabolomics resources have been developed for this species to facilitate the study of legume biology. Currently, over 60 proteomics studies of M. truncatula have been published. Many of these have focused upon the unique symbiosis formed between legumes and nitrogen fixing rhizobia bacteria, while others have focused on seed development and the specialized proteomes of distinct tissues/organs. These include the characterization of sub-cellular organelle proteomes such as nuclei and mitochondria, as well as proteins distributed in plasma or microsomal membranes from various tissues. The isolation of sub-cellular proteins typically requires a series of steps that are labor-intensive. Thus, efficient protocols for sub-cellular fractionation, purification, and enrichment are necessary for each cellular compartment. In addition, protein extraction, solubilization, separation, and digestion prior to mass spectral identification are important to enhance the detection of low abundance proteins and to increase the overall detectable proportion of the sub-cellular proteome. This review summarizes the sub-cellular proteomics studies in M. truncatula. PMID:23641248

  20. TESTLoc: protein subcellular localization prediction from EST data

    PubMed Central

    2010-01-01

    Background The eukaryotic cell has an intricate architecture with compartments and substructures dedicated to particular biological processes. Knowing the subcellular location of proteins not only indicates how bio-processes are organized in different cellular compartments, but also contributes to unravelling the function of individual proteins. Computational localization prediction is possible based on sequence information alone, and has been successfully applied to proteins from virtually all subcellular compartments and all domains of life. However, we realized that current prediction tools do not perform well on partial protein sequences such as those inferred from Expressed Sequence Tag (EST) data, limiting the exploitation of the large and taxonomically most comprehensive body of sequence information from eukaryotes. Results We developed a new predictor, TESTLoc, suited for subcellular localization prediction of proteins based on their partial sequence conceptually translated from ESTs (EST-peptides). Support Vector Machine (SVM) is used as computational method and EST-peptides are represented by different features such as amino acid composition and physicochemical properties. When TESTLoc was applied to the most challenging test case (plant data), it yielded high accuracy (~85%). Conclusions TESTLoc is a localization prediction tool tailored for EST data. It provides a variety of models for the users to choose from, and is available for download at http://megasun.bch.umontreal.ca/~shenyq/TESTLoc/TESTLoc.html PMID:21078192

  1. The subcellular distribution of small molecules: from pharmacokinetics to synthetic biology.

    PubMed

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

    2011-10-03

    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 review, we survey 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 points to many opportunities to advance the rational design of organelle-targeted chemical agents. These opportunities include (1) development of quantitative, non-fluorescence-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.

  2. New tools provide a second look at HDV ribozyme structure, dynamics and cleavage

    PubMed Central

    Kapral, Gary J.; Jain, Swati; Noeske, Jonas; Doudna, Jennifer A.; Richardson, David C.; Richardson, Jane S.

    2014-01-01

    The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA enzyme essential for processing viral transcripts during rolling circle viral replication. The first crystal structure of the cleaved ribozyme was solved in 1998, followed by structures of uncleaved, mutant-inhibited and ion-complexed forms. Recently, methods have been developed that make the task of modeling RNA structure and dynamics significantly easier and more reliable. We have used ERRASER and PHENIX to rebuild and re-refine the cleaved and cis-acting C75U-inhibited structures of the HDV ribozyme. The results correct local conformations and identify alternates for RNA residues, many in functionally important regions, leading to improved R values and model validation statistics for both structures. We compare the rebuilt structures to a higher resolution, trans-acting deoxy-inhibited structure of the ribozyme, and conclude that although both inhibited structures are consistent with the currently accepted hammerhead-like mechanism of cleavage, they do not add direct structural evidence to the biochemical and modeling data. However, the rebuilt structures (PDBs: 4PR6, 4PRF) provide a more robust starting point for research on the dynamics and catalytic mechanism of the HDV ribozyme and demonstrate the power of new techniques to make significant improvements in RNA structures that impact biologically relevant conclusions. PMID:25326328

  3. Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas

    SciTech Connect

    Nigam, S.K.; Towers, T. )

    1990-07-01

    Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with Stains-All. Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER.

  4. A Benchtop Fractionation Procedure for Subcellular Analysis of the Plant Metabolome

    PubMed Central

    Fürtauer, Lisa; Weckwerth, Wolfram; Nägele, Thomas

    2016-01-01

    Although compartmentation is a key feature of eukaryotic cells, biological research is frequently limited by methods allowing for the comprehensive subcellular resolution of the metabolome. It has been widely accepted that such a resolution would be necessary in order to approximate cellular biochemistry and metabolic regulation, yet technical challenges still limit both the reproducible subcellular fractionation and the sample throughput being necessary for a statistically robust analysis. Here, we present a method and a detailed protocol which is based on the non-aqueous fractionation technique enabling the assignment of metabolites to their subcellular localization. The presented benchtop method aims at unraveling subcellular metabolome dynamics in a precise and statistically robust manner using a relatively small amount of tissue material. The method is based on the separation of cellular fractions via density gradients consisting of organic, non-aqueous solvents. By determining the relative distribution of compartment-specific marker enzymes together with metabolite profiles over the density gradient it is possible to estimate compartment-specific metabolite concentrations by correlation. To support this correlation analysis, a spreadsheet is provided executing a calculation algorithm to determine the distribution of metabolites over subcellular compartments. The calculation algorithm performs correlation of marker enzyme activity and metabolite abundance accounting for technical errors, reproducibility and the resulting error propagation. The method was developed, tested and validated in three natural accessions of Arabidopsis thaliana showing different ability to acclimate to low temperature. Particularly, amino acids were strongly shuffled between subcellular compartments in a cold-sensitive accession while a cold-tolerant accession was characterized by a stable subcellular metabolic homeostasis. Finally, we conclude that subcellular metabolome analysis is

  5. Lactone-bound structures of cyclohexanone monooxygenase provide insight into the stereochemistry of catalysis.

    PubMed

    Yachnin, Brahm J; McEvoy, Michelle B; MacCuish, Roderick J D; Morley, Krista L; Lau, Peter C K; Berghuis, Albert M

    2014-12-19

    The Baeyer-Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO(Tight) and CHMO(Loose) structures. The CHMO(Tight) structure represents the enzyme state in which substrate acceptance and stereospecificity is determined, providing a foundation for engineering BVMOs with altered substrate spectra and/or stereospecificity. The CHMO(Loose) structure is the first structure where the product is solvent accessible. This structure represents the enzyme state upon binding and release of the substrate and product. In addition, the role of the invariant Arg329 in chaperoning the substrate/product during the catalytic cycle is highlighted. Overall, these data provide a structural framework for the engineering of BVMOs with altered substrate spectra and/or stereospecificity.

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

    PubMed Central

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

    2017-01-01

    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. PMID:27899614

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

    PubMed

    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.

  8. Structure of the Hantavirus Nucleoprotein Provides Insights into the Mechanism of RNA Encapsidation.

    PubMed

    Olal, Daniel; Daumke, Oliver

    2016-03-08

    Hantaviruses are etiological agents of life-threatening hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome. The nucleoprotein (N) of hantavirus is essential for viral transcription and replication, thus representing an attractive target for therapeutic intervention. We have determined the crystal structure of hantavirus N to 3.2 Å resolution. The structure reveals a two-lobed, mostly α-helical structure that is distantly related to that of orthobunyavirus Ns. A basic RNA binding pocket is located at the intersection between the two lobes. We provide evidence that oligomerization is mediated by amino- and C-terminal arms that bind to the adjacent monomers. Based on these findings, we suggest a model for the oligomeric ribonucleoprotein (RNP) complex. Our structure provides mechanistic insights into RNA encapsidation in the genus Hantavirus and constitutes a template for drug discovery efforts aimed at combating hantavirus infections.

  9. Vaccines based on structure-based design provide protection against infectious diseases.

    PubMed

    Thomas, Sunil; Luxon, Bruce A

    2013-11-01

    Vaccines elicit immune responses, provide protection against microorganisms and are considered as one of the most successful medical interventions against infectious diseases. Vaccines can be produced using attenuated virus or bacteria, recombinant proteins, bacterial polysaccharides, carbohydrates or plasmid DNA. Conventional vaccines rely on the induction of immune responses against antigenic proteins to be effective. The genetic diversity of microorganisms, coupled with the high degree of sequence variability in antigenic proteins, presents a challenge to developing broadly effective conventional vaccines. The observation that whole protein antigens are not necessarily essential for inducing immunity has led to the emergence of a new branch of vaccine design termed 'structural vaccinology'. Structure-based vaccines are designed on the rationale that protective epitopes should be sufficient to induce immune responses and provide protection against pathogens. Recent studies demonstrated that designing structure-based vaccine candidates with multiple epitopes induce a higher immune response. As yet there are no commercial vaccines available based on structure-based design and most of the structure-based vaccine candidates are in the preclinical stages of development. This review focuses on recent advances in structure-based vaccine candidates and their application in providing protection against infectious diseases.

  10. Neuronal Computations Made Visible with Subcellular Resolution.

    PubMed

    Kaschula, Richard; Salecker, Iris

    2016-06-30

    Sensory information is gradually processed within dedicated neural circuits to generate specific behaviors. In this issue, Yang et al. push technology boundaries to measure both voltage and calcium signals from subcellular compartments of genetically defined interconnected neurons and shed light on local neural computations critical for motion detection. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Measurement of Subcellular Force Generation in Neurons

    PubMed Central

    O’Toole, Matthew; Lamoureux, Phillip; Miller, Kyle E.

    2015-01-01

    Forces are important for neuronal outgrowth during the initial wiring of the nervous system and after trauma, yet subcellular force generation over the microtubule-rich region at the rear of the growth cone and along the axon has never, to our knowledge, been directly measured. Because previous studies have indicated microtubule polymerization and the microtubule-associated proteins Kinesin-1 and dynein all generate forces that push microtubules forward, a major question is whether the net forces in these regions are contractile or expansive. A challenge in addressing this is that measuring local subcellular force generation is difficult. Here we develop an analytical mathematical model that describes the relationship between unequal subcellular forces arranged in series within the neuron and the net overall tension measured externally. Using force-calibrated towing needles to measure and apply forces, in combination with docked mitochondria to monitor subcellular strain, we then directly measure force generation over the rear of the growth cone and along the axon of chick sensory neurons. We find the rear of the growth cone generates 2.0 nN of contractile force, the axon generates 0.6 nN of contractile force, and that the net overall tension generated by the neuron is 1.3 nN. This work suggests that the forward bulk flow of the cytoskeletal framework that occurs during axonal elongation and growth-cone pauses arises because strong contractile forces in the rear of the growth cone pull material forward. PMID:25762315

  12. Subcellular Biological Effects of Nanosecond Pulsed Electric Fields

    NASA Astrophysics Data System (ADS)

    Kolb, Juergen F.; Stacey, Michael

    Membranes of biological cells can be charged by exposure to pulsed electric fields. After the potential difference across the barrier reaches critical values on the order of 1 V, pores will form. For moderate pulse parameters of duration and amplitude, the effect is limited to the outer cell membrane. With the exposure to nanosecond pulses of several tens of kilovolts per centimeter, a similar effect is also expected for subcellular membranes and structures. Cells will respond to the disruption by different biochemical processes. This offers possibilities for the development of novel medical therapies, the manipulation of cells and microbiological decontamination.

  13. Crystal structure of microsomal prostaglandin E2 synthase provides insight into diversity in the MAPEG superfamily

    PubMed Central

    Sjögren, Tove; Nord, Johan; Ek, Margareta; Johansson, Patrik; Liu, Gang; Geschwindner, Stefan

    2013-01-01

    Prostaglandin E2 (PGE2) is a key mediator in inflammatory response. The main source of inducible PGE2, microsomal PGE2 synthase-1 (mPGES-1), has emerged as an interesting drug target for treatment of pain. To support inhibitor design, we have determined the crystal structure of human mPGES-1 to 1.2 Å resolution. The structure reveals three well-defined active site cavities within the membrane-spanning region in each monomer interface of the trimeric structure. An important determinant of the active site cavity is a small cytosolic domain inserted between transmembrane helices I and II. This extra domain is not observed in other structures of proteins within the MAPEG (Membrane-Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily but is likely to be present also in microsomal GST-1 based on sequence similarity. An unexpected feature of the structure is a 16-Å-deep cone-shaped cavity extending from the cytosolic side into the membrane-spanning region. We suggest a potential role for this cavity in substrate access. Based on the structure of the active site, we propose a catalytic mechanism in which serine 127 plays a key role. We have also determined the structure of mPGES-1 in complex with a glutathione-based analog, providing insight into mPGES-1 flexibility and potential for structure-based drug design. PMID:23431194

  14. Mesopores provide an amorphous state suitable for studying biomolecular structures at cryogenic temperatures

    PubMed Central

    Huang, Ya-Wen; Lai, Yei-Chen; Tsai, Chia-Jung; Chiang, Yun-Wei

    2011-01-01

    In nano-confinements, aqueous solutions can be found to remain in a liquid state at subfreezing temperatures. The finding provides a means of entering into previously inaccessible temperature regions for studying the dynamics and structure of bulk liquid. Here we show that studying biomolecular structures in nano-confinements improves the accuracy of cryostructures and provides better insight into the relationship between hydration water and biomolecules. Synthetic prion protein peptides are studied in two experimental conditions: (i) in confined nanochannels within mesoporous materials, and (ii) in vitrified bulk solvents, with a temperature range of 50–275 K, using cw/pulse ESR techniques. A large inhomogeneous lineshape broadening is only observed for the spectra from the vitrified bulk solvent below 70 K, suggesting a possible peptide clustering in the solution. The spin-counting and distance measurements by DEER-ESR provide further evidence that peptides are dispersed homogeneously in mesopores but heterogeneously in vitrified solvents wherein the biomolecular structure is disturbed due to heterogeneity in the bulk solvent structure. Our study demonstrates that the nanospace within mesoporous materials provides an amorphous environment that is better than vitrified bulk solvent for studying biostructures at cryogenic temperatures. PMID:21844377

  15. Stand structure influences nekton community composition and provides protection from natural disturbance in Micronesian mangroves

    Treesearch

    Richard A. MacKenzie; Nicole. Cormier

    2012-01-01

    Structurally complex mangrove roots are thought to provide foraging habitat, predation refugia, and typhoon protection for resident fish, shrimp, and crabs. The spatially compact nature of Micronesian mangroves results in model ecosystems to test these ideas. Tidal creek nekton assemblages were compared among mangrove forests impacted by Typhoon Sudal and differing in...

  16. Exercise When Young Provides Lifelong Benefits to Bone Structure and Strength

    SciTech Connect

    Warden,S.; Fuchs, R.; Castillo, A.; Nelson, I.; Turner, C.

    2007-01-01

    Short-term exercise in growing rodents provided lifelong benefits to bone structure, strength, and fatigue resistance. Consequently, exercise when young may reduce the risk for fractures later in life, and the old exercise adage of 'use it or lose it' may not be entirely applicable to the skeleton.

  17. Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation

    PubMed Central

    Lawrence, Sara L.; Feil, Susanne C.; Morton, Craig J.; Farrand, Allison J.; Mulhern, Terrence D.; Gorman, Michael A.; Wade, Kristin R.; Tweten, Rodney K.; Parker, Michael W.

    2015-01-01

    Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world’s leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface. PMID:26403197

  18. Crystal Structures of Phosphite Dehydrogenase Provide Insights into Nicotinamide Cofactor Regeneration

    SciTech Connect

    Zou, Yaozhong; Zhang, Houjin; Brunzelle, Joseph S.; Johannes, Tyler W.; Woodyer, Ryan; Hung, John E.; Nair, Nikhil; van der Donk, Wilfred A.; Zhao, Huimin; Nair, Satish K.

    2012-08-21

    The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD{sup +}-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been shown to conduct the enzymatic oxidation of phosphorus. Despite investigation for more than a decade into both the mechanism of its unusual reaction and its utility in cofactor regeneration, there has been a lack of any structural data for PTDH. Here we present the cocrystal structure of an engineered thermostable variant of PTDH bound to NAD{sup +} (1.7 {angstrom} resolution), as well as four other cocrystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 and 2.3 {angstrom} resolution). These structures provide a molecular framework for understanding prior mutational analysis and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.

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

    PubMed

    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.

  20. Discovery of GPCR Ligands by Molecular Docking Screening: Novel Opportunities Provided by Crystal Structures.

    PubMed

    Rodríguez, David; Ranganathan, Anirudh; Carlsson, Jens

    2015-01-01

    G protein-coupled receptors (GPCRs) constitute the largest group of human membrane proteins and have received significant attention in drug discovery for their important roles in physiological processes. Drug development for GPCRs has been remarkably successful and several of the most profitable pharmaceuticals on the market target members of this superfamily. Breakthroughs in structural biology for GPCRs have revealed how their binding sites recognize extracellular molecules at the atomic level. High-resolution crystal structures of GPCR-drug complexes capturing different receptor conformations are now available, which have provided insights into how ligands stabilize different functional states. Recently, the basis for subtype selectivity and novel allosteric binding sites has also been revealed by crystal structures. These accomplishments provide exciting opportunities to identify novel GPCR ligands using in silico structure-based methods such as molecular docking. Increased computational power now enables docking screens of large chemical libraries to identify molecules that complement GPCR binding sites, which may provide possibilities to identify ligands with tailored pharmacological properties. This review focuses on prospective docking screens against GPCRs and how this technique can be used to identify lead candidates with specific signaling or selectivity profiles. The current state of this field suggests that molecular docking, in combination with further understanding of GPCR signaling, will play an important role in future drug discovery.

  1. Structures of the Four Subfamilies of Phosphodiesterase-4 Provide Insight into the Selectivity of Their Inhibitors

    SciTech Connect

    Wang, H.; Peng, M; Chen , Y; Geng, J; Robinson, H; Houslay , M; Cai, J; Ke, H

    2007-01-01

    PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP 4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.

  2. Structures of the four subfamilies of phosphodiesterase-4 provide insight into the selectivity of their inhibitors.

    PubMed

    Wang, Huanchen; Peng, Ming-Sheng; Chen, Yi; Geng, Jie; Robinson, Howard; Houslay, Miles D; Cai, Jiwen; Ke, Hengming

    2007-12-01

    PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP {4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid} as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.

  3. Structures of parasite calreticulins provide insights into their flexibility and dual carbohydrate/peptide-binding properties

    PubMed Central

    Moreau, Christophe; Cioci, Gianluca; Iannello, Marina; Laffly, Emmanuelle; Chouquet, Anne; Ferreira, Arturo; Thielens, Nicole M.; Gaboriaud, Christine

    2016-01-01

    Calreticulin (CRT) is a multifaceted protein, initially discovered as an endoplasmic reticulum (ER) chaperone protein, that is essential in calcium metabolism. Various implications in cancer, early development and immunology have been discovered more recently for CRT, as well as its role as a dominant ‘eat-me’ prophagocytic signal. Intriguingly, cell-surface exposure/secretion of CRT is among the infective strategies used by parasites such as Trypanosoma cruzi, Entamoeba histolytica, Taenia solium, Leishmania donovani and Schistosoma mansoni. Because of the inherent flexibility of CRTs, their analysis by X-ray crystallography requires the design of recombinant constructs suitable for crystallization, and thus only the structures of two very similar mammalian CRT lectin domains are known. With the X-ray structures of two distant parasite CRTs, insights into species structural determinants that might be harnessed to fight against the parasites without affecting the functions of the host CRT are now provided. Moreover, although the hypothesis that CRT can exhibit both open and closed conformations has been proposed in relation to its chaperone function, only the open conformation has so far been observed in crystal structures. The first evidence is now provided of a complex conformational transition with the junction reoriented towards P-domain closure. SAXS experiments also provided additional information about the flexibility of T. cruzi CRT in solution, thus complementing crystallographic data on the open conformation. Finally, regarding the conserved lectin-domain structure and chaperone function, evidence is provided of its dual carbohydrate/protein specificity and a new scheme is proposed to interpret such unusual substrate-binding properties. These fascinating features are fully consistent with previous experimental observations, as discussed considering the broad spectrum of CRT sequence conservations and differences. PMID:27840680

  4. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    PubMed Central

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-01-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics. PMID:27323846

  5. Human Protein Subcellular Localization with Integrated Source and Multi-label Ensemble Classifier

    NASA Astrophysics Data System (ADS)

    Guo, Xiaotong; Liu, Fulin; Ju, Ying; Wang, Zhen; Wang, Chunyu

    2016-06-01

    Predicting protein subcellular location is necessary for understanding cell function. Several machine learning methods have been developed for computational prediction of primary protein sequences because wet experiments are costly and time consuming. However, two problems still exist in state-of-the-art methods. First, several proteins appear in different subcellular structures simultaneously, whereas current methods only predict one protein sequence in one subcellular structure. Second, most software tools are trained with obsolete data and the latest new databases are missed. We proposed a novel multi-label classification algorithm to solve the first problem and integrated several latest databases to improve prediction performance. Experiments proved the effectiveness of the proposed method. The present study would facilitate research on cellular proteomics.

  6. Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity.

    PubMed

    Bong, Seoung Min; Son, Kka-bi; Yang, Seung-Won; Park, Jae-Won; Cho, Jea-Won; Kim, Kyung-Tae; Kim, Hackyoung; Kim, Seung Jun; Kim, Young Jun; Lee, Byung Il

    2016-01-01

    Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dual-specificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)5R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P2 over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.

  7. Differential Site Accessibility Mechanistically Explains Subcellular-Specific N-Glycosylation Determinants

    PubMed Central

    Lee, Ling Yen; Lin, Chi-Hung; Fanayan, Susan; Packer, Nicolle H.; Thaysen-Andersen, Morten

    2014-01-01

    Glycoproteins perform extra- and intracellular functions in innate and adaptive immunity by lectin-based interactions to exposed glyco-determinants. Herein, we document and mechanistically explain the formation of subcellular-specific N-glycosylation determinants on glycoproteins trafficking through the shared biosynthetic machinery of human cells. LC-MS/MS-based quantitative glycomics showed that the secreted glycoproteins of eight human breast epithelial cells displaying diverse geno- and phenotypes consistently displayed more processed, primarily complex type, N-glycans than the high-mannose-rich microsomal glycoproteins. Detailed subcellular glycome profiling of proteins derived from three breast cell lines (MCF7/MDA468/MCF10A) demonstrated that secreted glycoproteins displayed significantly more α-sialylation and α1,6-fucosylation, but less α-mannosylation, than both the intermediately glycan-processed cell-surface glycoproteomes and the under-processed microsomal glycoproteomes. Subcellular proteomics and gene ontology revealed substantial presence of endoplasmic reticulum resident glycoproteins in the microsomes and confirmed significant enrichment of secreted and cell-surface glycoproteins in the respective subcellular fractions. The solvent accessibility of the glycosylation sites on maturely folded proteins of the 100 most abundant putative N-glycoproteins observed uniquely in the three subcellular glycoproteomes correlated with the glycan type processing thereby mechanistically explaining the formation of subcellular-specific N-glycosylation. In conclusion, human cells have developed mechanisms to simultaneously and reproducibly generate subcellular-specific N-glycosylation using a shared biosynthetic machinery. This aspect of protein-specific glycosylation is important for structural and functional glycobiology and discussed here in the context of the spatio-temporal interaction of glyco-determinants with lectins central to infection and immunity

  8. Genomic organization of the crested ibis MHC provides new insight into ancestral avian MHC structure

    PubMed Central

    Chen, Li-Cheng; Lan, Hong; Sun, Li; Deng, Yan-Li; Tang, Ke-Yi; Wan, Qiu-Hong

    2015-01-01

    The major histocompatibility complex (MHC) plays an important role in immune response. Avian MHCs are not well characterized, only reporting highly compact Galliformes MHCs and extensively fragmented zebra finch MHC. We report the first genomic structure of an endangered Pelecaniformes (crested ibis) MHC containing 54 genes in three regions spanning ~500 kb. In contrast to the loose BG (26 loci within 265 kb) and Class I (11 within 150) genomic structures, the Core Region is condensed (17 within 85). Furthermore, this Region exhibits a COL11A2 gene, followed by four tandem MHC class II αβ dyads retaining two suites of anciently duplicated “αβ” lineages. Thus, the crested ibis MHC structure is entirely different from the known avian MHC architectures but similar to that of mammalian MHCs, suggesting that the fundamental structure of ancestral avian class II MHCs should be “COL11A2-IIαβ1-IIαβ2.” The gene structures, residue characteristics, and expression levels of the five class I genes reveal inter-locus functional divergence. However, phylogenetic analysis indicates that these five genes generate a well-supported intra-species clade, showing evidence for recent duplications. Our analyses suggest dramatic structural variation among avian MHC lineages, help elucidate avian MHC evolution, and provide a foundation for future conservation studies. PMID:25608659

  9. Crystal structure of a claudin provides insight into the architecture of tight junctions.

    PubMed

    Suzuki, Hiroshi; Nishizawa, Tomohiro; Tani, Kazutoshi; Yamazaki, Yuji; Tamura, Atsushi; Ishitani, Ryuichiro; Dohmae, Naoshi; Tsukita, Sachiko; Nureki, Osamu; Fujiyoshi, Yoshinori

    2014-04-18

    Tight junctions are cell-cell adhesion structures in epithelial cell sheets that surround organ compartments in multicellular organisms and regulate the permeation of ions through the intercellular space. Claudins are the major constituents of tight junctions and form strands that mediate cell adhesion and function as paracellular barriers. We report the structure of mammalian claudin-15 at a resolution of 2.4 angstroms. The structure reveals a characteristic β-sheet fold comprising two extracellular segments, which is anchored to a transmembrane four-helix bundle by a consensus motif. Our analyses suggest potential paracellular pathways with distinctive charges on the extracellular surface, providing insight into the molecular basis of ion homeostasis across tight junctions.

  10. Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling.

    PubMed

    Zeev-Ben-Mordehai, Tzviya; Weberruß, Marion; Lorenz, Michael; Cheleski, Juliana; Hellberg, Teresa; Whittle, Cathy; El Omari, Kamel; Vasishtan, Daven; Dent, Kyle C; Harlos, Karl; Franzke, Kati; Hagen, Christoph; Klupp, Barbara G; Antonin, Wolfram; Mettenleiter, Thomas C; Grünewald, Kay

    2015-12-29

    Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling.

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

    PubMed Central

    Becker, Jordan T.

    2017-01-01

    ABSTRACT 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

  12. Cryogenic optical localization provides 3D protein structure data with Angstrom resolution.

    PubMed

    Weisenburger, Siegfried; Boening, Daniel; Schomburg, Benjamin; Giller, Karin; Becker, Stefan; Griesinger, Christian; Sandoghdar, Vahid

    2017-02-01

    We introduce Cryogenic Optical Localization in 3D (COLD), a method to localize multiple fluorescent sites within a single small protein with Angstrom resolution. We demonstrate COLD by determining the conformational state of the cytosolic Per-ARNT-Sim domain from the histidine kinase CitA of Geobacillus thermodenitrificans and resolving the four biotin sites of streptavidin. COLD provides quantitative 3D information about small- to medium-sized biomolecules on the Angstrom scale and complements other techniques in structural biology.

  13. Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism

    PubMed Central

    Fernandes, Carlos A. H.; Pazin, Wallance M.; Dreyer, Thiago R.; Bicev, Renata N.; Cavalcante, Walter L. G.; Fortes-Dias, Consuelo L.; Ito, Amando S.; Oliveira, Cristiano L. P.; Fernandez, Roberto Morato; Fontes, Marcos R. M.

    2017-01-01

    Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation. PMID:28256632

  14. Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism.

    PubMed

    Fernandes, Carlos A H; Pazin, Wallance M; Dreyer, Thiago R; Bicev, Renata N; Cavalcante, Walter L G; Fortes-Dias, Consuelo L; Ito, Amando S; Oliveira, Cristiano L P; Fernandez, Roberto Morato; Fontes, Marcos R M

    2017-03-03

    Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation.

  15. Stable preparations of tyrosine hydroxylase provide the solution structure of the full-length enzyme

    PubMed Central

    Bezem, Maria T.; Baumann, Anne; Skjærven, Lars; Meyer, Romain; Kursula, Petri; Martinez, Aurora; Flydal, Marte I.

    2016-01-01

    Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of catecholamine neurotransmitters. TH is a highly complex enzyme at mechanistic, structural, and regulatory levels, and the preparation of kinetically and conformationally stable enzyme for structural characterization has been challenging. Here, we report on improved protocols for purification of recombinant human TH isoform 1 (TH1), which provide large amounts of pure, stable, active TH1 with an intact N-terminus. TH1 purified through fusion with a His-tagged maltose-binding protein on amylose resin was representative of the iron-bound functional enzyme, showing high activity and stabilization by the natural feedback inhibitor dopamine. TH1 purified through fusion with a His-tagged ZZ domain on TALON is remarkably stable, as it was partially inhibited by resin-derived cobalt. This more stable enzyme preparation provided high-quality small-angle X-ray scattering (SAXS) data and reliable structural models of full-length tetrameric TH1. The SAXS-derived model reveals an elongated conformation (Dmax = 20 nm) for TH1, different arrangement of the catalytic domains compared with the crystal structure of truncated forms, and an N-terminal region with an unstructured tail that hosts the phosphorylation sites and a separated Ala-rich helical motif that may have a role in regulation of TH by interacting with binding partners. PMID:27462005

  16. Defining trauma in complex care management: Safety-net providers' perspectives on structural vulnerability and time.

    PubMed

    Thompson-Lastad, Ariana; Yen, Irene H; Fleming, Mark D; Van Natta, Meredith; Rubin, Sara; Shim, Janet K; Burke, Nancy J

    2017-08-01

    In this paper, we delineate how staff of two complex care management (CCM) programs in urban safety net hospitals in the United States understand trauma. We seek to (1) describe how staff in CCM programs talk about trauma in their patients' lives; (2) discuss how trauma concepts allow staff to understand patients' symptoms, health-related behaviors, and responses to care as results of structural conditions; and (3) delineate the mismatch between long-term needs of patients with histories of trauma and the short-term interventions that CCM programs provide. Observation and interview data gathered between February 2015 and August 2016 indicate that CCM providers define trauma expansively to include individual experiences of violence such as childhood abuse and neglect or recent assault, traumatization in the course of accessing health care and structural violence. Though CCM staff implement elements of trauma-informed care, the short-term design of CCM programs puts pressure on the staff to titrate their efforts, moving patients towards graduation or discharge. Trauma concepts enable clinicians to name structural violence in clinically legitimate language. As such, trauma-informed care and structural competency approaches can complement each other. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity

    PubMed Central

    Yates, Luke A.; Durrant, Benjamin P.; Fleurdépine, Sophie; Harlos, Karl; Norbury, Chris J.; Gilbert, Robert J.C.

    2015-01-01

    Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3′ ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164–N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes—for example by the binding of protein co-factors—may allow them alternatively to add single or multiple uridyl residues to the 3′ termini of RNA molecules. PMID:25712096

  18. Single-Molecule Chemo-Mechanical Spectroscopy Provides Structural Identity of Folding Intermediates

    PubMed Central

    Motlagh, Hesam N.; Toptygin, Dmitri; Kaiser, Christian M.; Hilser, Vincent J.

    2016-01-01

    Single-molecule force spectroscopy has emerged as a powerful tool for studying the folding of biological macromolecules. Mechanical manipulation has revealed a wealth of mechanistic information on transient and intermediate states. To date, the majority of state assignment of intermediates has relied on empirical demarcation. However, performing such experiments in the presence of different osmolytes provides an alternative approach that reports on the structural properties of intermediates. Here, we analyze the folding and unfolding of T4 lysozyme with optical tweezers under a chemo-mechanical perturbation by adding osmolytes. We find that two unrelated protective osmolytes, sorbitol and trimethylamine-n-oxide, function by marginally decelerating unfolding rates and specifically modulating early events in the folding process, stabilizing formation of an on-pathway intermediate. The chemo-mechanical perturbation provides access to two independent metrics of the relevant states during folding trajectories, the contour length, and the solvent-accessible surface area. We demonstrate that the dependence of the population of the intermediate in different osmolytes, in conjunction with its measured contour length, provides the ability to discriminate between potential structural models of intermediate states. Our study represents a general strategy that may be employed in the structural modeling of equilibrium intermediate states observed in single-molecule experiments. PMID:27028638

  19. Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs

    PubMed Central

    Wilk, Ronit; Hu, Jack; Blotsky, Dmitry; Krause, Henry M.

    2016-01-01

    In a previous analysis of 2300 mRNAs via whole-mount fluorescent in situ hybridization in cellularizing Drosophila embryos, we found that 70% of the transcripts exhibited some form of subcellular localization. To see whether this prevalence is unique to early Drosophila embryos, we examined ∼8000 transcripts over the full course of embryogenesis and ∼800 transcripts in late third instar larval tissues. The numbers and varieties of new subcellular localization patterns are both striking and revealing. In the much larger cells of the third instar larva, virtually all transcripts observed showed subcellular localization in at least one tissue. We also examined the prevalence and variety of localization mechanisms for >100 long noncoding RNAs. All of these were also found to be expressed and subcellularly localized. Thus, subcellular RNA localization appears to be the norm rather than the exception for both coding and noncoding RNAs. These results, which have been annotated and made available on a recompiled database, provide a rich and unique resource for functional gene analyses, some examples of which are provided. PMID:26944682

  20. Crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana provides insights into its product specificity.

    PubMed

    Zhang, Weiwei; Wang, Wenhe; Liu, Zihe; Xie, Yongchao; Wang, Hao; Mu, Yajuan; Huang, Yao; Feng, Yue

    2016-09-16

    Specifier proteins are important components of the glucosinolate-myrosinase system, which mediate plant defense against herbivory and pathogen attacks. Upon tissue disruption, glucosinolates are hydrolyzed to instable aglucones by myrosinases, and then aglucones will rearrange to form defensive isothiocyanates. Specifier proteins can redirect this reaction to form other products, such as simple nitriles, epithionitriles and organic thiocyanates instead of isothiocyanates based on the side chain structure of glucosinolate and the type of the specifier proteins. Nevertheless, the molecular mechanism underlying the different product spectrums of various specifier proteins was not fully understood. Here in this study, we solved the crystal structure of the Epithiospecifier Protein, ESP from Arabidopsis thaliana (AtESP) at 2.3 Å resolution. Structural comparisons with the previously solved structure of thiocyanate forming protein, TFP from Thlaspi arvense (TaTFP) reveal that AtESP shows a dimerization pattern different from TaTFP. Moreover, AtESP harbors a slightly larger active site pocket than TaTFP and several residues around the active site are different between the two proteins, which might account for the different product spectrums of the two proteins. Together, our structural study provides important insights into the molecular mechanisms of specifier proteins and shed light on the basis of their different product spectrums.

  1. Crystal structure of class III chitinase from pomegranate provides the insight into its metal storage capacity.

    PubMed

    Masuda, Taro; Zhao, Guanghua; Mikami, Bunzo

    2015-01-01

    Chitinase hydrolyzes the β-1,4-glycosidic bond in chitin. In higher plants, this enzyme has been regarded as a pathogenesis-related protein. Recently, we identified a class III chitinase, which functions as a calcium storage protein in pomegranate (Punica granatum) seed (PSC, pomegranate seed chitinase). Here, we solved a crystal structure of PSC at 1.6 Å resolution. Although its overall structure, including the structure of catalytic site and non-proline cis-peptides, was closely similar to those of other class III chitinases, PSC had some unique structural characteristics. First, there were some metal-binding sites with coordinated water molecules on the surface of PSC. Second, many unconserved aspartate residues were present in the PSC sequence which rendered the surface of PSC negatively charged. This acidic electrostatic property is in contrast to that of hevamine, well-characterized plant class III chitinase, which has rather a positively charged surface. Thus, the crystal structure provides a clue for metal association property of PSC.

  2. [Structural equation model for caregiving experience of families providing care for family members with mental disorders].

    PubMed

    Oh, In Ohg; Kim, Sunah

    2015-02-01

    This study was done to develop and test a structural model for caregiving experience including caregiving satisfaction and caregiving strain in families providing care for family members with a mental disorder. The Stress-appraisal-coping model was used as the conceptual framework and the structural equation model to confirm the path that explains what and how variables affect caregiving experience in these families. In this hypothesis model, exogenous variables were optimism, severity of illness and uncertainty. The endogenous variables were self efficacy, social support, caregiving satisfaction and caregiving strain. Data were collected using structured questionnaires. Optimism and caregiving self-efficacy had significant direct and indirect effects on caregiving satisfaction. Optimism, severity of illness and uncertainty had significant direct and indirect effects on caregiving strain. The modified path model explained effects of optimism on caregiving self-efficacy with social support in the path structure as a mediator. Also, there were direct and indirect effects of optimism and uncertainty on caregiving satisfaction with social support and caregiving self-efficacy in the path structure as a mediators. Results suggest the need to improve caregiving self-efficacy of these families, establish support systems such as a mental health professional support programs for caregiving self-efficacy. Optimism, severity of illness and uncertainty perceived by families need to be considered in the development of support programs in order to increase their effectiveness.

  3. Elastic network normal modes provide a basis for protein structure refinement

    NASA Astrophysics Data System (ADS)

    Gniewek, Pawel; Kolinski, Andrzej; Jernigan, Robert L.; Kloczkowski, Andrzej

    2012-05-01

    It is well recognized that thermal motions of atoms in the protein native state, the fluctuations about the minimum of the global free energy, are well reproduced by the simple elastic network models (ENMs) such as the anisotropic network model (ANM). Elastic network models represent protein dynamics as vibrations of a network of nodes (usually represented by positions of the heavy atoms or by the Cα atoms only for coarse-grained representations) in which the spatially close nodes are connected by harmonic springs. These models provide a reliable representation of the fluctuational dynamics of proteins and RNA, and explain various conformational changes in protein structures including those important for ligand binding. In the present paper, we study the problem of protein structure refinement by analyzing thermal motions of proteins in non-native states. We represent the conformational space close to the native state by a set of decoys generated by the I-TASSER protein structure prediction server utilizing template-free modeling. The protein substates are selected by hierarchical structure clustering. The main finding is that thermal motions for some substates, overlap significantly with the deformations necessary to reach the native state. Additionally, more mobile residues yield higher overlaps with the required deformations than do the less mobile ones. These findings suggest that structural refinement of poorly resolved protein models can be significantly enhanced by reduction of the conformational space to the motions imposed by the dominant normal modes.

  4. Multitask learning for protein subcellular location prediction.

    PubMed

    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.

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

    PubMed

    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.

  6. SIMS ion microscopy as a novel, practical tool for subcellular chemical imaging in cancer research

    NASA Astrophysics Data System (ADS)

    Chandra, S.

    2003-01-01

    The development of cryogenic sample preparations, subcellular image quantification schemes, and correlative confocal laser scanning microscopy and ion microscopy have made dynamic SIMS a versatile tool in biology and medicine. For example, ion microscopy can provide much needed, novel information on calcium influx and intracellular calcium stores at organelle resolution in normal and transformed cells in order to better understand the altered calcium signaling in malignant cells. 3-D SIMS imaging of cells revealed dynamic gradients of calcium in cells undergoing mitosis and cytokinesis. Studies of subcellular localization of anticancer drugs is another area of research where ion microscopy can provide novel observations in many types of cancers. Ion microscopy is already an essential tool in boron neutron capture therapy (BNCT) of brain cancer as it can be used to quantitatively image the subcellular location of boron in cells and tissues. This information is critically needed for testing the efficacy of boronated agents and for calculations of radiation dosimetry.

  7. Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA

    SciTech Connect

    Sidhu, Navdeep S.; Schreiber, Kathrin; Pröpper, Kevin; Becker, Stefan; Usón, Isabel; Sheldrick, George M.; Gärtner, Jutta; Krätzner, Ralph Steinfeld, Robert

    2014-05-01

    Mucopolysaccharidosis IIIA is a fatal neurodegenerative disease that typically manifests itself in childhood and is caused by mutations in the gene for the lysosomal enzyme sulfamidase. The first structure of this enzyme is presented, which provides insight into the molecular basis of disease-causing mutations, and the enzymatic mechanism is proposed. Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

  8. Scaling factors: transcription factors regulating subcellular domains.

    PubMed

    Mills, Jason C; Taghert, Paul H

    2012-01-01

    Developing cells acquire mature fates in part by selective (i.e. qualitatively different) expression of a few cell-specific genes. However, all cells share the same basic repertoire of molecular and subcellular building blocks. Therefore, cells must also specialize according to quantitative differences in cell-specific distributions of those common molecular resources. Here we propose the novel hypothesis that evolutionarily-conserved transcription factors called scaling factors (SFs) regulate quantitative differences among mature cell types. SFs: (1) are induced during late stages of cell maturation; (2) are dedicated to specific subcellular domains; and, thus, (3) allow cells to emphasize specific subcellular features. We identify candidate SFs and discuss one in detail: MIST1 (BHLHA15, vertebrates)/DIMM (CG8667, Drosophila); professional secretory cells use this SF to scale up regulated secretion. Because cells use SFs to develop their mature properties and also to adapt them to ever-changing environmental conditions, SF aberrations likely contribute to diseases of adult onset.

  9. 3-D Structure of the Slave and Rae Cratons Provides Clues to Their Construction

    NASA Astrophysics Data System (ADS)

    Snyder, D. B.

    2013-12-01

    Deep geologic structures within cratons that make up continental cores were long neglected. Recently acquired geophysical data from large observational arrays and geochemical data resulting from exploration for diamond has now made possible co-registration of large-scale (400-km depth), truly 3-dimensional data sets. P-waves, surface waves and magnetotelluric observations provide 3-D wavespeed and conductivity models. Multi-azimuthal receiver functions map seismic discontinuity surfaces in 3-D. Xenolith suites erupted in kimberlites provide rock samples at key lithospheric depths, albeit at sparsely distributed locations. These multi-disciplinary models are becoming available for several key cratons worldwide; here the deep structure of the Slave and Rae cratons of the Canadian Shield is described. Lithospheric layers with tapered, wedge-shaped margins are common. Slave craton layers are sub-horizontal and indicate construction of the craton core at 2.7 Ga by underthrusting and flat stacking of lithosphere. The central Rae craton has predominantly dipping discontinuities that indicate construction at 1.9 Ga by thrusting similar to that observed in crustal ';thick-skinned' fold-and-thrust belts. 3-D mapping of conductivity and metasomatism, the latter via mineral recrystallization and resetting of isotopic ages, overprints primary structures in both cratons. Distribution of more conductivitve mantle suggests that assumed causative pervasive metasomatism occurs at 100-200 km depths with ';chimneys' reaching to shallower depths, typically in locations where kimberlites or mineralization has occurred.

  10. Crystal structure of Manduca sexta prophenoloxidase provides insights into the mechanism of type 3 copper enzymes

    SciTech Connect

    Li, Yongchao; Wang, Yang; Jiang, Haobo; Deng, Junpeng

    2010-02-22

    Arthropod phenoloxidase (PO) generates quinones and other toxic compounds to sequester and kill pathogens during innate immune responses. It is also involved in wound healing and other physiological processes. Insect PO is activated from its inactive precursor, prophenoloxidase (PPO), by specific proteolysis via a serine protease cascade. Here, we report the crystal structure of PPO from a lepidopteran insect at a resolution of 1.97 {angstrom}, which is the initial structure for a PPO from the type 3 copper protein family. Manduca sexta PPO is a heterodimer consisting of 2 homologous polypeptide chains, PPO1 and PPO2. The active site of each subunit contains a canonical type 3 di-nuclear copper center, with each copper ion coordinated with 3 structurally conserved histidines. The acidic residue Glu-395 located at the active site of PPO2 may serve as a general base for deprotonation of monophenolic substrates, which is key to the ortho-hydroxylase activity of PO. The structure provides unique insights into the mechanism by which type 3 copper proteins differ in their enzymatic activities, albeit sharing a common active center. A drastic change in electrostatic surface induced on cleavage at Arg-51 allows us to propose a model for localized PPO activation in insects.

  11. Structure-function relationship of skeletal muscle provides inspiration for design of new artificial muscle

    NASA Astrophysics Data System (ADS)

    Gao, Yingxin; Zhang, Chi

    2015-03-01

    A variety of actuator technologies have been developed to mimic biological skeletal muscle that generates force in a controlled manner. Force generation process of skeletal muscle involves complicated biophysical and biochemical mechanisms; therefore, it is impossible to replace biological muscle. In biological skeletal muscle tissue, the force generation of a muscle depends not only on the force generation capacity of the muscle fiber, but also on many other important factors, including muscle fiber type, motor unit recruitment, architecture, structure and morphology of skeletal muscle, all of which have significant impact on the force generation of the whole muscle or force transmission from muscle fibers to the tendon. Such factors have often been overlooked, but can be incorporated in artificial muscle design, especially with the discovery of new smart materials and the development of innovative fabrication and manufacturing technologies. A better understanding of the physiology and structure-function relationship of skeletal muscle will therefore benefit the artificial muscle design. In this paper, factors that affect muscle force generation are reviewed. Mathematical models used to model the structure-function relationship of skeletal muscle are reviewed and discussed. We hope the review will provide inspiration for the design of a new generation of artificial muscle by incorporating the structure-function relationship of skeletal muscle into the design of artificial muscle.

  12. Stable Isotopes Provide Insight into Population Structure and Segregation in Eastern North Atlantic Sperm Whales

    PubMed Central

    Borrell, Asunción; Velásquez Vacca, Adriana; Pinela, Ana M.; Kinze, Carl; Lockyer, Christina H.; Vighi, Morgana; Aguilar, Alex

    2013-01-01

    In pelagic species inhabiting large oceans, genetic differentiation tends to be mild and populations devoid of structure. However, large cetaceans have provided many examples of structuring. Here we investigate whether the sperm whale, a pelagic species with large population sizes and reputedly highly mobile, shows indication of structuring in the eastern North Atlantic, an ocean basin in which a single population is believed to occur. To do so, we examined stable isotope values in sequential growth layer groups of teeth from individuals sampled in Denmark and NW Spain. In each layer we measured oxygen- isotope ratios (δ18O) in the inorganic component (hydroxyapatite), and nitrogen and carbon isotope ratios (δ15N: δ13C) in the organic component (primarily collagenous). We found significant differences between Denmark and NW Spain in δ15N and δ18O values in the layer deposited at age 3, considered to be the one best representing the baseline of the breeding ground, in δ15N, δ13C and δ18O values in the period up to age 20, and in the ontogenetic variation of δ15N and δ18O values. These differences evidence that diet composition, use of habitat and/or migratory destinations are dissimilar between whales from the two regions and suggest that the North Atlantic population of sperm whales is more structured than traditionally accepted. PMID:24324782

  13. Structure of sulfamidase provides insight into the molecular pathology of mucopolysaccharidosis IIIA.

    PubMed

    Sidhu, Navdeep S; Schreiber, Kathrin; Pröpper, Kevin; Becker, Stefan; Usón, Isabel; Sheldrick, George M; Gärtner, Jutta; Krätzner, Ralph; Steinfeld, Robert

    2014-05-01

    Mucopolysaccharidosis type IIIA (Sanfilippo A syndrome), a fatal childhood-onset neurodegenerative disease with mild facial, visceral and skeletal abnormalities, is caused by an inherited deficiency of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH; sulfamidase). More than 100 mutations in the SGSH gene have been found to reduce or eliminate its enzymatic activity. However, the molecular understanding of the effect of these mutations has been confined by a lack of structural data for this enzyme. Here, the crystal structure of glycosylated SGSH is presented at 2 Å resolution. Despite the low sequence identity between this unique N-sulfatase and the group of O-sulfatases, they share a similar overall fold and active-site architecture, including a catalytic formylglycine, a divalent metal-binding site and a sulfate-binding site. However, a highly conserved lysine in O-sulfatases is replaced in SGSH by an arginine (Arg282) that is positioned to bind the N-linked sulfate substrate. The structure also provides insight into the diverse effects of pathogenic mutations on SGSH function in mucopolysaccharidosis type IIIA and convincing evidence for the molecular consequences of many missense mutations. Further, the molecular characterization of SGSH mutations will lay the groundwork for the development of structure-based drug design for this devastating neurodegenerative disorder.

  14. Stable isotopes provide insight into population structure and segregation in eastern North Atlantic sperm whales.

    PubMed

    Borrell, Asunción; Velásquez Vacca, Adriana; Pinela, Ana M; Kinze, Carl; Lockyer, Christina H; Vighi, Morgana; Aguilar, Alex

    2013-01-01

    In pelagic species inhabiting large oceans, genetic differentiation tends to be mild and populations devoid of structure. However, large cetaceans have provided many examples of structuring. Here we investigate whether the sperm whale, a pelagic species with large population sizes and reputedly highly mobile, shows indication of structuring in the eastern North Atlantic, an ocean basin in which a single population is believed to occur. To do so, we examined stable isotope values in sequential growth layer groups of teeth from individuals sampled in Denmark and NW Spain. In each layer we measured oxygen- isotope ratios (δ(18)O) in the inorganic component (hydroxyapatite), and nitrogen and carbon isotope ratios (δ(15)N: δ(13)C) in the organic component (primarily collagenous). We found significant differences between Denmark and NW Spain in δ(15)N and δ(18)O values in the layer deposited at age 3, considered to be the one best representing the baseline of the breeding ground, in δ(15)N, δ(13)C and δ(18)O values in the period up to age 20, and in the ontogenetic variation of δ(15)N and δ(18)O values. These differences evidence that diet composition, use of habitat and/or migratory destinations are dissimilar between whales from the two regions and suggest that the North Atlantic population of sperm whales is more structured than traditionally accepted.

  15. Subcellular localization and regulation of coenzyme A synthase.

    PubMed

    Zhyvoloup, Alexander; Nemazanyy, Ivan; Panasyuk, Ganna; Valovka, Taras; Fenton, Tim; Rebholz, Heike; Wang, Mong-Lien; Foxon, Richard; Lyzogubov, Valeriy; Usenko, Vasylij; Kyyamova, Ramziya; Gorbenko, Olena; Matsuka, Genadiy; Filonenko, Valeriy; Gout, Ivan T

    2003-12-12

    CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.

  16. The crystal structure of two macrolide glycosyltransferases provides a blueprint for host cell antibiotic immunity.

    PubMed

    Bolam, David N; Roberts, Shirley; Proctor, Mark R; Turkenburg, Johan P; Dodson, Eleanor J; Martinez-Fleites, Carlos; Yang, Min; Davis, Benjamin G; Davies, Gideon J; Gilbert, Harry J

    2007-03-27

    Glycosylation of macrolide antibiotics confers host cell immunity from endogenous and exogenous agents. The Streptomyces antibioticus glycosyltransferases, OleI and OleD, glycosylate and inactivate oleandomycin and diverse macrolides including erythromycin, respectively. The structure of these enzyme-ligand complexes, in tandem with kinetic analysis of site-directed variants, provide insight into the interaction of macrolides with their synthetic apparatus. Erythromycin binds to OleD and the 23S RNA of its target ribosome in the same conformation and, although the antibiotic contains a large number of polar groups, its interaction with these macromolecules is primarily through hydrophobic contacts. Erythromycin and oleandomycin, when bound to OleD and OleI, respectively, adopt different conformations, reflecting a subtle effect on sugar positioning by virtue of a single change in the macrolide backbone. The data reported here provide structural insight into the mechanism of resistance to both endogenous and exogenous antibiotics, and will provide a platform for the future redesign of these catalysts for antibiotic remodelling.

  17. The crystal structure of two macrolide glycosyltransferases provides a blueprint for host cell antibiotic immunity

    PubMed Central

    Bolam, David N.; Roberts, Shirley; Proctor, Mark R.; Turkenburg, Johan P.; Dodson, Eleanor J.; Martinez-Fleites, Carlos; Yang, Min; Davis, Benjamin G.; Davies, Gideon J.; Gilbert, Harry J.

    2007-01-01

    Glycosylation of macrolide antibiotics confers host cell immunity from endogenous and exogenous agents. The Streptomyces antibioticus glycosyltransferases, OleI and OleD, glycosylate and inactivate oleandomycin and diverse macrolides including erythromycin, respectively. The structure of these enzyme–ligand complexes, in tandem with kinetic analysis of site-directed variants, provide insight into the interaction of macrolides with their synthetic apparatus. Erythromycin binds to OleD and the 23S RNA of its target ribosome in the same conformation and, although the antibiotic contains a large number of polar groups, its interaction with these macromolecules is primarily through hydrophobic contacts. Erythromycin and oleandomycin, when bound to OleD and OleI, respectively, adopt different conformations, reflecting a subtle effect on sugar positioning by virtue of a single change in the macrolide backbone. The data reported here provide structural insight into the mechanism of resistance to both endogenous and exogenous antibiotics, and will provide a platform for the future redesign of these catalysts for antibiotic remodelling. PMID:17376874

  18. The basic structure of community early intervention programs for children with autism: provider descriptions.

    PubMed

    Stahmer, Aubyn C

    2007-08-01

    Autism researchers have identified a set of common effective practice elements for early intervention (EI) (e.g., intensive programming). The current study examined the reported use of common elements of effective interventions in community EI settings. Eighty EI providers reported about their programs. The majority of participants reported using common effective elements, however, the depth and quality of the use of these elements was highly variable. Taking community program structure into account in future research will facilitate the development of methodologies, which immediately fit into the context of community programming rather than requiring program adaptation for use in the real world. Recommendations for using current community program structure to improve use of evidence-based practices are discussed.

  19. High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

    PubMed

    Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

    2015-06-16

    Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states.

  20. Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement

    PubMed Central

    Gruszczyk, Jakub; Lim, Nicholas T. Y.; Arnott, Alicia; He, Wen-Qiang; Nguitragool, Wang; Roobsoong, Wanlapa; Mok, Yee-Foong; Murphy, James M.; Smith, Katherine R.; Lee, Stuart; Bahlo, Melanie; Mueller, Ivo; Barry, Alyssa E.

    2016-01-01

    Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short β-hairpin, and, although the structural fold is similar to that of PfRh5—the essential invasion ligand in Plasmodium falciparum—its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase- and chymotrypsin-resistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection. PMID:26715754

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

    SciTech Connect

    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. - 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.

  2. Identifying subcellular localizations of mammalian protein complexes based on graph theory with a random forest algorithm.

    PubMed

    Li, Zhan-Chao; Lai, Yan-Hua; Chen, Li-Li; Chen, Chao; Xie, Yun; Dai, Zong; Zou, Xiao-Yong

    2013-04-05

    In the post-genome era, one of the most important and challenging tasks is to identify the subcellular localizations of protein complexes, and further elucidate their functions in human health with applications to understand disease mechanisms, diagnosis and therapy. Although various experimental approaches have been developed and employed to identify the subcellular localizations of protein complexes, the laboratory technologies fall far behind the rapid accumulation of protein complexes. Therefore, it is highly desirable to develop a computational method to rapidly and reliably identify the subcellular localizations of protein complexes. In this study, a novel method is proposed for predicting subcellular localizations of mammalian protein complexes based on graph theory with a random forest algorithm. Protein complexes are modeled as weighted graphs containing nodes and edges, where nodes represent proteins, edges represent protein-protein interactions and weights are descriptors of protein primary structures. Some topological structure features are proposed and adopted to characterize protein complexes based on graph theory. Random forest is employed to construct a model and predict subcellular localizations of protein complexes. Accuracies on a training set by a 10-fold cross-validation test for predicting plasma membrane/membrane attached, cytoplasm and nucleus are 84.78%, 71.30%, and 82.00%, respectively. And accuracies for the independent test set are 81.31%, 69.95% and 81.00%, respectively. These high prediction accuracies exhibit the state-of-the-art performance of the current method. It is anticipated that the proposed method may become a useful high-throughput tool and plays a complementary role to the existing experimental techniques in identifying subcellular localizations of mammalian protein complexes. The source code of Matlab and the dataset can be obtained freely on request from the authors.

  3. Providing mentorship support to general surgery residents: a model for structured group facilitation

    PubMed Central

    Champion, Caitlin; Bennett, Sean; Carver, David; El Tawil, Karim; Fabbro, Sarah; Howatt, Neil; Noei, Farahnaz; Rae, Rachel; Haggar, Fatima; Arnaout, Angel

    2015-01-01

    Summary Mentorship is foundational to surgical training, with recognized benefits for both mentees and mentors. The University of Ottawa General Surgery Mentorship Program was developed as a module-based group facilitation program to support inclusive personal and professional development of junior general surgery residents. The group format provided an opportunity for both vertical and horizontal mentorship relationships between staff mentors and resident mentees. Perceived benefits of program participants were evaluated at the conclusion of the first year of the program. The program was well-received by staff and resident participants and may provide a time-efficient and inclusive mentorship structure with the additional benefit of peer support. We review the development and implementation of the program to date and share our mentorship experience to encourage the growth of formal mentorship opportunities within general surgery training programs. PMID:26424687

  4. Structural Challenges of Providing Palliative Care for Patients with Breast Cancer

    PubMed Central

    Khoshnazar, Tahereh Alsadat Khoubbin; Rassouli, Maryam; Akbari, Mohammad Esmaeil; Lotfi-Kashani, Farah; Momenzadeh, Syrus; Haghighat, Shahpar; Sajjadi, Moosa

    2016-01-01

    Introduction: To establish a palliative care system (PCS) in Iran, it is necessary to identify the potential barriers. Aim: This study aims to highlight the views of stakeholders to know the challenges of providing palliative care for women with breast cancer. Materials and Methods: Semi-structured in-depth interviews are used with purposeful sampling conducted in Tehran, Iran; from January to June 2015. Twenty participants were included in the study: nine patients with breast cancer and ten health-care providers. The interviews were analyzed using qualitative directed content analysis based on Donabedian model. Data credibility was examined using the criteria of Lincoln and Guba. Results: Based on the pattern of Avedis Donabedian model, two main categories were identified: (1) palliative care services in the health system still remain undefined and (2) lack of adequate care providers. The subcategories emerged from the main categories are: (1) Inexistent home care, (2) specialized palliative care being in high demand, lack of: (a) Rehabilitation program and guidelines, (b) treatment/training protocols, (c) inefficient insurance and out-of-pocket costs, (d) patient referral system, (e) nontransparency of job description, and (f) weakness of teamwork. Discussion: The findings of the study identify views and perceptions of patients as well as the health professionals around the challenges of providing palliative care. To establish a structured PCS, we need to meet the challenges and remove perceived barriers to, including but not limited to, building up knowledge and awareness of health professionals, educating professional, and developing updated, well-defined, and standard treatment protocols, tailored to local conditions. PMID:27803569

  5. Human acid sphingomyelinase structures provide insight to molecular basis of Niemann–Pick disease

    SciTech Connect

    Zhou, Yan-Feng; Metcalf, Matthew C.; Garman, Scott C.; Edmunds, Tim; Qiu, Huawei; Wei, Ronnie R.

    2016-10-26

    Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann–Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients.

  6. Human acid sphingomyelinase structures provide insight to molecular basis of Niemann–Pick disease

    PubMed Central

    Zhou, Yan-Feng; Metcalf, Matthew C.; Garman, Scott C.; Edmunds, Tim; Qiu, Huawei; Wei, Ronnie R.

    2016-01-01

    Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and phosphocholine, essential components of myelin in neurons. Genetic alterations in ASM lead to ASM deficiency (ASMD) and have been linked to Niemann–Pick disease types A and B. Olipudase alfa, a recombinant form of human ASM, is being developed as enzyme replacement therapy to treat the non-neurological manifestations of ASMD. Here we present the human ASM holoenzyme and product bound structures encompassing all of the functional domains. The catalytic domain has a metallophosphatase fold, and two zinc ions and one reaction product phosphocholine are identified in a histidine-rich active site. The structures reveal the underlying catalytic mechanism, in which two zinc ions activate a water molecule for nucleophilic attack of the phosphodiester bond. Docking of sphingomyelin provides a model that allows insight into the selectivity of the enzyme and how the ASM domains collaborate to complete hydrolysis. Mapping of known mutations provides a basic understanding on correlations between enzyme dysfunction and phenotypes observed in ASMD patients. PMID:27725636

  7. Crystal Structure of the Herpesvirus Nuclear Egress Complex Provides Insights into Inner Nuclear Membrane Remodeling

    PubMed Central

    Zeev-Ben-Mordehai, Tzviya; Weberruß, Marion; Lorenz, Michael; Cheleski, Juliana; Hellberg, Teresa; Whittle, Cathy; El Omari, Kamel; Vasishtan, Daven; Dent, Kyle C.; Harlos, Karl; Franzke, Kati; Hagen, Christoph; Klupp, Barbara G.; Antonin, Wolfram; Mettenleiter, Thomas C.; Grünewald, Kay

    2015-01-01

    Summary Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Together, the conserved herpesvirus proteins pUL31 and pUL34 form the heterodimeric nuclear egress complex (NEC), which, in turn, mediates the formation of tight-fitting membrane vesicles around capsids at the inner nuclear membrane. Here, we present the crystal structure of the pseudorabies virus NEC. The structure revealed that a zinc finger motif in pUL31 and an extensive interaction network between the two proteins stabilize the complex. Comprehensive mutational analyses, characterized both in situ and in vitro, indicated that the interaction network is not redundant but rather complementary. Fitting of the NEC crystal structure into the recently determined cryoEM-derived hexagonal lattice, formed in situ by pUL31 and pUL34, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling. PMID:26711332

  8. The crystal structure of human GlnRS provides basis for the development of neurological disorders

    PubMed Central

    Ognjenović, Jana; Wu, Jiang; Matthies, Doreen; Baxa, Ulrich; Subramaniam, Sriram; Ling, Jiqiang; Simonović, Miljan

    2016-01-01

    Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggest that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. This report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases. PMID:26869582

  9. Crystal structures of enterovirus 71 (EV71) recombinant virus particles provide insights into vaccine design.

    PubMed

    Lyu, Ke; Wang, Guang-Chuan; He, Ya-Ling; Han, Jian-Feng; Ye, Qing; Qin, Cheng-Feng; Chen, Rong

    2015-02-06

    Hand-foot-and-mouth disease (HFMD) remains a major health concern in the Asia-Pacific regions, and its major causative agents include human enterovirus 71 (EV71) and coxsackievirus A16. A desirable vaccine against HFMD would be multivalent and able to elicit protective responses against multiple HFMD causative agents. Previously, we have demonstrated that a thermostable recombinant EV71 vaccine candidate can be produced by the insertion of a foreign peptide into the BC loop of VP1 without affecting viral replication. Here we present crystal structures of two different naturally occurring empty particles, one from a clinical C4 strain EV71 and the other from its recombinant virus containing an insertion in the VP1 BC loop. Crystal structure analysis demonstrated that the inserted foreign peptide is well exposed on the particle surface without significant structural changes in the capsid. Importantly, such insertions do not seem to affect the virus uncoating process as illustrated by the conformational similarity between an uncoating intermediate of another recombinant virus and that of EV71. Especially, at least 18 residues from the N terminus of VP1 are transiently externalized. Altogether, our study provides insights into vaccine development against HFMD.

  10. The crystal structure of human GlnRS provides basis for the development of neurological disorders

    SciTech Connect

    Ognjenovic, Jana; Wu, Jiang; Matthies, Doreen; Baxa, Ulrich; Subramaniam, Sriram; Ling, Jiqiang; Simonovic, Miljan

    2016-02-10

    Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggest that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. As a result, this report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases. Keywords

  11. The crystal structure of human GlnRS provides basis for the development of neurological disorders

    DOE PAGES

    Ognjenovic, Jana; Wu, Jiang; Matthies, Doreen; ...

    2016-02-10

    Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggestmore » that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. As a result, this report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases. Keywords« less

  12. Neristatin 1 provides critical insight into bryostatin 1 structure-function relationships.

    PubMed

    Kedei, Noemi; Kraft, Matthew B; Keck, Gary E; Herald, Cherry L; Melody, Noeleen; Pettit, George R; Blumberg, Peter M

    2015-04-24

    Bryostatin 1, a complex macrocyclic lactone isolated from Bugula neritina, has been the subject of multiple clinical trials for cancer. Although it functions as an activator of protein kinase C (PKC) in vitro, bryostatin 1 paradoxically antagonizes most responses to the prototypical PKC activator, the phorbol esters. The bottom half of the bryostatin 1 structure has been shown to be sufficient to confer binding to PKC. In contrast, we have previously shown that the top half of the bryostatin 1 structure is necessary for its unique biological behavior to antagonize phorbol ester responses. Neristatin 1 comprises a top half similar to that of bryostatin 1 together with a distinct bottom half that confers PKC binding. We report here that neristatin 1 is bryostatin 1-like, not phorbol ester-like, in its biological activity on U937 promyelocytic leukemia cells. We conclude that the top half of the bryostatin 1 structure is largely sufficient for bryostatin 1-like activity, provided the molecule also possesses an appropriate PKC binding domain.

  13. Identification of O-glycan Structures from Chicken Intestinal Mucins Provides Insight into Campylobactor jejuni Pathogenicity*

    PubMed Central

    Struwe, Weston B.; Gough, Ronan; Gallagher, Mary E.; Kenny, Diarmuid T.; Carrington, Stephen D.; Karlsson, Niclas G.; Rudd, Pauline M.

    2015-01-01

    The Gram-negative bacteria Campylobactor jejuni is the primary bacteria responsible for food poisoning in industrialized countries, and acute diarrheal illness is a leading cause of mortality among children in developing countries. C. jejuni are commensal in chickens. They are particularly abundant in the caecal crypts, and poultry products are commonly infected as a result of cross-contamination during processing. The interactions between C. jejuni and chicken intestinal tissues as well as the pathogenic molecular mechanisms of colonization in humans are unknown, but identifying these factors could provide potential targets to reduce the incidence of campylobacteriosis. Recently, purified chicken intestinal mucin was shown to attenuate adherence and invasion of C. jejuni in the human colorectal adenocarcinoma cell line HCT-8 in vitro, and this effect was attributed to mucin O-glycosylation. Mucins from different regions of the chicken intestine inhibited C. jejuni binding and internalization differentially, with large intestine>small intestine>caecum. Here, we use LC-MS to perform a detailed structural analysis of O-glycans released from mucins purified from chicken large intestine, small intestine, and caecum. The O-glycans identified were abundantly sulfated compared with the human intestines, and sulfate moieties were present throughout the chicken intestinal tract. Interestingly, alpha 1–2 linked fucose residues, which have a high binding affinity to C. jejuni, were identified in the small and large intestines. Additionally, N-glycolylneuraminic/N-acetylneuraminic acid containing structures present as Sda-like epitopes were identified in large intestine samples but not small intestine or caecum. O-glycan structural characterization of chicken intestinal mucins provides insights into adherence and invasion properties of C. jejuni, and may offer prospective candidate molecules aimed at reducing the incidence of infection. PMID:25776888

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

    DOE PAGES

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; ...

    2016-02-25

    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 (whichmore » 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. In conclusion, 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.« less

  15. Kinesin motors in plants: from subcellular dynamics to motility regulation.

    PubMed

    Lee, Yuh-Ru Julie; Qiu, Weihong; Liu, Bo

    2015-12-01

    Plants produce enormous forms of the microtubule (MT)-based motor kinesins that have been inspiring plant cell biologists to uncover their functions in relation to plant growth and development. Subcellular localization of kinesin proteins detected through live-cell imaging or immunofluorescence microscopy has provided great insights into the functions of these motors. Dozens of mitotic kinesins exhibit particularly splendid localization patterns from chromosomes and kinetochores to MT arrays like the preprophase band, spindle poles, the spindle midzone, phragmoplast distal ends, and the phragmoplast midzone. Different subcellular localizations indicate distinct functions of these motors that are yet to be characterized. The localization difference between plant kinesins and their animal counterparts implies mechanistic differences in mitosis and cytokinesis between the two kingdoms. When many forms of kinesins are present simultaneously, it becomes critical that their motility is differentially regulated with spatial and temporal precision. Insights into regulatory mechanisms of motors can often be brought about by in vitro single-molecule biophysical studies. Significant advances are expected in this area in the coming years owing to rapid technological advances that are being brought to various model plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Catchment structure that supports organic matter providing a natural control on rising river nutrient concentrations

    NASA Astrophysics Data System (ADS)

    Stutter, Marc; Ibiyemi, Adekunle; Wang, Chen

    2017-04-01

    The connectivity of sources of pollution in catchments has been well studied and brings concepts such as pollution hotspots and critical source areas. However, consideration of the placement of other structures combating rising pollution impacts has been less considered. One such area that is receiving developing focus is the layout of riparian management and buffer strips. However, there are wider aspects of connectivity and landscape structure that can bring benefits to delivery and in-stream processing of pollution. These include wetlands, forests and the distribution of soils of differing connectivity of organic matter varying in bioavailability. Organic matter is a great modulator of catchment processes from controlling the potential of land use (e.g. constraints of soil organic matter and wetness on agricultural use), to the amount and form of nutrients leached from soils, to controls of dissolved organic matter on in-stream biology that responds to nutrient concentrations. As the fundamental control of ecosystem energy available for many heterotrophic processes it mediates uptake, recycling and speciation of N, P at many stages of the catchment from soils to waters; as such DOM can be considered as a nature-based solution exerting a background level of control on inorganic nutrients. This poster explores the role of different structural aspects of catchments that provide beneficial organic matter inputs to rivers. At the fine scale the lability of riparian soil and leaf litter DOC are considered. At a riparian management scale the local changes in buffer strip soil C and DOC relative to field soils are considered. At the largest scale spatial data are explored for riparian structure, forests, wetlands and soils differing in delivery and forms of C across major Scottish rivers and used as co-variates to explain differences in in-stream processing of nutrients.

  17. Hydrophobic Core Variations Provide a Structural Framework for Tyrosine Kinase Evolution and Functional Specialization

    PubMed Central

    Kwon, Annie; Byrne, Dominic P.; Ferries, Samantha; Ruan, Zheng; Hanold, Laura E.; Katiyar, Samiksha; Kennedy, Eileen J.; Eyers, Patrick A.; Kannan, Natarajan

    2016-01-01

    Protein tyrosine kinases (PTKs) are a group of closely related enzymes that have evolutionarily diverged from serine/threonine kinases (STKs) to regulate pathways associated with multi-cellularity. Evolutionary divergence of PTKs from STKs has occurred through accumulation of mutations in the active site as well as in the commonly conserved hydrophobic core. While the functional significance of active site variations is well understood, relatively little is known about how hydrophobic core variations contribute to PTK evolutionary divergence. Here, using a combination of statistical sequence comparisons, molecular dynamics simulations, mutational analysis and in vitro thermostability and kinase assays, we investigate the structural and functional significance of key PTK-specific variations in the kinase core. We find that the nature of residues and interactions in the hydrophobic core of PTKs is strikingly different from other protein kinases, and PTK-specific variations in the core contribute to functional divergence by altering the stability and dynamics of the kinase domain. In particular, a functionally critical STK-conserved histidine that stabilizes the regulatory spine in STKs is selectively mutated to an alanine, serine or glutamate in PTKs, and this loss-of-function mutation is accommodated, in part, through compensatory PTK-specific interactions in the core. In particular, a PTK-conserved phenylalanine in the I-helix appears to structurally and functionally compensate for the loss of STK-histidine by interacting with the regulatory spine, which has far-reaching effects on enzyme activity, inhibitor sensing, and stability. We propose that hydrophobic core variations provide a selective advantage during PTK evolution by increasing the conformational flexibility, and therefore the allosteric potential of the kinase domain. Our studies also suggest that Tyrosine Kinase Like kinases such as RAF are intermediates in PTK evolutionary divergence inasmuch as they

  18. Trehalulose synthase native and carbohydrate complexed structures provide insights into sucrose isomerization.

    PubMed

    Ravaud, Stéphanie; Robert, Xavier; Watzlawick, Hildegard; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2007-09-21

    Various diseases related to the overconsumption of sugar make a growing need for sugar substitutes. Because sucrose is an inexpensive and readily available d-glucose donor, the industrial potential for enzymatic synthesis of the sucrose isomers trehalulose and/or isomaltulose from sucrose is large. The product specificity of sucrose isomerases that catalyze this reaction depends essentially on the possibility for tautomerization of sucrose, which is required for trehalulose formation. For optimal use of the enzyme, targeting controlled synthesis of these functional isomers, it is necessary to minimize the side reactions. This requires an extensive analysis of substrate binding modes and of the specificity-determining sites in the structure. The 1.6-2.2-A resolution three-dimensional structures of native and mutant complexes of a trehalulose synthase from Pseudomonas mesoacidophila MX-45 mimic successive states of the enzyme reaction. Combined with mutagenesis studies they give for the first time thorough insights into substrate recognition and processing and reaction specificities of these enzymes. Among the important outcomes of this study is the revelation of an aromatic clamp defined by Phe(256) and Phe(280) playing an essential role in substrate recognition and in controlling the reaction specificity, which is further supported by mutagenesis studies. Furthermore, this study highlights essential residues for binding the glucosyl and fructosyl moieties. The introduction of subtle changes informed by comparative three-dimensional structural data observed within our study can lead to fundamental modifications in the mode of action of sucrose isomerases and hence provide a template for industrial catalysts.

  19. Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking.

    PubMed

    Lázár, Enikő; Péterfi, Zalán; Sirokmány, Gábor; Kovács, Hajnal A; Klement, Eva; Medzihradszky, Katalin F; Geiszt, Miklós

    2015-06-01

    Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure-function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in "hot spots," which represent previously unknown locations of collagen IV crosslinking.

  20. A Near-Atomic Structure of the Dark Apoptosome Provides Insight into Assembly and Activation.

    PubMed

    Cheng, Tat Cheung; Akey, Ildikó V; Yuan, Shujun; Yu, Zhiheng; Ludtke, Steven J; Akey, Christopher W

    2017-01-03

    In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade β-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2' carbon of the deoxyribose ring. Interestingly, β-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to β-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Crystal structures of nitric oxide reductases provide key insights into functional conversion of respiratory enzymes.

    PubMed

    Tosha, Takehiko; Shiro, Yoshitsugu

    2013-03-01

    Respiration is an essential biological process to get bioenergy, ATP, for all kingdoms of life. Cytochrome c oxidase (COX) plays central role in aerobic respiration, catalyzing the reduction of O(2) coupled with pumping proton across the biological membrane. Nitric oxide reductase (NOR) involved in anaerobic nitrate respiration is suggested to be evolutionary related to COX and share the same progenitor with COX, on the basis of the amino acid sequence homology. Contrary to COX, NOR catalyzes the reduction of nitric oxide and shows no proton pumping ability. Thus, the respiratory enzyme acquires (or loses) proton pumping ability in addition to the conversion of the catalytic property along with the environmental change on earth. Recently, we solved the structures of two types of NORs, which provides novel insights into the functional conversion of the respiratory enzymes. In this review, we focus on the structural similarities and differences between COXs and NORs and discuss possible mechanism for the functional conversion of these enzymes during molecular evolution. Copyright © 2013 International Union of Biochemistry and Molecular Biology, Inc.

  2. Dynamic Nucleosome Movement Provides Structural Information of Topological Chromatin Domains in Living Human Cells

    PubMed Central

    Shinkai, Soya; Nozaki, Tadasu; Maeshima, Kazuhiro

    2016-01-01

    The mammalian genome is organized into submegabase-sized chromatin domains (CDs) including topologically associating domains, which have been identified using chromosome conformation capture-based methods. Single-nucleosome imaging in living mammalian cells has revealed subdiffusively dynamic nucleosome movement. It is unclear how single nucleosomes within CDs fluctuate and how the CD structure reflects the nucleosome movement. Here, we present a polymer model wherein CDs are characterized by fractal dimensions and the nucleosome fibers fluctuate in a viscoelastic medium with memory. We analytically show that the mean-squared displacement (MSD) of nucleosome fluctuations within CDs is subdiffusive. The diffusion coefficient and the subdiffusive exponent depend on the structural information of CDs. This analytical result enabled us to extract information from the single-nucleosome imaging data for HeLa cells. Our observation that the MSD is lower at the nuclear periphery region than the interior region indicates that CDs in the heterochromatin-rich nuclear periphery region are more compact than those in the euchromatin-rich interior region with respect to the fractal dimensions as well as the size. Finally, we evaluated that the average size of CDs is in the range of 100–500 nm and that the relaxation time of nucleosome movement within CDs is a few seconds. Our results provide physical and dynamic insights into the genome architecture in living cells. PMID:27764097

  3. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells

    PubMed Central

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  4. Protein subcellular localization prediction using multiple kernel learning based support vector machine.

    PubMed

    Hasan, Md Al Mehedi; Ahmad, Shamim; Molla, Md Khademul Islam

    2017-03-28

    Predicting the subcellular locations of proteins can provide useful hints that reveal their functions, increase our understanding of the mechanisms of some diseases, and finally aid in the development of novel drugs. As the number of newly discovered proteins has been growing exponentially, which in turns, makes the subcellular localization prediction by purely laboratory tests prohibitively laborious and expensive. In this context, to tackle the challenges, computational methods are being developed as an alternative choice to aid biologists in selecting target proteins and designing related experiments. However, the success of protein subcellular localization prediction is still a complicated and challenging issue, particularly, when query proteins have multi-label characteristics, i.e., if they exist simultaneously in more than one subcellular location or if they move between two or more different subcellular locations. To date, to address this problem, several types of subcellular localization prediction methods with different levels of accuracy have been proposed. The support vector machine (SVM) has been employed to provide potential solutions to the protein subcellular localization prediction problem. However, the practicability of an SVM is affected by the challenges of selecting an appropriate kernel and selecting the parameters of the selected kernel. To address this difficulty, in this study, we aimed to develop an efficient multi-label protein subcellular localization prediction system, named as MKLoc, by introducing multiple kernel learning (MKL) based SVM. We evaluated MKLoc using a combined dataset containing 5447 single-localized proteins (originally published as part of the Höglund dataset) and 3056 multi-localized proteins (originally published as part of the DBMLoc set). Note that this dataset was used by Briesemeister et al. in their extensive comparison of multi-localization prediction systems. Finally, our experimental results indicate that

  5. Ranking European regions as providers of structural riparian corridors for conservation and management purposes

    NASA Astrophysics Data System (ADS)

    Clerici, Nicola; Vogt, Peter

    2013-04-01

    Riparian zones are of utmost importance in providing a wide range of ecological and societal services. Among these, their role in maintaining landscape connectivity through ecological corridors for animals and plants is of major interest from a conservation and management perspective. This paper describes a methodology to identify European regions as providers of structural riparian corridors, and to rank them with reference to conservation priority. Physical riparian connectors among core habitat patches are identified through a recent segmentation technique, the Morphological Spatial Pattern Analysis. A multi-scale approach is followed by considering different edge distances to identify core and peripheral habitats for a range of hypothetical species. The ranking is performed using a simple set of indices that take into account the degree of environmental pressure and the presence of land protection schemes. An example for environmental reporting is carried out using European administrative regions and major rivers to summarize indices value. The approach is based on freely available software and simple metrics which can be easily reproduced in a GIS environment.

  6. The sub-cellular localization of Sulfolobus DNA replication.

    PubMed

    Gristwood, Tamzin; Duggin, Iain G; Wagner, Michaela; Albers, Sonja V; Bell, Stephen D

    2012-07-01

    Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.

  7. A structured assessment of emergency and acute care providers in Afghanistan during the current conflict.

    PubMed

    Rashid, Leeda; Afzali, Edris; Donaldson, Ross; Lazar, Paul; Bundesmann, Raghnild; Rashid, Samra

    2015-01-01

    Afghanistan has struggled with several decades of well-documented conflict, increasing the importance of providing emergency services to its citizens. However, little is known about the country's capacity to provide such care. Three native-speaking Afghan-American physicians performed an assessment of emergency care via combined quantitative and qualitative survey tools. Hospitals in Kabul, Afghanistan were selected based on probability proportional to size methodology, in which size was derived from prior work in the country and permission granted by the administering agency and the Ministry of Health. A written survey was given to physicians and nurses, followed by structured focus groups, and multiple days of observation per facility. A descriptive analysis was performed and data analyzed through a combination of variables in eight overarching categories relevant to emergency care. One hundred twenty-five surveys were completed from 9 hospitals. One third of respondents (32.8 %) worked full time in the emergency departments, with another 28.8 % working there at least three quarters of the time. Over 63 % of providers believed that the greatest delay for care in emergencies was in the prehospital setting. Differences were noted among the various types of facilities when looking at specific components of emergency care such as skill level of workers, frequencies of assaults in the hospitals, and other domains of service provision. Sum of squares between the different facility types were highest for areas of skill (SS = 210.3; p = .001), confidence in the system (SS = 156.5; p < .005), assault (SS = 487.6; p < .005), and feeling safe in the emergency departments (SS = 193.1, p < .005). Confidence negatively correlated to frequency of assaults (Pearson r = -.33; p < .005) but positively correlated with feeling safe (Pearson r = .51; p < .005) and reliability of equipment (Pearson r = .48; p < .005). The only correlation for access to services was prehospital care

  8. Tamoxifen Provides Structural and Functional Rescue in Murine Models of Photoreceptor Degeneration.

    PubMed

    Wang, Xu; Zhao, Lian; Zhang, Yikui; Ma, Wenxin; Gonzalez, Shaimar R; Fan, Jianguo; Kretschmer, Friedrich; Badea, Tudor C; Qian, Hao-Hua; Wong, Wai T

    2017-03-22

    Photoreceptor degeneration is a cause of irreversible vision loss in incurable blinding retinal diseases including retinitis pigmentosa (RP) and atrophic age-related macular degeneration. We found in two separate mouse models of photoreceptor degeneration that tamoxifen, a selective estrogen receptor modulator and a drug previously linked with retinal toxicity, paradoxically provided potent neuroprotective effects. In a light-induced degeneration model, tamoxifen prevented onset of photoreceptor apoptosis and atrophy and maintained near-normal levels of electroretinographic responses. Rescue effects were correlated with decreased microglial activation and inflammatory cytokine production in the retina in vivo and a reduction of microglia-mediated toxicity to photoreceptors in vitro, indicating a microglia-mediated mechanism of rescue. Tamoxifen also rescued degeneration in a genetic (Pde6b(rd10)) model of RP, significantly improving retinal structure, electrophysiological responses, and visual behavior. These prominent neuroprotective effects warrant the consideration of tamoxifen as a drug suitable for being repurposed to treat photoreceptor degenerative disease.SIGNIFICANCE STATEMENT Photoreceptor degeneration is a cause of irreversible blindness in a number of retinal diseases such as retinitis pigmentosa (RP) and atrophic age-related macular degeneration. Tamoxifen, a selective estrogen receptor modulator approved for the treatment of breast cancer and previously linked to a low incidence of retinal toxicity, was unexpectedly found to exert marked protective effects against photoreceptor degeneration. Structural and functional protective effects were found for an acute model of light-induced photoreceptor injury and for a genetic model for RP. The mechanism of protection involved the modulation of microglial activation and the production of inflammatory cytokines, highlighting the role of inflammatory mechanisms in photoreceptor degeneration. Tamoxifen may be

  9. Experimental Shielding Evaluation of the Radiation Protection Provided by Residential Structures

    NASA Astrophysics Data System (ADS)

    Dickson, Elijah D.

    The human health and environmental effects following a postulated accidental release of radioactive material to the environment has been a public and regulatory concern since the early development of nuclear technology and researched extensively to better understand the potential risks for accident mitigation and emergency planning purposes. The objective of this investigation is to research and develop the technical basis for contemporary building shielding factors for the U.S. housing stock. Building shielding factors quantify the protection a certain building-type provides from ionizing radiation. Much of the current data used to determine the quality of shielding around nuclear facilities and urban environments is based on simplistic point-kernel calculations for 1950's era suburbia and is no longer applicable to the densely populated urban environments seen today. To analyze a building's radiation shielding properties, the ideal approach would be to subject a variety of building-types to various radioactive materials and measure the radiation levels in and around the building. While this is not entirely practicable, this research uniquely analyzes the shielding effectiveness of a variety of likely U.S. residential buildings from a realistic source term in a laboratory setting. Results produced in the investigation provide a comparison between theory and experiment behind building shielding factor methodology by applying laboratory measurements to detailed computational models. These models are used to develop a series of validated building shielding factors for generic residential housing units using the computational code MCNP5. For these building shielding factors to be useful in radiologic consequence assessments and emergency response planning, two types of shielding factors have been developed for; (1) the shielding effectiveness of each structure within a semi-infinite cloud of radioactive material, and (2) the shielding effectiveness of each structure

  10. Observations of families in structured interactions: Parenting therapists provide reliable ratings of mothers’ parenting

    PubMed Central

    Storå, Bent; Hagtvet, Knut A; Heyerdahl, Sonja

    2014-01-01

    The reliability of observations of parenting by parenting therapists was assessed. An important predictor of externalizing behavior in children is quality of parenting. Data were videotapes of structured interactions in families with a child age 8–12 years referred to the evidence based Parent Management Training Oregon (PMTO) treatment program for child behavior problems. The therapists had clinical PMTO training but no training in systematic observation. PMTO observational coders with specific coder training were included as a reference for the therapists. Five therapists and two coders observed videotapes of 10 families and performed global evaluations of mothers’ parenting skills. They used the coder’s impression measure used in PMTO research. Scores were analyzed in a generalizability theory framework for the two groups of observers separately. Both observer types reliably rank-ordered the mothers and assessed the level of parenting skills. PMTO therapists without coder training provided reliable ratings of parenting constructs relevant to the clinical PMTO program in a manner comparable to that of the trained reference coders. PMID:24237459

  11. Geographic variation in the structure of oak hybrid zones provides insights into the dynamics of speciation.

    PubMed

    Zeng, Yan-Fei; Liao, Wan-Jin; Petit, Rémy J; Zhang, Da-Yong

    2011-12-01

    Studying geographic variation in the rate of hybridization between closely related species could provide a useful window on the evolution of reproductive isolation. Reinforcement theory predicts greater prezygotic isolation in areas of prolonged contact between recently diverged species than in areas of recent contact, which implies that old contact zones would be dominated by parental phenotypes with few hybrids (bimodal hybrid zones), whereas recent contact zones would be characterized by hybrid swarms (unimodal hybrid zones). Here, we investigate how the hybrid zones of two closely related Chinese oaks, Quercus mongolica and Q. liaotungensis, are structured geographically using both nuclear and chloroplast markers. We found that populations of Q. liaotungensis located around the Changbai Mountains in Northeast China, an inferred glacial refugium, were introgressed by genes from Q. mongolica, suggesting historical contact between the two species in this region. However, these introgressed populations form sharp bimodal hybrid zones with Q. mongolica. In contrast, populations of Q. liaotungensis located in North China, which show no sign of ancient introgression with Q. mongolica, form unimodal hybrid zones with Q. mongolica. These results are consistent with the hypothesis that selection against hybrids has had sufficient time to reinforce the reproductive barriers between Q. liaotungensis and Q. mongolica in Northeast China but not in North China.

  12. A Detailed Protocol for Subcellular RNA Sequencing (subRNA-seq).

    PubMed

    Mayer, Andreas; Churchman, L Stirling

    2017-10-02

    In eukaryotic cells, RNAs at various maturation and processing levels are distributed across cellular compartments. The standard approach to determine transcript abundance and identity in vivo is RNA sequencing (RNA-seq). RNA-seq relies on RNA isolation from whole-cell lysates and thus mainly captures fully processed, stable, and more abundant cytoplasmic RNAs over nascent, unstable, and nuclear RNAs. Here, we provide a step-by-step protocol for subcellular RNA-seq (subRNA-seq). subRNA-seq allows the quantitative measurement of RNA polymerase II-generated RNAs from the chromatin, nucleoplasm, and cytoplasm of mammalian cells. This approach relies on cell fractionation prior to RNA isolation and sequencing library preparation. High-throughput sequencing of the subcellular RNAs can then be used to reveal the identity, abundance, and subcellular distribution of transcripts, thus providing insights into RNA processing and maturation. Deep sequencing of the chromatin-associated RNAs further offers the opportunity to study nascent RNAs. Subcellular RNA-seq libraries are obtained within 5 days. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  13. Subcellular localization of the simian virus 40 agnoprotein.

    PubMed Central

    Nomura, S; Khoury, G; Jay, G

    1983-01-01

    The intracellular distribution of the simian virus 40 agnoprotein in infected cells was determined by indirect immunofluorescence and biochemical fractionation followed by indirect immunoprecipitation. The specific antibodies used in these studies were directed against either purified agnoprotein or a synthetic oligopeptide homologous to the N-terminus of the processed protein. Both procedures showed predominant localization of the agnoprotein to the cytosol and to the perinuclear region in association with the outer nuclear membrane. A minor but detectable fraction of the protein was also found in the nucleus. The definition of its subcellular distribution, as well as its high lability in vivo and affinity for nucleic acid, provide a basis for speculation on the function of this gene product. Images PMID:6296448

  14. Subcellular localization of the Schlafen protein family.

    PubMed

    Neumann, Brent; Zhao, Liang; Murphy, Kathleen; Gonda, Thomas J

    2008-05-23

    Although the first members of the Schlafen gene family were first described almost 10 years ago, the precise molecular/biochemical functions of the proteins they encode still remain largely unknown. Roles in cell growth, haematopoietic cell differentiation, and T cell development/maturation have, with some experimental support, been postulated, but none have been conclusively verified. Here, we have determined the subcellular localization of Schlafens 1, 2, 4, 5, 8, and 9, representing all three of the murine subgroups. We show that the proteins from subgroups I and II localize to the cytoplasm, while the longer forms in subgroup III localize exclusively to the nuclear compartment. We also demonstrate upregulation of Schlafen2 upon differentiation of haematopoietic cells and show this endogenous protein localizes to the cytoplasm. Thus, we propose the different subgroups of Schlafen proteins are likely to have functionally distinct roles, reflecting their differing localizations within the cell.

  15. Subcellular localization of the Schlafen protein family

    SciTech Connect

    Neumann, Brent; Zhao Liang; Murphy, Kathleen; Gonda, Thomas J.

    2008-05-23

    Although the first members of the Schlafen gene family were first described almost 10 years ago, the precise molecular/biochemical functions of the proteins they encode still remain largely unknown. Roles in cell growth, haematopoietic cell differentiation, and T cell development/maturation have, with some experimental support, been postulated, but none have been conclusively verified. Here, we have determined the subcellular localization of Schlafens 1, 2, 4, 5, 8, and 9, representing all three of the murine subgroups. We show that the proteins from subgroups I and II localize to the cytoplasm, while the longer forms in subgroup III localize exclusively to the nuclear compartment. We also demonstrate upregulation of Schlafen2 upon differentiation of haematopoietic cells and show this endogenous protein localizes to the cytoplasm. Thus, we propose the different subgroups of Schlafen proteins are likely to have functionally distinct roles, reflecting their differing localizations within the cell.

  16. Controlling subcellular delivery to optimize therapeutic effect

    PubMed Central

    Mossalam, Mohanad; Dixon, Andrew S; Lim, Carol S

    2010-01-01

    This article focuses on drug targeting to specific cellular organelles for therapeutic purposes. Drugs can be delivered to all major organelles of the cell (cytosol, endosome/lysosome, nucleus, nucleolus, mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes and proteasomes) where they exert specific effects in those particular subcellular compartments. Delivery can be achieved by chemical (e.g., polymeric) or biological (e.g., signal sequences) means. Unidirectional targeting to individual organelles has proven to be immensely successful for drug therapy. Newer technologies that accommodate multiple signals (e.g., protein switch and virus-like delivery systems) mimic nature and allow for a more sophisticated approach to drug delivery. Harnessing different methods of targeting multiple organelles in a cell will lead to better drug delivery and improvements in disease therapy. PMID:21113240

  17. Tuning the Catalytic Activity of Subcellular Nanoreactors.

    PubMed

    Jakobson, Christopher M; Chen, Yiqun; Slininger, Marilyn F; Valdivia, Elias; Kim, Edward Y; Tullman-Ercek, Danielle

    2016-07-31

    Bacterial microcompartments are naturally occurring subcellular organelles of bacteria and serve as a promising scaffold for the organization of heterologous biosynthetic pathways. A critical element in the design of custom biosynthetic organelles is quantitative control over the loading of heterologous enzymes to the interior of the organelles. We demonstrate that the loading of heterologous proteins to the 1,2-propanediol utilization microcompartment of Salmonella enterica can be controlled using two strategies: by modulating the transcriptional activation of the microcompartment container and by coordinating the expression of the microcompartment container and the heterologous cargo. These strategies allow general control over the loading of heterologous proteins localized by two different N-terminal targeting peptides and represent an important step toward tuning the catalytic activity of bacterial microcompartments for increased biosynthetic productivity. Copyright © 2016. Published by Elsevier Ltd.

  18. Quantitative analysis of glycerol in dicarboxylic acid-rich cutins provides insights into Arabidopsis cutin structure.

    PubMed

    Yang, Weili; Pollard, Mike; Li-Beisson, Yonghua; Ohlrogge, John

    2016-10-01

    Cutin is an extracellular lipid polymer that contributes to protective cuticle barrier functions against biotic and abiotic stresses in land plants. Glycerol has been reported as a component of cutin, contributing up to 14% by weight of total released monomers. Previous studies using partial hydrolysis of cuticle-enriched preparations established the presence of oligomers with glycerol-aliphatic ester links. Furthermore, glycerol-3-phosphate 2-O-acyltransferases (sn-2-GPATs) are essential for cutin biosynthesis. However, precise roles of glycerol in cutin assembly and structure remain uncertain. Here, a stable isotope-dilution assay was developed for the quantitative analysis of glycerol by GC/MS of triacetin with simultaneous determination of aliphatic monomers. To provide clues about the role of glycerol in dicarboxylic acid (DCA)-rich cutins, this methodology was applied to compare wild-type (WT) Arabidopsis cutin with a series of mutants that are defective in cutin synthesis. The molar ratio of glycerol to total DCAs in WT cutins was 2:1. Even when allowing for a small additional contribution from hydroxy fatty acids, this is a substantially higher glycerol to aliphatic monomer ratio than previously reported for any cutin. Glycerol content was strongly reduced in both stem and leaf cutin from all Arabidopsis mutants analyzed (gpat4/gpat8, att1-2 and lacs2-3). In addition, the molar reduction of glycerol was proportional to the molar reduction of total DCAs. These results suggest "glycerol-DCA-glycerol" may be the dominant motif in DCA-rich cutins. The ramifications and caveats for this hypothesis are presented.

  19. Application of femtosecond lasers for subcellular nanosurgery

    NASA Astrophysics Data System (ADS)

    Maxwell, Iva

    This dissertation offers a study of femtosecond laser disruption in single cells. Cells and tissues do not ordinarily absorb light in the near-IR wavelength range of femtosecond lasers. However, the peak intensity of a femtosecond laser pulse is very high and material disruption is possible through nonlinear absorption and plasma generation. Because the pulse duration is very short, it is possible to reach the intensity of optical breakdown at only nanojoules of energy per pulse. The low energy deposition and the high spatial localization of the nonlinear absorption, make femtosecond laser pulses an ideal tool for minimally disruptive subcellular nanosurgery. We show definitively that there can be bulk ablation within a single cell by studying the disrupted region under a transmission electron microscope. The width of the ablated area can be as small as 250 nm in diameter at energies near the ablation threshold. We also studied the effect of the laser repetition rate on the subcellular disruption threshold. We compared the pulse energies for kHz and MHz pulse trains, and found that in the MHz regime heat accumulation in the focal volume needs to be accounted for. For this repetition rate the minimum pulse energy necessary for disruption depends on the laser irradiation time. We used femtosecond laser nanosurgery to probe tension in actin stress fibers in living endothelial cells. By severing an individual stress fiber and visualizing its retraction, we showed that actin carries prestress in adherent, non-contractile cells. By plating the cells on softer, more compliant substrates, we measured the deflection of the substrate and extrapolated the force contribution of a stress filament on total amount of force exerted by the cell.

  20. SubCons: a new ensemble method for improved human subcellular localization predictions.

    PubMed

    Salvatore, M; Warholm, P; Shu, N; Basile, W; Elofsson, A

    2017-08-15

    Knowledge of the correct protein subcellular localization is necessary for understanding the function of a protein. Unfortunately large-scale experimental studies are limited in their accuracy. Therefore, the development of prediction methods has been limited by the amount of accurate experimental data. However, recently large-scale experimental studies have provided new data that can be used to evaluate the accuracy of subcellular predictions in human cells. Using this data we examined the performance of state of the art methods and developed SubCons, an ensemble method that combines four predictors using a Random Forest classifier. SubCons outperforms earlier methods in a dataset of proteins where two independent methods confirm the subcellular localization. Given nine subcellular localizations, SubCons achieves an F1-Score of 0.79 compared to 0.70 of the second best method. Furthermore, at a FPR of 1% the true positive rate (TPR) is over 58% for SubCons compared to less than 50% for the best individual predictor. SubCons is freely available as a webserver (http://subcons.bioinfo.se) and source code from https://bitbucket.org/salvatore_marco/subcons-web-server. The golden dataset as well is available from http://subcons.bioinfo.se/pred/download. arne@bioinfo.se. Supplementary data are available at Bioinformatics online.

  1. Integrative subcellular proteomic analysis allows accurate prediction of human disease-causing genes

    PubMed Central

    Zhao, Li; Chen, Yiyun; Bajaj, Amol Onkar; Eblimit, Aiden; Xu, Mingchu; Soens, Zachry T.; Wang, Feng; Ge, Zhongqi; Jung, Sung Yun; He, Feng; Li, Yumei; Wensel, Theodore G.; Qin, Jun; Chen, Rui

    2016-01-01

    Proteomic profiling on subcellular fractions provides invaluable information regarding both protein abundance and subcellular localization. When integrated with other data sets, it can greatly enhance our ability to predict gene function genome-wide. In this study, we performed a comprehensive proteomic analysis on the light-sensing compartment of photoreceptors called the outer segment (OS). By comparing with the protein profile obtained from the retina tissue depleted of OS, an enrichment score for each protein is calculated to quantify protein subcellular localization, and 84% accuracy is achieved compared with experimental data. By integrating the protein OS enrichment score, the protein abundance, and the retina transcriptome, the probability of a gene playing an essential function in photoreceptor cells is derived with high specificity and sensitivity. As a result, a list of genes that will likely result in human retinal disease when mutated was identified and validated by previous literature and/or animal model studies. Therefore, this new methodology demonstrates the synergy of combining subcellular fractionation proteomics with other omics data sets and is generally applicable to other tissues and diseases. PMID:26912414

  2. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    SciTech Connect

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

    2013-04-01

    The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  3. Age and Structural Lag: Society's Failure To Provide Meaningful Opportunities in Work, Family, and Leisure.

    ERIC Educational Resources Information Center

    Riley, Matilda White, Ed.; And Others

    This book is built around a single concept--structural lag (the tendency of social structures and norms to lag behind people's rapidly changing lives). Each of the 12 chapters explores implications of that concept in a different domain, and all reflect the authors' shared conviction of its importance, both for social theory and for practical…

  4. Organelle-Targeted H2S Probes Enable Visualization of the Subcellular Distribution of H2S Donors.

    PubMed

    Montoya, Leticia A; Pluth, Michael D

    2016-06-07

    Hydrogen sulfide (H2S) is an essential biological signaling molecule in diverse biological regulatory pathways. To provide new chemical tools for H2S imaging, we report here a fluorescent H2S detection platform (HSN2-BG) that is compatible with subcellular localization SNAP-tag fusion protein methodologies and use appropriate fusion protein constructs to demonstrate mitochondrial and lysosomal localization. We also demonstrate the efficacy of this detection platform to image endogenous H2S in Chinese hamster ovary (CHO) cells and use the developed constructs to report on the subcellular H2S distributions provided by common H2S donor molecules AP39, ADT-OH, GYY4137, and diallyltrisulfide (DATS). The developed constructs provide a platform poised to provide new insights into the subcellular distribution of common H2S donors and a useful tool for investigating H2S biochemistry.

  5. Apparatus and method for measuring single cell and sub-cellular photosynthetic efficiency

    DOEpatents

    Davis, Ryan Wesley; Singh, Seema; Wu, Huawen

    2013-07-09

    Devices for measuring single cell changes in photosynthetic efficiency in algal aquaculture are disclosed that include a combination of modulated LED trans-illumination of different intensities with synchronized through objective laser illumination and confocal detection. Synchronization and intensity modulation of a dual illumination scheme were provided using a custom microcontroller for a laser beam block and constant current LED driver. Therefore, single whole cell photosynthetic efficiency, and subcellular (diffraction limited) photosynthetic efficiency measurement modes are permitted. Wide field rapid light scanning actinic illumination is provided for both by an intensity modulated 470 nm LED. For the whole cell photosynthetic efficiency measurement, the same LED provides saturating pulses for generating photosynthetic induction curves. For the subcellular photosynthetic efficiency measurement, a switched through objective 488 nm laser provides saturating pulses for generating photosynthetic induction curves. A second near IR LED is employed to generate dark adapted states in the system under study.

  6. New mini- zincin structures provide a minimal scaffold for members of this metallopeptidase superfamily

    PubMed Central

    2014-01-01

    Background The Acel_2062 protein from Acidothermus cellulolyticus is a protein of unknown function. Initial sequence analysis predicted that it was a metallopeptidase from the presence of a motif conserved amongst the Asp-zincins, which are peptidases that contain a single, catalytic zinc ion ligated by the histidines and aspartic acid within the motif (HEXXHXXGXXD). The Acel_2062 protein was chosen by the Joint Center for Structural Genomics for crystal structure determination to explore novel protein sequence space and structure-based function annotation. Results The crystal structure confirmed that the Acel_2062 protein consisted of a single, zincin-like metallopeptidase-like domain. The Met-turn, a structural feature thought to be important for a Met-zincin because it stabilizes the active site, is absent, and its stabilizing role may have been conferred to the C-terminal Tyr113. In our crystallographic model there are two molecules in the asymmetric unit and from size-exclusion chromatography, the protein dimerizes in solution. A water molecule is present in the putative zinc-binding site in one monomer, which is replaced by one of two observed conformations of His95 in the other. Conclusions The Acel_2062 protein is structurally related to the zincins. It contains the minimum structural features of a member of this protein superfamily, and can be described as a “mini- zincin”. There is a striking parallel with the structure of a mini-Glu-zincin, which represents the minimum structure of a Glu-zincin (a metallopeptidase in which the third zinc ligand is a glutamic acid). Rather than being an ancestral state, phylogenetic analysis suggests that the mini-zincins are derived from larger proteins. PMID:24383880

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

    PubMed Central

    Yang, Pei-Chi; Boras, Britton W.; Jeng, Mao-Tsuen; Lewis, Timothy J.; McCulloch, Andrew D.; Harvey, Robert D.; Clancy, Colleen E.

    2016-01-01

    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 temporal

  8. 3D Printers Can Provide an Added Dimension for Teaching Structure-Energy Relationships

    ERIC Educational Resources Information Center

    Blauch, David N.; Carroll, Felix A.

    2014-01-01

    A 3D printer is used to prepare a variety of models representing potential energy as a function of two geometric coordinates. These models facilitate the teaching of structure-energy relationships in molecular conformations and in chemical reactions.

  9. 3D Printers Can Provide an Added Dimension for Teaching Structure-Energy Relationships

    ERIC Educational Resources Information Center

    Blauch, David N.; Carroll, Felix A.

    2014-01-01

    A 3D printer is used to prepare a variety of models representing potential energy as a function of two geometric coordinates. These models facilitate the teaching of structure-energy relationships in molecular conformations and in chemical reactions.

  10. Supercoiling as a Physical Process Providing Formation of Macroscopic Anisometric Supramolecular Structures.

    PubMed

    Skoblin, A A; Stovbun, S V

    2015-09-01

    Solutions of chiral and achiral trifluoroacetyl amino alcohols (TFAAA) contain anisometric structures with a diameter <1 nm and length ~7 nm. In homochiral solutions and xerogels, chiral TFAAA form strings with a diameter of ~30-100 nm and length more than ~1 μ, and achiral TFAAA condensate into isometric granule with diameter of ~1 μ. We conclude that molecular chirality determines helicity of strings within tens of nanometers or more. Stabilization of supramolecular structure of strings is presumably achieved via their supercoiling.

  11. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity.

    PubMed

    Sayer, Christopher; Isupov, Michail N; Westlake, Aaron; Littlechild, Jennifer A

    2013-04-01

    The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.

  12. Retrieving Backbone String Neighbors Provides Insights Into Structural Modeling of Membrane Proteins*

    PubMed Central

    Sun, Jiang-Ming; Li, Tong-Hua; Cong, Pei-Sheng; Tang, Sheng-Nan; Xiong, Wen-Wei

    2012-01-01

    Identification of protein structural neighbors to a query is fundamental in structure and function prediction. Here we present BS-align, a systematic method to retrieve backbone string neighbors from primary sequences as templates for protein modeling. The backbone conformation of a protein is represented by the backbone string, as defined in Ramachandran space. The backbone string of a query can be accurately predicted by two innovative technologies: a knowledge-driven sequence alignment and encoding of a backbone string element profile. Then, the predicted backbone string is employed to align against a backbone string database and retrieve a set of backbone string neighbors. The backbone string neighbors were shown to be close to native structures of query proteins. BS-align was successfully employed to predict models of 10 membrane proteins with lengths ranging between 229 and 595 residues, and whose high-resolution structural determinations were difficult to elucidate both by experiment and prediction. The obtained TM-scores and root mean square deviations of the models confirmed that the models based on the backbone string neighbors retrieved by the BS-align were very close to the native membrane structures although the query and the neighbor shared a very low sequence identity. The backbone string system represents a new road for the prediction of protein structure from sequence, and suggests that the similarity of the backbone string would be more informative than describing a protein as belonging to a fold. PMID:22415040

  13. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    PubMed Central

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

    2013-01-01

    The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-­aminotransferases. PMID:23519665

  14. Predicting eukaryotic protein subcellular location by fusing optimized evidence-theoretic K-Nearest Neighbor classifiers.

    PubMed

    Chou, Kuo-Chen; Shen, Hong-Bin

    2006-08-01

    Facing the explosion of newly generated protein sequences in the post genomic era, we are challenged to develop an automated method for fast and reliably annotating their subcellular locations. Knowledge of subcellular locations of proteins can provide useful hints for revealing their functions and understanding how they interact with each other in cellular networking. Unfortunately, it is both expensive and time-consuming to determine the localization of an uncharacterized protein in a living cell purely based on experiments. To tackle the challenge, a novel hybridization classifier was developed by fusing many basic individual classifiers through a voting system. The "engine" of these basic classifiers was operated by the OET-KNN (Optimized Evidence-Theoretic K-Nearest Neighbor) rule. As a demonstration, predictions were performed with the fusion classifier for proteins among the following 16 localizations: (1) cell wall, (2) centriole, (3) chloroplast, (4) cyanelle, (5) cytoplasm, (6) cytoskeleton, (7) endoplasmic reticulum, (8) extracell, (9) Golgi apparatus, (10) lysosome, (11) mitochondria, (12) nucleus, (13) peroxisome, (14) plasma membrane, (15) plastid, and (16) vacuole. To get rid of redundancy and homology bias, none of the proteins investigated here had >/=25% sequence identity to any other in a same subcellular location. The overall success rates thus obtained via the jack-knife cross-validation test and independent dataset test were 81.6% and 83.7%, respectively, which were 46 approximately 63% higher than those performed by the other existing methods on the same benchmark datasets. Also, it is clearly elucidated that the overwhelmingly high success rates obtained by the fusion classifier is by no means a trivial utilization of the GO annotations as prone to be misinterpreted because there is a huge number of proteins with given accession numbers and the corresponding GO numbers, but their subcellular locations are still unknown, and that the

  15. The In Vivo Architecture of the Exocyst Provides Structural Basis for Exocytosis.

    PubMed

    Picco, Andrea; Irastorza-Azcarate, Ibai; Specht, Tanja; Böke, Dominik; Pazos, Irene; Rivier-Cordey, Anne-Sophie; Devos, Damien P; Kaksonen, Marko; Gallego, Oriol

    2017-01-26

    The structural characterization of protein complexes in their native environment is challenging but crucial for understanding the mechanisms that mediate cellular processes. We developed an integrative approach to reconstruct the 3D architecture of protein complexes in vivo. We applied this approach to the exocyst, a hetero-octameric complex of unknown structure that is thought to tether secretory vesicles during exocytosis with a poorly understood mechanism. We engineered yeast cells to anchor the exocyst on defined landmarks and determined the position of its subunit termini at nanometer precision using fluorescence microscopy. We then integrated these positions with the structural properties of the subunits to reconstruct the exocyst together with a vesicle bound to it. The exocyst has an open hand conformation made of rod-shaped subunits that are interlaced in the core. The exocyst architecture explains how the complex can tether secretory vesicles, placing them in direct contact with the plasma membrane.

  16. Challenges and recommendations for obtaining chemical structures of industry-provided repurposing candidates.

    PubMed

    Southan, Christopher; Williams, Antony J; Ekins, Sean

    2013-01-01

    There is an expanding amount of interest directed at the repurposing and repositioning of drugs, as well as how in silico methods can assist these endeavors. Recent repurposing project tendering calls by the National Center for Advancing Translational Sciences (USA) and the Medical Research Council (UK) have included compound information and pharmacological data. However, none of the internal company development code names were assigned to chemical structures in the official documentation. This not only abrogates in silico analysis to support repurposing but consequently necessitates data gathering and curation to assign structures. Here, we describe the approaches, results and major challenges associated with this.

  17. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

    SciTech Connect

    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.

    2010-07-19

    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  18. Optical tomography of human skin with subcellular spatial and picosecond time resolution using intense near infrared femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Wollina, Uwe; Riemann, Iris; Peukert, Christiane; Halbhuber, Karl-Juergen; Konrad, Helga; Fischer, Peter; Fuenfstueck, Veronika; Fischer, Tobias W.; Elsner, Peter

    2002-06-01

    We describe the novel high resolution imaging tool DermaInspect 100 for non-invasive diagnosis of dermatological disorders based on multiphoton autofluorescence imaging (MAI)and second harmonic generation. Femtosecond laser pulses in the spectral range of 750 nm to 850 nm have been used to image in vitro and in vivo human skin with subcellular spatial and picosecond temporal resolution. The non-linear induced autofluorescence originates mainly from naturally endogenous fluorophores/protein structures like NAD(P)H, flavins, keratin, collagen, elastin, porphyrins and melanin. Second harmonic generation was observed in the stratum corneum and in the dermis. The system with a wavelength-tunable compact 80 MHz Ti:sapphire laser, a scan module with galvo scan mirrors, piezoelectric objective positioner, fast photon detector and time-resolved single photon counting unit was used to perform optical sectioning and 3D autofluorescence lifetime imaging (t-mapping). In addition, a modified femtosecond laser scanning microscope was involved in autofluorescence measurements. Tissues of patients with psoriasis, nevi, dermatitis, basalioma and melanoma have been investigated. Individual cells and skin structures could be clearly visualized. Intracellular components and connective tissue structures could be further characterized by tuning the excitation wavelength in the range of 750 nm to 850 nm and by calculation of mean fluorescence lifetimes per pixel and of particular regions of interest. The novel non-invasive imaging system provides 4D (x,y,z,t) optical biopsies with subcellular resolution and offers the possibility to introduce a further optical diagnostic method in dermatology.

  19. Amide I'-II' 2D IR spectroscopy provides enhanced protein secondary structural sensitivity.

    PubMed

    Deflores, Lauren P; Ganim, Ziad; Nicodemus, Rebecca A; Tokmakoff, Andrei

    2009-03-11

    We demonstrate how multimode 2D IR spectroscopy of the protein amide I' and II' vibrations can be used to distinguish protein secondary structure. Polarization-dependent amide I'-II' 2D IR experiments on poly-l-lysine in the beta-sheet, alpha-helix, and random coil conformations show that a combination of amide I' and II' diagonal and cross peaks can effectively distinguish between secondary structural content, where amide I' infrared spectroscopy alone cannot. The enhanced sensitivity arises from frequency and amplitude correlations between amide II' and amide I' spectra that reflect the symmetry of secondary structures. 2D IR surfaces are used to parametrize an excitonic model for the amide I'-II' manifold suitable to predict protein amide I'-II' spectra. This model reveals that the dominant vibrational interaction contributing to this sensitivity is a combination of negative amide II'-II' through-bond coupling and amide I'-II' coupling within the peptide unit. The empirically determined amide II'-II' couplings do not significantly vary with secondary structure: -8.5 cm(-1) for the beta sheet, -8.7 cm(-1) for the alpha helix, and -5 cm(-1) for the coil.

  20. Structure of an asymmetric ternary protein complex provides insight for membrane interaction.

    PubMed

    Dempsey, Brian R; Rezvanpour, Atoosa; Lee, Ting-Wai; Barber, Kathryn R; Junop, Murray S; Shaw, Gary S

    2012-10-10

    Plasma membrane repair involves the coordinated effort of proteins and the inner phospholipid surface to mend the rupture and return the cell back to homeostasis. Here, we present the three-dimensional structure of a multiprotein complex that includes S100A10, annexin A2, and AHNAK, which along with dysferlin, functions in muscle and cardiac tissue repair. The 3.5 Å resolution X-ray structure shows that a single region from the AHNAK C terminus is recruited by an S100A10-annexin A2 heterotetramer, forming an asymmetric ternary complex. The AHNAK peptide adopts a coil conformation that arches across the heterotetramer contacting both annexin A2 and S100A10 protomers with tight affinity (∼30 nM) and establishing a structural rationale whereby both S100A10 and annexin proteins are needed in AHNAK recruitment. The structure evokes a model whereby AHNAK is targeted to the membrane surface through sandwiching of the binding region between the S100A10/annexin A2 complex and the phospholipid membrane.

  1. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity

    USDA-ARS?s Scientific Manuscript database

    Botulinum neurotoxins (BoNTs) are highly potent oral poisons produced by Clostridium botulinum. BoNTs are secreted along with several auxiliary proteins forming progenitor toxin complexes (PTC). Here, we report the structure of a ~760 kDa 14-subunit PTC using a combination of X-ray crystallography a...

  2. Problems of interior structure, the solar dynamo and the role of SCADM in providing interior diagnostics

    NASA Technical Reports Server (NTRS)

    Weiss, N. O.

    1980-01-01

    What is already known about the structure of the Sun, the motion of its convective zone, and the solar cycle is reviewed. Topics discussed include solar variability, solar 'seismology', velocity patterns, magnetic fields, and the dynamo theory. Observations are needed to determine global properties (solar luminosity and radius), oscillations (p and g models), velocities (variation of rotation with time and depth), and magnetic fields.

  3. Do Clinical Clerks Provide Candidates with Adequate Formative Assessment during Objective Structured Clinical Examinations?

    ERIC Educational Resources Information Center

    Reiter, Harold I.; Rosenfeld, Jack; Nandagopal, Kiruthiga; Eva, Kevin W.

    2004-01-01

    Context: Various research studies have examined the question of whether expert or non-expert raters, faculty or students, evaluators or standardized patients, give more reliable and valid summative assessments of performance on Objective Structured Clinical Examinations (OSCEs). Less studied has been the question of whether or not non-faculty…

  4. Advantages of Providing Structured Supplemental Reading Instruction to Kindergarteners at Risk for Failure in Reading

    ERIC Educational Resources Information Center

    Helf, Shawnna; Cooke, Nancy L.; Konrad, Moira

    2014-01-01

    This study compared the reading gains of Kindergarten students who were at risk for reading failure who were taught with either a structured supplemental reading program or with teacher-designed or teacher-selected instruction. The authors used a quasi-experimental design with preexisting groups to examine changes from pretest to posttest.…

  5. Studies on cattle genomic structural variation provide insights into ruminant speciation and adaptation

    USDA-ARS?s Scientific Manuscript database

    Genomic structural variations, including segmental duplications (SD) and copy number variations (CNV), contribute significantly to individual health and disease in primates and rodents. As a part of the bovine genome annotation effort, we performed the first genome-wide analysis of SD in cattle usin...

  6. A high-resolution structure that provides insight into coiled-coil thiodepsipeptide dynamic chemistry.

    PubMed

    Dadon, Zehavit; Samiappan, Manickasundaram; Shahar, Anat; Zarivach, Raz; Ashkenasy, Gonen

    2013-09-16

    Stable and reactive: A crystal structure at 1.35 Å of a thioester coiled-coil protein reveals high similarity to all-peptide-bond proteins. In these assemblies, the thioester bonds are kept reactive towards thiol molecules in the mixture. This enables efficient domain exchange between proteins in response to changes in folding conditions or introduction of external templates.

  7. Hierarchical population structure in greater sage-grouse provides insight into management boundary delineation

    Treesearch

    Todd B. Cross; David E. Naugle; John C. Carlson; Michael K. Schwartz

    2016-01-01

    Understanding population structure is important for guiding ongoing conservation and restoration efforts. The greater sage-grouse (Centrocercus urophasianus) is a species of concern distributed across 1.2 million km2 of western North America. We genotyped 1499 greater sagegrouse from 297 leks across Montana, North Dakota and South Dakota using a 15 locus...

  8. The Spacing Effect for Structural Synaptic Plasticity Provides Specificity and Precision in Plastic Changes.

    PubMed

    San Martin, Alvaro; Rela, Lorena; Gelb, Bruce; Pagani, Mario Rafael

    2017-05-10

    In contrast to trials of training without intervals (massed training), training trials spaced over time (spaced training) induce a more persistent memory identified as long-term memory (LTM). This phenomenon, known as the spacing effect for memory, is poorly understood. LTM is supported by structural synaptic plasticity; however, how synapses integrate spaced stimuli remains elusive. Here, we analyzed events of structural synaptic plasticity at the single-synapse level after distinct patterns of stimulation in motoneurons of Drosophila We found that the spacing effect is a phenomenon detected at synaptic level, which determines the specificity and the precision in structural synaptic plasticity. Whereas a single pulse of stimulation (massed) induced structural synaptic plasticity, the same amount of stimulation divided in three spaced stimuli completely prevented it. This inhibitory effect was determined by the length of the interstimulus intervals. The inhibitory effect of the spacing was lost by suppressing the activity of Ras or mitogen-activated protein kinase, whereas the overexpression of Ras-WT enhanced it. Moreover, dividing the same total time of stimulation into five or more stimuli produced a higher precision in the number of events of plasticity. Ras mutations associated with intellectual disability abolished the spacing effect and led neurons to decode distinct stimulation patterns as massed stimulation. This evidence suggests that the spacing effect for memory may result from the effect of the spacing in synaptic plasticity, which appears to be a property not limited to neurons involved in learning and memory. We propose a model of spacing-dependent structural synaptic plasticity.SIGNIFICANCE STATEMENT Long-term memory (LTM) induced by repeated trials spaced over time is known as the spacing effect, a common property in the animal kingdom. Altered mechanisms in the spacing effect have been found in animal models of disorders with intellectual

  9. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-{angstrom}-resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-{angstrom}-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  10. An introduction to using the FORTRAN programs provided with Computational Nuclear Physics 1 Nuclear Structure

    NASA Technical Reports Server (NTRS)

    Boytos, Matthew A.; Norbury, John W.

    1992-01-01

    The authors of this paper have provided a set of ready-to-run FORTRAN programs that should be useful in the field of theoretical nuclear physics. The purpose of this document is to provide a simple synopsis of the programs and their use. A separate section is devoted to each program set and includes: abstract; files; compiling, linking, and running; obtaining results; and a tutorial.

  11. Tau regulates the subcellular localization of calmodulin

    SciTech Connect

    Barreda, Elena Gomez de

    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.

  12. Subcellular distribution of potassium in striated muscles

    SciTech Connect

    Edelmann, L.

    1984-01-01

    Microanalytical experiments have been performed to answer the question whether the main cellular cation, K+, follows the water distribution in the striated muscle cell or whether K+ follows the distribution of negative fixed charges (beta- and gamma-carboxyl groups of aspartic and glutamic acid residues). Subcellular localization of K and/or of the K surrogates Rb, Cs, and Tl has been investigated by the following methods: Chemical precipitation of K with tetraphenylborate. Autoradiography of alkali-metals and Tl in air-dried and frozen-hydrated preparations. TEM visualization of electron dense Cs and Tl in sections of freeze-dried and plastic embedded muscle. X-ray microanalysis of air-dried myofibrils and muscle cryosections. The experiments consistently show that K, Rb, Cs, and Tl do not follow the water distribution but are mainly accumulated in the A band, especially in the marginal regions, and at Z lines. The same sites preferentially accumulate Cs or uranyl cations when sections of freeze-dried, embedded muscle are exposed to these electron microscopic stains. It is concluded that the detected uneven distribution of K, Rb, Cs, and Tl in muscle is neither a freeze-drying artifact nor an embedding artifact and may result from a weak ion binding to the beta- and gamma-carboxyl groups of cellular proteins.

  13. Subcellular proteomics of Trypanosoma cruzi reservosomes

    PubMed Central

    Sant’Anna, Celso; Nakayasu, Ernesto S.; Pereira, Miria G.; Lourenço, Daniela; de Souza, Wanderley; Almeida, Igor C.; Cunha-e-Silva, Narcisa L.

    2009-01-01

    Reservosomes are the endpoint of the endocytic pathway in Trypanosoma cruzi epimastigotes. These organelles have the particular ability to concentrate proteins and lipids obtained from medium together with the main proteolytic enzymes originated from the secretory pathway, being at the same time a storage organelle and the main site of protein degradation. Subcellular proteomics have been extensively used for profiling organelles in different cell types. Here, we combine cell fractionation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify reservosome-resident proteins. Starting from a purified reservosome fraction, we established a protocol to isolate reservosome membranes. Transmission electron microscopy was applied to confirm the purity of the fractions. To achieve a better coverage of identified proteins we analyzed the fractions separately and combined the results. LC-MS/MS analysis identified in total 709 T. cruzi-specific proteins; of these, 456 had predicted function and 253 were classified as hypothetical proteins. We could confirm the presence of most of the proteins validated by previous work and identify new proteins from different classes such as enzymes, proton pumps, transport proteins and others. The definition of the reservosome protein profile is a good tool to assess their molecular signature, identify molecular markers, and understand their relationship with different organelles. PMID:19288526

  14. Structure of Ljungan virus provides insight into genome packaging of this picornavirus

    NASA Astrophysics Data System (ADS)

    Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Porta, Claudine; Wenham, Hannah; Ekström, Jens-Ola; Panjwani, Anusha; Knowles, Nick J.; Kotecha, Abhay; Siebert, C. Alistair; Lindberg, A. Michael; Fry, Elizabeth E.; Rao, Zihe; Tuthill, Tobias J.; Stuart, David I.

    2015-10-01

    Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.

  15. Preparing a Health Care White Paper: Providing Structure to the Writing Process.

    PubMed

    Rotarius, Timothy; Rotarius, Velmarie

    2016-01-01

    Health care leaders operate in a very complex and turbulent business environment. Both government regulations and market forces are very active in the industry. Thus, health care managers have many multifaceted and, sometimes, contradictory expectations placed upon them and their organizations. To ensure professional accountability, health care executives often join professional associations and strive for licenses and certifications that are intended to place the professional above the rest. One important avenue to achieve various licensing and certification accomplishments involves writing a white paper about a specific topic of interest to the industry and organization. Presented herein are structural processes that facilitate the creation and preparation of a health care white paper. Both conceptual and empirical structures of white papers are presented, with the similarities and the differences between conceptual and empirical papers highlighted.

  16. Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy.

    PubMed

    Islam, Mohammad Monirul; Yohda, Masafumi; Kidokoro, Shun-Ichi; Kuroda, Yutaka

    2017-03-07

    We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19-24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein-water hydrogen bonds and improving the hydration shells around the alanine's methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration.

  17. Structure of Concatenated HAMP Domains Provides a Mechanism for Signal Transduction

    SciTech Connect

    Airola, Michael V.; Watts, Kylie J.; Bilwes, Alexandrine M.; Crane, Brian R.

    2010-08-23

    HAMP domains are widespread prokaryotic signaling modules found as single domains or poly-HAMP chains in both transmembrane and soluble proteins. The crystal structure of a three-unit poly-HAMP chain from the Pseudomonas aeruginosa soluble receptor Aer2 defines a universal parallel four-helix bundle architecture for diverse HAMP domains. Two contiguous domains integrate to form a concatenated di-HAMP structure. The three HAMP domains display two distinct conformations that differ by changes in helical register, crossing angle, and rotation. These conformations are stabilized by different subsets of conserved residues. Known signals delivered to HAMP would be expected to switch the relative stability of the two conformations and the position of a coiled-coil phase stutter at the junction with downstream helices. We propose that the two conformations represent opposing HAMP signaling states and suggest a signaling mechanism whereby HAMP domains interconvert between the two states, which alternate down a poly-HAMP chain.

  18. Structure of Ljungan virus provides insight into genome packaging of this picornavirus

    PubMed Central

    Zhu, Ling; Wang, Xiangxi; Ren, Jingshan; Porta, Claudine; Wenham, Hannah; Ekström, Jens-Ola; Panjwani, Anusha; Knowles, Nick J.; Kotecha, Abhay; Siebert, C. Alistair; Lindberg, A. Michael; Fry, Elizabeth E.; Rao, Zihe; Tuthill, Tobias J.; Stuart, David I.

    2015-01-01

    Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses. PMID:26446437

  19. Providing structural modules with self-integrity monitoring software user's manual

    NASA Technical Reports Server (NTRS)

    1990-01-01

    National Aeronautics and Space Administration (NASA) Contract NAS7-961 (A Small Business Innovation and Research (SBIR) contract from NASA) involved research dealing with remote structural damage detection using the concept of substructures. Several approaches were developed. The main two were: (1) the module (substructure) transfer function matrix (MTFM) approach; and (2) modal strain energy distribution method (MSEDM). Either method can be used with a global structure; however, the focus was on substructures. As part of the research contract, computer software was to be developed which would implement the developed methods. This was done and it was used to process all the finite element generated numerical data for the research. The software was written for the IBM AT personal computer. Copies of it were placed on floppy disks. This report serves as a user's manual for the two sets of damage detection software. Sections 2.0 and 3.0 discuss the use of the MTFM and MSEDM software, respectively.

  20. Structural Similarities between Brain and Linguistic Data Provide Evidence of Semantic Relations in the Brain

    PubMed Central

    Crangle, Colleen E.; Perreau-Guimaraes, Marcos; Suppes, Patrick

    2013-01-01

    This paper presents a new method of analysis by which structural similarities between brain data and linguistic data can be assessed at the semantic level. It shows how to measure the strength of these structural similarities and so determine the relatively better fit of the brain data with one semantic model over another. The first model is derived from WordNet, a lexical database of English compiled by language experts. The second is given by the corpus-based statistical technique of latent semantic analysis (LSA), which detects relations between words that are latent or hidden in text. The brain data are drawn from experiments in which statements about the geography of Europe were presented auditorily to participants who were asked to determine their truth or falsity while electroencephalographic (EEG) recordings were made. The theoretical framework for the analysis of the brain and semantic data derives from axiomatizations of theories such as the theory of differences in utility preference. Using brain-data samples from individual trials time-locked to the presentation of each word, ordinal relations of similarity differences are computed for the brain data and for the linguistic data. In each case those relations that are invariant with respect to the brain and linguistic data, and are correlated with sufficient statistical strength, amount to structural similarities between the brain and linguistic data. Results show that many more statistically significant structural similarities can be found between the brain data and the WordNet-derived data than the LSA-derived data. The work reported here is placed within the context of other recent studies of semantics and the brain. The main contribution of this paper is the new method it presents for the study of semantics and the brain and the focus it permits on networks of relations detected in brain data and represented by a semantic model. PMID:23799009

  1. Homology modeling and molecular dynamics provide structural insights into tospovirus nucleoprotein.

    PubMed

    Lima, Rayane Nunes; Faheem, Muhammad; Barbosa, João Alexandre Ribeiro Gonçalves; Polêto, Marcelo Depólo; Verli, Hugo; Melo, Fernando Lucas; Resende, Renato Oliveira

    2016-12-15

    Tospovirus is a plant-infecting genus within the family Bunyaviridae, which also includes four animal-infecting genera: Hantavirus, Nairovirus, Phlebovirus and Orthobunyavirus. Compared to these members, the structures of Tospovirus proteins still are poorly understood. Despite multiple studies have attempted to identify candidate N protein regions involved in RNA binding and protein multimerization for tospovirus using yeast two-hybrid systems (Y2HS) and site-directed mutagenesis, the tospovirus ribonucleocapsids (RNPs) remains largely uncharacterized at the molecular level and the lack of structural information prevents detailed insight into these interactions. Here we used the nucleoprotein structure of LACV (La Crosse virus-Orthobunyavirus) and molecular dynamics simulations to access the structure and dynamics of the nucleoprotein from tospovirus GRSV (Groundnut ringspot virus). The resulting model is a monomer composed by a flexible N-terminal and C-terminal arms and a globular domain with a positively charged groove in which RNA is deeply encompassed. This model allowed identifying the candidate amino acids residues involved in RNA interaction and N-N multimerization. Moreover, most residues predicted to be involved in these interactions are highly conserved among tospoviruses. Crucially, the interaction model proposed here for GRSV N is further corroborated by the all available mutational studies on TSWV (Tomato spotted wilt virus) N, so far. Our data will help designing further and more accurate mutational and functional studies of tospovirus N proteins. In addition, the proposed model may shed light on the mechanisms of RNP shaping and could allow the identification of essential amino acid residues as potential targets for tospovirus control strategies.

  2. Further constraints of the North American Craton structure provided by the recent Oklahoma earthquake

    NASA Astrophysics Data System (ADS)

    Chu, R.; Helmberger, D. V.

    2012-12-01

    Seismic velocities and attenuation are essential for understanding the structure of continental lithosphere and geodynamics of the earth. Chu et al. (2012abc) used USArray waveform data from small regional earthquakes in Quebec and Virginia to investigate the upper-mantle P velocity structure (CR) beneath the North American Craton. These results suggest a lithospheric thickness of ~200 km with an 8° discontinuity at a depth of ~120 km. Here we present results from the 2011 Oklahoma earthquake to investigate the S-velocity structure and seismic attenuation. We first obtain the source parameters of the earthquake using the Cut-and-Paste method. Next we use this source to model the regional waveform data. This strike-slip event produced excellent profiles of broadband tangential motions to the edge of the array (14°). The crossover of crustal S to Sn is well observed where SmS has a critical distance of ~2°. The strength of this critical-angle reflection allows the crustal multiples to setup and be strong out to 14° before crossing into the stronger crustal Love waves. These waveforms are modeled broadband (2~100 s) by assuming a three-layer crust and CR-type mantle. The best model is accessed through a grid-search approach as used above. Our results favor a weak low-velocity layer in the lower crust with a shear velocity of 3.6 km/s. This low-velocity zone appear to be responsible for the high frequency shaking at larger distances in eastern United States. Below the Moho, S-velocity slightly decreases with depth, which agrees with the CR model. Synthetic calculations using various undulating Moho depths and sediment thickness suggest that sediment structures have a strong impact on the crustal multiples, which may be why those multiples are not commonly observed. Preliminary work on the attenuation suggests that the Q in the craton is relatively high (Q~600).

  3. Metastable structures and isotope exchange reactions in polyoxometalate ions provide a molecular view of oxide dissolution.

    PubMed

    Rustad, James R; Casey, William H

    2012-01-10

    Reactions involving minerals and glasses in water are slow and difficult to probe spectroscopically but are fundamental to the performance of oxide materials in green technologies such as automotive thermoelectric power generation, CO2 capture and storage and water-oxidation catalysis; these must be made from geochemically common elements and operate in hydrous environments. Polyoxometalate ions (POMs) have structures similar to condensed oxide phases and can be used as molecular models of the oxide/water interface. Oxygen atoms in POM exchange isotopes at different rates, but, at present, there is no basis for predicting how the coordination environment and metal substitution influences rates and mechanisms. Here we identify low-energy metastable configurations that form from the breaking of weak bonds between metals and underlying highly coordinated oxygen atoms, followed by facile hydroxide, hydronium or water addition. The mediation of oxygen exchange by these stuffed structures suggests a new view of the relationship between structure and reactivity at the oxide/solution interface.

  4. A Structural Model for a Self-Assembled Nanotube Provides Insight into Its Exciton Dynamics

    PubMed Central

    2016-01-01

    The design and synthesis of functional self-assembled nanostructures is frequently an empirical process fraught with critical knowledge gaps about atomic-level structure in these noncovalent systems. Here, we report a structural model for a semiconductor nanotube formed via the self-assembly of naphthalenediimide-lysine (NDI-Lys) building blocks determined using experimental 13C–13C and 13C–15N distance restraints from solid-state nuclear magnetic resonance supplemented by electron microscopy and X-ray powder diffraction data. The structural model reveals a two-dimensional-crystal-like architecture of stacked monolayer rings each containing ∼50 NDI-Lys molecules, with significant π-stacking interactions occurring both within the confines of the ring and along the long axis of the tube. Excited-state delocalization and energy transfer are simulated for the nanotube based on time-dependent density functional theory and an incoherent hopping model. Remarkably, these calculations reveal efficient energy migration from the excitonic bright state, which is in agreement with the rapid energy transfer within NDI-Lys nanotubes observed previously using fluorescence spectroscopy. PMID:26120375

  5. Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding

    PubMed Central

    Appleton, Brent A; Wu, Ping; Maloney, Janice; Yin, JianPing; Liang, Wei-Ching; Stawicki, Scott; Mortara, Kyle; Bowman, Krista K; Elliott, J Michael; Desmarais, William; Bazan, J Fernando; Bagri, Anil; Tessier-Lavigne, Marc; Koch, Alexander W; Wu, Yan; Watts, Ryan J; Wiesmann, Christian

    2007-01-01

    Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins. PMID:17989695

  6. Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression

    PubMed Central

    Martin-Arevalillo, Raquel; Nanao, Max H.; Vinos-Poyo, Thomas; Mast, David; Galvan-Ampudia, Carlos; Brunoud, Géraldine; Dumas, Renaud

    2017-01-01

    Transcriptional repression involves a class of proteins called corepressors that link transcription factors to chromatin remodeling complexes. In plants such as Arabidopsis thaliana, the most prominent corepressor is TOPLESS (TPL), which plays a key role in hormone signaling and development. Here we present the crystallographic structure of the Arabidopsis TPL N-terminal region comprising the LisH and CTLH (C-terminal to LisH) domains and a newly identified third region, which corresponds to a CRA domain. Comparing the structure of TPL with the mammalian TBL1, which shares a similar domain structure and performs a parallel corepressor function, revealed that the plant TPLs have evolved a new tetramerization interface and unique and highly conserved surface for interaction with repressors. Using site-directed mutagenesis, we validated those surfaces in vitro and in vivo and showed that TPL tetramerization and repressor binding are interdependent. Our results illustrate how evolution used a common set of protein domains to create a diversity of corepressors, achieving similar properties with different molecular solutions. PMID:28698367

  7. Toponome imaging system: in situ protein network mapping in normal and cancerous colon from the same patient reveals more than five-thousand cancer specific protein clusters and their subcellular annotation by using a three symbol code.

    PubMed

    Bhattacharya, Sayantan; Mathew, George; Ruban, Ernie; Epstein, David B A; Krusche, Andreas; Hillert, Reyk; Schubert, Walter; Khan, Michael

    2010-12-03

    In a proof of principle study, we have applied an automated fluorescence toponome imaging system (TIS) to examine whether TIS can find protein network structures, distinguishing cancerous from normal colon tissue present in a surgical sample from the same patient. By using a three symbol code and a power of combinatorial molecular discrimination (PCMD) of 2(21) per subcellular data point in one single tissue section, we demonstrate an in situ protein network structure, visualized as a mosaic of 6813 protein clusters (combinatorial molecular phenotype or CMPs), in the cancerous part of the colon. By contrast, in the histologically normal colon, TIS identifies nearly 5 times the number of protein clusters as compared to the cancerous part (32 009). By subcellular visualization procedures, we found that many cell surface membrane molecules were closely associated with the cell cytoskeleton as unique CMPs in the normal part of the colon, while the same molecules were disassembled in the cancerous part, suggesting the presence of dysfunctional cytoskeleton-membrane complexes. As expected, glandular and stromal cell signatures were found, but interestingly also found were potentially TIS signatures identifying a very restricted subset of cells expressing several putative stem cell markers, all restricted to the cancerous tissue. The detection of these signatures is based on the extreme searching depth, high degree of dimensionality, and subcellular resolution capacity of TIS. These findings provide the technological rationale for the feasibility of a complete colon cancer toponome to be established by massive parallel high throughput/high content TIS mapping.

  8. Cross-bridge cycling gives rise to spatiotemporal heterogeneity of dynamic subcellular mechanics in cardiac myocytes probed with atomic force microscopy.

    PubMed

    Azeloglu, Evren U; Costa, Kevin D

    2010-03-01

    To study how the dynamic subcellular mechanical properties of the heart relate to the fundamental underlying process of actin-myosin cross-bridge cycling, we developed a novel atomic force microscope elastography technique for mapping spatiotemporal stiffness of isolated, spontaneously beating neonatal rat cardiomyocytes. Cells were indented repeatedly at a rate close but unequal to their contractile frequency. The resultant changes in pointwise apparent elastic modulus cycled at a predictable envelope frequency between a systolic value of 26.2 +/- 5.1 kPa and a diastolic value of 7.8 +/- 4.1 kPa at a representative depth of 400 nm. In cells probed along their major axis, spatiotemporal changes in systolic stiffness displayed a heterogeneous pattern, reflecting the banded sarcomeric structure of underlying myofibrils. Treatment with blebbistatin eliminated contractile activity and resulted in a uniform apparent modulus of 6.5 +/- 4.8 kPa. This study represents the first quantitative dynamic mechanical mapping of beating cardiomyocytes. The technique provides a means of probing the micromechanical effects of disease processes and pharmacological treatments on beating cardiomyocytes, providing new insights and relating subcellular cardiac structure and function.

  9. Imaging of Caenorhabditis elegans samples and sub-cellular localization of new generation photosensitizers for photodynamic therapy, using non-linear microscopy

    NASA Astrophysics Data System (ADS)

    Filippidis, G.; Kouloumentas, C.; Kapsokalyvas, D.; Voglis, G.; Tavernarakis, N.; Papazoglou, T. G.

    2005-08-01

    Two-photon excitation fluorescence (TPEF) and second-harmonic generation (SHG) are relatively new promising tools for the imaging and mapping of biological structures and processes at the microscopic level. The combination of the two image-contrast modes in a single instrument can provide unique and complementary information concerning the structure and the function of tissues and individual cells. The extended application of this novel, innovative technique by the biological community is limited due to the high price of commercial multiphoton microscopes. In this study, a compact, inexpensive and reliable setup utilizing femtosecond pulses for excitation was developed for the TPEF and SHG imaging of biological samples. Specific cell types of the nematode Caenorhabditis elegans were imaged. Detection of the endogenous structural proteins of the worm, which are responsible for observation of SHG signals, was achieved. Additionally, the binding of different photosensitizers in the HL-60 cell line was investigated, using non-linear microscopy. The sub-cellular localization of photosensitizers of a new generation, very promising for photodynamic therapy (PDT), (Hypericum perforatum L. extracts) was achieved. The sub-cellular localization of these novel photosensitizers was linked with their photodynamic action during PDT, and the possible mechanisms for cell killing have been elucidated.

  10. Discriminative Motif Finding for Predicting Protein Subcellular Localization

    PubMed Central

    Lin, Tien-ho; Murphy, Robert F.; Bar-Joseph, Ziv

    2010-01-01

    Many methods have been described to predict the subcellular location of proteins from sequence information. However, most of these methods either rely on global sequence properties or use a set of known protein targeting motifs to predict protein localization. Here we develop and test a novel method that identifies potential targeting motifs using a discriminative approach based on hidden Markov models (discriminative HMMs). These models search for motifs that are present in a compartment but absent in other, nearby, compartments by utilizing an hierarchical structure that mimics the protein sorting mechanism. We show that both discriminative motif finding and the hierarchical structure improves localization prediction on a benchmark dataset of yeast proteins. The motifs identified can be mapped to known targeting motifs and they are more conserved than the average protein sequence. Using our motif-based predictions we can identify potential annotation errors in public databases for the location of some of the proteins. A software implementation and the dataset described in this paper are available from http://murphylab.web.cmu.edu/software/2009_TCBB_motif/ PMID:21233524

  11. Apollo 17 Lunar Sounder Data provide Insight into Aitken Crater's Subsurface Structure

    NASA Technical Reports Server (NTRS)

    Cooper, Bonnie L.

    2007-01-01

    In preparation for the forthcoming avalanche of data from Lunar Reconnaissance Orbiter (LRO), we conducted a pilot study to demonstrate integration of multiple geophysical data sets. We applied methods of data integration that are used by the commercial mineral exploration industry to enhance the value of historical data sets and to provide a roadmap for future efforts.

  12. After-School All-Stars: Providing Structured Health and Physical Activity Programs in Urban Environments

    ERIC Educational Resources Information Center

    Thompson, Walter R.

    2009-01-01

    Physical education time has been reduced or even eliminated in middle and high schools in favor of more time for standardized test preparation, especially in urban schools and inner cities. One way to replace the time lost is by providing it after school as part of a comprehensive program. After-School All-Stars (ASAS) is such a program, networked…

  13. Predicting multisite protein subcellular locations: progress and challenges.

    PubMed

    Du, Pufeng; Xu, Chao

    2013-06-01

    In the last two decades, predicting protein subcellular locations has become a hot topic in bioinformatics. A number of algorithms and online services have been developed to computationally assign a subcellular location to a given protein sequence. With the progress of many proteome projects, more and more proteins are annotated with more than one subcellular location. However, multisite prediction has only been considered in a handful of recent studies, in which there are several common challenges. In this special report, the authors discuss what these challenges are, why these challenges are important and how the existing studies gave their solutions. Finally, a vision of the future of predicting multisite protein subcellular locations is given.

  14. Trimethyltin retinopathy: relationship of subcellular response to neuronal subspecialization

    SciTech Connect

    Bouldin, T.W.; Goines, N.D.; Krigman, M.R.

    1984-03-01

    Retinal neurons from rats acutely intoxicated with trimethyltin (TMT) were examined by light and electron microscopy to determine if there is a relationship between the subcellular response of a neuron to TMT and its morphologic subspecialization. Subcellular pathologic alterations were present in neurons from all three cellular layers of the sensory retina. However, the type and degree of subcellular response varied among the highly subspecialized neurons of the different retinal layers. Clusters of dense-cored vesicles and tubules were mainly limited to neurons of the ganglion-cell layer, large accumulations of dense bodies were mainly limited to neurons of the inner nuclear layer, and neuronal necrosis was mainly limited to the photoreceptor cells. The inner segment of the photoreceptor cell shared with the perikaryon of more conventional neurons a special vulnerability to TMT cytotoxicity. Our results suggest that the subspecialization of neurons affects the type and the degree of subcellular response to TMT.

  15. Structures of human SRP72 complexes provide insights into SRP RNA remodeling and ribosome interaction

    PubMed Central

    Becker, Matthias M. M.; Lapouge, Karine; Segnitz, Bernd; Wild, Klemens; Sinning, Irmgard

    2017-01-01

    Co-translational protein targeting and membrane protein insertion is a fundamental process and depends on the signal recognition particle (SRP). In mammals, SRP is composed of the SRP RNA crucial for SRP assembly and function and six proteins. The two largest proteins SRP68 and SRP72 form a heterodimer and bind to a regulatory site of the SRP RNA. Despite their essential roles in the SRP pathway, structural information has been available only for the SRP68 RNA-binding domain (RBD). Here we present the crystal structures of the SRP68 protein-binding domain (PBD) in complex with SRP72-PBD and of the SRP72-RBD bound to the SRP S domain (SRP RNA, SRP19 and SRP68) detailing all interactions of SRP72 within SRP. The SRP72-PBD is a tetratricopeptide repeat, which binds an extended linear motif of SRP68 with high affinity. The SRP72-RBD is a flexible peptide crawling along the 5e- and 5f-loops of SRP RNA. A conserved tryptophan inserts into the 5e-loop forming a novel type of RNA kink-turn stabilized by a potassium ion, which we define as K+-turn. In addition, SRP72-RBD remodels the 5f-loop involved in ribosome binding and visualizes SRP RNA plasticity. Docking of the S domain structure into cryo-electron microscopy density maps reveals multiple contact sites between SRP68/72 and the ribosome, and explains the role of SRP72 in the SRP pathway. PMID:27899666

  16. Crystal structure of a maltogenic amylase provides insights into a catalytic versatility.

    PubMed

    Kim, J S; Cha, S S; Kim, H J; Kim, T J; Ha, N C; Oh, S T; Cho, H S; Cho, M J; Kim, M J; Lee, H S; Kim, J W; Choi, K Y; Park, K H; Oh, B H

    1999-09-10

    Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. Compared with many different amylases that are able to hydrolyze only alpha-D-(1,4)-glycosidic bonds, maltogenic amylases exhibit catalytic versatility: hydrolysis of alpha-D-(1,4)- and alpha-D-(1,6)-glycosidic bonds and transglycosylation of oligosaccharides to C3-, C4-, or C6-hydroxyl groups of various acceptor mono- or disaccharides. It has been speculated that the catalytic property of the enzymes is linked to the additional approximately 130 residues at the N terminus that are absent in other typical alpha-amylases. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2.8 A. The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (alpha/beta)(8) barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical alpha-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transglycosylation and to allow binding of a branched oligosaccharide for hydrolysis of alpha-D-(1,4)-glycosidic or alpha-D-(1,6)-glycosidic bond. The (alpha/beta)(8) barrel of the enzyme is the preserved scaffold in all the known amylases. The structure represents a novel example of how an enzyme acquires a different substrate profile and a catalytic versatility from a common active site and represents a framework for explaining the catalytic activities of transglycosylation and hydrolysis of alpha-D-(1,6)-glycosidic bond.

  17. Extortion provides alternative routes to the evolution of cooperation in structured populations

    NASA Astrophysics Data System (ADS)

    Xu, Xiongrui; Rong, Zhihai; Wu, Zhi-Xi; Zhou, Tao; Tse, Chi Kong

    2017-05-01

    In this paper, we study the evolution of cooperation in structured populations (individuals are located on either a regular lattice or a scale-free network) in the context of repeated games by involving three types of strategies, namely, unconditional cooperation, unconditional defection, and extortion. The strategy updating of the players is ruled by the replicator-like dynamics. We find that extortion strategies can act as catalysts to promote the emergence of cooperation in structured populations via different mechanisms. Specifically, on regular lattice, extortioners behave as both a shield, which can enwrap cooperators inside and keep them away from defectors, and a spear, which can defeat those surrounding defectors with the help of the neighboring cooperators. Particularly, the enhancement of cooperation displays a resonance-like behavior, suggesting the existence of optimal extortion strength mostly favoring the evolution of cooperation, which is in good agreement with the predictions from the generalized mean-field approximation theory. On scale-free network, the hubs, who are likely occupied by extortioners or defectors at the very beginning, are then prone to be conquered by cooperators on small-degree nodes as time elapses, thus establishing a bottom-up mechanism for the emergence and maintenance of cooperation.

  18. Crystal structures of highly simplified BPTIs provide insights into hydration-driven increase of unfolding enthalpy

    PubMed Central

    Islam, Mohammad Monirul; Yohda, Masafumi; Kidokoro, Shun-ichi; Kuroda, Yutaka

    2017-01-01

    We report a thermodynamic and structural analysis of six extensively simplified bovine pancreatic trypsin inhibitor (BPTI) variants containing 19–24 alanines out of 58 residues. Differential scanning calorimetry indicated a two-state thermal unfolding, typical of a native protein with densely packed interior. Surprisingly, increasing the number of alanines induced enthalpy stabilization, which was however over-compensated by entropy destabilization. X-ray crystallography indicated that the alanine substitutions caused the recruitment of novel water molecules facilitating the formation of protein–water hydrogen bonds and improving the hydration shells around the alanine’s methyl groups, both of which presumably contributed to enthalpy stabilization. There was a strong correlation between the number of water molecules and the thermodynamic parameters. Overall, our results demonstrate that, in contrast to our initial expectation, a protein sequence in which over 40% of the residues are alanines can retain a densely packed structure and undergo thermal denaturation with a large enthalpy change, mainly contributed by hydration. PMID:28266637

  19. Structural characterization of encapsulated ferritin provides insight into iron storage in bacterial nanocompartments

    PubMed Central

    He, Didi; Hughes, Sam; Vanden-Hehir, Sally; Georgiev, Atanas; Altenbach, Kirsten; Tarrant, Emma; Mackay, C Logan; Waldron, Kevin J; Clarke, David J; Marles-Wright, Jon

    2016-01-01

    Ferritins are ubiquitous proteins that oxidise and store iron within a protein shell to protect cells from oxidative damage. We have characterized the structure and function of a new member of the ferritin superfamily that is sequestered within an encapsulin capsid. We show that this encapsulated ferritin (EncFtn) has two main alpha helices, which assemble in a metal dependent manner to form a ferroxidase center at a dimer interface. EncFtn adopts an open decameric structure that is topologically distinct from other ferritins. While EncFtn acts as a ferroxidase, it cannot mineralize iron. Conversely, the encapsulin shell associates with iron, but is not enzymatically active, and we demonstrate that EncFtn must be housed within the encapsulin for iron storage. This encapsulin nanocompartment is widely distributed in bacteria and archaea and represents a distinct class of iron storage system, where the oxidation and mineralization of iron are distributed between two proteins. DOI: http://dx.doi.org/10.7554/eLife.18972.001 PMID:27529188

  20. The picobirnavirus crystal structure provides functional insights into virion assembly and cell entry

    PubMed Central

    Duquerroy, Stéphane; Da Costa, Bruno; Henry, Céline; Vigouroux, Armelle; Libersou, Sonia; Lepault, Jean; Navaza, Jorge; Delmas, Bernard; Rey, Félix A

    2009-01-01

    Double-stranded (ds) RNA virus particles are organized around a central icosahedral core capsid made of 120 identical subunits. This core capsid is unable to invade cells from outside, and animal dsRNA viruses have acquired surrounding capsid layers that are used to deliver a transcriptionally active core particle across the membrane during cell entry. In contrast, dsRNA viruses infecting primitive eukaryotes have only a simple core capsid, and as a consequence are transmitted only vertically. Here, we report the 3.4 Å X-ray structure of a picobirnavirus—an animal dsRNA virus associated with diarrhoea and gastroenteritis in humans. The structure shows a simple core capsid with a distinctive icosahedral arrangement, displaying 60 two-fold symmetric dimers of a coat protein (CP) with a new 3D-fold. We show that, as many non-enveloped animal viruses, CP undergoes an autoproteolytic cleavage, releasing a post-translationally modified peptide that remains associated with nucleic acid within the capsid. Our data also show that picobirnavirus particles are capable of disrupting biological membranes in vitro, indicating that its simple 120-subunits capsid has evolved animal cell invasion properties. PMID:19407816

  1. Fish species introductions provide novel insights into the patterns and drivers of phylogenetic structure in freshwaters

    PubMed Central

    Strecker, Angela L.; Olden, Julian D.

    2014-01-01

    Despite long-standing interest of terrestrial ecologists, freshwater ecosystems are a fertile, yet unappreciated, testing ground for applying community phylogenetics to uncover mechanisms of species assembly. We quantify phylogenetic clustering and overdispersion of native and non-native fishes of a large river basin in the American Southwest to test for the mechanisms (environmental filtering versus competitive exclusion) and spatial scales influencing community structure. Contrary to expectations, non-native species were phylogenetically clustered and related to natural environmental conditions, whereas native species were not phylogenetically structured, likely reflecting human-related changes to the basin. The species that are most invasive (in terms of ecological impacts) tended to be the most phylogenetically divergent from natives across watersheds, but not within watersheds, supporting the hypothesis that Darwin's naturalization conundrum is driven by the spatial scale. Phylogenetic distinctiveness may facilitate non-native establishment at regional scales, but environmental filtering restricts local membership to closely related species with physiological tolerances for current environments. By contrast, native species may have been phylogenetically clustered in historical times, but species loss from contemporary populations by anthropogenic activities has likely shaped the phylogenetic signal. Our study implies that fundamental mechanisms of community assembly have changed, with fundamental consequences for the biogeography of both native and non-native species. PMID:24452027

  2. Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism.

    PubMed

    Keren-Kaplan, Tal; Zeev Peters, Lee; Levin-Kravets, Olga; Attali, Ilan; Kleifeld, Oded; Shohat, Noa; Artzi, Shay; Zucker, Ori; Pilzer, Inbar; Reis, Noa; Glickman, Michael H; Ben-Aroya, Shay; Prag, Gali

    2016-10-04

    Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors.

  3. Structural basis for interdomain communication in SHIP2 providing high phosphatase activity

    PubMed Central

    Le Coq, Johanne; Camacho-Artacho, Marta; Velázquez, José Vicente; Santiveri, Clara M; Gallego, Luis Heredia; Campos-Olivas, Ramón; Dölker, Nicole; Lietha, Daniel

    2017-01-01

    SH2-containing-inositol-5-phosphatases (SHIPs) dephosphorylate the 5-phosphate of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) and play important roles in regulating the PI3K/Akt pathway in physiology and disease. Aiming to uncover interdomain regulatory mechanisms in SHIP2, we determined crystal structures containing the 5-phosphatase and a proximal region adopting a C2 fold. This reveals an extensive interface between the two domains, which results in significant structural changes in the phosphatase domain. Both the phosphatase and C2 domains bind phosphatidylserine lipids, which likely helps to position the active site towards its substrate. Although located distant to the active site, the C2 domain greatly enhances catalytic turnover. Employing molecular dynamics, mutagenesis and cell biology, we identify two distinct allosteric signaling pathways, emanating from hydrophobic or polar interdomain interactions, differentially affecting lipid chain or headgroup moieties of PI(3,4,5)P3. Together, this study reveals details of multilayered C2-mediated effects important for SHIP2 activity and points towards interesting new possibilities for therapeutic interventions. DOI: http://dx.doi.org/10.7554/eLife.26640.001 PMID:28792888

  4. Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation

    PubMed Central

    Guillet, Valérie; Knibiehler, Martine; Gregory-Pauron, Lynn; Remy, Marie-Hélène; Chemin, Cécile; Raynaud-Messina, Brigitte; Bon, Cécile; Kollman, Justin M; Agard, David A; Merdes, Andreas; Mourey, Lionel

    2013-01-01

    Microtubule nucleation in all eukaryotes involves γ-tubulin small complexes (γTuSCs) that comprise two molecules of γ-tubulin bound to γ-tubulin complex proteins (GCPs) GCP2 and GCP3. In many eukaryotes, multiple γTuSCs associate with GCP4, GCP5 and GCP6 into large γ-tubulin ring complexes (γTuRCs). Recent cryo-EM studies indicate that a scaffold similar to γTuRCs is formed by lateral association of γTuSCs, with the C-terminal regions of GCP2 and GCP3 binding γ-tubulin molecules. However, the exact role of GCPs in microtubule nucleation remains unknown. Here we report the crystal structure of human GCP4 and show that its C-terminal domain binds directly to γ-tubulin. The human GCP4 structure is the prototype for all GCPs, as it can be precisely positioned within the γTuSC envelope, revealing the nature of protein-protein interactions and conformational changes regulating nucleation activity. PMID:21725292

  5. Der p 5 Crystal Structure Provides Insight into the Group 5 Dust Mite Allergens

    SciTech Connect

    Mueller, G.; Gosavi, R; Krahn, J; Edwards, L; Cuneo, M; Glesner, J; Pomes, A; Chapman, M; London, R; Pedersen, L

    2010-01-01

    Group 5 allergens from house dust mites elicit strong IgE antibody binding in mite-allergic patients. The structure of Der p 5 was determined by x-ray crystallography to better understand the IgE epitopes, to investigate the biologic function in mites, and to compare with the conflicting published Blo t 5 structures, designated 2JMH and 2JRK in the Protein Data Bank. Der p 5 is a three-helical bundle similar to Blo t 5, but the interactions of the helices are more similar to 2JMH than 2JRK. The crystallographic asymmetric unit contains three dimers of Der p 5 that are not exactly alike. Solution scattering techniques were used to assess the multimeric state of Der p 5 in vitro and showed that the predominant state was monomeric, similar to Blo t 5, but larger multimeric species are also present. In the crystal, the formation of the Der p 5 dimer creates a large hydrophobic cavity of {approx}3000 {angstrom}{sup 3} that could be a ligand-binding site. Many allergens are known to bind hydrophobic ligands, which are thought to stimulate the innate immune system and have adjuvant-like effects on IgE-mediated inflammatory responses.

  6. Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism

    PubMed Central

    Keren-Kaplan, Tal; Zeev Peters, Lee; Levin-Kravets, Olga; Attali, Ilan; Kleifeld, Oded; Shohat, Noa; Artzi, Shay; Zucker, Ori; Pilzer, Inbar; Reis, Noa; Glickman, Michael H.; Ben-Aroya, Shay; Prag, Gali

    2016-01-01

    Ubiquitin receptors decode ubiquitin signals into many cellular responses. Ubiquitin receptors also undergo coupled monoubiquitylation, and rapid deubiquitylation has hampered the characterization of the ubiquitylated state. Using bacteria that express a ubiquitylation apparatus, we purified and determined the crystal structure of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state. The structure shows a novel ubiquitin-binding patch that directs K84 ubiquitylation. Superimposition of ubiquitylated-Rpn10 onto electron-microscopy models of proteasomes indicates that the Rpn10-conjugated ubiquitin clashes with Rpn9, suggesting that ubiquitylation might be involved in releasing Rpn10 from the proteasome. Indeed, ubiquitylation on immobilized proteasomes dissociates the modified Rpn10 from the complex, while unmodified Rpn10 mainly remains associated. In vivo experiments indicate that contrary to wild type, Rpn10-K84R is stably associated with the proteasomal subunit Rpn9. Similarly Rpn10, but not ubiquitylated-Rpn10, binds Rpn9 in vitro. Thus we suggest that ubiquitylation functions to dissociate modified ubiquitin receptors from their targets, a function that promotes cyclic activity of ubiquitin receptors. PMID:27698474

  7. Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

    PubMed Central

    Jin, Lei; Le, Thi Tuc Nghi; Cheng, Luisa W.; Strotmeier, Jasmin; Kruel, Anna Magdalena; Yao, Guorui; Perry, Kay; Rummel, Andreas; Jin, Rongsheng

    2013-01-01

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry. PMID:24130488

  8. Permeation properties of the hair cell mechanotransducer channel provide insight into its molecular structure

    PubMed Central

    Pan, B.; Waguespack, J.; Schnee, M. E.; LeBlanc, C.

    2012-01-01

    Mechanoelectric transducer (MET) channels, located near stereocilia tips, are opened by deflecting the hair bundle of sensory hair cells. Defects in this process result in deafness. Despite this critical function, the molecular identity of MET channels remains a mystery. Inherent channel properties, particularly those associated with permeation, provide the backbone for the molecular identification of ion channels. Here, a novel channel rectification mechanism is identified, resulting in a reduced pore size at positive potentials. The apparent difference in pore dimensions results from Ca2+ binding within the pore, occluding permeation. Driving force for permeation at hyperpolarized potentials is increased because Ca2+ can more easily be removed from binding within the pore due to the presence of an electronegative external vestibule that dehydrates and concentrates permeating ions. Alterations in Ca2+ binding may underlie tonotopic and Ca2+-dependent variations in channel conductance. This Ca2+-dependent rectification provides targets for identifying the molecular components of the MET channel. PMID:22323630

  9. Subcellular analysis by laser ablation electrospray ionization mass spectrometry

    DOEpatents

    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.

  10. The structure of C290A:C393A Aurora A provides structural insights into kinase regulation

    PubMed Central

    Burgess, Selena G.; Bayliss, Richard

    2015-01-01

    Aurora A is a Ser/Thr protein kinase that functions in cell-cycle regulation and is implicated in cancer development. During mitosis, Aurora A is activated by autophosphorylation on its activation loop at Thr288. The Aurora A catalytic domain (amino acids 122–403) expressed in Escherichia coli autophosphorylates on two activation-loop threonine residues (Thr288 and Thr287), whereas a C290A,C393A double point mutant of the Aurora A catalytic domain autophosphorylates only on Thr288. The structure of the complex of this mutant with ADP and magnesium was determined to 2.1 Å resolution using molecular replacement. This is an improvement on the existing 2.75 Å resolution structure of the equivalent wild-type complex. The structure confirms that single phosphorylation of the activation loop on Thr288 is insufficient to stabilize a ‘fully active’ conformation of the activation loop in the absence of binding to TPX2. PMID:25760707

  11. Synthesis and structural characterization of soluble neuromelanin analogs provides important clues to its biosynthesis.

    PubMed

    Ferrari, Emanuele; Engelen, Mireille; Monzani, Enrico; Sturini, Michela; Girotto, Stefania; Bubacco, Luigi; Zecca, Luigi; Casella, Luigi

    2013-01-01

    Elucidating the structure and biosynthesis of neuromelanin (NM) would be an important step towards understanding its putative role in the pathogenesis of Parkinson's disease. A useful complement to studies aimed at unraveling the origin and properties of this essentially insoluble natural substance is the preparation of synthetic derivatives that resemble NM. With this aim in mind, water-soluble conjugates between dopamine-derived melanin and bovine serum albumin (BSA) were synthesized. Melanin-BSA adducts were prepared with both eumelanic oligomers obtained through the oxidative polymerization of dopamine and pheomelanic oligomers obtained under the same conditions from dopamine and cysteine. Iron ions were added during the synthesis to understand the interaction between the pigment and this metal ion, as the NM in neurons in several human brain regions contains significant amounts of iron. The structures of the conjugates were analyzed by (1)H NMR spectroscopy and controlled proteolysis/MS experiments. The binding of iron(III) ions was evaluated by ICP analysis and EPR spectroscopy. The EPR signal from bound iron(III) indicated high-spin octahedral sites and, as also seen for NM, the signal is coupled to a signal from a radical associated with the melanic components of the conjugates. However, the intensity of the EPR signal from iron suggested a reduced fraction of the total iron, indicating that most of the iron is strongly coupled in clusters within the matrix. The amount of paramagnetic, mononuclear iron(III) was greater in the pheomelanin-BSA conjugates, suggesting that iron clustering is reduced in the sulfur-containing pigment. Thus, the melanin-BSA conjugates appear to be good models for the natural pigment.

  12. Conformational Variations of Both Phosphodiesterase-5 and Inhibitors Provide the Structural Basis for the Physiological Effects of Vardenafil and Sildenafil

    SciTech Connect

    Wang, H.; Ye, M; Robinson, H; Fransis, S; Ke, H

    2007-01-01

    Vardenafil has higher affinity to phosphodiesterase-5 (PDE5) than sildenafil and lower administered dosage for the treatment of erectile dysfunction. However, the molecular basis for these differences is puzzling because two drugs have similar chemical structures. Reported here is a crystal structure of the fully active and nonmutated PDE5A1 catalytic domain in complex with vardenafil. The structure shows that the conformation of the H-loop in the PDE5A1-vardenafil complex is different from those of any known structures of the unliganded PDE5 and its complexes with the inhibitors. In addition, the molecular configuration of vardenafil differs from that of sildenafil when bound to PDE5. It is noteworthy that the binding of vardenafil causes loss of the divalent metal ions that have been observed in all the previously published PDE structures. The conformational variation of both PDE5 and the inhibitors provides structural insight into the different potencies of the drugs.

  13. Conformational Variations of Both Phosphodiesterase-5 and Inhibitors Provide the Structural Basis for the Physiological Effects of Verdenafil and Sildenafil

    SciTech Connect

    Wang,H.; Ye, M.; Robinson, H.; Francis, S.; Ke, H.

    2008-01-01

    Vardenafil has higher affinity to phosphodiesterase-5 (PDE5) than sildenafil and lower administered dosage for the treatment of erectile dysfunction. However, the molecular basis for these differences is puzzling because two drugs have similar chemical structures. Reported here is a crystal structure of the fully active and nonmutated PDE5A1 catalytic domain in complex with vardenafil. The structure shows that the conformation of the H-loop in the PDE5A1-vardenafil complex is different from those of any known structures of the unliganded PDE5 and its complexes with the inhibitors. In addition, the molecular configuration of vardenafil differs from that of sildenafil when bound to PDE5. It is noteworthy that the binding of vardenafil causes loss of the divalent metal ions that have been observed in all the previously published PDE structures. The conformational variation of both PDE5 and the inhibitors provides structural insight into the different potencies of the drugs.

  14. Direct Imaging of Single Cells and Tissue at Subcellular Spatial Resolution Using Transmission Geometry MALDI MS

    PubMed Central

    Zavalin, Andre; Todd, Erik M.; Rawhouser, Patrick D.; Yang, Junhai; Norris, Jeremy L.; Caprioli, Richard M.

    2012-01-01

    The need of cellular and sub-cellular spatial resolution in LDI / MALDI Imaging Mass Spectrometry (IMS) necessitates micron and sub-micron laser spot sizes at biologically relevant sensitivities, introducing significant challenges for MS technology. To this end we have developed a transmission geometry vacuum ion source that allows the laser beam to irradiate the back side of the sample. This arrangement obviates the mechanical / ion optic complications in the source by completely separating the optical lens and ion optic structures. We have experimentally demonstrated the viability of transmission geometry MALDI MS for imaging biological tissues and cells with sub-cellular spatial resolution. Furthermore, we demonstrate that in conjunction with new sample preparation protocols, the sensitivity of this instrument is sufficient to obtain molecular images at sub-micron spatial resolution. PMID:23147833

  15. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells

    PubMed Central

    1980-01-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates. PMID:7372714

  16. Subcellular compartmentalization of saccharide moieties in cultured normal and malignant cells.

    PubMed

    Virtanen, I; Ekblom, P; Laurila, P

    1980-05-01

    We studied subcellular localization of saccharide moieties in cultured normal and malignant cells fixed in paraformaldehyde and treated with a nonionic detergent, using lectins specific for various surgar residues as probes in fluorescence microscopy. In normal cells, concanavalin A and Lens culinaris agglutinin, specific for mannose-rich carbohydrate cores in glycoproteins, labeled the endoplasmic reticulum as a wide perinuclear region. Other lectins, on the other hand, stained the Golgi apparatus as a juxtanuclear reticular structure. A similar compartmentalization was also seen in all malignant cells studied, although the Golgi apparatus in these cells was distinctly vesicular in appearance. Our results indicate that saccharide moieties in both normal and malignant cells are similarly compartmentalized, and thus speak in favor of a unidirectional subcellular flow for both membrane and secreted glycoconjugates.

  17. The influence of care provider access to structural empowerment on individualized care in long-term-care facilities.

    PubMed

    Caspar, Sienna; O'Rourke, Norm

    2008-07-01

    Implementing management initiatives that enable formal caregivers to provide quality, individualized care to older adults in long-term-care (LTC) facilities is increasingly important given that the number of LTC residents is projected to triple by 2031. The objective of this study was to explore the relationship between care provider access to structural empowerment and the provision of individualized care in LTC. We computed structural equation models separately for registered nurses and licensed practical nurses (n = 242) and care aides (n = 326) to examine the relationship between access to empowerment structures (i.e., informal power, formal power, information, support, resources, opportunity) and the provision of individualized care. We subsequently undertook invariance analyses to determine if the association between empowerment structures and reported provision of individualized care differed between caregiver groups. Access to structural empowerment had a statistically significant, positive association with provision of individualized care for both groups. For registered nurses/licensed practical nurses and care aides, empowerment explained 50% and 45% of observed variance in individualized care, respectively. These notable percentages did not differ significantly between caregiver groups. Of the empowerment structures, support, especially in the form of access to educational opportunities and recognition for a job well done, seems to be particularly significant to care providers. Findings from this study suggest that provision of individualized care in LTC may be enhanced when formal caregivers have appreciable access to empowerment structures.

  18. Cloning of human PEX cDNA. Expression, subcellular localization, and endopeptidase activity.

    PubMed

    Lipman, M L; Panda, D; Bennett, H P; Henderson, J E; Shane, E; Shen, Y; Goltzman, D; Karaplis, A C

    1998-05-29

    Mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets. To gain insight into the role of PEX in normal physiology we have cloned the human full-length cDNA and studied its tissue expression, subcellular localization, and peptidase activity. We show that the cDNA encodes a 749-amino acid protein structurally related to a family of neutral endopeptidases that include neprilysin as prototype. By Northern blot analysis, the size of the full-length PEX transcript is 6.5 kilobases. PEX expression, as determined by semi-quantitative polymerase chain reaction, is high in bone and in tumor tissue associated with the paraneoplastic syndrome of renal phosphate wasting. PEX is glycosylated in the presence of canine microsomal membranes and partitions exclusively in the detergent phase from Triton X-114 extractions of transiently transfected COS cells. Immunofluorescence studies in A293 cells expressing PEX tagged with a c-myc epitope show a predominant cell-surface location for the protein with its COOH-terminal domain in the extracellular compartment, substantiating the assumption that PEX, like other members of the neutral endopeptidase family, is a type II integral membrane glycoprotein. Cell membranes from cultured COS cells transiently expressing PEX efficiently degrade exogenously added parathyroid hormone-derived peptides, demonstrating for the first time that recombinant PEX can function as an endopeptidase. PEX peptidase activity may provide a convenient target for pharmacological intervention in states of altered phosphate homeostasis and in metabolic bone diseases.

  19. Nanosecond pulsed electric field generators for the study of subcellular effects.

    PubMed

    Kolb, Juergen F; Kono, Susumu; Schoenbach, Karl H

    2006-04-01

    Modeling and experimental studies have shown that pulsed electric fields of nanosecond duration and megavolt per meter amplitude affect subcellular structures but do not lead to the formation of large pores in the outer membrane. This "intracellular electromanipulation" requires the use of pulse generators which provide extremely high power but low energy pulses. In this study, we describe the concept of the required pulsed power sources, their design, operation, and the necessary diagnostics. Two types of pulse generators based on the Blumlein line principle have been developed and are described here. One system is designed to treat a large number of cells in cuvettes holding volumes from 0.1 to 0.8 ml. Pulses of up to 40 kV amplitude, with a duration of 10 ns and a rise time close to 1 ns can be applied to the cuvette. For an electrode gap of 1 mm this voltage corresponds to an average electric field of 40 MV/m. The second system allows for real time observation of individual cells under a microscope. It generates pulses of 10-300 ns duration with a rise time of 3.5 ns and voltage amplitudes up to 1 kV. Connected to a microreactor with an electrode gap of 100 microm, electric fields up to 10 MV/m are applied.

  20. Rapid Morphological Change in the Masticatory Structures of an Important Ecosystem Service Provider

    PubMed Central

    Doudna, John W.; Danielson, Brent J.

    2015-01-01

    Humans have altered the biotic and abiotic environmental conditions of most organisms. In some cases, such as intensive agriculture, an organism’s entire ecosystem is converted to novel conditions. Thus, it is striking that some species continue to thrive under such conditions. The prairie deer mouse (Peromyscus maniculatus bairdii) is an example of such an organism, and so we sought to understand what role evolutionary adaptation played in the success of this species, with particular interest in adaptations to novel foods. In order to understand the evolutionary history of this species’ masticatory structures, we examined the maxilla, zygomatic plate, and mandible of historic specimens collected prior to 1910 to specimens collected in 2012 and 2013. We found that mandibles, zygomatic plates, and maxilla have all changed significantly since 1910, and that morphological development has shifted significantly. We present compelling evidence that these differences are due to natural selection as a response to a novel and ubiquitous food source, waste grain (corn, Zea mays and soybean, Glycine max). PMID:26061880

  1. Rapid Morphological Change in the Masticatory Structures of an Important Ecosystem Service Provider.

    PubMed

    Doudna, John W; Danielson, Brent J

    2015-01-01

    Humans have altered the biotic and abiotic environmental conditions of most organisms. In some cases, such as intensive agriculture, an organism's entire ecosystem is converted to novel conditions. Thus, it is striking that some species continue to thrive under such conditions. The prairie deer mouse (Peromyscus maniculatus bairdii) is an example of such an organism, and so we sought to understand what role evolutionary adaptation played in the success of this species, with particular interest in adaptations to novel foods. In order to understand the evolutionary history of this species' masticatory structures, we examined the maxilla, zygomatic plate, and mandible of historic specimens collected prior to 1910 to specimens collected in 2012 and 2013. We found that mandibles, zygomatic plates, and maxilla have all changed significantly since 1910, and that morphological development has shifted significantly. We present compelling evidence that these differences are due to natural selection as a response to a novel and ubiquitous food source, waste grain (corn, Zea mays and soybean, Glycine max).

  2. Catalysis and Structure of Zebrafish Urate Oxidase Provide Insights into the Origin of Hyperuricemia in Hominoids

    PubMed Central

    Marchetti, Marialaura; Liuzzi, Anastasia; Fermi, Beatrice; Corsini, Romina; Folli, Claudia; Speranzini, Valentina; Gandolfi, Francesco; Bettati, Stefano; Ronda, Luca; Cendron, Laura; Berni, Rodolfo; Zanotti, Giuseppe; Percudani, Riccardo

    2016-01-01

    Urate oxidase (Uox) catalyses the first reaction of oxidative uricolysis, a three-step enzymatic pathway that allows some animals to eliminate purine nitrogen through a water-soluble compound. Inactivation of the pathway in hominoids leads to elevated levels of sparingly soluble urate and puts humans at risk of hyperuricemia and gout. The uricolytic activities lost during evolution can be replaced by enzyme therapy. Here we report on the functional and structural characterization of Uox from zebrafish and the effects on the enzyme of the missense mutation (F216S) that preceded Uox pseudogenization in hominoids. Using a kinetic assay based on the enzymatic suppression of the spectroscopic interference of the Uox reaction product, we found that the F216S mutant has the same turnover number of the wild-type enzyme but a much-reduced affinity for the urate substrate and xanthine inhibitor. Our results indicate that the last functioning Uox in hominoid evolution had an increased Michaelis constant, possibly near to upper end of the normal range of urate in the human serum (~300 μM). Changes in the renal handling of urate during primate evolution can explain the genetic modification of uricolytic activities in the hominoid lineage without the need of assuming fixation of deleterious mutations. PMID:27922051

  3. Submicron structures provide preferential spots for carbon and nitrogen sequestration in soils

    PubMed Central

    Vogel, Cordula; Mueller, Carsten W.; Höschen, Carmen; Buegger, Franz; Heister, Katja; Schulz, Stefanie; Schloter, Michael; Kögel-Knabner, Ingrid

    2014-01-01

    The sequestration of carbon and nitrogen by clay-sized particles in soils is well established, and clay content or mineral surface area has been used to estimate the sequestration potential of soils. Here, via incubation of a sieved (<2 mm) topsoil with labelled litter, we find that only some of the clay-sized surfaces bind organic matter (OM). Surprisingly, <19% of the visible mineral areas show an OM attachment. OM is preferentially associated with organo-mineral clusters with rough surfaces. By combining nano-scale secondary ion mass spectrometry and isotopic tracing, we distinguish between new labelled and pre-existing OM and show that new OM is preferentially attached to already present organo-mineral clusters. These results, which provide evidence that only a limited proportion of the clay-sized surfaces contribute to OM sequestration, revolutionize our view of carbon sequestration in soils and the widely used carbon saturation estimates. PMID:24399306

  4. The structure of a food product assortment modulates the effect of providing choice on food intake.

    PubMed

    Parizel, Odile; Sulmont-Rossé, Claire; Fromentin, Gilles; Delarue, Julien; Labouré, Hélène; Benamouzig, Robert; Marsset-Baglieri, Agnès

    2016-09-01

    Several authors showed that providing choice may increase food liking and food intake. However, the impact of choice may be modulated by assortment's characteristics, such as the number of alternatives or their dissimilarity. The present study compared the impact of choice on food liking and intake under the two following conditions: (1) when choosing a product to consume from among similar products versus dissimilar products; and (2) when choosing a product to consume from among pleasant products versus unpleasant products. Two experiments were carried out using the same design: the "apple puree" experiment (n = 80), where the volunteers choose from among similar products (apple purees varying in texture) and the "dessert" experiment (n = 80), where the volunteers choose from among dissimilar products (fruit dessert, dairy dessert, custard, pudding). During the first session, participants rated their liking for 12 products (apples purees or desserts). Then the participants were divided into a "pleasant" group (n = 40) in which volunteers were assigned three pleasant products, and an "unpleasant" group (n = 40) in which volunteers were assigned three unpleasant products. Finally, all of the volunteers participated in a choice session - volunteers were presented with their three assigned products and asked to choose one of the products, and a no-choice session - volunteers were served with one product that was randomly selected from among their three assigned products. Providing choice led to an increase in food liking in both experiments and an increase in food intake only for the desserts, namely only when the volunteers chose the product to consume from among "not too similar" alternatives. No effect of assortment's pleasantness was observed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Subcellular and intranuclear localization of neptunium-237 (V) in rat liver.

    PubMed

    Paquet, F; Verry, M; Grillon, G; Landesman, C; Masse, R; Taylor, D M

    1995-08-01

    The present investigation was aimed at establishing the distribution of 237Np within the different structures of hepatocytes. Rats were contaminated experimentally by intravenous injection of 237Np (V) and the subcellular structures of the liver were separated by ultracentrifugation. Twenty-four hours after contamination, the nuclear and cytosolic fractions bound 54 and 32%, respectively, of the total radionuclide. Purification of the nuclei followed by dissociation of the protein components in medium of increasing ionic strength showed a specific binding of neptunium to the structural proteins of the nuclear matrix.

  6. Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass

    PubMed Central

    Chai, Kian Piaw; Othman, Noor Farhan Binti; Teh, Aik-Hong; Ho, Kok Lian; Chan, Kok-Gan; Shamsir, Mohd Shahir; Goh, Kian Mau; Ng, Chyan Leong

    2016-01-01

    A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca2+ ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca2+ ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite. PMID:26975884

  7. Crystal structure of Anoxybacillus α-amylase provides insights into maltose binding of a new glycosyl hydrolase subclass.

    PubMed

    Chai, Kian Piaw; Othman, Noor Farhan Binti; Teh, Aik-Hong; Ho, Kok Lian; Chan, Kok-Gan; Shamsir, Mohd Shahir; Goh, Kian Mau; Ng, Chyan Leong

    2016-03-15

    A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo, and GtamyII), Bacillus aquimaris (BaqA), and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca(2+) ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca(2+) ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.

  8. 'Unite and conquer': enhanced prediction of protein subcellular localization by integrating multiple specialized tools

    PubMed Central

    Shen, Yao Qing; Burger, Gertraud

    2007-01-01

    Background Knowing the subcellular location of proteins provides clues to their function as well as the interconnectivity of biological processes. Dozens of tools are available for predicting protein location in the eukaryotic cell. Each tool performs well on certain data sets, but their predictions often disagree for a given protein. Since the individual tools each have particular strengths, we set out to integrate them in a way that optimally exploits their potential. The method we present here is applicable to various subcellular locations, but tailored for predicting whether or not a protein is localized in mitochondria. Knowledge of the mitochondrial proteome is relevant to understanding the role of this organelle in global cellular processes. Results In order to develop a method for enhanced prediction of subcellular localization, we integrated the outputs of available localization prediction tools by several strategies, and tested the performance of each strategy with known mitochondrial proteins. The accuracy obtained (up to 92%) surpasses by far the individual tools. The method of integration proved crucial to the performance. For the prediction of mitochondrion-located proteins, integration via a two-layer decision tree clearly outperforms simpler methods, as it allows emphasis of biologically relevant features such as the mitochondrial targeting peptide and transmembrane domains. Conclusion We developed an approach that enhances the prediction accuracy of mitochondrial proteins by uniting the strength of specialized tools. The combination of machine-learning based integration with biological expert knowledge leads to improved performance. This approach also alleviates the conundrum of how to choose between conflicting predictions. Our approach is easy to implement, and applicable to predicting subcellular locations other than mitochondria, as well as other biological features. For a trial of our approach, we provide a webservice for mitochondrial protein

  9. Xylans Provide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat.

    PubMed

    Ralet, Marie-Christine; Crépeau, Marie-Jeanne; Vigouroux, Jacqueline; Tran, Joseph; Berger, Adeline; Sallé, Christine; Granier, Fabienne; Botran, Lucy; North, Helen M

    2016-05-01

    Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells produce large amounts of mucilage that is released upon imbibition. This mucilage is structured into two domains: an outer diffuse layer that can be easily removed by agitation and an inner layer that remains attached to the outer seed coat. Both layers are composed primarily of pectic rhamnogalacturonan I (RG-I), the inner layer also containing rays of cellulose that extend from the top of each columella. Perturbation in cellulosic ray formation has systematically been associated with a redistribution of pectic mucilage from the inner to the outer layer, in agreement with cellulose-pectin interactions, the nature of which remained unknown. Here, by analyzing the outer layer composition of a series of mutant alleles, a tight proportionality of xylose, galacturonic acid, and rhamnose was evidenced, except for mucilage modified5-1 (mum5-1; a mutant showing a redistribution of mucilage pectin from the inner adherent layer to the outer soluble one), for which the rhamnose-xylose ratio was increased drastically. Biochemical and in vitro binding assay data demonstrated that xylan chains are attached to RG-I chains and mediate the adsorption of mucilage to cellulose microfibrils. mum5-1 mucilage exhibited very weak adsorption to cellulose. MUM5 was identified as a putative xylosyl transferase recently characterized as MUCI21. Together, these findings suggest that the binding affinity of xylose ramifications on RG-I to a cellulose scaffold is one of the factors involved in the formation of the adherent mucilage layer. © 2016 American Society of Plant Biologists. All Rights Reserved.

  10. Xylans Provide the Structural Driving Force for Mucilage Adhesion to the Arabidopsis Seed Coat1

    PubMed Central

    Crépeau, Marie-Jeanne; Vigouroux, Jacqueline; Berger, Adeline; Sallé, Christine; Botran, Lucy

    2016-01-01

    Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells produce large amounts of mucilage that is released upon imbibition. This mucilage is structured into two domains: an outer diffuse layer that can be easily removed by agitation and an inner layer that remains attached to the outer seed coat. Both layers are composed primarily of pectic rhamnogalacturonan I (RG-I), the inner layer also containing rays of cellulose that extend from the top of each columella. Perturbation in cellulosic ray formation has systematically been associated with a redistribution of pectic mucilage from the inner to the outer layer, in agreement with cellulose-pectin interactions, the nature of which remained unknown. Here, by analyzing the outer layer composition of a series of mutant alleles, a tight proportionality of xylose, galacturonic acid, and rhamnose was evidenced, except for mucilage modified5-1 (mum5-1; a mutant showing a redistribution of mucilage pectin from the inner adherent layer to the outer soluble one), for which the rhamnose-xylose ratio was increased drastically. Biochemical and in vitro binding assay data demonstrated that xylan chains are attached to RG-I chains and mediate the adsorption of mucilage to cellulose microfibrils. mum5-1 mucilage exhibited very weak adsorption to cellulose. MUM5 was identified as a putative xylosyl transferase recently characterized as MUCI21. Together, these findings suggest that the binding affinity of xylose ramifications on RG-I to a cellulose scaffold is one of the factors involved in the formation of the adherent mucilage layer. PMID:26979331

  11. Structures of Metal-Substituted Human Histone Deacetylase 8 Provide Mechanistic Inferences on Biological Function

    SciTech Connect

    Dowling, Daniel P.; Gattis, Samuel G.; Fierke, Carol A.; Christianson, David W.

    2010-08-23

    The metal-dependent histone deacetylases (HDACs) adopt an {alpha}/{beta} protein fold first identified in rat liver arginase. Despite insignificant overall amino acid sequence identity, these enzymes share a strictly conserved metal binding site with divergent metal specificity and stoichiometry. HDAC8, originally thought to be a Zn{sup 2+}-metallohydrolase, exhibits increased activity with Co{sup 2+} and Fe{sup 2+} cofactors based on k{sub cat}/K{sub M} (Gantt, S. L., Gattis, S. G., and Fierke, C. A. (2006) Biochemistry 45, 6170-6178). Here, we report the first X-ray crystal structures of metallo-substituted HDAC8, Co{sup 2+}-HDAC8, D101L Co{sup 2+}-HDAC8, D101L Mn{sup 2+}-HDAC8, and D101L Fe{sup 2+}-HDAC8, each complexed with the inhibitor M344. Metal content of protein samples in solution is confirmed by inductively coupled plasma mass spectrometry. For the crystalline enzymes, peaks in Bijvoet difference Fourier maps calculated from X-ray diffraction data collected near the respective elemental absorption edges confirm metal substitution. Additional solution studies confirm incorporation of Cu{sup 2+}; Fe{sup 3+} and Ni{sup 2+} do not bind under conditions tested. The metal dependence of the substrate K{sub M} values and the K{sub i} values of hydroxamate inhibitors that chelate the active site metal are consistent with substrate-metal coordination in the precatalytic Michaelis complex that enhances catalysis. Additionally, although HDAC8 binds Zn{sup 2+} nearly 106-fold more tightly than Fe{sup 2+}, the affinities for both metal ions are comparable to the readily exchangeable metal concentrations estimated in living cells, suggesting that HDAC8 could bind either or both Fe{sup 2+} or Zn{sup 2+} in vivo.

  12. Inter-basin movements of Mediterranean sperm whales provide insight into their population structure and conservation

    NASA Astrophysics Data System (ADS)

    Frantzis, A.; Airoldi, S.; Notarbartolo-di-Sciara, G.; Johnson, C.; Mazzariol, S.

    2011-04-01

    The sperm whale is one of the very few deep diving mammal species in the Mediterranean Sea. Following a rare mass stranding of male sperm whales in the Adriatic Sea in December 2009, photo-identification methods were used in order to investigate previous sightings of the stranded whales in the region. Fluke photos of the stranded whales were compared with those of 153 and 128 free-ranging individuals photographed in the western and eastern Mediterranean basins, respectively. Three out of the seven stranded whales had been previously photo-identified and some of them more than once. To reach the stranding place, two of these re-identified whales performed long-range inter-basin movements of about 1600-2100 km (in a straight line) either through the Strait of Sicily or the Strait of Messina. In addition, comparisons among all whales photographed in the two Mediterranean basins revealed that one more individual first photographed in the western basin (1991) was re-identified 13 years later in the eastern basin (2004). These three cases provide the first conclusive evidence of inter-basin movement of sperm whales in the Mediterranean Sea. Inter-basin gene flow is important for the survival of the small and endangered Mediterranean sperm whale population. Mitigating the disturbance created by human activities in the straits area is crucial for its conservation.

  13. Genetic Structure of Water Chestnut Beetle: Providing Evidence for Origin of Water Chestnut.

    PubMed

    Tang, Xiao-Tian; Zheng, Fu-Shan; Qin, Jing; Lu, Ming-Xing; Du, Yu-Zhou

    2016-01-01

    Water chestnut beetle (Galerucella birmanica Jacoby) is a pest of the water chestnut (Trapa natans L.). To analyze the phylogeny and biogeography of the beetle and provide evidence for the origin of T. natans in China, we conducted this by using three mitochondrial genes (COI, COII and Cytb) and nuclear ITS2 ribosomal DNA of G. birmanica. As for mtDNA genes, the beetle could be subdivided into three groups: northeastern China (NEC), central-northern-southern China (CC-NC-SC) and southwestern China (SWC) based on SAMOVA, phylogenetic analyses and haplotype networks. But for ITS2, no obvious lineages were obtained but individuals which were from NEC region clustered into one clade, which might be due to sequence conservation of ITS2. Significant genetic variation was observed among the three groups with infrequent gene flow between groups, which may have been restricted due to natural barriers and events in the Late Pleistocene. Based on our analyses of genetic variation in the CC-NC-SC geographical region, the star-like haplotype networks, approximate Bayesian computation, niche modelling and phylogeographic variation of the beetle, we concluded that the beetle population has been lasting in the lower, central reaches of the Yangtze River Basin with its host plant, water chestnut, which is consistent with archaeological records. Moreover, we speculate that the CC-NC-SC population of G. birmanica may have undergone a period of expansion coincident with domestication of the water chestnut approximately 113,900-126,500 years ago.

  14. Pulse energy dependence of subcellular dissection by femtosecond laser pulses

    NASA Technical Reports Server (NTRS)

    Heisterkamp, A.; Maxwell, I. Z.; Mazur, E.; Underwood, J. M.; Nickerson, J. A.; Kumar, S.; Ingber, D. E.

    2005-01-01

    Precise dissection of cells with ultrashort laser pulses requires a clear understanding of how the onset and extent of ablation (i.e., the removal of material) depends on pulse energy. We carried out a systematic study of the energy dependence of the plasma-mediated ablation of fluorescently-labeled subcellular structures in the cytoskeleton and nuclei of fixed endothelial cells using femtosecond, near-infrared laser pulses focused through a high-numerical aperture objective lens (1.4 NA). We find that the energy threshold for photobleaching lies between 0.9 and 1.7 nJ. By comparing the changes in fluorescence with the actual material loss determined by electron microscopy, we find that the threshold for true material ablation is about 20% higher than the photobleaching threshold. This information makes it possible to use the fluorescence to determine the onset of true material ablation without resorting to electron microscopy. We confirm the precision of this technique by severing a single microtubule without disrupting the neighboring microtubules, less than 1 micrometer away. c2005 Optical Society of America.

  15. Imaging subcellular features of a sectioned rat brain using time-of-flight secondary ion mass spectrometry and scanning probe microscopy

    NASA Astrophysics Data System (ADS)

    Nie, H.-Y.; Francis, J. T.; Taylor, A. R.; Walzak, M. J.; Chang, W. H.; MacFabe, D. F.; Lau, W. M.

    2008-12-01

    Coronal sections of unfixed rat brain samples were prepared on a flat substrate in order to reveal hippocampal formation (CA1-4 pyramidal neurons) and adjacent neocortical white matter. We demonstrate the feasibility of using surface sensitive techniques such as time-of-flight secondary ion mass spectrometry (ToF-SIMS) and scanning probe microscopy (SPM) to probe lipid distribution, as well as the subcellular features of neurons. In the same anatomical areas, the phase shift image in SPM is especially useful in revealing the cross-section of subcellular structures. We show that the phase shift images reveal distinctive subcellular features and ion images of CN - and PO 2- fragments from ToF-SIMS appear to define some of the subcellular features.

  16. Genetic Structure of Water Chestnut Beetle: Providing Evidence for Origin of Water Chestnut

    PubMed Central

    Qin, Jing; Lu, Ming-Xing; Du, Yu-Zhou

    2016-01-01

    Water chestnut beetle (Galerucella birmanica Jacoby) is a pest of the water chestnut (Trapa natans L.). To analyze the phylogeny and biogeography of the beetle and provide evidence for the origin of T. natans in China, we conducted this by using three mitochondrial genes (COI, COII and Cytb) and nuclear ITS2 ribosomal DNA of G. birmanica. As for mtDNA genes, the beetle could be subdivided into three groups: northeastern China (NEC), central-northern-southern China (CC-NC-SC) and southwestern China (SWC) based on SAMOVA, phylogenetic analyses and haplotype networks. But for ITS2, no obvious lineages were obtained but individuals which were from NEC region clustered into one clade, which might be due to sequence conservation of ITS2. Significant genetic variation was observed among the three groups with infrequent gene flow between groups, which may have been restricted due to natural barriers and events in the Late Pleistocene. Based on our analyses of genetic variation in the CC-NC-SC geographical region, the star-like haplotype networks, approximate Bayesian computation, niche modelling and phylogeographic variation of the beetle, we concluded that the beetle population has been lasting in the lower, central reaches of the Yangtze River Basin with its host plant, water chestnut, which is consistent with archaeological records. Moreover, we speculate that the CC-NC-SC population of G. birmanica may have undergone a period of expansion coincident with domestication of the water chestnut approximately 113,900–126,500 years ago. PMID:27459279

  17. Near-planar Solution Structures of Mannose-binding Lectin Oligomers Provide Insight on Activation of Lectin Pathway of Complement

    PubMed Central

    Miller, Ami; Phillips, Anna; Gor, Jayesh; Wallis, Russell; Perkins, Stephen J.

    2012-01-01

    The complement system is a fundamental component of innate immunity that orchestrates complex immunological and inflammatory processes. Complement comprises over 30 proteins that eliminate invading microorganisms while maintaining host cell integrity. Protein-carbohydrate interactions play critical roles in both the activation and regulation of complement. Mannose-binding lectin (MBL) activates the lectin pathway of complement via the recognition of sugar arrays on pathogenic surfaces. To determine the solution structure of MBL, synchrotron x-ray scattering and analytical ultracentrifugation experiments showed that the carbohydrate-recognition domains in the MBL dimer, trimer, and tetramer are positioned close to each other in near-planar fan-like structures. These data were subjected to constrained modeling fits. A bent structure for the MBL monomer was identified starting from two crystal structures for its carbohydrate-recognition domain and its triple helical region. The MBL monomer structure was used to identify 10–12 near-planar solution structures for each of the MBL dimers, trimers, and tetramers starting from 900 to 6,859 randomized structures for each. These near-planar fan-like solution structures joined at an N-terminal hub clarified how the carbohydrate-recognition domain of MBL binds to pathogenic surfaces. They also provided insight on how MBL presents a structural template for the binding and auto-activation of the MBL-associated serine proteases to initiate the lectin pathway of complement activation. PMID:22167201

  18. The dynamic subcellular localization of ERK: mechanisms of translocation and role in various organelles.

    PubMed

    Wainstein, Ehud; Seger, Rony

    2016-04-01

    The dynamic subcellular localization of ERK in resting and stimulated cells plays an important role in its regulation. In resting cells, ERK localizes in the cytoplasm, and upon stimulation, it translocates to its target substrates and organelles. ERK signaling initiated from different places in resting cells has distinct outcomes. In this review, we summarize the mechanisms of ERK1/2 translocation to the nucleus and mitochondria, and of ERK1c to the Golgi. We also show that ERK1/2 translocation to the nucleus is a useful anti cancer target. Unraveling the complex subcellular localization of ERK and its dynamic changes upon stimulation provides a better understanding of the regulation of ERK signaling and may result in the development of new strategies to combat ERK-related diseases.

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

    PubMed Central

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

    2017-01-01

    Recent advances in upconversion nanophotosensitizers (UCNPs-PS) excited by near-infrared (NIR) 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. It is of vital importance for these nanophotosensitizers to reach specifically the organelles and to perform PDT with precise time control. To do so, we have in this work traced the dynamic subcellular distribution, especially in organelles such as lysosomes and mitochondria, of the poly(allylamine)-modified and dual-loaded nanophotosensitizers. The apoptosis of the cancer cells induced by PDT with the dependence of the distribution status of the nanophotosensitizers in organelles was obtained, which has provided an in-depth picture of intracellular trafficking of organelle-targeted nanophotosensitizers. Our results shall facilitate the improvement of nanotechnology assisted photodynamic therapy of cancers. PMID:28361967

  20. Predicting apoptosis protein subcellular location with PseAAC by incorporating tripeptide composition.

    PubMed

    Liao, Bo; Jiang, Jun-Bao; Zeng, Qing-Guang; Zhu, Wen

    2011-11-01

    The function of the protein is closely correlated with its subcellular localization. Probing into the mechanism of protein sorting and predicting protein subcellular location can provide important clues or insights for understanding the function of proteins. In this paper, we introduce a new PseAAC approach to encode the protein sequence based on the physicochemical properties of amino acid residues. Each of the protein samples was defined as a 146D (dimensional) vector including the 20 amino acid composition components and 126 adjacent triune residues contents. To evaluate the effectiveness of this encoding scheme, we did jackknife tests on three datasets using the support vector machine algorithm. The total prediction accuracies are 84.9%, 91.2%, and 92.6%, respectively. The satisfactory results indicate that our method could be a useful tool in the area of bioinformatics and proteomics.

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

    PubMed

    Doughty, David M; Dieterle, Michael; Sessions, Alex L; Fischer, Woodward W; Newman, Dianne K

    2014-01-01

    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 (2)H-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.

  2. A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions

    SciTech Connect

    Brown, Roslyn N.; Sanford, James A.; Park, Jea H.; Deatherage, Brooke L.; Champion, Boyd L.; Smith, Richard D.; Heffron, Fred; Adkins, Joshua N.

    2012-06-01

    Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and infection-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of over 30% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB, PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein location for Salmonella and a framework for further investigations using computational modeling.

  3. A Sub-Cellular Viscoelastic Model for Cell Population Mechanics

    PubMed Central

    Jamali, Yousef; Azimi, Mohammad; Mofrad, Mohammad R. K.

    2010-01-01

    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 communication

  4. Symbiotic functioning, structural adaptation, and subcellular organization of root nodules from Psoralea pinnata (L.) plants grown naturally under wetland and upland conditions in the Cape Fynbos of South Africa.

    PubMed

    Kanu, Sheku A; Dakora, Felix D

    2017-01-01

    In the Cape Fynbos of South Africa, Psoralea pinnata (L.) plants occur naturally in both wetland and well-drained soils and yet effectively fix N2 under the two contrasting conditions. In this study, nodule structure and functioning in P. pinnata plants from the two habitats were evaluated using light and transmission electron microscopy (TEM), as well as the (15)N natural abundance technique. The results showed that, structurally, fully developed P. pinnata nodules were spherical in shape with six components (namely, lenticels, periderm, outer cortex, middle cortex, inner cortex, and a central bacteria-infected medulla region). Morphometric analysis revealed 44 and 84 % increase in cell area and volume of wetland nodules compared to those from upland. The percentage area of nodules occupied by the middle cortex in wetland nodules was twice that of upland nodules. As a result, the size of the medulla region in wetland nodules was significantly reduced compared to upland nodules. Additionally, the average area of medulla occupied by intercellular air spaces in wetland nodules was about five times that of upland nodules (about 431 % increase in wetland over upland nodules). TEM data also showed more bacteroids in symbiosomes of upland nodules when compared to wetland nodules. However, isotopic analysis of above-ground plant parts revealed no differences in symbiotic parameters such as N concentration, ∂(15)N and %Ndfa between wetland and upland P. pinnata plants. These results suggest that, under limiting O2 conditions especially in wetlands, nodules make structural and functional adjustments to meet the O2 demands of N2-fixing bacteroids.

  5. The subcellular sites of sphingomyelin synthesis in BHK cells.

    PubMed

    Miro Obradors, M J; Sillence, D; Howitt, S; Allan, D

    1997-10-30

    The subcellular distributions of the enzymes which synthesise sphingomyelin (SM) and glucosylceramide (GluCer) from ceramide have been assessed in BHK cells. On a sucrose density gradient GluCer synthase (a marker of the cis/medial Golgi apparatus) and the trans-Golgi marker galactosyltransferase showed an similar monotonic distribution. In contrast, SM synthase showed two peaks of activity, a minor one which migrated with the Golgi markers and a major one which had a density close to that of plasma membrane markers (sphingomyelin, cholesterol, PtdSer, ganglioside GM3 and alkaline phosphodiesterase). When cell homogenates were treated with digitonin, the sedimentation characteristics of the Golgi markers was largely unaffected whereas the plasma membrane markers and the main peak of SM synthase activity were shifted to higher density. In contrast, when cells were treated with brefeldin A (BFA) the Golgi markers were shifted to higher density but not the plasma membrane markers or the main peak of SM synthase. These results suggest that the bulk of SM synthase activity in BHK cells is not associated with the Golgi cisternae but with a cell compartment which is relatively rich in cholesterol (e.g., plasma membrane, endosomes or trans-Golgi network.) Further experiments in which cells were treated with sphingomyelinase provided evidence that SM synthase activity was in an internal compartment and not at the plasma membrane.

  6. Automated learning of generative models for subcellular location: building blocks for systems biology.

    PubMed

    Zhao, Ting; Murphy, Robert F

    2007-12-01

    The goal of location proteomics is the systematic and comprehensive study of protein subcellular location. We have previously developed automated, quantitative methods to identify protein subcellular location families, but there have been no effective means of communicating their patterns to integrate them with other information for building cell models. We built generative models of subcellular location that are learned from a collection of images so that they not only represent the pattern, but also capture its variation from cell to cell. Our models contain three components: a nuclear model, a cell shape model and a protein-containing object model. We built models for six patterns that consist primarily of discrete structures. To validate the generated images, we showed that they are recognized with reasonable accuracy by a classifier trained on real images. We also showed that the model parameters themselves can be used as features to discriminate the classes. The models allow the synthesis of images with the expectation that they are drawn from the same underlying statistical distribution as the images used to train them. They can potentially be combined for many proteins to yield a high resolution location map in support of systems biology.

  7. The Work Experience Survey (WES) Manual: A Structured Interview for Identifying Barriers to Career Maintenance. A Service Provider's Guide.

    ERIC Educational Resources Information Center

    Roessler, Richard T.; And Others

    This manual provides information on and instructions for administering the Work Experience Survey (WES), a 30- to 60-minute structured interview methodology to help individuals with disabilities direct their own accommodation planning. Section 1 describes the WES, which consists of six sections: (1) background information on the respondent, (2) an…

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

    PubMed Central

    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

  9. Topological constraints are major determinants of tRNA tertiary structure and dynamics and provide basis for tertiary folding cooperativity

    PubMed Central

    Mustoe, Anthony M.; Brooks, Charles L.; Al-Hashimi, Hashim M.

    2014-01-01

    Recent studies have shown that basic steric and connectivity constraints encoded at the secondary structure level are key determinants of 3D structure and dynamics in simple two-way RNA junctions. However, the role of these topological constraints in higher order RNA junctions remains poorly understood. Here, we use a specialized coarse-grained molecular dynamics model to directly probe the thermodynamic contributions of topological constraints in defining the 3D architecture and dynamics of transfer RNA (tRNA). Topological constraints alone restrict tRNA's allowed conformational space by over an order of magnitude and strongly discriminate against formation of non-native tertiary contacts, providing a sequence independent source of folding specificity. Topological constraints also give rise to long-range correlations between the relative orientation of tRNA's helices, which in turn provides a mechanism for encoding thermodynamic cooperativity between distinct tertiary interactions. These aspects of topological constraints make it such that only several tertiary interactions are needed to confine tRNA to its native global structure and specify functionally important 3D dynamics. We further show that topological constraints are conserved across tRNA's different naturally occurring secondary structures. Taken together, our results emphasize the central role of secondary-structure-encoded topological constraints in defining RNA 3D structure, dynamics and folding. PMID:25217593

  10. Three-dimensional mid-infrared tomographic imaging of endogenous and exogenous molecules in a single intact cell with subcellular resolution.

    PubMed

    Quaroni, Luca; Obst, Martin; Nowak, Marcus; Zobi, Fabio

    2015-01-02

    Microscopy in the mid-infrared spectral range provides detailed chemical information on a sample at moderate spatial resolution and is being used increasingly in the characterization of biological entities as challenging as single cells. However, a conventional cellular 2D imaging measurement is limited in its ability to associate specific compositional information to subcellular structures because of the interference from the complex topography of the sample. Herein we provide a method and protocols that overcome this challenge in which tilt-series infrared tomography is used with a standard benchtop infrared microscope. This approach gives access to the quantitative 3D distribution of molecular components based on the intrinsic contrast provided by the sample. We demonstrate the method by quantifying the distribution of an exogenous metal carbonyl complex throughout the cell and by reporting changes in its coordination sphere in different locations in the cell. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Assessment of the structural validity of the domestic violence healthcare providers' survey questionnaire using a Nigerian sample.

    PubMed

    John, Ime Akpan; Lawoko, Stephen

    2010-06-01

    There has been increased advocacy to involve healthcare providers in the prevention of intimate partner violence (IPV) through screening for it in healthcare. Yet, only one in ten providers screen for IPV, suggesting barriers. Understanding the readiness of healthcare providers to screen for IPV is therefore paramount. The Domestic Violence Healthcare Provider Survey Scales (DVHPSS) is a previously validated, comprehensive scale to study readiness of healthcare providers to screen for IPV. However, an understanding of its usefulness in the Sub-Saharan African context remains elusive. The current study undertook to examine the structural validity of the DVHPSS in Nigeria. Exploratory factor analysis and Cronbach's Alpha were run to reveal the factorial structure and reliability of the instrument/subscales respectively. Established thresholds were used to determine significant factor loadings and alphas coefficient. A six factor model emerged, with 2 factors similar to the original scale, another two differing slightly and a further two factors resulting from a splitting up of the original combination of victim/provider safety to having distinct victim and provider safety subscales. With slight modifications, the DVHPSS can be use to study IPV screening among Nigerian healthcare professionals. Introducing screening protocols could promote better understanding of crucial questions that were lost in the analysis. ‎

  12. Divergent Roles of CAAX Motif-signaled Posttranslational Modifications in the Regulation and Subcellular Localization of Ral GTPases*

    PubMed Central

    Gentry, Leanna R.; Nishimura, Akiyuki; Cox, Adrienne D.; Martin, Timothy D.; Tsygankov, Denis; Nishida, Motohiro; Elston, Timothy C.; Der, Channing J.

    2015-01-01

    The Ras-like small GTPases RalA and RalB are well validated effectors of RAS oncogene-driven human cancer growth, and pharmacologic inhibitors of Ral function may provide an effective anti-Ras therapeutic strategy. Intriguingly, although RalA and RalB share strong overall amino acid sequence identity, exhibit essentially identical structural and biochemical properties, and can utilize the same downstream effectors, they also exhibit divergent and sometimes opposing roles in the tumorigenic and metastatic growth of different cancer types. These distinct biological functions have been attributed largely to sequence divergence in their carboxyl-terminal hypervariable regions. However, the role of posttranslational modifications signaled by the hypervariable region carboxyl-terminal tetrapeptide CAAX motif (C = cysteine, A = aliphatic amino acid, X = terminal residue) in Ral isoform-selective functions has not been addressed. We determined that these modifications have distinct roles and consequences. Both RalA and RalB require Ras converting CAAX endopeptidase 1 (RCE1) for association with the plasma membrane, albeit not with endomembranes, and loss of RCE1 caused mislocalization as well as sustained activation of both RalA and RalB. In contrast, isoprenylcysteine carboxylmethyltransferase (ICMT) deficiency disrupted plasma membrane localization only of RalB, whereas RalA depended on ICMT for efficient endosomal localization. Furthermore, the absence of ICMT increased stability of RalB but not RalA protein. Finally, palmitoylation was critical for subcellular localization of RalB but not RalA. In summary, we have identified striking isoform-specific consequences of distinct CAAX-signaled posttranslational modifications that contribute to the divergent subcellular localization and activity of RalA and RalB. PMID:26216878

  13. Structure of putrescine aminotransferase from Escherichia coli provides insights into the substrate specificity among class III aminotransferases.

    PubMed

    Cha, Hyung Jin; Jeong, Jae-Hee; Rojviriya, Catleya; Kim, Yeon-Gil

    2014-01-01

    YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 Å resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

  14. SNAP-Tag-Based Subcellular Protein Labeling and Fluorescent Imaging with Naphthalimides.

    PubMed

    Wang, Chao; Song, Xinbo; Xiao, Yi

    2017-09-05

    Genetically encoded technologies provide methods for the specific labeling and imaging of proteins, which is essential to understand the subcellular localization of these proteins and their function. Herein, we employed naphthalimide, an efficient two-photon fluorophore, to develop O(6) -benzylguanine (BG) derivatives for specific labeling of subcellular proteins and fluorescent imaging through the SNAP-tag. Three naphthalimide-BG derivatives, TNI-BG, QNI-BG, and ONI-BG, were conveniently synthesized through modular "click chemistry" in high yields. All of them showed high labeling efficiency with SNAP-tag in solution (≈1-2×10(3)  s(-1)  m(-1) ) and in bacteria. Among them, ONI-BG showed high specificity to diffused, histone H2B and mitochondria COX8A targeted SNAP-tag in mammalian cells. The protein-labeled naphthalimides exhibited high two-photon absorption cross-sections, which indicated their potential application in protein-specific two-photon fluorescent imaging, such as two-photon fluorescent lifetime imaging and two-photon multicolor imaging. Therefore, ONI-BG is a versatile tool that can be used to track subcellular proteins through multiple fluorescent techniques. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Subcellular RNA profiling links splicing and nuclear DICER1 to alternative cleavage and polyadenylation

    PubMed Central

    Neve, Jonathan; Burger, Kaspar; Li, Wencheng; Hoque, Mainul; Patel, Radhika; Tian, Bin; Gullerova, Monika; Furger, Andre

    2016-01-01

    Alternative cleavage and polyadenylation (APA) plays a crucial role in the regulation of gene expression across eukaryotes. Although APA is extensively studied, its regulation within cellular compartments and its physiological impact remains largely enigmatic. Here, we used a rigorous subcellular fractionation approach to compare APA profiles of cytoplasmic and nuclear RNA fractions from human cell lines. This approach allowed us to extract APA isoforms that are subjected to differential regulation and provided us with a platform to interrogate the molecular regulatory pathways that shape APA profiles in different subcellular locations. Here, we show that APA isoforms with shorter 3′ UTRs tend to be overrepresented in the cytoplasm and appear to be cell-type–specific events. Nuclear retention of longer APA isoforms occurs and is partly a result of incomplete splicing contributing to the observed cytoplasmic bias of transcripts with shorter 3′ UTRs. We demonstrate that the endoribonuclease III, DICER1, contributes to the establishment of subcellular APA profiles not only by expected cytoplasmic miRNA-mediated destabilization of APA mRNA isoforms, but also by affecting polyadenylation site choice. PMID:26546131

  16. iTRAQ-based quantitative subcellular proteomic analysis of Avibirnavirus-infected cells.

    PubMed

    Sun, Yanting; Hu, Boli; Fan, Chengfei; Jia, Lu; Zhang, Yina; Du, Aifang; Zheng, Xiaojuan; Zhou, Jiyong

    2015-07-01

    Infectious bursal disease virus (IBDV) enters the host cells via endocytic pathway to achieve viral replication in the cytoplasm. Here, we performed LC-MS/MS coupled with isobaric tags for relative and absolute quantification labeling of differentially abundant proteins of IBDV-infected cells using a subcellular fractionation strategy. We show that the viral infection regulates the abundance and/or subcellular localization of 3211 proteins during early infection. In total, 23 cellular proteins in the cytoplasmic proteome and 34 in the nuclear proteome were significantly altered after virus infection. These differentially abundant proteins are involved in such biological processes as immune response, signal transduction, RNA processing, macromolecular biosynthesis, energy metabolism, virus binding, and cellular apoptosis. Moreover, transcriptional profiles of the 25 genes corresponding to the identified proteins were analyzed by quantitative real-time RT-PCR. Ingenuity Pathway Analysis clustered the differentially abundant proteins primarily into the mTOR pathway, PI3K/Akt pathway, and interferon-β signaling cascades. Confocal microscopy showed colocalization of the viral protein VP3 with host proteins heterogeneous nuclear ribonucleoprotein H1, nuclear factor 45, apoptosis inhibitor 5, nuclear protein localization protein 4 and DEAD-box RNA helicase 42 during the virus infection. Together, these identified subcellular constituents provide important information for understanding host-IBDV interactions and underlying mechanisms of IBDV infection and pathogenesis.

  17. Examining clinicians’ experiences providing sexual health services for LGBTQ youth: considering social and structural determinants of health in clinical practice

    PubMed Central

    Knight, R. E.; Shoveller, J. A.; Carson, A. M.; Contreras-Whitney, J. G.

    2014-01-01

    Although barriers related to lesbian, gay, bisexual, transgender and queer (LGBTQ) youth’s experiences accessing sexual health services have been examined in detail, research into the experiences and perceptions of clinicians providing these services has been conspicuously absent. The aim of this article is to explore the perceptions and experiences of clinicians providing sexual health services for LGBTQ youth. Drawing on in-depth, semi-structured interviews, this study examines 24 clinicians’ experiences providing sexual health services to LGBTQ youth in five communities in British Columbia, Canada. Our findings reveal how many clinicians provide services to LGBTQ youth with a lack of cultural competency—either implicitly (e.g. by describing heteronormative practices) or explicitly (e.g. by expressing frustration that they had not been sufficiently provided with appropriate training related to LGBTQ youth sexual health). Institutional norms and values were identified as the dominant barriers in the effective provision of LGBTQ-tailored services. Many clinicians find themselves unprepared to provide culturally competent sexual health services that have both the capacity to address individual-level issues (e.g. promoting condom use) while considering (and adapting services to) the broader socio-cultural and structural conditions that can render LGBTQ youth socially vulnerable. PMID:24412811

  18. Examining clinicians' experiences providing sexual health services for LGBTQ youth: considering social and structural determinants of health in clinical practice.

    PubMed

    Knight, R E; Shoveller, J A; Carson, A M; Contreras-Whitney, J G

    2014-08-01

    Although barriers related to lesbian, gay, bisexual, transgender and queer (LGBTQ) youth's experiences accessing sexual health services have been examined in detail, research into the experiences and perceptions of clinicians providing these services has been conspicuously absent. The aim of this article is to explore the perceptions and experiences of clinicians providing sexual health services for LGBTQ youth. Drawing on in-depth, semi-structured interviews, this study examines 24 clinicians' experiences providing sexual health services to LGBTQ youth in five communities in British Columbia, Canada. Our findings reveal how many clinicians provide services to LGBTQ youth with a lack of cultural competency-either implicitly (e.g., by describing heteronormative practices) or explicitly (e.g., by expressing frustration that they had not been sufficiently provided with appropriate training related to LGBTQ youth sexual health). Institutional norms and values were identified as the dominant barriers in the effective provision of LGBTQ-tailored services. Many clinicians find themselves unprepared to provide culturally competent sexual health services that have both the capacity to address individual-level issues (e.g. promoting condom use) while considering (and adapting services to) the broader socio-cultural and structural conditions that can render LGBTQ youth socially vulnerable. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  19. Investigation of the functional properties and subcellular localization of alpha human and rainbow trout estrogen receptors within a unique yeast cellular context.

    PubMed

    Le Grand, Adélaïde; Bouter, Anthony; Couturier, Anne; Mulner-Lorillon, Odile; Le Goff, Xavier; Chesnel, Franck; Sire, Olivier; Le Tilly, Véronique

    2015-05-01

    Estrogens are steroid hormones that play a pivotal role in growth, differentiation and function of reproductive and non-reproductive tissues, mediated through estrogen receptors (ERs). Estrogens are involved in different genomic and non-genomic cell signaling pathways which involve well-defined subcellular ER localizations. Thus, ER activity results from complex interplays between intrinsic binding properties and specific subcellular localization. Since these two factors are deeply intricate, we carried out, in a unique yeast cell context, a comparative study to better understand structure/function/subcellular distribution relationships. This was carried out by comparing two ERs: the human ER α subtype (hERα) and the short form of the α isoform of the rainbow trout ER (rtERαS). Their distinct binding properties to agonist and antagonist ligands and subcellular localizations were characterized in Saccharomyces cerevisiae yeast cells. An unexpected partial agonistic effect of ICI 182-780 was observed for rtERαS. Concomitant to distinct binding properties, distinct subcellular localizations were observed before and after ligand stimulation. Due to the unique cell context, the link between ERs intrinsic binding properties and subcellular localizations is partly unveiled and issues are hypothesized based on the role of cytoplasmic transient complexes which play a role in the ER cytoplasmic/nuclear partition, which in turn is critical for the recruitment of co-regulators in the nucleus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Feature Fusion Based SVM Classifier for Protein Subcellular Localization Prediction.

    PubMed

    Rahman, Julia; Mondal, Md Nazrul Islam; Islam, Md Khaled Ben; Hasan, Md Al Mehedi

    2016-12-18

    For the importance of protein subcellular localization in different branches of life science and drug discovery, researchers have focused their attentions on protein subcellular localization prediction. Effective representation of features from protein sequences plays a most vital role in protein subcellular localization prediction specially in case of machine learning techniques. Single feature representation-like pseudo amino acid composition (PseAAC), physiochemical property models (PPM), and amino acid index distribution (AAID) contains insufficient information from protein sequences. To deal with such problems, we have proposed two feature fusion representations, AAIDPAAC and PPMPAAC, to work with Support Vector Machine classifiers, which fused PseAAC with PPM and AAID accordingly. We have evaluated the performance for both single and fused feature representation of a Gram-negative bacterial dataset. We have got at least 3% more actual accuracy by AAIDPAAC and 2% more locative accuracy by PPMPAAC than single feature representation.

  1. Compressed learning and its applications to subcellular localization.

    PubMed

    Zheng, Zhong-Long; Guo, Li; Jia, Jiong; Xie, Chen-Mao; Zeng, Wen-Cai; Yang, Jie

    2011-09-01

    One of the main challenges faced by biological applications is to predict protein subcellular localization in automatic fashion accurately. To achieve this in these applications, a wide variety of machine learning methods have been proposed in recent years. Most of them focus on finding the optimal classification scheme and less of them take the simplifying the complexity of biological systems into account. Traditionally, such bio-data are analyzed by first performing a feature selection before classification. Motivated by CS (Compressed Sensing) theory, we propose the methodology which performs compressed learning with a sparseness criterion such that feature selection and dimension reduction are merged into one analysis. The proposed methodology decreases the complexity of biological system, while increases protein subcellular localization accuracy. Experimental results are quite encouraging, indicating that the aforementioned sparse methods are quite promising in dealing with complicated biological problems, such as predicting the subcellular localization of Gram-negative bacterial proteins.

  2. Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation.

    PubMed

    Matsumoto, Shunsuke; Shimada, Atsushi; Nyirenda, James; Igura, Mayumi; Kawano, Yoshiaki; Kohda, Daisuke

    2013-10-29

    Oligosaccharyltransferase transfers an oligosaccharide chain to the asparagine residues in proteins. The archaeal and eubacterial oligosaccharyltransferases are single subunit membrane enzymes, referred to as "AglB" (archaeal glycosylation B) and "PglB" (protein glycosylation B), respectively. Only one crystal structure of a full-length PglB has been solved. Here we report the crystal structures of the full-length AglB from a hyperthermophilic archaeon, Archaeoglobus fulgidus. The AglB and PglB proteins share the common overall topology of the 13 transmembrane helices, and a characteristic long plastic loop in the transmembrane region. This is the structural basis for the formation of the catalytic center, consisting of conserved acidic residues coordinating a divalent metal ion. In one crystal form, a sulfate ion was bound next to the metal ion. This structure appears to represent a dolichol-phosphate binding state, and suggests the release mechanism for the glycosylated product. The structure in the other crystal form corresponds to the resting state conformation with the well-ordered plastic loop in the transmembrane region. The overall structural similarity between the distantly related AglB and PglB proteins strongly indicates the conserved catalytic mechanism in the eukaryotic counterpart, the STT3 (stauroporine and temperature sensitivity 3) protein. The detailed structural comparison provided the dynamic view of the N-glycosylation reaction, involving the conversion between the structured and unstructured states of the plastic loop in the transmembrane region and the formation and collapse of the Ser/Thr-binding pocket in the C-terminal globular domain.

  3. The shades of gray of the chromatin fiber: recent literature provides new insights into the structure of chromatin.

    PubMed

    Ausió, Juan

    2015-01-01

    The chromatin fiber consists of a string of nucleosomes connected by linker DNA regions. The hierarchy of folding of this fiber within the cell has long been controversial, and the existence of an originally described 30 nm fiber has been debated and reviewed extensively. This review contextualizes two recent papers on this topic that suggest the 30 nm fiber to be an over-simplification. The idealized model from the first study provides good insight into the constraints and histone participation in the maintenance of the fiber structure. The second paper provides a theoretical description of a more realistic view of the highly heterogeneous and dynamic chromatin organization in the in vivo setting. It is now time to abandon the highly regular "one start" solenoidal 30 nm structure and replace it with a more realistic highly dynamic, polymorphic fiber.

  4. Enhancing a Pathway-Genome Database (PGDB) to Capture Subcellular Localization of Metabolites and Enzymes: The Nucleotide-Sugar Biosynthetic Pathways of Populus trichocarpa

    SciTech Connect

    Nag, A.; Karpinets, T. V.; Chang, C. H.; Bar-Peled, M.

    2012-01-01

    Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s).

  5. Polyallelic structural variants can provide accurate, highly informative genetic markers focused on diagnosis and therapeutic targets: Accuracy vs. Precision.

    PubMed

    Roses, A D

    2016-02-01

    Structural variants (SVs) include all insertions, deletions, and rearrangements in the genome, with several common types of nucleotide repeats including single sequence repeats, short tandem repeats, and insertion-deletion length variants. Polyallelic SVs provide highly informative markers for association studies with well-phenotyped cohorts. SVs can influence gene regulation by affecting epigenetics, transcription, splicing, and/or translation. Accurate assays of polyallelic SV loci are required to define the range and allele frequency of variable length alleles.

  6. Structural Analysis of the Myo1c and Neph1 Complex Provides Insight into the Intracellular Movement of Neph1

    PubMed Central

    Arif, Ehtesham; Sharma, Pankaj; Solanki, Ashish; Mallik, Leena; Rathore, Yogendra S.; Twal, Waleed O.; Nath, Samir K.; Gandhi, Darpan; Holzman, Lawrence B.; Ostap, E. Michael

    2016-01-01

    The Myo1c motor functions as a cargo transporter supporting various cellular events, including vesicular trafficking, cell migration, and stereociliary movements of hair cells. Although its partial crystal structures were recently described, the structural details of its interaction with cargo proteins remain unknown. This study presents the first structural demonstration of a cargo protein, Neph1, attached to Myo1c, providing novel insights into the role of Myo1c in intracellular movements of this critical slit diaphragm protein. Using small angle X-ray scattering studies, models of predominant solution conformation of unliganded full-length Myo1c and Myo1c bound to Neph1 were constructed. The resulting structures show an extended S-shaped Myo1c with Neph1 attached to its C-terminal tail. Importantly, binding of Neph1 did not induce a significant shape change in Myo1c, indicating this as a spontaneous process or event. Analysis of interaction surfaces led to the identification of a critical residue in Neph1 involved in binding to Myo1c. Indeed, a point mutant from this site abolished interaction between Neph1 and Myo1c when tested in the in vitro and in live-cell binding assays. Live-cell imaging, including fluorescence recovery after photobleaching, provided further support for the role of Myo1c in intracellular vesicular movement of Neph1 and its turnover at the membrane. PMID:27044863

  7. Subcellular analysis of starch metabolism in developing barley seeds using a non-aqueous fractionation method

    PubMed Central

    Tiessen, Axel; Nerlich, Annika; Faix, Benjamin; Hümmer, Christine; Fox, Simon; Trafford, Kay; Weber, Hans; Weschke, Winfriede; Geigenberger, Peter

    2012-01-01

    Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80–90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published Km values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective Km values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass–action ratios

  8. Crystal Structures of Malonyl-Coenzyme A Decarboxylase Provide Insights into Its Catalytic Mechanism and Disease-Causing Mutations

    PubMed Central

    Froese, D. Sean; Forouhar, Farhad; Tran, Timothy H.; Vollmar, Melanie; Kim, Yi Seul; Lew, Scott; Neely, Helen; Seetharaman, Jayaraman; Shen, Yang; Xiao, Rong; Acton, Thomas B.; Everett, John K.; Cannone, Giuseppe; Puranik, Sriharsha; Savitsky, Pavel; Krojer, Tobias; Pilka, Ewa S.; Kiyani, Wasim; Lee, Wen Hwa; Marsden, Brian D.; von Delft, Frank; Allerston, Charles K.; Spagnolo, Laura; Gileadi, Opher; Montelione, Gaetano T.; Oppermann, Udo; Yue, Wyatt W.; Tong, Liang

    2013-01-01

    Summary Malonyl-coenzyme A decarboxylase (MCD) is found from bacteria to humans, has important roles in regulating fatty acid metabolism and food intake, and is an attractive target for drug discovery. We report here four crystal structures of MCD from human, Rhodopseudomonas palustris, Agrobacterium vitis, and Cupriavidus metallidurans at up to 2.3 Å resolution. The MCD monomer contains an N-terminal helical domain involved in oligomerization and a C-terminal catalytic domain. The four structures exhibit substantial differences in the organization of the helical domains and, consequently, the oligomeric states and intersubunit interfaces. Unexpectedly, the MCD catalytic domain is structurally homologous to those of the GCN5-related N-acetyltransferase superfamily, especially the curacin A polyketide synthase catalytic module, with a conserved His-Ser/Thr dyad important for catalysis. Our structures, along with mutagenesis and kinetic studies, provide a molecular basis for understanding pathogenic mutations and catalysis, as well as a template for structure-based drug design. PMID:23791943

  9. Functional Probes of Drug–Receptor Interactions Implicated by Structural Studies: Cys-Loop Receptors Provide a Fertile Testing Ground

    PubMed Central

    2015-01-01

    Structures of integral membrane receptors provide valuable models for drug–receptor interactions across many important classes of drug targets and have become much more widely available in recent years. However, it remains to be determined to what extent these images are relevant to human receptors in their biological context and how subtle issues such as subtype selectivity can be informed by them. The high precision structural modifications enabled by unnatural amino acid mutagenesis on mammalian receptors expressed in vertebrate cells allow detailed tests of predictions from structural studies. Using the Cys-loop superfamily of ligand-gated ion channels, we show that functional studies lead to detailed binding models that, at times, are significantly at odds with the structural studies on related invertebrate proteins. Importantly, broad variations in binding interactions are seen for very closely related receptor subtypes and for varying drugs at a given binding site. These studies highlight the essential interplay between structural studies and functional studies that can guide efforts to develop new pharmaceuticals. PMID:24568098

  10. Crystal structures of the CERT START domain with inhibitors provide insights into the mechanism of ceramide transfer.

    PubMed

    Kudo, Norio; Kumagai, Keigo; Matsubara, Ryosuke; Kobayashi, Shu; Hanada, Kentaro; Wakatsuki, Soichi; Kato, Ryuichi

    2010-02-19

    The cytosolic protein CERT transfers ceramide from the endoplasmic reticulum to the Golgi apparatus where ceramide is converted to SM. The C-terminal START (steroidogenic acute regulatory protein-related lipid transfer) domain of CERT binds one ceramide molecule in its central amphiphilic cavity. (1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide (HPA), a synthesized analogue of ceramide, inhibits ceramide transfer by CERT. Here we report crystal structures of the CERT START domain in complex with HPAs of varying acyl chain lengths. In these structures, one HPA molecule is buried in the amphiphilic cavity where the amide and hydroxyl groups of HPA form a hydrogen-bond network with specific amino acid residues. The Omega1 loop, which has been suggested to function as a gate of the cavity, adopts a different conformation when bound to HPA than when bound to ceramide. In the Omega1 loop region, Trp473 shows the largest difference between these two structures. This residue exists inside of the cavity in HPA-bound structures, while it is exposed to the outside of the protein in the apo-form and ceramide-bound complex structures. Surface plasmon resonance experiments confirmed that Trp473 is important for interaction with membranes. These results provide insights into not only the molecular mechanism of inhibition by HPAs but also possible mechanisms by which CERT interacts with ceramide. 2009 Elsevier Ltd. All rights reserved.

  11. Functional probes of drug-receptor interactions implicated by structural studies: Cys-loop receptors provide a fertile testing ground.

    PubMed

    Van Arnam, Ethan B; Dougherty, Dennis A

    2014-08-14

    Structures of integral membrane receptors provide valuable models for drug-receptor interactions across many important classes of drug targets and have become much more widely available in recent years. However, it remains to be determined to what extent these images are relevant to human receptors in their biological context and how subtle issues such as subtype selectivity can be informed by them. The high precision structural modifications enabled by unnatural amino acid mutagenesis on mammalian receptors expressed in vertebrate cells allow detailed tests of predictions from structural studies. Using the Cys-loop superfamily of ligand-gated ion channels, we show that functional studies lead to detailed binding models that, at times, are significantly at odds with the structural studies on related invertebrate proteins. Importantly, broad variations in binding interactions are seen for very closely related receptor subtypes and for varying drugs at a given binding site. These studies highlight the essential interplay between structural studies and functional studies that can guide efforts to develop new pharmaceuticals.

  12. Crystallographic analysis of NHERF1–PLCβ3 interaction provides structural basis for CXCR2 signaling in pancreatic cancer

    SciTech Connect

    Jiang, Yuanyuan; Wang, Shuo; Holcomb, Joshua; Trescott, Laura; Guan, Xiaoqing; Hou, Yuning; Brunzelle, Joseph; Sirinupong, Nualpun; Li, Chunying; Yang, Zhe

    2014-04-04

    Highlights: • CXCR2–NHERF1–PLCβ3 complex regulates CXCR2 signaling in pancreatic cancer. • The crystal structure of the NHERF1 PDZ1 domain in complex with PLCβ3. • The structure reveals specificity determinants of PDZ1–PLCβ3 interaction. • Endogenous PLCβ3 in pancreatic cancer cells interacts with both PDZ1 and PDZ2. • Structural basis of the PDZ1–PLCβ3 interaction is valuable in selective drug design. - Abstract: The formation of CXCR2–NHERF1–PLCβ3 macromolecular complex in pancreatic cancer cells regulates CXCR2 signaling activity and plays an important role in tumor proliferation and invasion. We previously have shown that disruption of the NHERF1-mediated CXCR2–PLCβ3 interaction abolishes the CXCR2 signaling cascade and inhibits pancreatic tumor growth in vitro and in vivo. Here we report the crystal structure of the NHERF1 PDZ1 domain in complex with the C-terminal PLCβ3 sequence. The structure reveals that the PDZ1–PLCβ3 binding specificity is achieved by numerous hydrogen bonds and hydrophobic contacts with the last four PLCβ3 residues contributing to specific interactions. We also show that PLCβ3 can bind both NHERF1 PDZ1 and PDZ2 in pancreatic cancer cells, consistent with the observation that the peptide binding pockets of these PDZ domains are highly structurally conserved. This study provides an understanding of the structural basis for the PDZ-mediated NHERF1–PLCβ3 interaction that could prove valuable in selective drug design against CXCR2-related cancers.

  13. Regional public health preparedness teams in North Carolina: an analysis of their structural capacity and impact on services provided.

    PubMed

    Horney, Jennifer A; Markiewicz, Milissa; Meyer, Anne Marie; Casani, Julie; Hegle, Jennifer; MacDonald, Pia D M

    2011-01-01

    In December 2001, the North Carolina Division of Public Health established Public Health Regional Surveillance Teams (PHRSTs) to build local public health capacity to prevent, prepare for, respond to, and recover from public health incidents and events. Seven PHRSTs are colocated at local health departments (LHDs) around the state. The authors assessed structural capacity of the PHRSTs and analyzed the relationship between structural capacity and the frequency of support and services provided to LHDs by PHRSTs. Five categories of structural capacity were measured: human, fiscal, informational, physical, and organizational resources. In addition, variation in structural capacity among teams was also examined. The most variation was seen in human resources. Although each team was originally designed to include a physician/epidemiologist, industrial hygienist, nurse/epidemiologist, and administrative support technician, team composition varied such that only the administrative support technician is common to all teams. Variation in team composition was associated with differences in the support and services that PHRSTs provide to LHDs. Teams that reported having a medical doctor or a doctor of osteopathic medicine (chi2 = 9.95; p < 0.01) or an epidemiologist (chi2 = 5.35; p < 0.02) had larger budgets and provided more support and services, and teams that housed a pharmacist reported more partners (chi2 = 52.34; p < 0.01). Teams that received directives from more groups (such as LHDs) also provided more support and services in planning (Z = 21.71; p < 0.01), communication and liaison (Z = 12.11; p < 0.01), epidemiology and surveillance (Z = 5.09; p < 0.01), consultation and technical support (Z = 2.25; p = 0.02), H1N1 outbreak assistance (Z = 10.25; p < 0.01), and public health event response (Z = 2.19; p = 0.03). In the last 10 years, significant variation in structural capacity, particularly in human resources, has been introduced among PHRSTs. These differences

  14. DIRECT secure messaging as a common transport layer for reporting structured and unstructured lab results to outpatient providers.

    PubMed

    Sujansky, Walter; Wilson, Tom

    2015-04-01

    This report describes a grant-funded project to explore the use of DIRECT secure messaging for the electronic delivery of laboratory test results to outpatient physicians and electronic health record systems. The project seeks to leverage the inherent attributes of DIRECT secure messaging and electronic provider directories to overcome certain barriers to the delivery of lab test results in the outpatient setting. The described system enables laboratories that generate test results as HL7 messages to deliver these results as structured or unstructured documents attached to DIRECT secure messages. The system automatically analyzes generated HL7 messages and consults an electronic provider directory to determine the appropriate DIRECT address and delivery format for each indicated recipient. The system also enables lab results delivered to providers as structured attachments to be consumed by HL7 interface engines and incorporated into electronic health record systems. Lab results delivered as unstructured attachments may be printed or incorporated into patient records as PDF files. The system receives and logs acknowledgement messages to document the status of each transmitted lab result, and a graphical interface allows searching and review of this logged information. The described system is a fully implemented prototype that has been tested in a laboratory setting. Although this approach is promising, further work is required to pilot test the system in production settings with clinical laboratories and outpatient provider organizations. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Physical activity recommendations for patients with electrophysiologic and structural congenital heart disease: a survey of Canadian health care providers.

    PubMed

    Roston, Thomas M; De Souza, Astrid M; Sandor, George G S; Sanatani, Shubhayan; Potts, James E

    2013-08-01

    Determining safe levels of physical activity for children and adolescents with electrophysiologic and structural congenital heart disease is a challenging clinical problem. The body of evidence for making these recommendations is limited and likely based on expert opinion, medicolegal concerns, and perceived risks of sudden cardiac death (SCD) with activity. The Bethesda Conference has established consensus guidelines for determining the eligibility of athletes with cardiovascular abnormalities for competitive sports and their disqualification from them. However, literature on guidelines for noncompetitive physical activity is not available. A survey was designed to determine practice patterns for patients with electrophysiologic and structural congenital heart disease. Between July 2011 and December 2011, approximately 350 health care providers working with this group of patients were recruited by email or while attending professional meetings. The survey received 81 responses, primarily from pediatric cardiologists (70 %). The findings indicate that the majority of Canadian cardiac care providers surveyed are only partially implementing current recommendations. Areas of variance included physical activity recommendations for hypertrophic cardiomyopathy, long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, and heart transplantation, among others. The development of comprehensive consensus guidelines for activity recommendations was supported by 96 % of the respondents. The heterogeneity of responses may be attributable to conflicting and poorly evidenced information in the literature, a lack of emphasis on recreational activity, an entrenched tendency toward bed rest in the cardiology community, and a lack of awareness by cardiac care providers regarding the actual risk associated with physical activity in electrophysiologic and structural congenital heart disease. A balanced discussion is required in considering both the significant benefit of

  16. Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

    SciTech Connect

    Albright, Seth; Chen Bin; Holbrook, Kristen; Jain, Nitin U.

    2008-04-04

    CD14 functions as a key pattern recognition receptor for a diverse array of Gram-negative and Gram-positive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy, the binding of three different endotoxin ligands, lipopolysaccharide, lipoteichoic acid, and a PGN-derived compound, muramyl dipeptide to a {sup 15}N isotopically labeled 152-residue N-terminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern of residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site.

  17. Regulating Subcellular Metal Homeostasis: The Key to Crop Improvement

    PubMed Central

    Bashir, Khurram; Rasheed, Sultana; Kobayashi, Takanori; Seki, Motoaki; Nishizawa, Naoko K.

    2016-01-01

    Iron (Fe), zinc (Zn), manganese (Mn), and copper (Cu) are essential micronutrient mineral elements for living organisms, as they regulate essential cellular processes, such as chlorophyll synthesis and photosynthesis (Fe, Cu, and Mn), respiration (Fe and Cu), and transcription (Zn). The storage and distribution of these minerals in various cellular organelles is strictly regulated to ensure optimal metabolic rates. Alteration of the balance in uptake, distribution, and/or storage of these minerals severely impairs cellular metabolism and significantly affects plant growth and development. Thus, any change in the metal profile of a cellular compartment significantly affects metabolism. Different subcellular compartments are suggested to be linked through complex retrograde signaling networks to regulate cellular metal homeostasis. Various genes regulating cellular and subcellular metal distribution have been identified and characterized. Understanding the role of these transporters is extremely important to elaborate the signaling between various subcellular compartments. Moreover, modulation of the proteins involved in cellular metal homeostasis may help in the regulation of metabolism, adaptability to a diverse range of environmental conditions, and biofortification. Here, we review progress in the understanding of different subcellular metal transport components in plants and discuss the prospects of regulating cellular metabolism and strategies to develop biofortified crop plants. PMID:27547212

  18. Bioavailability of purified subcellular metals to a marine fish.

    PubMed

    Guo, Feng; Yao, Jie; Wang, Wen-Xiong

    2013-09-01

    In the present study, the authors used a supply of naturally contaminated oysters to investigate how the subcellular metal distribution and the metal burden in prey affected the transfer of metals to a marine fish, the grunt Terapon jarbua. The oysters, Crassostrea hongkongensis, each with different contamination histories, were collected and separated into 3 subcellular fractions: 1) metal-rich granules, 2) cellular debris, and 3) a combined fraction of organelles, heat-denatured proteins, and metallothionein-like proteins, defined as the trophically available metal (TAM). These purified fractions showed a wide range of metal concentrations and were fed to the fish for a period of 7 d at a daily comparable feeding rate of 3% of fish body weight. After 7 d exposure, the newly absorbed metals were mainly distributed in the intestine and liver, indicating a significant tissue-specific trophic transfer, especially for Cd and Cu. The trophic transfer factors (TTFs) showed a sequence of cellular debris >TAM > metal-rich granules, suggesting the impact of subcellular distribution in prey on metal bioavailability. However, significant inverse relationships between the TTFs and the metal concentrations in diets were also found in the present study, especially for Cd and Zn. The subcellular metal compartmentalization might be less important than the metal concentration in prey influencing the trophic transfer. The authors' results have important implications for bioavailability and environmental assessment of dietary metals.

  19. The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport.

    PubMed

    McCoy, Jason G; Ren, Zhenning; Stanevich, Vitali; Lee, Jumin; Mitra, Sharmistha; Levin, Elena J; Poget, Sebastien; Quick, Matthias; Im, Wonpil; Zhou, Ming

    2016-06-07

    The phosphoenolpyruvate:carbohydrate phosphotransferase systems are found in bacteria, where they play central roles in sugar uptake and regulation of cellular uptake processes. Little is known about how the membrane-embedded components (EIICs) selectively mediate the passage of carbohydrates across the membrane. Here we report the functional characterization and 2.55-Å resolution structure of a maltose transporter, bcMalT, belonging to the glucose superfamily of EIIC transporters. bcMalT crystallized in an outward-facing occluded conformation, in contrast to the structure of another glucose superfamily EIIC, bcChbC, which crystallized in an inward-facing occluded conformation. The structures differ in the position of a structurally conserved substrate-binding domain that is suggested to play a central role in sugar transport. In addition, molecular dynamics simulations suggest a potential pathway for substrate entry from the periplasm into the bcMalT substrate-binding site. These results provide a mechanistic framework for understanding substrate recognition and translocation for the glucose superfamily EIIC transporters.

  20. Structure of the Tuberous Sclerosis Complex 2 (TSC2) N Terminus Provides Insight into Complex Assembly and Tuberous Sclerosis Pathogenesis.

    PubMed

    Zech, Reinhard; Kiontke, Stephan; Mueller, Uwe; Oeckinghaus, Andrea; Kümmel, Daniel

    2016-09-16

    Tuberous sclerosis complex (TSC) is caused by mutations in the TSC1 and TSC2 tumor suppressor genes. The gene products hamartin and tuberin form the TSC complex that acts as GTPase-activating protein for Rheb and negatively regulates the mammalian target of rapamycin complex 1 (mTORC1). Tuberin contains a RapGAP homology domain responsible for inactivation of Rheb, but functions of other protein domains remain elusive. Here we show that the TSC2 N terminus interacts with the TSC1 C terminus to mediate complex formation. The structure of the TSC2 N-terminal domain from Chaetomium thermophilum and a homology model of the human tuberin N terminus are presented. We characterize the molecular requirements for TSC1-TSC2 interactions and analyze pathological point mutations in tuberin. Many mutations are structural and produce improperly folded protein, explaining their effect in pathology, but we identify one point mutant that abrogates complex formation without affecting protein structure. We provide the first structural information on TSC2/tuberin with novel insight into the molecular function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. The structure of apo ArnA features an unexpected central binding pocket and provides an explanation for enzymatic cooperativity.

    PubMed

    Fischer, Utz; Hertlein, Simon; Grimm, Clemens

    2015-03-01

    The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD(+)-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.

  2. Native-state hydrogen-exchange studies of a fragment complex can provide structural information about the isolated fragments

    PubMed Central

    Chakshusmathi, G.; Ratnaparkhi, Girish S.; Madhu, P. K.; Varadarajan, R.

    1999-01-01

    Ordered protein complexes are often formed from partially ordered fragments that are difficult to structurally characterize by conventional NMR and crystallographic techniques. We show that concentration-dependent hydrogen exchange studies of a fragment complex can provide structural information about the solution structures of the isolated fragments. This general methodology can be applied to any bimolecular or multimeric system. The experimental system used here consists of Ribonuclease S, a complex of two fragments of Ribonuclease A. Ribonuclease S and Ribonuclease A have identical three-dimensional structures but exhibit significant differences in their dynamics and stability. We show that the apparent large dynamic differences between Ribonuclease A and Ribonuclease S are caused by small amounts of free fragments in equilibrium with the folded complex, and that amide exchange rates in Ribonuclease S can be used to determine corresponding rates in the isolated fragments. The studies suggest that folded RNase A and the RNase S complex exhibit very similar dynamic behavior. Thus cleavage of a protein chain at a single site need not be accompanied by a large increase in flexibility of the complex relative to that of the uncleaved protein. PMID:10393919

  3. The changing health care marketplace: current industry trends, new provider organizational structures, and effects on plastic surgeons.

    PubMed

    Krieger, L M

    1998-09-01

    Current market forces are driving the health care industry in new directions. The managed care industry is currently undergoing a market shakeout, as manifested by consolidation, increased competition, and lower profits. Medicare is fighting to remain solvent by lowering fees paid to providers, driving patients into managed care plans, and cracking down on billing irregularities. For providers, the combined effect of these trends is lower fees, increased risk-sharing, and increased overhead. Plastic surgeons face new demands in this environment. They must increase their efficiency and form new alliances with other providers. These alliances allow plastic surgeons to maintain a steady stream of patients, to manage risk, to negotiate more lucrative contracts with managed care organizations, and to increase efficiency. To achieve these alliances, plastic surgeons must alter the organizational structure of their practices. Several corporate practice models are becoming more prevalent; these include large group practices, physician practice management companies, and integrated delivery systems. Each structure has advantages for plastic surgeons, but each also requires plastic surgeons to trade varying degrees of financial and professional autonomy for market strength.

  4. RNASTAR: An RNA STructural Alignment Repository that provides insight into the evolution of natural and artificial RNAs

    PubMed Central

    Widmann, Jeremy; Stombaugh, Jesse; McDonald, Daniel; Chocholousova, Jana; Gardner, Paul; Iyer, Matthew K.; Liu, Zongzhi; Lozupone, Catherine A.; Quinn, John; Smit, Sandra; Wikman, Shandy; Zaneveld, Jesse R.R.; Knight, Rob

    2012-01-01

    Automated RNA alignment algorithms often fail to recapture the essential conserved sites that are critical for function. To assist in the refinement of these algorithms, we manually curated a set of 148 alignments with a total of 9600 unique sequences, in which each alignment was backed by at least one crystal or NMR structure. These alignments included both naturally and artificially selected molecules. We used principles of isostericity to improve the alignments from an average of 83%–94% isosteric base pairs. We expect that this alignment collection will assist in a wide range of benchmarking efforts and provide new insight into evolutionary principles governing change in RNA structural motifs. The improved alignments have been contributed to the Rfam database. PMID:22645380

  5. RNASTAR: an RNA STructural Alignment Repository that provides insight into the evolution of natural and artificial RNAs.

    PubMed

    Widmann, Jeremy; Stombaugh, Jesse; McDonald, Daniel; Chocholousova, Jana; Gardner, Paul; Iyer, Matthew K; Liu, Zongzhi; Lozupone, Catherine A; Quinn, John; Smit, Sandra; Wikman, Shandy; Zaneveld, Jesse R R; Knight, Rob

    2012-07-01

    Automated RNA alignment algorithms often fail to recapture the essential conserved sites that are critical for function. To assist in the refinement of these algorithms, we manually curated a set of 148 alignments with a total of 9600 unique sequences, in which each alignment was backed by at least one crystal or NMR structure. These alignments included both naturally and artificially selected molecules. We used principles of isostericity to improve the alignments from an average of 83%-94% isosteric base pairs. We expect that this alignment collection will assist in a wide range of benchmarking efforts and provide new insight into evolutionary principles governing change in RNA structural motifs. The improved alignments have been contributed to the Rfam database.

  6. Structure of myosin-1c tail bound to calmodulin provides insights into calcium-mediated conformational coupling.

    PubMed

    Lu, Qing; Li, Jianchao; Ye, Fei; Zhang, Mingjie

    2015-01-01

    Class I myosins can sense cellular mechanical forces and function as tension-sensitive anchors or transporters. How mechanical load is transduced from the membrane-binding tail to the force-generating head in myosin-1 is unknown. Here we determined the crystal structure of the entire tail of mouse myosin-1c in complex with apocalmodulin, showing that myosin-1c adopts a stable monomer conformation suited for force transduction. The lever-arm helix and the C-terminal extended PH domain of the motor are coupled by a stable post-IQ domain bound to calmodulin in a highly unusual mode. Ca(2+) binding to calmodulin induces major conformational changes in both IQ motifs and the post-IQ domain and increases flexibility of the myosin-1c tail. Our study provides a structural blueprint for the neck and tail domains of myosin-1 and expands the target binding modes of the master Ca(2+)-signal regulator calmodulin.

  7. CeFra-seq: Systematic mapping of RNA subcellular distribution properties through cell fractionation coupled to deep-sequencing.

    PubMed

    Lefebvre, Fabio Alexis; Cody, Neal A L; Bouvrette, Louis Philip Benoit; Bergalet, Julie; Wang, Xiaofeng; Lécuyer, Eric

    2017-08-15

    The subcellular trafficking of RNA molecules is a conserved feature of eukaryotic cells and plays key functions in diverse processes implicating polarised cellular activities. Large-scale imaging and subcellular transcriptomic studies suggest that regulated RNA localization is a highly prevalent process that appears to be disrupted in several neuromuscular disorders. These features underline the importance and usefulness of implementing procedures to assess global transcriptome subcellular distribution properties. Here, we present a method combining biochemical fractionation of cells and high-throughput RNA sequencing (CeFra-seq) that enables rapid and efficient systematic mapping of RNA cytotopic distributions in cells. The described procedure involves biochemical fractionation to derive extracts of nuclear, cytosolic, endomembrane, cytoplasmic insoluble and extracellular material from cell culture lines. The RNA content of each fraction can then be profiled by deep-sequencing, revealing global subcellular signatures. We provide a detailed protocol for the CeFra-seq procedure along with relevant validation steps and data analysis guidelines to graphically represent RNA spatial distribution features. As a complement to imaging approaches, CeFra-seq represents a powerful and scalable tool to investigate global alterations in RNA trafficking. Copyright © 2017. Published by Elsevier Inc.

  8. Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast*

    PubMed Central

    Lowman, Douglas W.; Greene, Rachel R.; Bearden, Daniel W.; Kruppa, Michael D.; Pottier, Max; Monteiro, Mario A.; Soldatov, Dmitriy V.; Ensley, Harry E.; Cheng, Shih-Chin; Netea, Mihai G.; Williams, David L.

    2014-01-01

    The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. 1H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae. PMID:24344127

  9. Midwives' perceptions of organisational structures and processes influencing their ability to provide caseload care to socially disadvantaged and vulnerable women.

    PubMed

    Menke, Jane; Fenwick, Jennifer; Gamble, Jenny; Brittain, Hazel; Creedy, Debra K

    2014-10-01

    This study examined midwives' perceptions of organisational structures and processes of care when working in a caseload model (Midwifery Group Practice MGP) for socially disadvantaged and vulnerable childbearing women. This study used Donabedian's theoretical framework for evaluating the quality of health care provision. Of the 17 eligible midwives, 15 participated in focus group discussions and two others provided written comments. Thematic analysis was guided by three headings; clinical outcomes, processes of care and organisational structure. Midwives believed they provided an excellent service to socially disadvantaged and vulnerable childbearing women. Midwives gained satisfaction from working in partnership with women, working across their full scope of practice, and making a difference to the women. However the midwives perceived the MGP was situated within an organisation that was hostile to the caseload model of care. Midwives felt frustrated and distressed by a lack of organisational support for the model and a culture of blame dominated by medicine. A lack of material resources and no identified office space created feelings akin to 'homelessness'. Together these challenges threatened the cohesiveness of the MGP and undermined midwives' ability to advocate for women and keep birth normal. If access to caseload midwifery care for women with diverse backgrounds and circumstances is to be enhanced, then mechanisms need to be implemented to ensure organisational structures and processes are developed to sustain midwives in the provision of 'best practice' maternity care. Women accessing midwifery caseload care have excellent maternal and newborn outcomes. However there remains limited understanding of the impact of organisational structures and processes of care on clinical outcomes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Crystal structure of a chimera of human and Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferases provides insights into oligomerization.

    PubMed

    Gayathri, P; Sujay Subbayya, I N; Ashok, Chethan S; Selvi, T Senthamizh; Balaram, Hemalatha; Murthy, M R N

    2008-12-01

    The crystal structure of a chimera of Plasmodium falciparum (Pf) and human hypoxanthine guanine phosphoribosyltransferases (HGPRT), which consists of the core of the protein from the human enzyme and the hood region from the Pf enzyme, has been determined as a complex with the product guanosine monophosphate (GMP). The chimera can utilize hypoxanthine, guanine, and xanthine as substrates, similar to the Pf enzyme. It exists as a monomer-dimer mixture in solution, but shifts to a tetramer on addition of phosphoribosyl pyrophosphate (PRPP). The structural studies reveal that the asymmetric unit of the crystal consists of two monomers of the chimeric HGPRT. Surprisingly, the dimer interface of the chimera is the less extensive AC interface of the parent HGPRTs. An analysis of the crystal structures of the various human HGPRTs provides an explanation for the oligomeric characteristics of the chimera. Pro93 and Tyr197 form part of crucial interactions holding together the AB interface in the unliganded or GMP-bound forms of HGPRT, while Pro93 and His26 interact at the interface after binding of PRPP. Replacement of Tyr197 of human HGPRT by Ile207 in the chimera disrupts the interaction at the AB interface in the absence of PRPP. In the presence of PRPP, the interaction between Pro93 and His26 could restore the AB interface, shifting the chimeric enzyme to a tetrameric state. The structure provides valuable insights into the differences in the AB interface between Pf and human HGPRTs, which may be useful for designing selective inhibitors against the parasite enzyme.

  11. Evidence that Osteoblasts are Specialized Citrate-producing Cells that Provide the Citrate for Incorporation into the Structure of Bone

    PubMed Central

    Franklin, Renty B.; Chellaiah, Meena; Zou, Jing; Reynolds, Mark A.; Costello, Leslie C.

    2015-01-01

    Citrate is a major component of bone in all vertebrates, but its implications in bone have remained largely unknown. Recent studies identified that citrate is incorporated into the structure of the hydroxyapatite nanocrystal/collagen complex; and is essential for the important biomechanical properties of bone. This raises the important question, “What is the source of citrate for incorporation into bone?”; A question that heretofore had remained unresolved. Studies in this report were designed to determine the plausibility of our concept that the osteoblasts are specialized citrate-producing cells, which provide the citrate that is incorporated into the structure of bone; and that osteogenic differentiation of mesenchyme cells leads to the development of the citrate-producing osteoblasts. The results demonstrated that primary human osteoblasts exhibit the capability of citrate-production. Undifferentiated mesenchyme cells do not exhibit the capability of citrate production; and osteogenic differentiation results in citrate-producing osteoblasts. The up-regulation of zinc uptake transporter ZIP1 is essential for the manifestation of the citrate-producing capability of the osteoblasts. We determined that osteoblast transport of citrate from plasma is not a likely source of citrate in bone. Thus, this study establishes for the first time that the osteoblasts are specialized citrate-producing cells that provide the citrate for incorporation into the structure of bone; and that mesenchyme cell osteogenesis leads to differentiated citrate-producing osteoblasts. This is a new understanding; which must include the osteogenic development of citrate-producing osteoblasts, and the process of “citration” in concert with mineralization during bone formation. It also provides a new understanding of the role of bone in the homeostatic maintenance of plasma citrate concentration. PMID:25745519

  12. Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility

    PubMed Central

    McIntosh, Victoria J.; Abou Samra, Abdul B.; Mohammad, Ramzi M.; Lasley, Robert D.

    2016-01-01

    Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility. Methods: Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-β-cyclodextrin (MβCD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed. Results: Treatment with MβCD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of MβCD was on p38 subcellular distribution of p38α with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i. Conclusions: These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility. PMID:27441649

  13. Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism

    PubMed Central

    Soldano, Anabel; Klinke, Sebastián; Otero, Lisandro H.; Rivera, Mario; Catalano-Dupuy, Daniela L.

    2017-01-01

    Heme oxygenase from Leptospira interrogans is an important virulence factor. During catalysis, redox equivalents are provided to this enzyme by the plastidic-type ferredoxin-NADP+ reductase also found in L. interrogans. This process may have evolved to aid this bacterial pathogen to obtain heme-iron from their host and enable successful colonization. Herein we report the crystal structure of the heme oxygenase-heme complex at 1.73 Å resolution. The structure reveals several distinctive features related to its function. A hydrogen bonded network of structural water molecules that extends from the catalytic site to the protein surface was cleared observed. A depression on the surface appears to be the H+ network entrance from the aqueous environment to the catalytic site for O2 activation, a key step in the heme oxygenase reaction. We have performed a mutational analysis of the F157, located at the above-mentioned depression. The mutant enzymes were unable to carry out the complete degradation of heme to biliverdin since the reaction was arrested at the verdoheme stage. We also observed that the stability of the oxyferrous complex, the efficiency of heme hydroxylation and the subsequent conversion to verdoheme was adversely affected. These findings underscore a long-range communication between the outer fringes of the hydrogen-bonded network of structural waters and the heme active site during catalysis. Finally, by analyzing the crystal structures of ferredoxin-NADP+ reductase and heme oxygenase, we propose a model for the productive association of these proteins. PMID:28771589

  14. The Structure of Allophanate Hydrolase from Granulibacter bethesdensis Provides Insights into Substrate Specificity in the Amidase Signature Family

    SciTech Connect

    Lin, Yi; Maurice, Martin

    2013-01-02

    Allophanate hydrolase (AH) catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2. AH belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cis-Ser-Lys catalytic triad. In this study, the first structures of AH fromGranulibacter bethesdensis were determined, with and without the substrate analogue malonate, to 2.2 and 2.8 Å, respectively. The structures confirm the identity of the catalytic triad residues and reveal an altered dimerization interface that is not conserved in the amidase signature family. The structures also provide insights into previously unrecognized substrate specificity determinants in AH. Two residues, Tyr299 and Arg307, are within hydrogen bonding distance of a carboxylate moiety of malonate. Both Tyr299 and Arg307 were mutated, and the resulting modified enzymes revealed >3 order of magnitude reductions in both catalytic efficiency and substrate stringency. It is proposed that Tyr299 and Arg307 serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser172. The structure further suggests the presence of a unique C-terminal domain in AH. While this domain is conserved, it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Analysis of the AH active site architecture offers new insights into common determinants of catalysis and specificity among divergent members of the amidase signature family.

  15. Team Objective Structured Bedside Assessment (TOSBA): a novel and feasible way of providing formative teaching and assessment.

    PubMed

    Miller, S D W; Butler, M W; Meagher, F; Costello, R W; McElvaney, N G

    2007-03-01

    It can be challenging to teach and assess medical students successfully in the setting of a hospital ward using real patients. We describe a novel method of providing weekly formative clinical assessment and teaching to final year students on an acute medical ward: The Team Objective Structured Bedside Assessment (TOSBA). The TOSBA involves three groups of five students rotating through three ward-based stations (each station consists of an inpatient and facilitator). Each group spends 25 minutes at a bedside station where the facilitator asks consecutive students to perform one of five clinical tasks. Every student receives a standardised grade and is provided with educational feedback at each of the three stations. We report our 15-month experience using the TOSBA format to assess and teach a large number of medical students on a weekly basis. We discuss the advantages and potential drawbacks of our approach.

  16. Data-independent acquisition (MSE) with ion mobility provides a systematic method for analysis of a bacteriophage structural proteome.

    PubMed

    Moran, Deborah; Cross, Trevor; Brown, Lewis M; Colligan, Ryan M; Dunbar, David

    2014-01-01

    In this work, a method was developed to study the structural proteome of mycobacteriophage Marvin, a recent isolate from soil with 107 predicted coding sequences. This prototype method was applied for semi-quantitative analysis of the composition of this mycobacteriophage virion using ion mobility spectrometry and data-independent acquisition (MS(E)-IMS). MS(E)-IMS was compared to a more conventional proteomics technique employing mass spectrometry with a data-dependent acquisition strategy. MS(E)-IMS provided broad coverage of the virion proteome and high sequence coverage for individual proteins. This shotgun method does not depend on the limited sensitivity of visualization of protein bands by staining reagents inherent in gel-based methods. The method is comprehensive, provides high sequence coverage and is proposed as a particularly efficient method for the study of bacteriophage proteomes. Copyright © 2013 Elsevier B.V. All rights reserved.

  17. A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules.

    PubMed

    Sahasrabuddhe, Amogh A; Bajpai, Virendra K; Gupta, Chhitar M

    2004-03-01

    To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 10(6) copies per cell of this protein (M(r) 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed.

  18. Subcellular Fractionation Analysis of the Extraction of Ubiquitinated Polytopic Membrane Substrate during ER-Associated Degradation.

    PubMed

    Nakatsukasa, Kunio; Kamura, Takumi

    2016-01-01

    During ER-associated degradation (ERAD), misfolded polytopic membrane proteins are ubiquitinated and retrotranslocated to the cytosol for proteasomal degradation. However, our understanding as to how polytopic membrane proteins are extracted from the ER to the cytosol remains largely unclear. To better define the localization and physical properties of ubiquitinated polytopic membrane substrates in vivo, we performed subcellular fractionation analysis of Ste6*, a twelve transmembrane protein that is ubiquitinated primarily by Doa10 E3 ligase in yeast. Consistent with previous in vitro studies, ubiquitinated Ste6* was extracted from P20 (20,000 g pellet) fraction to S20 (20,000 g supernatant) fraction in a Cdc48/p97-dependent manner. Similarly, Ubx2p, which recruits Cdc48/p97 to the ER, facilitated the extraction of Ste6*. By contrast, lipid droplet formation, which was suggested to be dispensable for the degradation of Hrd1-substrates in yeast, was not required for the degradation of Ste6*. Intriguingly, we found that ubiquitinated Ste6* in the S20 fraction could be enriched by further centrifugation at 100,000 g. Although it is currently uncertain whether ubiquitinated Ste6* in P100 fraction is completely free from any lipids, membrane flotation analysis suggested the existence of two distinct populations of ubiquitinated Ste6* with different states of membrane association. Together, these results imply that ubiquitinated Ste6* may be sequestered into a putative quality control sub-structure by Cdc48/p97. Fractionation assays developed in the present study provide a means to further dissect the ill-defined post-ubiquitination step during ERAD of polytopic membrane substrates.

  19. Lead tolerance mechanism in Conyza canadensis: subcellular distribution, ultrastructure, antioxidative defense system, and phytochelatins.

    PubMed

    Li, Ying; Zhou, Chuifan; Huang, Meiying; Luo, Jiewen; Hou, Xiaolong; Wu, Pengfei; Ma, Xiangqing

    2016-03-01

    We used hydroponic experiments to examine the effects of different concentrations of lead (Pb) on the performance of the Pb-tolerable plant Conyza canadensis. In these experiments, most of the Pb was accumulated in the roots; there was very little Pb accumulated in stems and leaves. C. canadensis is able to take up significant amounts of Pb whilst greatly restricting its transportation to specific parts of the aboveground biomass. High Pb concentrations inhibited plant growth, increased membrane permeability, elevated antioxidant enzyme activity in roots, and caused a significant increase in root H2O2 and malondialdehyde content. Analysis of Pb content at the subcellular level showed that most Pb was associated with the cell wall fraction, followed by the nucleus-rich fraction, and with a minority present in the mitochondrial and soluble fractions. Furthermore, transmission electron microscopy and energy dispersive X-ray analysis of root cells revealed that the cell wall and intercellular space in C. canadensis roots are the main locations of Pb accumulation. Additionally, high Pb concentrations adversely affected the cellular structure of C. canadensis roots. The increased enzyme activity suggests that the antioxidant system may play an important role in eliminating or alleviating Pb toxicity in C. canadensis roots. However, the levels of non-protein sulfhydryl compounds, glutathione, and phytochelatin did not significantly change in either the roots or leaves under Pb-contaminated treatments. Our results provide strong evidence that cell walls restrict Pb uptake into the root and act as an important barrier protecting root cells, while demonstrating that antioxidant enzyme levels are correlated with Pb exposure. These findings demonstrate the roles played by these detoxification mechanisms in supporting Pb tolerance in C. canadensis.

  20. Leaf oil body functions as a subcellular factory for the production of a phytoalexin in Arabidopsis.

    PubMed

    Shimada, Takashi L; Takano, Yoshitaka; Shimada, Tomoo; Fujiwara, Masayuki; Fukao, Yoichiro; Mori, Masashi; Okazaki, Yozo; Saito, Kazuki; Sasaki, Ryosuke; Aoki, Koh; Hara-Nishimura, Ikuko

    2014-01-01

    Oil bodies are intracellular structures present in the seed and leaf cells of many land plants. Seed oil bodies are known to function as storage compartments for lipids. However, the physiological function of leaf oil bodies is unknown. Here, we show that leaf oil bodies function as subcellular factories for the production of a stable phytoalexin in response to fungal infection and senescence. Proteomic analysis of oil bodies prepared from Arabidopsis (Arabidopsis thaliana) leaves identified caleosin (CLO3) and α-dioxygenase (α-DOX1). Both CLO3 and α-DOX1 were localized on the surface of oil bodies. Infection with the pathogenic fungus Colletotrichum higginsianum promoted the formation of CLO3- and α-DOX1-positive oil bodies in perilesional areas surrounding the site of infection. α-DOX1 catalyzes the reaction from α-linolenic acid (a major fatty acid component of oil bodies) to an unstable compound, 2-hydroperoxy-octadecatrienoic acid (2-HPOT). Intriguingly, a combination of α-DOX1 and CLO3 produced a stable compound, 2-hydroxy-octadecatrienoic acid (2-HOT), from α-linolenic acid. This suggests that the colocalization of α-DOX1 and CLO3 on oil bodies might prevent the degradation of unstable 2-HPOT by efficiently converting 2-HPOT into the stable compound 2-HOT. We found that 2-HOT had antifungal activity against members of the genus Colletotrichum and that infection with C. higginsianum induced 2-HOT production. These results defined 2-HOT as an Arabidopsis phytoalexin. This study provides, to our knowledge, the first evidence that leaf oil bodies produce a phytoalexin under a pathological condition, which suggests a new mechanism of plant defense.

  1. X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid.

    PubMed

    Lizak, Christian; Worrall, Liam J; Baumann, Lars; Pfleiderer, Moritz M; Volkers, Gesa; Sun, Tianjun; Sim, Lyann; Wakarchuk, Warren; Withers, Stephen G; Strynadka, Natalie C J

    2017-07-19

    Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

  2. Crystal structure of the amyloid-β p3 fragment provides a model for oligomer formation in Alzheimer's disease.

    PubMed

    Streltsov, Victor A; Varghese, Joseph N; Masters, Colin L; Nuttall, Stewart D

    2011-01-26

    Alzheimer's disease is a progressive neurodegenerative disorder associated with the presence of amyloid-β (Aβ) peptide fibrillar plaques in the brain. However, current evidence suggests that soluble nonfibrillar Aβ oligomers may be the major drivers of Aβ-mediated synaptic dysfunction. Structural information on these Aβ species has been very limited because of their noncrystalline and unstable nature. Here, we describe a crystal structure of amylogenic residues 18-41 of the Aβ peptide (equivalent to the p3 α/γ-secretase fragment of amyloid precursor protein) presented within the CDR3 loop region of a shark Ig new antigen receptor (IgNAR) single variable domain antibody. The predominant oligomeric species is a tightly associated Aβ dimer, with paired dimers forming a tetramer in the crystal caged within four IgNAR domains, preventing uncontrolled amyloid formation. Our structure correlates with independently observed features of small nonfibrillar Aβ oligomers and reveals conserved elements consistent with residues and motifs predicted as critical in Aβ folding and oligomerization, thus potentially providing a model system for nonfibrillar oligomer formation in Alzheimer's disease.

  3. Oligomeric state in the crystal structure of modular FAD synthetase provides insights into its sequential catalysis in prokaryotes.

    PubMed

    Herguedas, Beatriz; Martínez-Júlvez, Marta; Frago, Susana; Medina, Milagros; Hermoso, Juan A

    2010-07-09

    The crystal structure of the modular flavin adenine dinucleotide (FAD) synthetase from Corynebacterium ammoniagenes has been solved at 1.95 A resolution. The structure of C. ammoniagenes FAD synthetase presents two catalytic modules-a C-terminus with ATP-riboflavin kinase activity and an N-terminus with ATP-flavin mononucleotide (FMN) adenylyltransferase activity-that are responsible for the synthesis of FAD from riboflavin in two sequential steps. In the monomeric structure, the active sites from both modules are placed 40 A away, preventing the direct transfer of the product from the first reaction (FMN) to the second catalytic site, where it acts as substrate. Crystallographic and biophysical studies revealed a hexameric assembly formed by the interaction of two trimers. Each trimer presents a head-tail configuration, with FMN adenylyltransferase and riboflavin kinase modules from different protomers approaching the active sites and allowing the direct transfer of FMN. Experimental results provide molecular-level evidences of the mechanism of the synthesis of FMN and FAD in prokaryotes in which the oligomeric state could be involved in the regulation of the catalytic efficiency of the modular enzyme. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  4. Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase.

    PubMed

    Kumar, Vidya P; Cisneros, Jose A; Frey, Kathleen M; Castellanos-Gonzalez, Alejandro; Wang, Yiqiang; Gangjee, Aleem; White, A Clinton; Jorgensen, William L; Anderson, Karen S

    2014-09-01

    Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors. Copyright © 2014. Published by Elsevier Ltd.

  5. Exploration of insights, opportunities and caveats provided by the X-ray structures of hSERT.

    PubMed

    Topiol, Sid; Bang-Andersen, Benny; Sanchez, Connie; Bøgesø, Klaus P

    2016-10-15

    The recently reported X-ray structures of the human serotonin (5-HT) transporter SERT with bound inhibitors open new opportunities for drug discovery at SERT, selectivity design with respect to other neurotransmitter sodium transporters, and enhanced understanding of the molecular events involved in SERT action. Through computational and structural analyses, we explore the binding and migration of 5-HT at SERT. Consistent with earlier studies of leucine migration at the bacterial homolog of SERT, LeuT, we find multiple potential 'stopover' sites for 5-HT binding at SERT including the two (transmembrane S1 and extracellular vestibule S2) seen in the binding of the SSRI (S)-citalopram (S-Cit) to SERT, as well as other sites. Docking studies reveal the possibility of both hetero- (S-Cit+5-HT) and homo-dimeric (5-HT+5-HT) co-binding at both these sites which may explain earlier published allosteric activity observations and provide novel design strategies. Comparisons with substrate bound X-ray structures of the dopamine transporter reveal a number of potential sources of selectivity, some of which may be 'artificial' including target based, species related, experimental design related, and ligand dependent examples including substrate versus inhibitor related features.

  6. Phosphodiesterases and subcellular compartmentalized cAMP signaling in the cardiovascular system.

    PubMed

    Stangherlin, Alessandra; Zaccolo, Manuela

    2012-01-01

    Phosphodiesterases are key enzymes in the cAMP signaling cascade. They convert cAMP in its inactive form 5'-AMP and critically regulate the intensity and the duration of cAMP-mediated signals. Multiple isoforms exist that possess different intracellular distributions, different affinities for cAMP, and different catalytic and regulatory properties. This complex repertoire of enzymes provides a multiplicity of ways to modulate cAMP levels, to integrate more signaling pathways, and to respond to the specific needs of the cell within distinct subcellular domains. In this review we summarize key findings on phosphodiesterase compartmentalization in the cardiovascular system.

  7. Survey of social health insurance structure in selected countries; providing framework for basic health insurance in Iran

    PubMed Central

    Mohammadi, Effat; Raissi, Ahmad Reza; Barooni, Mohsen; Ferdoosi, Massoud; Nuhi, Mojtaba

    2014-01-01

    is based on the nationality or residence, which the insured by paying the insurance premiums within 6-10% of their income and employment status, are entitled to use the services. Providing services to the insured are performed by indirect forms. Payments to the service providers for the fee of inpatient and outpatient services are conservative and the related diagnostic groups system. Conclusions: Paying attention to the importance of modification of the fragmented health insurance system and financing the country's healthcare can reduce much of the failure of the health system, including the access of the public to health services. The countries according to the degree of development, governmental, and private insurance companies and existing rules must use the appropriate structure, comprehensive approach to the structure, and financing of the health social insurance on the investigated basis and careful attention to the intersections and differentiation. Studied structures, using them in the proposed approach and taking advantages of the perspectives of different beneficiaries about discussed topics can be important and efficient in order to achieve the goals of the health social insurance. PMID:25540789

  8. Survey of social health insurance structure in selected countries; providing framework for basic health insurance in Iran.

    PubMed

    Mohammadi, Effat; Raissi, Ahmad Reza; Barooni, Mohsen; Ferdoosi, Massoud; Nuhi, Mojtaba

    2014-01-01

    paying the insurance premiums within 6-10% of their income and employment status, are entitled to use the services. Providing services to the insured are performed by indirect forms. Payments to the service providers for the fee of inpatient and outpatient services are conservative and the related diagnostic groups system. Paying attention to the importance of modification of the fragmented health insurance system and financing the country's healthcare can reduce much of the failure of the health system, including the access of the public to health services. The countries according to the degree of development, governmental, and private insurance companies and existing rules must use the appropriate structure, comprehensive approach to the structure, and financing of the health social insurance on the investigated basis and careful attention to the intersections and differentiation. Studied structures, using them in the proposed approach and taking advantages of the perspectives of different beneficiaries about discussed topics can be important and efficient in order to achieve the goals of the health social insurance.

  9. Crystal structure of peroxide stress regulator from Streptococcus pyogenes provides functional insights into the mechanism of oxidative stress sensing.

    PubMed

    Makthal, Nishanth; Rastegari, Sheila; Sanson, Misu; Ma, Zhen; Olsen, Randall J; Helmann, John D; Musser, James M; Kumaraswami, Muthiah

    2013-06-21

    Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.

  10. Crystal Structure of Peroxide Stress Regulator from Streptococcus pyogenes Provides Functional Insights into the Mechanism of Oxidative Stress Sensing*

    PubMed Central

    Makthal, Nishanth; Rastegari, Sheila; Sanson, Misu; Ma, Zhen; Olsen, Randall J.; Helmann, John D.; Musser, James M.; Kumaraswami, Muthiah

    2013-01-01

    Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR. PMID:23645680

  11. Structure of the extracellular portion of CD46 provides insights into its interactions with complement proteins and pathogens.

    PubMed

    Persson, B David; Schmitz, Nikolaus B; Santiago, César; Zocher, Georg; Larvie, Mykol; Scheu, Ulrike; Casasnovas, José M; Stehle, Thilo

    2010-09-30

    The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.

  12. Understanding health care provider barriers to hospital affiliated medical fitness center facility referral: a questionnaire survey and semi structured interviews.

    PubMed

    Smock, Carissa; Alemagno, Sonia

    2017-08-03

    The purpose of this study is to understand health care provider barriers to referring patients to Medical Fitness Center Facilities within an affiliated teaching hospital system using referral of diabetic services as an example. The aims of this study include: (1) to assess health care providers' awareness and use of facilities, (2) to determine barriers to referring patients to facilities, (3) identify current and needed resources and/or changes to increase referral to facilities. A 20-item electronic survey and requests for semi-structured interviews were administered to hospital system directors and managers (n = 51). Directors and managers instructed physicians and staff to complete the survey and interviews as applicable. Perceived barriers, knowledge, utilization, and referral of patients to Medical Fitness Center Facilities were collected and examined. Descriptive statistics were generated regarding practice characteristics, provider characteristics, and referral. Of the health care providers surveyed and interviewed (n = 25) 40% indicated verbally suggesting use of facilities, 24% provided a flyer about the facilities. No respondents indicated that they directly referred patients to the facilities. However, 16% referred patients to other locations for physical activity - including their own department's management and prevention services. 20% do not refer to Medical Fitness Center Facilities or any other lifestyle programs/locations. Lack of time (92%) and lack of standard guidelines and operating procedures (88%) are barriers to referral. All respondents indicated a strong ability to refer patients to Medical Fitness Center Facilities if given education about referral programs available as well as standard clinical guidelines and protocol for delivery. The results of this study indicate that, although few healthcare providers are currently referring patients to Medical Fitness Center Facilities, health care providers with an affiliated Medical Fitness

  13. Subcellular investigation of photosynthesis-driven carbon assimilation in the symbiotic reef coral Pocillopora damicornis.

    PubMed

    Kopp, Christophe; Domart-Coulon, Isabelle; Escrig, Stephane; Humbel, Bruno M; Hignette, Michel; Meibom, Anders

    2015-02-10

    Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [(13)C]bicarbonate and [(15)N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. Our results provide detailed in situ subcellular visualization of the fate of photosynthesis-derived carbon and nitrogen in the coral-dinoflagellate endosymbiosis. We directly demonstrate that lipid droplets and glycogen granules in the coral tissue are sinks for translocated carbon photosynthates by dinoflagellates and confirm their key role in the trophic interactions within the coral-dinoflagellate association. Copyright © 2015 Kopp et al.

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

    PubMed

    Johnson, Gregory R; Li, Jieyue; Shariff, Aabid; Rohde, Gustavo K; Murphy, Robert F

    2015-12-01

    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.

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

    PubMed Central

    Johnson, Gregory R.; Li, Jieyue; Shariff, Aabid; Rohde, Gustavo K.; Murphy, Robert F.

    2015-01-01

    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. PMID:26624011

  16. Subcellular components of the amphibian egg - Insights provided by gravitational studies

    NASA Technical Reports Server (NTRS)

    Neff, A. W.; Ritzenthaler, J. D.; Rosenbaum, J. F.

    1989-01-01

    The variability in the response of Xenopus laevis eggs to a given force environment is studied. The roles of cytoplasmic organelle, the yolk platelets, and cytoskeletal components in varying in cytoplasmic mobility are examined. The data reveal that the packing of yolk platelets is not a major factor in causing cytoplasmic mobility differences and microtubules may affect cytoplasmic mobility.

  17. A Neighbourhood Algorithm Analysis of the Constraints Provided by Surface Wave Dispersion Data on Upper Mantle Structure

    NASA Astrophysics Data System (ADS)

    Beghein, C.; Lebedev, S.; van der Hilst, R.

    2005-12-01

    Interstation dispersion curves can be used to obtain regional 1D profiles of the crust and upper mantle. Unlike phase velocity maps, dispersion curves can be determined with small errors and for a broad frequency band. We want to determine what features interstation surface wave dispersion curves can constrain. Using synthetic data and the Neighbourhood Algorithm, a direct search approach that provides a full statistical assessment of model uncertainites and trade-offs, we investigate how well crustal and upper mantle structure can be recovered with fundamental Love and Rayleigh waves. We also determine how strong are the trade-offs between the different parameters and what depth resolution can we expect to achieve with the current level of precision of this type of data. Synthetic dispersion curves between approximately 7 and 340s were assigned realistic error bars, i.e. an increase of the relative uncertainty with the period but with an amplitude consistent with the one achieve in ``real'' measurements. These dispersion curves were generated by two types of isotropic model differing only by their crustal structure. One represents an oceanic region (shallow Moho) and the other corresponds to an archean continental area with a larger Moho depth. Preliminary results show that while the Moho depth, the shear-velocity structure in the transition zone, between 200 and 410km depth, and between the base of the crust and 50km depth are generally well recovered, crustal structure and Vs between between 50 and 200km depth are more difficult to constrain with Love waves or Rayleigh waves alone because of some trade-off between the two layers. When these two layers are put together, the resolution of Vs between 50 and 100km depth apperas to improve. Stucture deeper than the transition zone is not constrained by the data because of a lack of sensitivity. We explore the possibility of differentiating between an upper and lower crust as well, and we investigate whether a joint

  18. Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies

    PubMed Central

    Thapa, Dharendra; Shepherd, Danielle L.

    2014-01-01

    Cardiac tissue contains discrete pools of mitochondria that are characterized by their subcellular spatial arrangement. Subsarcolemmal mitochondria (SSM) exist below the cell membrane, interfibrillar mitochondria (IFM) reside in rows between the myofibrils, and perinuclear mitochondria are situated at the nuclear poles. Microstructural imaging of heart tissue coupled with the development of differential isolation techniques designed to sequentially separate spatially distinct mitochondrial subpopulations have revealed differences in morphological features including shape, absolute size, and internal cristae arrangement. These findings have been complemented by functional studies indicating differences in biochemical parameters and, potentially, functional roles for the ATP generated, based upon subcellular location. Consequently, mitochondrial subpopulations appear to be influenced differently during cardiac pathologies including ischemia/reperfusion, heart failure, aging, exercise, and diabetes mellitus. These influences may be the result of specific structural and functional disparities between mitochondrial subpopulations such that the stress elicited by a given cardiac insult differentially impacts subcellular locales and the mitochondria contained within. The goal of this review is to highlight some of the inherent structural and functional differences that exist between spatially distinct cardiac mitochondrial subpopulations as well as provide an overview of the differential impact of various cardiac pathologies on spatially distinct mitochondrial subpopulations. As an outcome, we will instill a basis for incorporating subcellular spatial location when evaluating the impact of cardiac pathologies on the mitochondrion. Incorporation of subcellular spatial location may offer the greatest potential for delineating the influence of cardiac pathology on this critical organelle. PMID:24778166

  19. Measuring factors affecting implementation of health innovations: a systematic review of structural, organizational, provider, patient, and innovation level measures.

    PubMed

    Chaudoir, Stephenie R; Dugan, Alicia G; Barr, Colin H I

    2013-02-17

    Two of the current methodological barriers to implementation science efforts are the lack of agreement regarding constructs hypothesized to affect implementation success and identifiable measures of these constructs. In order to address these gaps, the main goals of this paper were to identify a multi-level framework that captures the predominant factors that impact implementation outcomes, conduct a systematic review of available measures assessing constructs subsumed within these primary factors, and determine the criterion validity of these measures in the search articles. We conducted a systematic literature review to identify articles reporting the use or development of measures designed to assess constructs that predict the implementation of evidence-based health innovations. Articles published through 12 August 2012 were identified through MEDLINE, CINAHL, PsycINFO and the journal Implementation Science. We then utilized a modified five-factor framework in order to code whether each measure contained items that assess constructs representing structural, organizational, provider, patient, and innovation level factors. Further, we coded the criterion validity of each measure within the search articles obtained. Our review identified 62 measures. Results indicate that organization, provider, and innovation-level constructs have the greatest number of measures available for use, whereas structural and patient-level constructs have the least. Additionally, relatively few measures demonstrated criterion validity, or reliable association with an implementation outcome (e.g., fidelity). In light of these findings, our discussion centers on strategies that researchers can utilize in order to identify, adapt, and improve extant measures for use in their own implementation research. In total, our literature review and resulting measures compendium increases the capacity of researchers to conceptualize and measure implementation-related constructs in their ongoing and

  20. Measuring factors affecting implementation of health innovations: a systematic review of structural, organizational, provider, patient, and innovation level measures

    PubMed Central

    2013-01-01

    Background Two of the current methodological barriers to implementation science efforts are the lack of agreement regarding constructs hypothesized to affect implementation success and identifiable measures of these constructs. In order to address these gaps, the main goals of this paper were to identify a multi-level framework that captures the predominant factors that impact implementation outcomes, conduct a systematic review of available measures assessing constructs subsumed within these primary factors, and determine the criterion validity of these measures in the search articles. Method We conducted a systematic literature review to identify articles reporting the use or development of measures designed to assess constructs that predict the implementation of evidence-based health innovations. Articles published through 12 August 2012 were identified through MEDLINE, CINAHL, PsycINFO and the journal Implementation Science. We then utilized a modified five-factor framework in order to code whether each measure contained items that assess constructs representing structural, organizational, provider, patient, and innovation level factors. Further, we coded the criterion validity of each measure within the search articles obtained. Results Our review identified 62 measures. Results indicate that organization, provider, and innovation-level constructs have the greatest number of measures available for use, whereas structural and patient-level constructs have the least. Additionally, relatively few measures demonstrated criterion validity, or reliable association with an implementation outcome (e.g., fidelity). Discussion In light of these findings, our discussion centers on strategies that researchers can utilize in order to identify, adapt, and improve extant measures for use in their own implementation research. In total, our literature review and resulting measures compendium increases the capacity of researchers to conceptualize and measure implementation

  1. The Mammalian Adult Neurogenesis Gene Ontology (MANGO) Provides a Structural Framework for Published Information on Genes Regulating Adult Hippocampal Neurogenesis

    PubMed Central

    Overall, Rupert W.; Paszkowski-Rogacz, Maciej; Kempermann, Gerd

    2012-01-01

    Background Adult hippocampal neurogenesis is not a single phenotype, but consists of a number of sub-processes, each of which is under complex genetic control. Interpretation of gene expression studies using existing resources often does not lead to results that address the interrelatedness of these processes. Formal structure, such as provided by ontologies, is essential in any field for comprehensive interpretation of existing knowledge but, until now, such a structure has been lacking for adult neurogenesis. Methodology/Principal Findings We have created a resource with three components 1. A structured ontology describing the key stages in the development of adult hippocampal neural stem cells into functional granule cell neurons. 2. A comprehensive survey of the literature to annotate the results of all published reports on gene function in adult hippocampal neurogenesis (257 manuscripts covering 228 genes) to the appropriate terms in our ontology. 3. An easy-to-use searchable interface to the resulting database made freely available online. The manuscript presents an overview of the database highlighting global trends such as the current bias towards research on early proliferative stages, and an example gene set enrichment analysis. A limitation of the resource is the current scope of the literature which, however, is growing by around 100 publications per year. With the ontology and database in place, new findings can be rapidly annotated and regular updates of the database will be made publicly available. Conclusions/Significance The resource we present allows relevant interpretation of gene expression screens in terms of defined stages of postnatal neuronal development. Annotation of genes by hand from the adult neurogenesis literature ensures the data are directly applicable to the system under study. We believe this approach could also serve as an example to other fields in a ‘bottom-up’ community effort complementing the already successful

  2. The mammalian adult neurogenesis gene ontology (MANGO) provides a structural framework for published information on genes regulating adult hippocampal neurogenesis.

    PubMed

    Overall, Rupert W; Paszkowski-Rogacz, Maciej; Kempermann, Gerd

    2012-01-01

    Adult hippocampal neurogenesis is not a single phenotype, but consists of a number of sub-processes, each of which is under complex genetic control. Interpretation of gene expression studies using existing resources often does not lead to results that address the interrelatedness of these processes. Formal structure, such as provided by ontologies, is essential in any field for comprehensive interpretation of existing knowledge but, until now, such a structure has been lacking for adult neurogenesis. We have created a resource with three components 1. A structured ontology describing the key stages in the development of adult hippocampal neural stem cells into functional granule cell neurons. 2. A comprehensive survey of the literature to annotate the results of all published reports on gene function in adult hippocampal neurogenesis (257 manuscripts covering 228 genes) to the appropriate terms in our ontology. 3. An easy-to-use searchable interface to the resulting database made freely available online. The manuscript presents an overview of the database highlighting global trends such as the current bias towards research on early proliferative stages, and an example gene set enrichment analysis. A limitation of the resource is the current scope of the literature which, however, is growing by around 100 publications per year. With the ontology and database in place, new findings can be rapidly annotated and regular updates of the database will be made publicly available. The resource we present allows relevant interpretation of gene expression screens in terms of defined stages of postnatal neuronal development. Annotation of genes by hand from the adult neurogenesis literature ensures the data are directly applicable to the system under study. We believe this approach could also serve as an example to other fields in a 'bottom-up' community effort complementing the already successful 'top-down' approach of the Gene Ontology.

  3. N-terminal domain of alphaB-crystallin provides a conformational switch for multimerization and structural heterogeneity.

    PubMed

    Jehle, Stefan; Vollmar, Breanna S; Bardiaux, Benjamin; Dove, Katja K; Rajagopal, Ponni; Gonen, Tamir; Oschkinat, Hartmut; Klevit, Rachel E

    2011-04-19

    The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.

  4. Insights into remodeling events during eukaryotic large ribosomal subunit assembly provided by high resolution cryo-EM structures.

    PubMed

    Biedka, Stephanie; Wu, Shan; LaPeruta, Amber J; Gao, Ning; Woolford, John L

    2017-03-07

    Ribosomes are responsible for translating the genome, in the form of mRNA, into the proteome in all organisms. Biogenesis of ribosomes in eukaryotes is a complex process involving numerous remodeling events driven in part by the concerted actions of hundreds of protein assembly factors. A major challenge in studying eukaryotic ribosome assembly has, until recently, been a lack of structural data to facilitate understanding of the conformational and compositional changes the pre-ribosome undergoes during its construction. Cryo-electron microscopy (cryo-EM) has begun filling these gaps; recent advances in cryo-EM have enabled the determination of several high resolution pre-ribosome structures. This review focuses mainly on lessons learned from the study of pre-60S particles purified from yeast using the assembly factor Nog2 as bait. These Nog2 particles provide insight into many aspects of nuclear stages of 60S subunit assembly, including construction of major 60S subunit functional centers and processing of the ITS2 spacer RNA.

  5. Novel UDP-GalNAc Derivative Structures Provide Insight into the Donor Specificity of Human Blood Group Glycosyltransferase.

    PubMed

    Wagner, Gerd K; Pesnot, Thomas; Palcic, Monica M; Jørgensen, Rene

    2015-12-25

    Two closely related glycosyltransferases are responsible for the final step of the biosynthesis of ABO(H) human blood group A and B antigens. The two enzymes differ by only four amino acid residues, which determine whether the enzymes transfer GalNAc from UDP-GalNAc or Gal from UDP-Gal to the H-antigen acceptor. The enzymes belong to the class of GT-A folded enzymes, grouped as GT6 in the CAZy database, and are characterized by a single domain with a metal dependent retaining reaction mechanism. However, the exact role of the four amino acid residues in the specificity of the enzymes is still unresolved. In this study, we report the first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative. Interestingly, the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the "tucked under" conformation previously observed for the UDP-Gal donor. In addition, we show that this UDP-GalNAc derivative in complex with the H-antigen acceptor provokes the same unusual binding pocket closure as seen for the corresponding UDP-Gal derivative. Despite this, the two derivatives show vastly different kinetic properties. Our results provide a important structural insight into the donor substrate specificity and utilization in blood group biosynthesis, which can very likely be exploited for the development of new glycosyltransferase inhibitors and probes.

  6. Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

    PubMed Central

    Marcaida, María José; Prieto, Jesús; Redondo, Pilar; Nadra, Alejandro D.; Alibés, Andreu; Serrano, Luis; Grizot, Sylvestre; Duchateau, Philippe; Pâques, Frédéric; Blanco, Francisco J.; Montoya, Guillermo

    2008-01-01

    Homing endonucleases, also known as meganucleases, are sequence-specific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold. PMID:18974222

  7. Predicting protein subcellular locations with feature selection and analysis.

    PubMed

    Cai, Yudong; He, Jianfeng; Li, Xinlei; Feng, Kaiyan; Lu, Lin; Feng, Kairui; Kong, Xiangyin; Lu, Wencong

    2010-04-01

    In this paper, we propose a strategy to predict the subcellular locations of proteins by combining various feature selection methods. Firstly, proteins are coded by amino-acid composition and physicochemical properties, then these features are arranged by Minimum Redundancy Maximum Relevance method and further filtered by feature selection procedure. Nearest Neighbor Algorithm is used as a prediction model to predict the protein subcellular locations, and gains a correct prediction rate of 70.63%, evaluated by Jackknife cross-validation. Results of feature selection also enable us to identify the most important protein properties. The prediction software is available for public access on the website http://chemdata.shu.edu.cn/sub22/, which may play a important complementary role to a series of web-server predictors summarized recently in a review by Chou and Shen (Chou, K.C., Shen, H.B. Natural Science, 2009, 2, 63-92, http://www.scirp.org/journal/NS/).

  8. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    NASA Astrophysics Data System (ADS)

    Weng, Shinuo; Shao, Yue; Chen, Weiqiang; Fu, Jianping

    2016-09-01

    Mechanical homeostasis--a fundamental process by which cells maintain stable states under environmental perturbations--is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs). Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths through either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modelling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviours in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight the previously underappreciated physical nature of the mechanical homeostasis of cells.

  9. Mechanosensitive subcellular rheostasis drives emergent single-cell mechanical homeostasis

    PubMed Central

    Chen, Weiqiang; Fu, Jianping

    2016-01-01

    Mechanical homeostasis - a fundamental process by which cells maintain stable states under environmental perturbations - is regulated by two subcellular mechanotransducers: cytoskeleton tension and integrin-mediated focal adhesions (FAs)1-5. Here, we show that single-cell mechanical homeostasis is collectively driven by the distinct, graduated dynamics (rheostasis) of subcellular cytoskeleton tension and FAs. Such rheostasis involves a mechanosensitive pattern wherein ground states of cytoskeleton tension and FA determine their distinct reactive paths via either relaxation or reinforcement. Pharmacological perturbations of the cytoskeleton and molecularly modulated integrin catch-slip bonds biased the rheostasis and induced non-homeostasis of FAs, but not of cytoskeleton tension, suggesting a unique sensitivity of FAs in regulating homeostasis. Theoretical modeling revealed myosin-mediated cytoskeleton contractility and catch-slip-bond-like behaviors in FAs and the cytoskeleton as sufficient and necessary mechanisms for quantitatively recapitulating mechanosensitive rheostasis. Our findings highlight previously underappreciated physical nature of the mechanical homeostasis of cells. PMID:27240108

  10. Hepatic Subcellular Distribution of (3H)T-2 Toxin

    DTIC Science & Technology

    1989-02-10

    glucuronide conjugates. Glucuronide conjugates accounted for radiolabel eliminated via the bile. The time course for distri ution of radiolabel in liver...subcellular distribution of T-2 mycotoxin and its metabolites was studied in isolated rat livers perfused with ( 3 HJT-2 toxin. After a 120-min perfusion, the...associated with HT-2, 4-deacetylneosolaniol, T-2 tetraol, and glucuronide conjugates. Glucuronide conjugates accounted for radiolabel eliminated via the

  11. Intracellular And Subcellular Partitioning Of Nickel In Aureococcus Anophagefferens

    NASA Astrophysics Data System (ADS)

    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.

  12. Motion compensation for in vivo subcellular optical microscopy.

    PubMed

    Lucotte, B; Balaban, R S

    2014-04-01

    In this review, we focus on the impact of tissue motion on attempting to conduct subcellular resolution optical microscopy, in vivo. Our position is that tissue motion is one of the major barriers in conducting these studies along with light induced damage, optical probe loading as well as absorbing and scattering effects on the excitation point spread function and collection of emitted light. Recent developments in the speed of image acquisition have reached the limit, in most cases, where the signal from a subcellular voxel limits the speed and not the scanning rate of the microscope. Different schemes for compensating for tissue displacements due to rigid body and deformation are presented from tissue restriction, gating, adaptive gating and active tissue tracking. We argue that methods that minimally impact the natural physiological motion of the tissue are desirable because the major reason to perform in vivo studies is to evaluate normal physiological functions. Towards this goal, active tracking using the optical imaging data itself to monitor tissue displacement and either prospectively or retrospectively correct for the motion without affecting physiological processes is desirable. Critical for this development was the implementation of near real time image processing in conjunction with the control of the microscope imaging parameters. Clearly, the continuing development of methods of motion compensation as well as significant technological solutions to the other barriers to tissue subcellular optical imaging in vivo, including optical aberrations and overall signal-to-noise ratio, will make major contributions to the understanding of cell biology within the body.

  13. Analysis of Ras/ERK Compartmentalization by Subcellular Fractionation.

    PubMed

    Agudo-Ibañez, Lorena; Crespo, Piero; Casar, Berta

    2017-01-01

    A vast number of stimuli use the Ras/Raf/MEK/ERK signaling cascade to transmit signals from their cognate receptors, in order to regulate multiple cellular functions, including key processes such as proliferation, cell cycle progression, differentiation, and survival. The duration, intensity and specificity of the responses are, in part, controlled by the compartmentalization/subcellular localization of the signaling intermediaries. Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site-specific regulatory mechanisms, distinctively engaging effector pathways and switching-on diverse genetic programs to generate a multitude of biological responses. The Ras effector pathway leading to ERKs activation is also subject to space-related regulatory processes. About half of ERK1/2 substrates are found in the nucleus and function mainly as transcription factors. The other half resides in the cytosol and other cellular organelles. Such subcellular distribution enhances the complexity of the Ras/ERK cascade and constitutes an essential mechanism to endow variability to its signals, which enables their participation in the regulation of a broad variety of functions. Thus, analyzing the subcellular compartmentalization of the members of the Ras/ERK cascade constitutes an important factor to be taken into account when studying specific biological responses evoked by Ras/ERK signals. Herein, we describe methods for such purpose.

  14. Dynamics of subcellular proteomes during brain development.

    PubMed

    McClatchy, Daniel B; Liao, Lujian; Lee, Ji Hyoung; Park, Sung Kyu; Yates, John R

    2012-04-06

    Many neurological disorders are caused by perturbations during brain development, but these perturbations cannot be readily identified until there is comprehensive description of the development process. In this study, we performed mass spectrometry analysis of the synaptosomal and mitochondrial fractions from three rat brain regions at four postnatal time points. To quantitate our analysis, we employed (15)N labeled rat brains using a technique called SILAM (stable isotope labeling in mammals). We quantified 167429 peptides and identified over 5000 statistically significant changes during development including known disease-associated proteins. Global analysis revealed distinct trends between the synaptic and nonsynaptic mitochondrial proteomes and common protein networks between regions each consisting of a unique array of expression patterns. Finally, we identified novel regulators of neurodevelopment that possess the identical temporal pattern of known regulators of neurodevelopment. Overall, this study is the most comprehensive quantitative analysis of the developing brain proteome to date, providing an important resource for neurobiologists.

  15. Fluorescence lifetime imaging to quantify sub-cellular oxygen measurements in live macrophage during bacterial invasion

    NASA Astrophysics Data System (ADS)

    Dragavon, Joe; Amiri, Megdouda; Marteyn, Benoit; Sansonetti, Philipe; Shorte, Spencer

    2011-03-01

    Fluorophore concentration, the surrounding microenvironment, and photobleaching greatly influence the fluorescence intensity of a fluorophore, increasing the difficulty to directly observe micro-environmental factors such as pH and oxygen. However, the fluorescence lifetime of a fluorophore is essentially independent of both the fluorophore concentration and photobleaching, providing a viable alternative to intensity measurements. The development of fluorescence lifetime imaging (FLI) allows for the direct measurement of the microenvironment surrounding a fluorophore. Pt-porphyrin is a fluorophore whose optical properties include a very stable triplet excited state. This energy level overlaps strongly with the ground triplet state of oxygen, making the phosphorescent lifetime directly proportional to the surrounding oxygen concentration. Initial experiments using this fluorophore involved the use of individual microwells coated with the porphyrin. Cells were allowed to enter the micro-wells before being sealed to create a diffusionally isolated volume. The decrease in the extracellular oxygen concentration was observed using FLI. However, this isolation technique provides only the consumption rate but cannot indicate the subcellular oxygen distribution. To improve upon this, live macrophages are loaded with the porphyrin and the fluorescence lifetime determined using a Lambert Instruments Lifa-X FLI system. Initial results indicate that an increase in subcellular oxygen is observed upon initial exposure to invasive bacteria. A substantial decrease in oxygen is observed after about 1 hour of exposure. The cells remain in this deoxygenated state until the bacteria are removed or cell death occurs.

  16. Chemotactic recruitment of adult neural progenitor cells into multifunctional hydrogels providing sustained SDF-1α release and compatible structural support.

    PubMed

    Lim, Teck Chuan; Rokkappanavar, Swetha; Toh, Wei Seong; Wang, Li-Shan; Kurisawa, Motoichi; Spector, Myron

    2013-03-01

    Without chemotactic cues and structural support, cavitary brain lesions typically fail to recruit endogenous neural progenitor cells (NPCs). Toward resolving this, we engineered multifunctional biomaterials comprising injectable gelatin-hydroxyphenylpropionic acid (Gtn-HPA) hydrogels and dextran sulfate/chitosan polyelectrolyte complex nanoparticles (PCNs) that delivered stromal cell-derived factor-1α (SDF-1α). Over 7 d of interface with in vitro tissue simulant containing adult rat hippocampal NPCs (aNPCs) and their neuronal progeny, Gtn-HPA/SDF-1α-PCN hydrogels promoted chemotactic recruitment to enhance infiltration of aNPCs by 3- to 45-fold relative to hydrogels that lacked SDF-1α or vehicles to sustain SDF-1α release. When cross-linked with 0.85-0.95 mM HO, Gtn-HPA/SDF-1α-PCN hydrogels provided optimally permissive structural support for migration of aNPCs. Specific matrix metalloproteinase (MMP) inhibitors revealed that 42, 30, and 55% of cell migration into Gtn-HPA/SDF-1α-PCN hydrogels involved MMP-2, 3, and 9, respectively, demonstrating the hydrogels to be compatible toward homing endogenous NPCs, given their expression of similar MMPs. Interestingly, PCNs utilized FGF-2 found in situ to induce chemokinesis, potentiate SDF-1α chemotactic recruitment, and increase proliferation of recruited cells, which collectively orchestrated a higher number of migrated aNPCs. Overall, Gtn-HPA/SDF-1α-PCN hydrogels prove to be promising biomaterials for injection into cavitary brain lesions to recruit endogenous NPCs and enhance neural tissue repair/regeneration.

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

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash

    2008-12-01

    provided a rule-of-thumb criterion for the validation of sample preparation. The fractured freeze-dried cells allowed 3-D SIMS imaging and localization of 13C 15N labeled molecules and therapeutic drugs containing an elemental tag. Examples are shown to demonstrate that both diffusible elements and molecules are prone to artifact-induced relocation at subcellular scale if the sample preparation is compromised. The sample preparation is problem dependent and may vary widely between the diverse sample types of biological systems and the type of instrument used for SIMS analysis. The sample preparation, however, must be validated so that SIMS can be applied with confidence in biology and medicine.

  18. The effect of organelle discovery upon sub-cellular protein localisation.

    PubMed

    Breckels, L M; Gatto, L; Christoforou, A; Groen, A J; Lilley, K S; Trotter, M W B

    2013-08-02

    Prediction of protein sub-cellular localisation by employing quantitative mass spectrometry experiments is an expanding field. Several methods have led to the assignment of proteins to specific subcellular localisations by partial separation of organelles across a fractionation scheme coupled with computational analysis. Methods developed to analyse organelle data have largely employed supervised machine learning algorithms to map unannotated abundance profiles to known protein-organelle associations. Such approaches are likely to make association errors if organelle-related groupings present in experimental output are not included in data used to create a protein-organelle classifier. Currently, there is no automated way to detect organelle-specific clusters within such datasets. In order to address the above issues we adapted a phenotype discovery algorithm, originally created to filter image-based output for RNAi screens, to identify putative subcellular groupings in organelle proteomics experiments. We were able to mine datasets to a deeper level and extract interesting phenotype clusters for more comprehensive evaluation in an unbiased fashion upon application of this approach. Organelle-related protein clusters were identified beyond those sufficiently annotated for use as training data. Furthermore, we propose avenues for the incorporation of observations made into general practice for the classification of protein-organelle membership from quantitative MS experiments. Protein sub-cellular localisation plays an important role in molecular interactions, signalling and transport mechanisms. The prediction of protein localisation by quantitative mass-spectrometry (MS) proteomics is a growing field and an important endeavour in improving protein annotation. Several such approaches use gradient-based separation of cellular organelle content to measure relative protein abundance across distinct gradient fractions. The distribution profiles are commonly mapped in

  19. The Diversification of the LIM Superclass at the Base of the Metazoa Increased Subcellular Complexity and Promoted Multicellular Specialization

    PubMed Central

    Baxevanis, Andreas D.

    2012-01-01

    Background Throughout evolution, the LIM domain has been deployed in many different domain configurations, which has led to the formation of a large and distinct group of proteins. LIM proteins are involved in relaying stimuli received at the cell surface to the nucleus in order to regulate cell structure, motility, and division. Despite their fundamental roles in cellular processes and human disease, little is known about the evolution of the LIM superclass. Results We have identified and characterized all known LIM domain-containing proteins in six metazoans and three non-metazoans. In addition, we performed a phylogenetic analysis on all LIM domains and, in the process, have identified a number of novel non-LIM domains and motifs in each of these proteins. Based on these results, we have formalized a classification system for LIM proteins, provided reasonable timing for class and family origin events; and identified lineage-specific loss events. Our analysis is the first detailed description of the full set of LIM proteins from the non-bilaterian species examined in this study. Conclusion Six of the 14 LIM classes originated in the stem lineage of the Metazoa. The expansion of the LIM superclass at the base of the Metazoa undoubtedly contributed to the increase in subcellular complexity required for the transition from a unicellular to multicellular lifestyle and, as such, was a critically important event in the history of animal multicellularity. PMID:22438907

  20. Three-dimensional optical method for integrated visualization of mouse islet microstructure and vascular network with subcellular-level resolution

    NASA Astrophysics Data System (ADS)

    Fu, Ya-Yuan; Lu, Chih-Hsuan; Lin, Chi-Wen; Juang, Jyuhn-Huarng; Enikolopov, Grigori; Sibley, Eric; Chiang, Ann-Shyn; Tang, Shiue-Cheng

    2010-07-01

    Microscopic visualization of islets of Langerhans under normal and diabetic conditions is essential for understanding the pathophysiology of the disease. The intrinsic opacity of pancreata, however, limits optical accessibility for high-resolution light microscopy of islets in situ. Because the standard microtome-based, 2-D tissue analysis confines visualization of the islet architecture at a specific cut plane, 3-D representation of image data is preferable for islet assessment. We applied optical clearing to minimize the random light scattering in the mouse pancreatic tissue. The optical-cleared pancreas allowed penetrative, 3-D microscopic imaging of the islet microstructure and vasculature. Specifically, the islet vasculature was revealed by vessel painting-lipophilic dye labeling of blood vessels-for confocal microscopy. The voxel-based confocal micrographs were digitally processed with projection algorithms for 3-D visualization. Unlike the microtome-based tissue imaging, this optical method for penetrative imaging of mouse islets yielded clear, continuous optical sections for an integrated visualization of the islet microstructure and vasculature with subcellular-level resolution. We thus provide a useful imaging approach to change our conventional planar view of the islet structure into a 3-D panorama for better understanding of the islet physiology.

  1. Predicting protein subcellular localization based on information content of gene ontology terms.

    PubMed

    Zhang, Shu-Bo; Tang, Qiang-Rong

    2016-12-01

    Predicting the location where a protein resides within a cell is important in cell biology. Computational approaches to this issue have attracted more and more attentions from the community of biomedicine. Among the protein features used to predict the subcellular localization of proteins, the feature derived from Gene Ontology (GO) has been shown to be superior to others. However, most of the sights in this field are set on the presence or absence of some predefined GO terms. We proposed a method to derive information from the intrinsic structure of the GO graph. The feature vector was constructed with each element in it representing the information content of the GO term annotating to a protein investigated, and the support vector machines was used as classifier to test our extracted features. Evaluation experiments were conducted on three protein datasets and the results show that our method can enhance eukaryotic and human subcellular location prediction accuracy by up to 1.1% better than previous studies that also used GO-based features. Especially in the scenario where the cellular component annotation is absent, our method can achieved satisfied results with an overall accuracy of more than 87%.

  2. Protein expression and subcellular localization of the general purine transporter UapC from Aspergillus nidulans.

    PubMed

    Valdez-Taubas, J; Diallinas, G; Scazzocchio, C; Rosa, A L

    2000-07-01

    The uapC gene of Aspergillus nidulans belongs to a family of nucleobase-specific transporters conserved in prokaryotic and eucaryotic organisms. We report the use of immunological and green fluorescent protein based strategies to study protein expression and subcellular distribution of UapC. A chimeric protein containing a plant-adapted green fluorescent protein (sGFP) fused to the C-terminus of UapC was shown to be functional in vivo, as it complements a triple mutant (i.e., uapC(-) uapA(-) azgA(-)) unable to grow on uric acid as the sole nitrogen source. UapC-GFP is located in the plasma membrane and, secondarily, in internal structures observed as fluorescent dots. A strong correlation was found between cellular levels of UapC-GFP fluorescence and known patterns of uapC gene expression. This work represents the first in vivo study of protein expression and subcellular localization of a filamentous fungal nucleobase transporter.

  3. Plasmodium berghei MAPK1 Displays Differential and Dynamic Subcellular Localizations during Liver Stage Development

    PubMed Central

    Wierk, Jannika Katharina; Langbehn, Annette; Kamper, Maria; Richter, Stefanie; Burda, Paul-Christian; Heussler, Volker Theo; Deschermeier, Christina

    2013-01-01

    Mitogen-activated protein kinases (MAPKs) regulate key signaling events in eukaryotic cells. In the genomes of protozoan Plasmodium parasites, the causative agents of malaria, two genes encoding kinases with significant homology to other eukaryotic MAPKs have been identified (mapk1, mapk2). In this work, we show that both genes are transcribed during Plasmodium berghei liver stage development, and analyze expression and subcellular localization of the PbMAPK1 protein in liver stage parasites. Live cell imaging of transgenic parasites expressing GFP-tagged PbMAPK1 revealed a nuclear localization of PbMAPK1 in the early schizont stage mediated by nuclear localization signals in the C-terminal domain. In contrast, a distinct localization of PbMAPK1 in comma/ring-shaped structures in proximity to the parasite’s nuclei and the invaginating parasite membrane was observed during the cytomere stage of parasite development as well as in immature blood stage schizonts. The PbMAPK1 localization was found to be independent of integrity of a motif putatively involved in ATP binding, integrity of the putative activation motif and the presence of a predicted coiled-coil domain in the C-terminal domain. Although PbMAPK1 knock out parasites showed normal liver stage development, the kinase may still fulfill a dual function in both schizogony and merogony of liver stage parasites regulated by its dynamic and stage-dependent subcellular localization. PMID:23544094

  4. Isolation of plasma membranes from murine ependymoblastoma and subcellular distribution of amphotericin B in this tumor.

    PubMed

    Laurent, G; Doriaux, M; Hildebrand, J

    1977-04-01

    A method for the isolation of plasma membranes from an experimental murine ependymoblastoma is described. In this procedure, 5'-nucleotidase was used as the plasma membrane marker, since cytochemical methods demonstrated that the enzyme was present on this subcellular structure only. The final plasma membrane preparation showed a 15-fold enrichment in 5'-nucleotidase activity and a 17-fold enrichment in the activity of phosphodiesterase I, another plasma membrane marker. The specific activity of beta-glucuronidase (lysosomal enzyme) was twice that of the whole homogenate, the specific activity of arylesterase (microsomal enzyme) was similar to that of the whole homogenate and succinate dehydrogenase (mitochondrial marker) was not detected. Electron microscopy of this fraction showed vesicles on which 5'-nucleotidase activity could be demonstrated. The subcellular distribution of [3H]amphotericin B per mg of protein was similar in the plasma membrane preparation and in the whole homogenate. It is concluded that, in ependymoblastoma, amphotericin B shows no selective affinity for the plasma membrane.

  5. Mono- and Dinuclear Phosphorescent Rhenium(I) Complexes: Impact of Subcellular Localization on Anticancer Mechanisms.

    PubMed

    Ye, Rui-Rong; Tan, Cai-Ping; Chen, Mu-He; Hao, Liang; Ji, Liang-Nian; Mao, Zong-Wan

    2016-06-01

    Elucidation of relationship among chemical structure, cellular uptake, localization, and biological activity of anticancer metal complexes is important for the understanding of their mechanisms of action. Organometallic rhenium(I) tricarbonyl compounds have emerged as potential multifunctional anticancer drug candidates that can integrate therapeutic and imaging capabilities in a single molecule. Herein, two mononuclear phosphorescent rhenium(I) complexes (Re1 and Re2), along with their corresponding dinuclear complexes (Re3 and Re4), were designed and synthesized as potent anticancer agents. The subcellular accumulation of Re1-Re4 was conveniently analyzed by confocal microscopy in situ in live cells by utilizing their intrinsic phosphorescence. We found that increased lipophilicity of the bidentate ligands could enhance their cellular uptake, leading to improved anticancer efficacy. The dinuclear complexes were more potent than the mononuclear counterparts. The molecular anticancer mechanisms of action evoked by Re3 and Re4 were explored in detail. Re3 with a lower lipophilicity localizes to lysosomes and induces caspase-independent apoptosis, whereas Re4 with higher lipophilicity specially accumulates in mitochondria and induces caspase-independent paraptosis in cancer cells. Our study demonstrates that subcellular localization is crucial for the anticancer mechanisms of these phosphorescent rhenium(I) complexes.

  6. Analysis of potato virus X replicase and TGBp3 subcellular locations

    SciTech Connect

    Bamunusinghe, Devinka; Hemenway, Cynthia L.; Nelson, Richard S.; Sanderfoot, Anton A.; Ye, Chang M.; Silva, Muniwarage A.T.; Payton, M.; Verchot-Lubicz, Jeanmarie

    2009-10-25

    Potato virus X (PVX) infection leads to certain cytopathological modifications of the host endomembrane system. The subcellular location of the PVX replicase was previously unknown while the PVX TGBp3 protein was previously reported to reside in the ER. Using PVX infectious clones expressing the green fluorescent protein reporter, and antisera detecting the PVX replicase and host membrane markers, we examined the subcellular distribution of the PVX replicase in relation to the TGBp3. Confocal and electron microscopic observations revealed that the replicase localizes in membrane bound structures that derive from the ER. A subset of TGBp3 resides in the ER at the same location as the replicase. Sucrose gradient fractionation showed that the PVX replicase and TGBp3 proteins co-fractionate with ER marker proteins. This localization represents a region where both proteins may be synthesized and/or function. There is no evidence to indicate that either PVX protein moves into the Golgi apparatus. Cerulenin, a drug that inhibits de novo membrane synthesis, also inhibited PVX replication. These combined data indicate that PVX replication relies on ER-derived membrane recruitment and membrane proliferation.

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

    PubMed Central

    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. PMID:24744697

  8. Changes in architecture of the Golgi complex and other subcellular organelles during myogenesis

    PubMed Central

    1993-01-01

    Myogenesis involves changes in both gene expression and cellular architecture. Little is known of the organization, in muscle in vivo, of the subcellular organelles involved in protein synthesis despite the potential importance of targeted protein synthesis for formation and maintenance of functional domains such as the neuromuscular junction. A panel of antibodies to markers of the ER, the Golgi complex, and the centrosome were used to localize these organelles by immunofluorescence in myoblasts and myotubes of the mouse muscle cell line C2 in vitro, and in intact single muscle fibers from the rat flexor digitorum brevis. Antibodies to the ER stained structures throughout the cytoplasm of both C2 myoblasts and myotubes. In contrast, the spatial relationship between nucleus, centrosome, and Golgi complex was dramatically altered. These changes could also be observed in a low- calcium medium that allowed differentiation while preventing myoblast fusion. Muscle fibers in vivo resembled myotubes except that the ER occupied a smaller volume of cytoplasm and no staining was found for one of the Golgi complex markers, the enzyme alpha-mannosidase II. Electron microscopy, however, clearly showed the presence of stacks of Golgi cisternae in both junctional and extrajunctional regions of muscle fibers. The perinuclear distribution of the Golgi complex was also observed in live muscle fibers stained with a fluorescent lipid. Thus, the distribution of subcellular organelles of the secretory pathway was found to be similar in myotubes and muscle fibers, and all organelles were found in both junctional and extrajunctional areas of muscle. PMID:7678420

  9. Incoordination among Subcellular Compartments Is Associated with Depression-Like Behavior Induced by Chronic Mild Stress

    PubMed Central

    Xu, Aiping; Cui, Shan

    2016-01-01

    Background: Major depressive disorder is characterized as persistent low mood. A chronically stressful life in genetically susceptible individuals is presumably the major etiology that leads to dysfunctions of monoamine and hypothalamus-pituitary-adrenal axis. These pathogenic factors cause neuron atrophy in the limbic system for major depressive disorder. Cell-specific pathophysiology is unclear, so we investigated prelimbic cortical GABAergic neurons and their interaction with glutamatergic neurons in depression-like mice. Methods: Mice were treated with chronic unpredictable mild stress for 3 weeks until they expressed depression-like behaviors confirmed by sucrose preference, Y-maze, and forced swimming tests. The structures and functions of GABAergic and glutamatergic units in prelimbic cortices were studied by cell imaging and electrophysiology in chronic unpredictable mild stress-induced depression mice vs controls. Results: In depression-like mice, prelimbic cortical GABAergic neurons show incoordination among the subcellular compartments, such as decreased excitability and synaptic outputs as well as increased reception from excitatory inputs. GABAergic synapses on glutamatergic cells demonstrate decreased presynaptic innervation and increased postsynaptic responsiveness. Conclusions: Chronic unpredictable mild stress-induced incoordination in prelimbic cortical GABAergic and glutamatergic neurons dysregulates their target neurons, which may be the pathological basis for depressive mood. The rebalance of compatibility among subcellular compartments would be an ideal strategy to treat neural disorders. PMID:26506857

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

    PubMed

    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.

  11. Spatially extensive microbial biogeography of the Indian Ocean provides insights into the unique community structure of a pristine coral atoll

    PubMed Central

    Jeffries, Thomas C.; Ostrowski, Martin; Williams, Rohan B.; Xie, Chao; Jensen, Rachelle M.; Grzymski, Joseph J.; Senstius, Svend Jacob; Givskov, Michael; Hoeke, Ron; Philip, Gayle K.; Neches, Russell Y.; Drautz-Moses, Daniela I.; Chénard, Caroline; Paulsen, Ian T.; Lauro, Federico M.

    2015-01-01

    Microorganisms act both as drivers and indicators of perturbations in the marine environment. In an effort to establish baselines to predict the response of marine habitats to environmental change, here we report a broad survey of microbial diversity across the Indian Ocean, including the first microbial samples collected in the pristine lagoon of Salomon Islands, Chagos Archipelago. This was the first large-scale ecogenomic survey aboard a private yacht employing a ‘citizen oceanography’ approach and tools and protocols easily adapted to ocean going sailboats. Our data highlighted biogeographic patterns in microbial community composition across the Indian Ocean. Samples from within the Salomon Islands lagoon contained a community which was different even from adjacent samples despite constant water exchange, driven by the dominance of the photosynthetic cyanobacterium Synechococcus. In the lagoon, Synechococcus was also responsible for driving shifts in the metatranscriptional profiles. Enrichment of transcripts related to photosynthesis and nutrient cycling indicated bottom-up controls of community structure. However a five-fold increase in viral transcripts within the lagoon during the day, suggested a concomitant top-down control by bacteriophages. Indeed, genome recruitment against Synechococcus reference genomes suggested a role of viruses in providing the ecological filter for determining the β-diversity patterns in this system. PMID:26481089

  12. Spatially extensive microbial biogeography of the Indian Ocean provides insights into the unique community structure of a pristine coral atoll

    NASA Astrophysics Data System (ADS)

    Jeffries, Thomas C.; Ostrowski, Martin; Williams, Rohan B.; Xie, Chao; Jensen, Rachelle M.; Grzymski, Joseph J.; Senstius, Svend Jacob; Givskov, Michael; Hoeke, Ron; Philip, Gayle K.; Neches, Russell Y.; Drautz-Moses, Daniela I.; Chénard, Caroline; Paulsen, Ian T.; Lauro, Federico M.

    2015-10-01

    Microorganisms act both as drivers and indicators of perturbations in the marine environment. In an effort to establish baselines to predict the response of marine habitats to environmental change, here we report a broad survey of microbial diversity across the Indian Ocean, including the first microbial samples collected in the pristine lagoon of Salomon Islands, Chagos Archipelago. This was the first large-scale ecogenomic survey aboard a private yacht employing a ‘citizen oceanography’ approach and tools and protocols easily adapted to ocean going sailboats. Our data highlighted biogeographic patterns in microbial community composition across the Indian Ocean. Samples from within the Salomon Islands lagoon contained a community which was different even from adjacent samples despite constant water exchange, driven by the dominance of the photosynthetic cyanobacterium Synechococcus. In the lagoon, Synechococcus was also responsible for driving shifts in the metatranscriptional profiles. Enrichment of transcripts related to photosynthesis and nutrient cycling indicated bottom-up controls of community structure. However a five-fold increase in viral transcripts within the lagoon during the day, suggested a concomitant top-down control by bacteriophages. Indeed, genome recruitment against Synechococcus reference genomes suggested a role of viruses in providing the ecological filter for determining the β-diversity patterns in this system.

  13. The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress.

    PubMed

    Yunta, Cristina; Martínez-Ripoll, Martín; Zhu, Jian-Kang; Albert, Armando

    2011-11-18

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.

  14. The Structure of Arabidopsis thaliana OST1 Provides Insights into the Kinase Regulation Mechanism in Response to Osmotic Stress

    PubMed Central

    Yunta, Cristina; Martínez-Ripoll, Martín; Zhu, Jian-Kang; Albert, Armando

    2013-01-01

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. PMID:21983340

  15. Spatially extensive microbial biogeography of the Indian Ocean provides insights into the unique community structure of a pristine coral atoll.

    PubMed

    Jeffries, Thomas C; Ostrowski, Martin; Williams, Rohan B; Xie, Chao; Jensen, Rachelle M; Grzymski, Joseph J; Senstius, Svend Jacob; Givskov, Michael; Hoeke, Ron; Philip, Gayle K; Neches, Russell Y; Drautz-Moses, Daniela I; Chénard, Caroline; Paulsen, Ian T; Lauro, Federico M

    2015-10-20

    Microorganisms act both as drivers and indicators of perturbations in the marine environment. In an effort to establish baselines to predict the response of marine habitats to environmental change, here we report a broad survey of microbial diversity across the Indian Ocean, including the first microbial samples collected in the pristine lagoon of Salomon Islands, Chagos Archipelago. This was the first large-scale ecogenomic survey aboard a private yacht employing a 'citizen oceanography' approach and tools and protocols easily adapted to ocean going sailboats. Our data highlighted biogeographic patterns in microbial community composition across the Indian Ocean. Samples from within the Salomon Islands lagoon contained a community which was different even from adjacent samples despite constant water exchange, driven by the dominance of the photosynthetic cyanobacterium Synechococcus. In the lagoon, Synechococcus was also responsible for driving shifts in the metatranscriptional profiles. Enrichment of transcripts related to photosynthesis and nutrient cycling indicated bottom-up controls of community structure. However a five-fold increase in viral transcripts within the lagoon during the day, suggested a concomitant top-down control by bacteriophages. Indeed, genome recruitment against Synechococcus reference genomes suggested a role of viruses in providing the ecological filter for determining the β-diversity patterns in this system.

  16. Structure of the CED-4-CED-9 Complex Provides Insights into Programmed Cell Death in Caenorhabditis elegans

    SciTech Connect

    Yan,N.; Chai, J.; Lee, E.; Gu, L.; Liu, Q.; He, J.; Wu, J.; Kokel, D.; Li, H.; et al.

    2005-01-01

    Interplay among four genes-egl-1, ced-9, ced-4 and ced-3-controls the onset of programmed cell death in the nematode Caenorhabditis elegans. Activation of the cell-killing protease CED-3 requires CED-4. However, CED-4 is constitutively inhibited by CED-9 until its release by EGL-1. Here we report the crystal structure of the CED-4-CED-9 complex at 2.6 Angstrom resolution, and a complete reconstitution of the CED-3 activation pathway using homogeneous proteins of CED-4, CED-9 and EGL-1. One molecule of CED-9 binds to an asymmetric dimer of CED-4, but specifically recognizes only one of the two CED-4 molecules. This specific interaction prevents CED-4 from activating CED-3. EGL-1 binding induces pronounced conformational changes in CED-9 that result in the dissociation of the CED-4 dimer from CED-9. The released CED-4 dimer further dimerizes to form a tetramer, which facilitates the autoactivation of CED-3. Together, our studies provide important insights into the regulation of cell death activation in C. elegans.

  17. Structural and functional diversity of cadherin at the adherens junction

    PubMed Central

    2011-01-01

    Adhesion between cells is essential to the evolution of multicellularity. Indeed, morphogenesis in animals requires firm but flexible intercellular adhesions that are mediated by subcellular structures like the adherens junction (AJ). A key component of AJs is classical cadherins, a group of transmembrane proteins that maintain dynamic cell–cell associations in many animal species. An evolutionary reconstruction of cadherin structure and function provides a comprehensive framework with which to appreciate the diversity of morphogenetic mechanisms in animals. PMID:21708975

  18. The COOLER Code: A Novel Analytical Approach to Calculate Subcellular Energy Deposition by Internal Electron Emitters.

    PubMed

    Siragusa, Mattia; Baiocco, Giorgio; Fredericia, Pil M; Friedland, Werner; Groesser, Torsten; Ottolenghi, Andrea; Jensen, Mikael

    2017-08-01

    COmputation Of Local Electron Release (COOLER), a software program has been designed for dosimetry assessment at the cellular/subcellular scale, with a given distribution of administered low-energy electron-emitting radionuclides in cellular compartments, which remains a critical step in risk/benefit analysis for advancements in internal radiotherapy. The software is intended to overcome the main limitations of the medical internal radiation dose (MIRD) formalism for calculations of cellular S-values (i.e., dose to a target region in the cell per decay in a given source region), namely, the use of the continuous slowing down approximation (CSDA) and the assumption of a spherical cell geometry. To this aim, we developed an analytical approach, entrusted to a MATLAB-based program, using as input simulated data for electron spatial energy deposition directly derived from full Monte Carlo track structure calculations with PARTRAC. Results from PARTRAC calculations on electron range, stopping power and residual energy versus traveled distance curves are presented and, when useful for implementation in COOLER, analytical fit functions are given. Example configurations for cells in different culture conditions (V79 cells in suspension or adherent culture) with realistic geometrical parameters are implemented for use in the tool. Finally, cellular S-value predictions by the newly developed code are presented for different cellular geometries and activity distributions (uniform activity in the nucleus, in the entire cell or on the cell surface), validated against full Monte Carlo calculations with PARTRAC, and compared to MIRD standards, as well as results based on different track structure calculations (Geant4-DNA). The largest discrepancies between COOLER and MIRD predictions were generally found for electrons between 25 and 30 keV, where the magnitude of disagreement in S-values can vary from 50 to 100%, depending on the activity distribution. In calculations for

  19. An exo-β-(1→3)-D-galactanase from Streptomyces sp. provides insights into type II arabinogalactan structure

    PubMed Central

    Ling, Naomi X.-Y.; Lee, Joanne; Ellis, Miriam; Liao, Ming-Long; Mau, Shaio-Lim; Guest, David; Janssen, Peter H.; Kováč, Pavol; Bacic, Antony; Pettolino, Filomena A.

    2012-01-01

    An exo-β-(1→3)-D-galactanase (SGalase1) that specifically cleaves the β-(1→3)-D-galactan backbone of arabinogalactan-proteins (AGPs) was isolated from culture filtrates of a soil Streptomyces sp. Internal peptide sequence information was used to clone and recombinantly express the gene in E. coli. The molecular mass of the isolated enzyme was ~45 kDa, similar to the 48.2 kDa mass predicted from the amino acid sequence. The pI, pH and temperature optima for the enzyme were ~7.45, 3.8 and 48 °C, respectively. The native and recombinant enzymes specifically hydrolysed β-(1→3)-D-galacto-oligo- or poly-saccharides from the upstream (non-reducing) end, typical of an exo-acting enzyme. A second homologous Streptomyces gene (SGalase2) was also cloned and expressed. SGalase2 was similar in size (47.9 kDa) and enzyme activity to SGalase1 but differed in its pH optimum (pH 5). Both SGalase1 and SGalase2 are predicted to belong to the CAZy glycosyl hydrolase family GH 43 based on activity, sequence homology and phylogenetic analysis. The Km and Vmax of the native exo-β-(1→3)-D-galactanase for de-arabinosylated gum arabic (dGA) were 19 mg/ml and 9.7 μmol D-Gal/min/mg protein, respectively. The activity of these enzymes is well suited for the study of type II galactan structures and provides an important tool for the investigation of the biological role of AGPs in plants. De-arabinosylated gum arabic (dGA) was used as a model to investigate the use of these enzymes in defining type II galactan structure. Exhaustive hydrolysis of dGA resulted in a limited number of oligosaccharide products with a trisaccharide of Gal2GlcA1 predominating. PMID:22464224

  20. Combined glucosamine and chondroitin sulfate provides functional and structural benefit in the anterior cruciate ligament transection model.

    PubMed

    Silva, Francisco Saraiva; Yoshinari, Natalino Hajime; Castro, Rondinelle Ribeiro; Girão, Virgínia Cláudia Carneiro; Pompeu, Margarida Maria Lima; Feitosa, Judith Pessoa de Andrade; Rocha, Francisco Airton Castro

    2009-02-01

    Evidence that combined glucosamine sulfate and chondroitin sulfate (Gluchon) or isolated glucosamine (Glu) modifies joint damage in osteoarthritis (OA) is still lacking. We studied joint pain and cartilage damage using the anterior cruciate ligament transection (ACLT) model. Wistar rats were subjected to ACLT of the right knee (OA) or sham operation. Groups received either Glu (500 mg/kg), Gluchon (500 mg/kg glucosamine +400 mg/kg chondroitin) or vehicle (non-treated--NT) per os starting 7 days prior to ACLT until sacrifice at 70 days. Joint pain was evaluated daily using the rat-knee joint articular incapacitation test. Structural joint damage was assessed using histology and biochemistry as the chondroitin sulfate (CS) content of cartilage by densitometry (microgram per milligram dried cartilage), comparing to standard CS. The molar weight (Mw) of the CS samples, used as a qualitative biochemical parameter, was obtained by comparing their relative mobility on a polyacrylamide gel electrophoresis to standard CS. Gluchon, but not Glu, significantly reduced joint pain (P < 0.05) compared to NT. There was an increase in CS content in the OA group (77.7 +/- 8.3 microg/mg) compared to sham (53.5 +/- 11.2 microg/mg) (P < 0.05). The CS from OA samples had higher Mw (4:62 +/- 10(4) g/mol) compared to sham (4:18 +/- 0.19 x 10(4) g/mol) (P < 0.05). Gluchon administration significantly reversed both the increases in CS content (54.4 +/- 12.1 microg/mg) and Mw (4:18 +/- 0.2 x 10(4) g/mol) as compared to NT. Isolated Glu decreased CS content though not reaching statistical significance. Cartilage histology alterations were also significantly prevented by Gluchon administration. Gluchon provides clinical (analgesia) and structural benefits in the ACLT model. This is the first demonstration that biochemical alterations occurring in parallel to histological damage in OA are prevented by Gluchon administration.

  1. Subcellular Investigation of Photosynthesis-Driven Carbon Assimilation in the Symbiotic Reef Coral Pocillopora damicornis

    PubMed Central

    Domart-Coulon, Isabelle; Escrig, Stephane; Humbel, Bruno M.; Hignette, Michel

    2015-01-01

    ABSTRACT  Reef-building corals form essential, mutualistic endosymbiotic associations with photosynthetic Symbiodinium dinoflagellates, providing their animal host partner with photosynthetically derived nutrients that allow the coral to thrive in oligotrophic waters. However, little is known about the dynamics of these nutritional interactions at the (sub)cellular level. Here, we visualize with submicrometer spatial resolution the carbon and nitrogen fluxes in the intact coral-dinoflagellate association from the reef coral Pocillopora damicornis by combining nanoscale secondary ion mass spectrometry (NanoSIMS) and transmission electron microscopy with pulse-chase isotopic labeling using [13C]bicarbonate and [15N]nitrate. This allows us to observe that (i) through light-driven photosynthesis, dinoflagellates rapidly assimilate inorganic bicarbonate and nitrate, temporarily storing carbon within lipid droplets and starch granules for remobilization in nighttime, along with carbon and nitrogen incorporation into other subcellular compartments for dinoflagellate growth and maintenance, (ii) carbon-containing photosynthates are translocated to all four coral tissue layers, where they accumulate after only 15 min in coral lipid droplets from the oral gastroderm and within 6 h in glycogen granules from the oral epiderm, and (iii) the translocation of nitrogen-containing photosynthates is delayed by 3 h. PMID:25670779

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

    PubMed Central

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

    2014-01-01

    Purpose Manganese (Mn) is an effective contrast agent and biologically active metal, which has been widely utilized for Mn-enhanced MRI (MEMRI). The purpose of this study was to develop and test a Mn binding protein for use as an genetic reporter for MEMRI. Methods 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 and MR imaging and relaxometry. Results 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. Conclusion 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. PMID:25522343

  3. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development1[OPEN

    PubMed Central

    2016-01-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time. PMID:27406170

  4. Calcium signaling in live cells on elastic gels under mechanical vibration at subcellular levels.

    PubMed

    Nishitani, Wagner Shin; Saif, Taher A; Wang, Yingxiao

    2011-01-01

    A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels.

  5. Calcium Signaling in Live Cells on Elastic Gels under Mechanical Vibration at Subcellular Levels

    PubMed Central

    Nishitani, Wagner Shin; Saif, Taher A.; Wang, Yingxiao

    2011-01-01

    A new device was designed to generate a localized mechanical vibration of flexible gels where human umbilical vein endothelial cells (HUVECs) were cultured to mechanically stimulate these cells at subcellular locations. A Fluorescence Resonance Energy Transfer (FRET)-based calcium biosensor (an improved Cameleon) was used to monitor the spatiotemporal distribution of intracellular calcium concentrations in the cells upon this mechanical stimulation. A clear increase in intracellular calcium concentrations over the whole cell body (global) can be observed in the majority of cells under mechanical stimulation. The chelation of extracellular calcium with EGTA or the blockage of stretch-activated calcium channels on the plasma membrane with streptomycin or gadolinium chloride significantly inhibited the calcium responses upon mechanical stimulation. Thapsigargin, an endoplasmic reticulum (ER) calcium pump inhibitor, or U73122, a phospholipase C (PLC) inhibitor, resulted in mainly local calcium responses occurring at regions close to the stimulation site. The disruption of actin filaments with cytochalasin D or inhibition of actomyosin contractility with ML-7 also inhibited the global calcium responses. Therefore, the global calcium response in HUVEC depends on the influx of calcium through membrane stretch-activated channels, followed by the release of inositol trisphosphate (IP3) via PLC activation to trigger the ER calcium release. Our newly developed mechanical stimulation device can also provide a powerful tool for the study of molecular mechanism by which cells perceive the mechanical cues at subcellular levels. PMID:22053183

  6. A Balance Between Two Nuclear Localization Sequences and a Nuclear Export Sequence Governs Extradenticle Subcellular Localization

    PubMed Central

    Stevens, Katherine E.; Mann, Richard S.

    2007-01-01

    During animal development, transcription factor activities are modulated by several means, including subcellular localization. The Hox cofactor Extradenticle (Exd) has a dynamic subcellular localization, such that Exd is cytoplasmic by default, but is nuclear when complexed with another homeodomain protein, Homothorax (Hth). These observations raise the question of whether dimerization with Hth simply induces Exd's nuclear localization or, alternatively, if Hth is also necessary for Exd activity. To address this question, we analyzed the nuclear transport signals in Exd, including a divergent nuclear export signal (NES) and two nuclear localization signals (NLSs). We show that, although these signals are weak compared to canonical signals, they balance each other in Exd. We also provide evidence that Exd contains an NLS mask that contributes to its cytoplasmic localization. With these signals characterized, we generated forms of Exd that are nuclear localized in the absence of Hth. Surprisingly, although these Exd forms are functional, they do not phenocopy Hth overexpression. These findings suggest that Hth is required for Exd activity, not simply for inducing its nuclear localization. PMID:17277370

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

    PubMed

    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.

  8. Classification of Subcellular Phenotype Images by Decision Templates for Classifier Ensemble

    NASA Astrophysics Data System (ADS)

    Zhang, Bailing

    2010-01-01

    Subcellular localization is a key functional characteristic of proteins. An automatic, reliable and efficient prediction system for protein subcellular localization is needed for large-scale genome analysis. The automated cell phenotype image classification problem is an interesting "bioimage informatics" application. It can be used for establishing knowledge of the spatial distribution of proteins within living cells and permits to screen systems for drug discovery or for early diagnosis of a disease. In this paper, three well-known texture feature extraction methods including local binary patterns (LBP), Gabor filtering and Gray Level Coocurrence Matrix (GLCM) have been applied to cell phenotype images and the multiple layer perceptron (MLP) method has been used to classify cell phenotype image. After classification of the extracted features, decision-templates ensemble algorithm (DT) is used to combine base classifiers built on the different feature sets. Different texture feature sets can provide sufficient diversity among base classifiers, which is known as a necessary condition for improvement in ensemble performance. For the HeLa cells, the human classification error rate on this task is of 17% as reported in previous publications. We obtain with our method an error rate of 4.8%.

  9. Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

    PubMed

    Bascom, Carlisle S; Wu, Shu-Zon; Nelson, Katherine; Oakey, John; Bezanilla, Magdalena

    2016-09-01

    Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers. Because the PDMS chambers are bonded to a coverslip, it is possible to image P. patens development at high resolution over long time periods. Using PDMS chambers, we report that wild-type protonemal tissue grows at the same rate as previously reported for growth on solid medium. Using long-term imaging, we highlight key developmental events, demonstrate compatibility with high-resolution confocal microscopy, and obtain growth rates for a slow-growing mutant. By coupling the powerful genetic tools available to P. patens with long-term growth and imaging provided by PDMS microfluidic chambers, we demonstrate the capability to study cellular and subcellular developmental events in plants directly and in real time.

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

    PubMed Central

    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

  11. Analysis of subcellular localization of auxin carriers PIN, AUX/LAX and PGP in Sorghum bicolor

    PubMed Central

    Wang, SuiKang; Shen, ChenJia; Zhang, SaiNa; Xu, YanXia; Jiang, DeAn; Qi, YanHua

    2011-01-01

    Auxin transport at least correlates to the three gene families: efflux carriers PIN-formed (PIN), p-glycoprotein (PGP), and influx carrier auxin resistant 1/like aux1(AUX/LAX) in Arabidopsis thaliana. In monocotyledon Sorghum bicolor, the biological function of these genes retains unclear. Our previous study reported that the member analysis, organ-specific expression and expression profiles of the auxin transporter PIN, PGP and AUX/LAX gene families in Sorghum bicolor under IAA, brassinosteroid, polar auxin transport inhibitors and abiotic stresses. Here we further supply the prediction of subcellular localization of SbPIN, SbLAX and SbPGP proteins and discuss the potential relationship between the subcellular localization and stress response. The predicted results showed that the most of SbPIN, SbLAX and SbPGP proteins are localized to the plasma membrane, except few localized to vacuolar membrane and endoplasmic reticulum. This data set provides novel information for investigation of auxin transporters in Sorghum bicolor. PMID:22112459

  12. Protein subcellular localization of fluorescence imagery using spatial and transform domain features.

    PubMed

    Tahir, Muhammad; Khan, Asifullah; Majid, Abdul

    2012-01-01

    Subcellular localization of proteins is one of the most significant characteristics of living cells. Prediction of protein subcellular locations is crucial to the understanding of various protein functions. Therefore, an accurate, computationally efficient and reliable prediction system is required. In this article, the predictions of various Support Vector Machine (SVM) models have been combined through majority voting. The proposed ensemble SVM-SubLoc has achieved the highest success rates of 99.7% using hybrid features of Haralick textures and local binary patterns (HarLBP), 99.4% using hybrid features of Haralick textures and Local Ternary Patterns (HarLTP). In addition, SVM-SubLoc has yielded 99.0% accuracy using only local ternary patterns (LTPs) based features. The dimensionality of HarLBP feature vector is 581 compared with 78 and 52 for HarLTP and LTPs, respectively. Hence, SVM-SubLoc in conjunction with LTPs is fast, sufficiently accurate and simple predictive system. The proposed SVM-SubLoc approach thus provides superior prediction performance using the reduced feature space compared with existing approaches. A web server accompanying the proposed prediction scheme is available at http://111.68.99.218/ SVM-SubLoc asif@pieas.edu.pk; khan.asifullah@gmail.com Supplementary data are available at Bioinformatics online.

  13. Seascape genomics provides evidence for thermal adaptation and current-mediated population structure in American lobster (Homarus americanus).

    PubMed

    Benestan, Laura; Quinn, Brady K; Maaroufi, Halim; Laporte, Martin; Clark, Fraser K; Greenwood, Spencer J; Rochette, Rémy; Bernatchez, Louis

    2016-10-01

    Investigating how environmental features shape the genetic structure of populations is crucial for understanding how they are potentially adapted to their habitats, as well as for sound management. In this study, we assessed the relative importance of spatial distribution, ocean currents and sea surface temperature (SST) on patterns of putatively neutral and adaptive genetic variation among American lobster from 19 locations using population differentiation (PD) approaches combined with environmental association (EA) analyses. First, PD approaches (using bayescan, arlequin and outflank) found 28 outlier SNPs putatively under divergent selection and 9770 neutral SNPs in common. Redundancy analysis revealed that spatial distribution, ocean current-mediated larval connectivity and SST explained 31.7% of the neutral genetic differentiation, with ocean currents driving the majority of this relationship (21.0%). After removing the influence of spatial distribution, no SST were significant for putatively neutral genetic variation whereas minimum annual SST still had a significant impact and explained 8.1% of the putatively adaptive genetic variation. Second, EA analyses (using Pearson correlation tests, bayescenv and lfmm) jointly identified seven SNPs as candidates for thermal adaptation. Covariation at these SNPs was assessed with a spatial multivariate analysis that highlighted a significant temperature association, after accounting for the influence of spatial distribution. Among the 505 candidate SNPs detected by at least one of the three approaches, we discovered three polymorphisms located in genes previously shown to play a role in thermal adaptation. Our results have implications for the management of the American lobster and provide a foundation on which to predict how this species will cope with climate change.

  14. Could structural similarity of specific domains between animal globins and plant antenna proteins provide hints important for the photoprotection mechanism?

    PubMed

    Ioannidis, Nikolaos E; Kotzabasis, Kiriakos

    2015-01-07

    Non photochemical quenching is a fundamental mechanism in photosynthesis, which protects plants against excess excitation energy and is of crucial importance for their survival and fitness. In the last decades hundreds of papers have appeared that describe the role of antenna regulation in protection or the so called qE response. However, the exact quenching site is still obscure. Previously overlooked features of the antenna may provide hints towards the elucidation of its functionality and of the quenching mechanism. Recently it was demonstrated that the catalytic domain of human myoglobin that binds the pigment (i.e. heme) is similar in structure to the domain of the light harvesting complex II of pea that binds Chl a 614 (former known as b3). In addition, it is well accepted that conformational changes of the chlorophyll macrocycle result in reversible changes of fluorescence (the lowest fluorescence corresponds to non planar macrocycle). Here we put forward a hypothesis regarding the molecular mechanism that leads to the formation of a quenching center inside the antenna proteins. Our main suggestion is that a conformational change of helix H5 (known also as helix D) forces conformational changes in the macrocycle of Chl a 614 is implicated in the ΔA535 absorbance change and quenching during photoprotective qE. The specific features (some of them similar to those of heme domain of globins) of the b3 domain account for these traits. The model predicts that antenna proteins having b3 pigments (i.e. LHCII, CP29, CP26) can act as potential quenchers.

  15. The crystal structure of the intact E. coli RelBE toxin-antitoxin complex provides the structural basis for conditional cooperativity.

    PubMed

    Bøggild, Andreas; Sofos, Nicholas; Andersen, Kasper R; Feddersen, Ane; Easter, Ashley D; Passmore, Lori A; Brodersen, Ditlev E

    2012-10-10

    The bacterial relBE locus encodes a toxin-antitoxin complex in which the toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally. The antitoxin, RelB, both binds and inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. Here, we present the crystal structure of the intact Escherichia coli RelB2E2 complex at 2.8 Å resolution, comprising both the RelB-inhibited RelE and the RelB dimerization domain that binds DNA. RelE and RelB associate into a V-shaped heterotetrameric complex with the ribbon-helix-helix (RHH) dimerization domain at the apex. Our structure supports a model in which relO is optimally bound by two adjacent RelB2E heterotrimeric units, and is not compatible with concomitant binding of two RelB2E2 heterotetramers. The results thus provide a firm basis for understanding the model of conditional cooperativity at the molecular level.

  16. The Crystal Structure of the Intact E. coli RelBE Toxin-Antitoxin Complex Provides the Structural Basis for Conditional Cooperativity

    PubMed Central

    Bøggild, Andreas; Sofos, Nicholas; Andersen, Kasper R.; Feddersen, Ane; Easter, Ashley D.; Passmore, Lori A.; Brodersen, Ditlev E.

    2012-01-01

    Summary The bacterial relBE locus encodes a toxin-antitoxin complex in which the toxin, RelE, is capable of cleaving mRNA in the ribosomal A site cotranslationally. The antitoxin, RelB, both binds and inhibits RelE, and regulates transcription through operator binding and conditional cooperativity controlled by RelE. Here, we present the crystal structure of the intact Escherichia coli RelB2E2 complex at 2.8 Å resolution, comprising both the RelB-inhibited RelE and the RelB dimerization domain that binds DNA. RelE and RelB associate into a V-shaped heterotetrameric complex with the ribbon-helix-helix (RHH) dimerization domain at the apex. Our structure supports a model in which relO is optimally bound by two adjacent RelB2E heterotrimeric units, and is not compatible with concomitant binding of two RelB2E2 heterotetramers. The results thus provide a firm basis for understanding the model of conditional cooperativity at the molecular level. PMID:22981948

  17. Subcellular characterization of glucose uptake in coronary endothelial cells.

    PubMed

    Gaudreault, N; Scriven, D R L; Laher, I; Moore, E D W

    2008-01-01

    Despite all the evidence linking glucose toxicity to an increased risk of cardiovascular diseases, very little is known about the regulation of glucose uptake in endothelial cells. We have previously reported an asymmetric distribution of the GLUTs (1-5) and SGLT-1 in en face preparations of rat coronary artery endothelia [Gaudreault N., Scriven D.R., Moore E.D., 2004. Characterisation of glucose transporters in the intact coronary artery endothelium in rats: GLUT-2 upregulated by long-term hyperglycaemia. Diabetologia 47(12),2081-2092]. We assessed this time, through immunocytochemistry and wide field fluorescence microscopy coupled to deconvolution, the presence and subcellular distribution of glucose transporters in cultures of human coronary artery endothelial cells (HCAECs). HCAECs express GLUT-1 to 5 and SGLT-1, but their subcellular distribution lacks the luminal/abluminal asymmetry and the proximity to cell-to-cell junctions observed in intact endothelium. To determine the impact of the transporters' distribution on intracellular glucose accumulation, a fluorescent glucose analog (2-NBDG) was used in conjunction with confocal microscopy to monitor uptake in individual cells; the arteries were mounted in an arteriograph chamber with physiological flow rates. The uptake in both preparations was inhibited by cytochalasin-B and d-glucose and stimulated by insulin, but the distribution of the incorporated 2-NBDG mirrored that of the transporters. In HCAEC it was distributed throughout the cell and in the intact arterial endothelium it was restricted to the narrow cytosolic volume adjacent to the cell-to-cell junctions. We suggest that the latter subcellular organization and compartmentalization may facilitate transendothelial transport of glucose in intact coronary artery.

  18. Immunocytochemical determination of the subcellular distribution of ascorbate in plants

    PubMed Central

    Stumpe, M.; Mauch, F.

    2010-01-01

    Ascorbate is an important antioxidant in plants and fulfills many functions related to plant defense, redox signaling and modulation of gene expression. We have analyzed the subcellular distribution of reduced and oxidized ascorbate in leaf cells of Arabidopsis thaliana and Nicotiana tabacum by high-resolution immuno electron microscopy. The accuracy and specificity of the applied method is supported by several observations. First, preadsorption of the ascorbate antisera with ascorbic acid or dehydroascorbic acid resulted in the reduction of the labeling to background levels. Second, the overall labeling density was reduced between 50 and 61% in the ascorbate-deficient Arabidopsis mutants vtc1-2 and vtc2-1, which correlated well with biochemical measurements. The highest ascorbate-specific labeling was detected in nuclei and the cytosol whereas the lowest levels were found in vacuoles. Intermediate labeling was observed in chloroplasts, mitochondria and peroxisomes. This method was used to determine the subcellular ascorbate distribution in leaf cells of plants exposed to high light intensity, a stress factor that is well known to cause an increase in cellular ascorbate concentration. High light intensities resulted in a strong increase in overall labeling density. Interestingly, the strongest compartment-specific increase was found in vacuoles (fourfold) and in plastids (twofold). Ascorbate-specific labeling was restricted to the matrix of mitochondria and to the stroma of chloroplasts in control plants but was also detected in the lumen of thylakoids after high light exposure. In summary, this study reveals an improved insight into the subcellular distribution of ascorbate in plants and the method can now be applied to determine compartment-specific changes in ascorbate in response to various stress situations. Electronic supplementary material The online version of this article (doi:10.1007/s00425-010-1275-x) contains supplementary material, which is available

  19. Subcellular localization of ubiquitin and ubiquitinated proteins in Arabidopsis thaliana.

    PubMed

    Beers, E P; Moreno, T N; Callis, J

    1992-08-05

    Ubiquitin is a highly conserved, 76-amino acid, eukaryotic protein. Its widely accepted role as a proteolytic cofactor depends on its unique ability to covalently ligate to other cellular proteins. While there is good evidence for the existence of such ubiquitinated proteins in the cytosolic and nuclear compartments, relatively little is known about the presence of free ubiquitin and ubiquitinated proteins in other subcellular compartments. This is especially true of higher plants, which have not previously been the subject of extensive biochemical subcellular localizations of ubiquitinated proteins. We extracted cell wall proteins and purified nuclei, vacuoles, chloroplasts, and microsomes from chlorophyllous tissues of Arabidopsis. Immunoblot analyses were used to compare the profiles of ubiquitinated proteins from purified subcellular fractions to those from unfractionated extracts. Purified nuclei contained, in addition to a complex mixture of high molecular mass ubiquitinated proteins, a strongly immunoreactive 28-kDa protein. In the apoplastic extract, we did not detect any ubiquitinated proteins enriched above the background level of those due to cytosolic contamination. Vacuoles appeared to contribute significantly to the ubiquitinated proteins present in the whole protoplast extract. At least three high molecular mass ubiquitinated proteins were unique to the vacuolar extract. Chloroplast stromal proteins did not react specifically with anti-ubiquitin antibodies. When microsomal ubiquitinated proteins were compared to those found in a whole protoplast extract, a distinct pattern was evident. Microsomal ubiquitinated proteins were not visible in the 10,000 x g supernatant used to prepare the 100,000 x g pellet, indicating that they were probably low abundance proteins in the protoplast extract.

  20. Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.

    PubMed

    Kim, Hyun-Jun; Kwon, Hye-Rim; Bae, Chang-Dae; Park, Joobae; Hong, Kyung U

    2010-05-15

    During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.

  1. Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals.

    PubMed

    Arakaki, Atsushi; Kikuchi, Daiki; Tanaka, Masayoshi; Yamagishi, Ayana; Yoda, Takuto; Matsunaga, Tadashi

    2016-10-15

    The magnetosome is an organelle specialized for inorganic magnetite crystal synthesis in magnetotactic bacteria. The complex mechanism of magnetosome formation is regulated by magnetosome proteins in a stepwise manner. Protein localization is a key step for magnetosome development; however, a global study of magnetosome protein localization remains to be conducted. Here, we comparatively analyzed the subcellular localization of a series of green fluorescent protein (GFP)-tagged magnetosome proteins. The protein localizations were categorized into 5 groups (short-length linear, middle-length linear, long-length linear, cell membrane, and intracellular dispersing), which were related to the protein functions. Mms6, which regulates magnetite crystal growth, localized along magnetosome chain structures under magnetite-forming (microaerobic) conditions but was dispersed in the cell under nonforming (aerobic) conditions. Correlative fluorescence and electron microscopy analyses revealed that Mms6 preferentially localized to magnetosomes enclosing magnetite crystals. We suggest that a highly organized spatial regulation mechanism controls magnetosome protein localization during magnetosome formation in magnetotactic bacteria. Magnetotactic bacteria synthesize magnetite (Fe3O4) nanocrystals in a prokaryotic organelle called the magnetosome. This organelle is formed using various magnetosome proteins in multiple steps, including vesicle formation, magnetosome alignment, and magnetite crystal formation, to provide compartmentalized nanospaces for the regulation of iron concentrations and redox conditions, enabling the synthesis of a morphologically controlled magnetite crystal. Thus, to rationalize the complex organelle development, the localization of magnetosome proteins is considered to be highly regulated; however, the mechanisms remain largely unknown. Here, we performed comparative localization analysis of magnetosome proteins that revealed the presence of a spatial

  2. Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals

    PubMed Central

    Arakaki, Atsushi; Kikuchi, Daiki; Tanaka, Masayoshi; Yamagishi, Ayana; Yoda, Takuto

    2016-01-01

    ABSTRACT The magnetosome is an organelle specialized for inorganic magnetite crystal synthesis in magnetotactic bacteria. The complex mechanism of magnetosome formation is regulated by magnetosome proteins in a stepwise manner. Protein localization is a key step for magnetosome development; however, a global study of magnetosome protein localization remains to be conducted. Here, we comparatively analyzed the subcellular localization of a series of green fluorescent protein (GFP)-tagged magnetosome proteins. The protein localizations were categorized into 5 groups (short-length linear, middle-length linear, long-length linear, cell membrane, and intracellular dispersing), which were related to the protein functions. Mms6, which regulates magnetite crystal growth, localized along magnetosome chain structures under magnetite-forming (microaerobic) conditions but was dispersed in the cell under nonforming (aerobic) conditions. Correlative fluorescence and electron microscopy analyses revealed that Mms6 preferentially localized to magnetosomes enclosing magnetite crystals. We suggest that a highly organized spatial regulation mechanism controls magnetosome protein localization during magnetosome formation in magnetotactic bacteria. IMPORTANCE Magnetotactic bacteria synthesize magnetite (Fe3O4) nanocrystals in a prokaryotic organelle called the magnetosome. This organelle is formed using various magnetosome proteins in multiple steps, including vesicle formation, magnetosome alignment, and magnetite crystal formation, to provide compartmentalized nanospaces for the regulation of iron concentrations and redox conditions, enabling the synthesis of a morphologically controlled magnetite crystal. Thus, to rationalize the complex organelle development, the localization of magnetosome proteins is considered to be highly regulated; however, the mechanisms remain largely unknown. Here, we performed comparative localization analysis of magnetosome proteins that revealed the

  3. Dynamic neuroanatomy at subcellular resolution in the zebrafish.

    PubMed

    Faucherre, Adèle; López-Schier, Hernán

    2014-01-01

    Genetic means to visualize and manipulate neuronal circuits in the intact animal have revolutionized neurobiology. "Dynamic neuroanatomy" defines a range of approaches aimed at quantifying the architecture or subcellular organization of neurons over time during their development, regeneration, or degeneration. A general feature of these approaches is their reliance on the optical isolation of defined neurons in toto by genetically expressing markers in one or few cells. Here we use the afferent neurons of the lateral line as an example to describe a simple method for the dynamic neuroanatomical study of axon terminals in the zebrafish by laser-scanning confocal microscopy.

  4. Self-calibrating viscosity probes: design and subcellular localization.

    PubMed

    Dakanali, Marianna; Do, Thai H; Horn, Austin; Chongchivivat, Akaraphon; Jarusreni, Tuptim; Lichlyter, Darcy; Guizzunti, Gianni; Haidekker, Mark A; Theodorakis, Emmanuel A

    2012-07-15

    We describe the design, synthesis and fluorescence profiles of new self-calibrating viscosity dyes in which a coumarin (reference fluorophore) has been covalently linked with a molecular rotor (viscosity sensor). Characterization of their fluorescence properties was made with separate excitation of the units and through resonance energy transfer from the reference to the sensor dye. We have modified the linker and the substitution of the rotor in order to change the hydrophilicity of these probes thereby altering their subcellular localization. For instance, hydrophilic dye 12 shows a homogeneous distribution inside the cell and represents a suitable probe for viscosity measurements in the cytoplasm.

  5. Modeling the Structure of Partnership Between Researchers and Front-Line Service Providers: Strengthening Collaborative Public Health Research.

    PubMed

    Pinto, Rogério M; Wall, Melanie M; Spector, Anya Y

    2014-01-01

    Partnerships between HIV researchers and service providers are essential for reducing the gap between research and practice. Community-Based Participatory Research principles guided this cross-sectional study, combining 40 in-depth interviews with surveys of 141 providers in 24 social service agencies in New York City. We generated the Provider-Researcher Partnership Model to account for provider- and agency-level factors' influence on intentions to form partnerships with researchers. Providers preferred "balanced partnerships" in which researchers and providers allocated research tasks and procedures to reflect diverse knowledge/skill sets. An organizational culture that values research can help enhance providers' intentions to partner. Providers' intentions and priorities found in this study may encourage researchers to engage in and policy makers to fund collaborative research.

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

    PubMed Central

    Koukourakis, Michael I.; Kalamida, Dimitra; Giatromanolaki, Alexandra; Zois, Christos E.; Sivridis, Efthimios; Pouliliou, Stamatia; Mitrakas, Achilleas; Gatter, Kevin C.; Harris, Adrian L.

    2015-01-01

    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. PMID:26378792

  7. In vivo imaging of specific drug target binding at subcellular resolution

    PubMed Central

    Dubach, J.M.; Vinegoni, C.; Mazitschek, R.; Fumene Feruglio, P.; Cameron, L.A.; Weissleder, R.

    2015-01-01