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Sample records for protein comp patterns

  1. Proteomic Analysis of Tendon Extracellular Matrix Reveals Disease Stage-specific Fragmentation and Differential Cleavage of COMP (Cartilage Oligomeric Matrix Protein)*

    PubMed Central

    Dakin, Stephanie Georgina; Smith, Roger Kenneth Whealands; Heinegård, Dick; Önnerfjord, Patrik; Khabut, Areej; Dudhia, Jayesh

    2014-01-01

    During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1β and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1β enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease. PMID:24398684

  2. Novel cartilage oligomeric matrix protein (COMP) neoepitopes identified in synovial fluids from patients with joint diseases using affinity chromatography and mass spectrometry.

    PubMed

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J; Dahlberg, Leif E; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-07-25

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDSPAGE followed by in-gel digestion and mass spectrometric identification and characterization.Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.

  3. Novel Cartilage Oligomeric Matrix Protein (COMP) Neoepitopes Identified in Synovial Fluids from Patients with Joint Diseases Using Affinity Chromatography and Mass Spectrometry*

    PubMed Central

    Åhrman, Emma; Lorenzo, Pilar; Holmgren, Kristin; Grodzinsky, Alan J.; Dahlberg, Leif E.; Saxne, Tore; Heinegård, Dick; Önnerfjord, Patrik

    2014-01-01

    To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDS-PAGE followed by in-gel digestion and mass spectrometric identification and characterization. Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided. PMID:24917676

  4. Ultrastructural immunolocalization of cartilage oligomeric matrix protein (COMP) in the articular cartilage on the equine third carpal bone in trained and untrained horses.

    PubMed

    Skiöldebrand, E; Ekman, S; Heinegård, D; Hultenby, K

    2010-04-01

    The present study was designed to delineate the presence of COMP at the ultrastructural level comparing concentrations between two areas of articular cartilage from the equine third carpal bone, subjected to different loading, from trained and untrained horses. We also analyzed the fibril thickness of collagen type II in the same compartments and zones. Samples were collected from high load-bearing areas of the dorsal radial facet (intermittent high load) and an area of the palmar condyle (low constant load) in five non-trained and three trained young racehorses. The data show that COMP is much less abundant in the matrix in intermittent high loaded areas of articular cartilage from trained horses as compared to the untrained horses (p=0.036). On the other hand, the untrained horses often displayed a higher immunolabeling in loaded areas compared to unloaded areas, indicating that an adequate dynamic load promotes COMP synthesis and/or retention, while an excessive load may have an opposite effect. The collagen fibril diameter showed marked variation between individuals. The present study indicates that dynamic in vivo compression at high load and frequency lowers matrix content of COMP in the articular cartilage of the third carpal bone. It also indicates that the collagen network is influenced by mechanical load following by strenuous exercise.

  5. Protein patterning by a DNA origami framework.

    PubMed

    Aslan, Hüsnü; Krissanaprasit, Abhichart; Besenbacher, Flemming; Gothelf, Kurt V; Dong, Mingdong

    2016-08-18

    A spatial arrangement of proteins provides structural and functional advantages in vast technological applications as well as fundamental research. Most protein patterning procedures employ complicated, time consuming and very costly nanofabrication techniques. As an alternative route, we developed a fully biomolecular self-assembly method using DNA Origami Frames (DOF) as a template for both small and large scale protein patterning. We employed a triangular DOF (tDOF) to arrange the Bovine Serum Albumin (BSA) protein. Our in situ protein patterning strategy provides a novel, fully organic platform using a fast and low-cost surface approach with possible utilization in fundamental science and technological applications.

  6. NDT-COMP9 microcomputer

    SciTech Connect

    Dodd, C.V.; Cowan, R.F.

    1980-09-01

    An 8080-based microcomputer system, the NDT-COMP9, has been designed for instrumentation control and data analysis in eddy-current tests. The NDT-COMP9 represents a significantly more powerful computer system than the NDT-COMP8 microcomputer from which it was developed. The NDT-COMP9 system is contained on a 240- by 120-mm (9.5- by 4.8-in.) circuit board and will fit in a four-wide Nuclear Instrumentation Module (NIM) BIN with 26-pin edge connectors. In addition to the 8080-compatible central processing unit (CPU), an arithmetic processing unit (APU) is available to provide up to 32-bit fixed- or floating-point, basic or transcendental math functions. The 16K of read only memory (ROM) and random access memory (RAM), one serial input-output (I/O) port (RS-232-C at a maximum speed of 9600 baud), and 72 parallel I/O ports are available. The baud rate is under software control. A system monitor and math package are available for use with the microcomputer.

  7. Geometry-induced protein pattern formation.

    PubMed

    Thalmeier, Dominik; Halatek, Jacob; Frey, Erwin

    2016-01-19

    Protein patterns are known to adapt to cell shape and serve as spatial templates that choreograph downstream processes like cell polarity or cell division. However, how can pattern-forming proteins sense and respond to the geometry of a cell, and what mechanistic principles underlie pattern formation? Current models invoke mechanisms based on dynamic instabilities arising from nonlinear interactions between proteins but neglect the influence of the spatial geometry itself. Here, we show that patterns can emerge as a direct result of adaptation to cell geometry, in the absence of dynamical instability. We present a generic reaction module that allows protein densities robustly to adapt to the symmetry of the spatial geometry. The key component is an NTPase protein that cycles between nucleotide-dependent membrane-bound and cytosolic states. For elongated cells, we find that the protein dynamics generically leads to a bipolar pattern, which vanishes as the geometry becomes spherically symmetrical. We show that such a reaction module facilitates universal adaptation to cell geometry by sensing the local ratio of membrane area to cytosolic volume. This sensing mechanism is controlled by the membrane affinities of the different states. We apply the theory to explain AtMinD bipolar patterns in [Formula: see text] EcMinDE Escherichia coli. Due to its generic nature, the mechanism could also serve as a hitherto-unrecognized spatial template in many other bacterial systems. Moreover, the robustness of the mechanism enables self-organized optimization of protein patterns by evolutionary processes. Finally, the proposed module can be used to establish geometry-sensitive protein gradients in synthetic biological systems.

  8. Pattern Recognition of Adsorbing HP Lattice Proteins

    NASA Astrophysics Data System (ADS)

    Wilson, Matthew S.; Shi, Guangjie; Wüst, Thomas; Landau, David P.; Schmid, Friederike

    2015-03-01

    Protein adsorption is relevant in fields ranging from medicine to industry, and the qualitative behavior exhibited by course-grained models could shed insight for further research in such fields. Our study on the selective adsorption of lattice proteins utilizes the Wang-Landau algorithm to simulate the Hydrophobic-Polar (H-P) model with an efficient set of Monte Carlo moves. Each substrate is modeled as a square pattern of 9 lattice sites which attract either H or P monomers, and are located on an otherwise neutral surface. The fully enumerated set of 102 unique surfaces is simulated with each protein sequence. A collection of 27-monomer sequences is used- each of which is non-degenerate and protein-like. Thermodynamic quantities such as the specific heat and free energy are calculated from the density of states, and are used to investigate the adsorption of lattice proteins on patterned substrates. Research supported by NSF.

  9. Patterning proteins and cells using soft lithography.

    PubMed

    Kane, R S; Takayama, S; Ostuni, E; Ingber, D E; Whitesides, G M

    1999-12-01

    This review describes the pattering of proteins and cells using a non-photolithographic microfabrication technology, which we call 'soft lithography' because it consists of a set of related techniques, each of which uses stamps or channels fabricated in an elastomeric ('soft') material for pattern transfer. The review covers three soft lithographic techniques: microcontact printing, patterning using microfluidic channels, and laminar flow patterning. These soft lithographic techniques are inexpensive, are procedurally simple, and can be used to pattern a variety of planar and non-planar substrates. Their successful application does not require stringent regulation of the laboratory environment, and they can be used to pattern surfaces with delicate ligands. They provide control over both the surface chemistry and the cellular environment. We discuss both the procedures for patterning based on these soft lithographic techniques, and their applications in biosensor technology, in tissue engineering, and for fundamental studies in cell biology.

  10. Protein patterns as endpoints in environmental remediation

    SciTech Connect

    Bradley, B.; Brown, D.

    1995-12-31

    Biological endpoints can complement chemical analyses in monitoring environmental remediation. In some cases the levels of chemical detection are so low that the costs of clean-up to no detection would be prohibitive. And chemical tests do not indicate the availability of the contaminants to the biota. On the other hand many if not most biological tests lack specificity. The authors have investigated a protein expression assay to establish an endpoint for clean-up of sulfur mustard and breakdown products. Earthworms (Lumbricus terrestris) were exposed to sulfur mustard (SM), a breakdown product thiodiethanol (TDE), and ethylene glycol, the solvent for the two chemicals. Tissue from the lining of the coelomic cavity was taken from each of 6 worms in each treatment class. Soluble proteins were extracted and separated on one and two-dimensional (1D and 2D) gels. The 1 D gels showed no difference by eye but the patterns from control and solvent control worms on 2D gels differed from those of worms exposed to TDE and SM. The 1D gel data were digitized and analyzed by pattern recognition using artificial neural networks. The protein patterns under the two treatments and the two controls were learned in one set of data and successfully recognized in a second. This indicated that what was learned was useful in recognizing patterns induced by SM and TDE. Thus a possible endpoint for remediation would be the protein pattern at no effect levels of chemicals of interest.

  11. Finding protein-protein interaction patterns by contact map matching.

    PubMed

    Melo, R C; Ribeiro, C; Murray, C S; Veloso, C J M; da Silveira, C H; Neshich, G; Meira, W; Carceroni, R L; Santoro, M M

    2007-10-05

    We propose a novel method for defining patterns of contacts present in protein-protein complexes. A new use of the traditional contact maps (more frequently used for representation of the intra-chain contacts) is presented for analysis of inter-chain contacts. Using an algorithm based on image processing techniques, we can compare protein-protein interaction maps and also obtain a dissimilarity score between them. The same algorithm used to compare the maps can align the contacts of all the complexes and be helpful in the determination of a pattern of conserved interactions at the interfaces. We present an example for the application of this method by analyzing the pattern of interaction of bovine pancreatic trypsin inhibitors and trypsins, chymotrypsins, a thrombin, a matriptase, and a kallikrein - all classified as serine proteases. We found 20 contacts conserved in trypsins and chymotrypsins and 3 specific ones are present in all the serine protease complexes studied. The method was able to identify important contacts for the protein family studied and the results are in agreement with the literature.

  12. Sedimentation Patterns of Rapidly Reversible Protein Interactions

    PubMed Central

    Schuck, Peter

    2010-01-01

    Abstract The transport behavior of macromolecular mixtures with rapidly reversible complex formation is of great interest in the study of protein interactions by many different methods. Complicated transport patterns arise even for simple bimolecular reactions, when all species exhibit different migration velocities. Although partial differential equations are available to describe the spatial and temporal evolution of the interacting system given particular initial conditions, a general overview of the phase behavior of the systems in parameter space has not yet been reported. In the case of sedimentation of two-component mixtures, this study presents simple analytical solutions that solve the underlying equations in the diffusion-free limit previously subject to Gilbert-Jenkins theory. The new expressions describe, with high precision, the average sedimentation coefficients and composition of each boundary, which allow the examination of features of the whole parameter space at once, and may be used for experimental design and robust analysis of experimental boundary patterns to derive the stoichiometry and affinity of the complex. This study finds previously unrecognized features, including a phase transition between boundary patterns. The model reveals that the time-average velocities of all components in the reaction mixture must match—a condition that suggests an intuitive physical picture of an effective particle of the coupled cosedimentation of an interacting system. Adding to the existing numerical solutions of the relevant partial differential equations, the effective particle model provides physical insights into the relationships of the parameters that govern sedimentation patterns. PMID:20441765

  13. DETECTION OF TOPOLOGICAL PATTERNS IN PROTEIN NETWORKS.

    SciTech Connect

    MASLOV,S.SNEPPEN,K.

    2003-11-17

    property of many biological networks that was recently brought to attention of the scientific community [3, 4, 5] is an extremely broad distribution of node connectivities defined as the number of immediate neighbors of a given node in the network. While the majority of nodes have just a few edges connecting them to other nodes in the network, there exist some nodes, that we will refer to as ''hubs'', with an unusually large number of neighbors. The connectivity of the most connected hub in such a network is typically several orders of magnitude larger than the average connectivity in the network. Often the distribution of connectivities of individual nodes can be approximated by a scale-free power law form [3] in which case the network is referred to as scale-free. Among biological networks distributions of node connectivities in metabolic [4], protein interaction [5], and brain functional [6] networks can be reasonably approximated by a power law extending for several orders of magnitude. The set of connectivities of individual nodes is an example of a low-level (single-node) topological property of a network. While it answers the question about how many neighbors a given node has, it gives no information about the identity of those neighbors. It is clear that most functional properties of networks are defined at a higher topological level in the exact pattern of connections of nodes to each other. However, such multi-node connectivity patterns are rather difficult to quantify and compare between networks. In this work we concentrate on multi-node topological properties of protein networks. These networks (as any other biological networks) lack the top-down design. Instead, selective forces of biological evolution shape them from raw material provided by random events such as mutations within individual genes, and gene duplications. As a result their connections are characterized by a large degree of randomness. One may wonder which connectivity patterns are indeed

  14. COMP and Col9A3 mutations and their relationship to the pseudoachondroplasia phenotype.

    PubMed

    Jung, Woon-Won; Balce, Gracia Cielo; Cho, Jae-Woo; Jung, Sung-Chul; Hong, Suk-Joo; Song, Hae-Ryong

    2010-12-01

    While pseudoachondroplasia (PSACH) is almost exclusively caused by cartilage oligomeric matrix protein (COMP) mutations, many patients identified with the PSACH phenotype do not have this mutation, suggesting gene and locus heterogeneity. In order to further characterize this entity, we studied 32 clinically and radiographically diagnosed PSACH patients, among 19 families. COMP and collagen (Col) IX (A1, A2 and A3) mutations, were determined. Patients who tested negative for pathological gene mutations but who were identified with the PSACH phenotype, were included. The phenotypes were characterized according to height deviation (cm) from normal, lower extremity mechanical axis deviation (MAD), cervical and thoracolumbar spine involvement, pelvic index, as well as hip, knee, ankle and hand involvement. We report an 81% mutation detection rate for PSACH, of which COMP+Col9A3 mutations were more prevalent (61%) than COMP mutations alone (30%). Of our PSACH patients, 19% tested negative for both COMP and Col9A3 mutations, and they presented with the greatest mean height deviations, but the least mean MADs. While all the PSACH mutations consistently produced the severe phenotype, the V426A mutation in Col9A3 produced the most severe. Mother-daughter and father-son phenotypic similarities were noted in the COMP+Col9A3 families. Col9A3 and gender play confounding roles in the phenotypic severity of PSACH. The presence of the PSACH phenotype in patients who tested negative for known mutations further confirms the genetic heterogeneity of this condition.

  15. Adsorption of HP Lattice Proteins on Patterned Surfaces

    NASA Astrophysics Data System (ADS)

    Wilson, Matthew; Shi, Guangjie; Landau, David P.; Li, Ying Wai; Wuest, Thomas

    2014-03-01

    The HP lattice model[2] is a course-grained, yet useful tool for modeling protein sequences where amino acids are treated as either hydrophobic (H) or polar (P) monomers. With the use of Wang-Landau sampling and an efficient set of Monte-Carlo moves[3], HP lattice proteins adsorbed on patterned surfaces are studied. Each substrate is modeled as a periodically bounded pattern of lattice sites that interact with either H or P monomers in the lattice protein, where the energy contributions of the surface are determined by assigned coupling strengths. By analyzing energy degeneracies, along with the thermodynamic and structural quantities of the protein, both the protein folding and surface adsorption can be observed. The adsorption behavior of the lattice proteins on patterned surfaces will be compared to those interacting with uniform surfaces. Research supported by NSF.

  16. Encoding protein-ligand interaction patterns in fingerprints and graphs.

    PubMed

    Desaphy, Jérémy; Raimbaud, Eric; Ducrot, Pierre; Rognan, Didier

    2013-03-25

    We herewith present a novel and universal method to convert protein-ligand coordinates into a simple fingerprint of 210 integers registering the corresponding molecular interaction pattern. Each interaction (hydrophobic, aromatic, hydrogen bond, ionic bond, metal complexation) is detected on the fly and physically described by a pseudoatom centered either on the interacting ligand atom, the interacting protein atom, or the geometric center of both interacting atoms. Counting all possible triplets of interaction pseudoatoms within six distance ranges, and pruning the full integer vector to keep the most frequent triplets enables the definition of a simple (210 integers) and coordinate frame-invariant interaction pattern descriptor (TIFP) that can be applied to compare any pair of protein-ligand complexes. TIFP fingerprints have been calculated for ca. 10,000 druggable protein-ligand complexes therefore enabling a wide comparison of relationships between interaction pattern similarity and ligand or binding site pairwise similarity. We notably show that interaction pattern similarity strongly depends on binding site similarity. In addition to the TIFP fingerprint which registers intermolecular interactions between a ligand and its target protein, we developed two tools (Ishape, Grim) to align protein-ligand complexes from their interaction patterns. Ishape is based on the overlap of interaction pseudoatoms using a smooth Gaussian function, whereas Grim utilizes a standard clique detection algorithm to match interaction pattern graphs. Both tools are complementary and enable protein-ligand complex alignments capitalizing on both global and local pattern similarities. The new fingerprint and companion alignment tools have been successfully used in three scenarios: (i) interaction-biased alignment of protein-ligand complexes, (ii) postprocessing docking poses according to known interaction patterns for a particular target, and (iii) virtual screening for bioisosteric

  17. Directed self-assembly of proteins into discrete radial patterns

    PubMed Central

    Thakur, Garima; Prashanthi, Kovur; Thundat, Thomas

    2013-01-01

    Unlike physical patterning of materials at nanometer scale, manipulating soft matter such as biomolecules into patterns is still in its infancy. Self-assembled monolayer (SAM) with surface density gradient has the capability to drive biomolecules in specific directions to create hierarchical and discrete structures. Here, we report on a two-step process of self-assembly of the human serum albumin (HSA) protein into discrete ring structures based on density gradient of SAM. The methodology involves first creating a 2-dimensional (2D) polyethylene glycol (PEG) islands with responsive carboxyl functionalities. Incubation of proteins on such pre-patterned surfaces results in direct self-assembly of protein molecules around PEG islands. Immobilization and adsorption of protein on such structures over time evolve into the self-assembled patterns. PMID:23719678

  18. Mining the characteristic interaction patterns on protein-protein binding interfaces.

    PubMed

    Li, Yan; Liu, Zhihai; Han, Li; Li, Chengke; Wang, Renxiao

    2013-09-23

    Protein-protein interactions are observed in various biological processes. They are important for understanding the underlying molecular mechanisms and can be potential targets for developing small-molecule regulators of such processes. Previous studies suggest that certain residues on protein-protein binding interfaces are "hot spots". As an extension to this concept, we have developed a residue-based method to identify the characteristic interaction patterns (CIPs) on protein-protein binding interfaces, in which each pattern is a cluster of four contacting residues. Systematic analysis was conducted on a nonredundant set of 1,222 protein-protein binding interfaces selected out of the entire Protein Data Bank. Favored interaction patterns across different protein-protein binding interfaces were retrieved by considering both geometrical and chemical conservations. As demonstrated on two test tests, our method was able to predict hot spot residues on protein-protein binding interfaces with good recall scores and acceptable precision scores. By analyzing the function annotations and the evolutionary tree of the protein-protein complexes in our data set, we also observed that protein-protein interfaces sharing common characteristic interaction patterns are normally associated with identical or similar biological functions.

  19. Elastohydrodynamics and Kinetics of Protein Patterning in the Immunological Synapse.

    PubMed

    Carlson, Andreas; Mahadevan, L

    2015-12-01

    We propose a minimal mathematical model for the physical basis of membrane protein patterning in the immunological synapse (IS), which encompass membrane mechanics, protein binding kinetics and motion, and fluid flow in the synaptic cleft. Our theory leads to simple predictions for the spatial and temporal scales of protein cluster formation, growth and arrest as a function of membrane stiffness, rigidity and kinetics of the adhesive proteins, and the fluid flow in the synaptic cleft. Numerical simulations complement these scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment shows that passive elastohydrodynamics and kinetics of protein binding in the synaptic cleft can describe the short-time formation and organization of protein clusters, without evoking any active processes in the cytoskeleton. Despite the apparent complexity of the process, our analysis shows that just two dimensionless parameters characterize the spatial and temporal evolution of the protein pattern: a ratio of membrane elasticity to protein stiffness, and the ratio of a hydrodynamic time scale for fluid flow relative to the protein binding rate. A simple phase diagram encompasses the variety of patterns that can arise.

  20. Elastohydrodynamics and Kinetics of Protein Patterning in the Immunological Synapse

    PubMed Central

    Carlson, Andreas; Mahadevan, L.

    2015-01-01

    We propose a minimal mathematical model for the physical basis of membrane protein patterning in the immunological synapse (IS), which encompass membrane mechanics, protein binding kinetics and motion, and fluid flow in the synaptic cleft. Our theory leads to simple predictions for the spatial and temporal scales of protein cluster formation, growth and arrest as a function of membrane stiffness, rigidity and kinetics of the adhesive proteins, and the fluid flow in the synaptic cleft. Numerical simulations complement these scaling laws by quantifying the nucleation, growth and stabilization of proteins domains on the size of the cell. Direct comparison with experiment shows that passive elastohydrodynamics and kinetics of protein binding in the synaptic cleft can describe the short-time formation and organization of protein clusters, without evoking any active processes in the cytoskeleton. Despite the apparent complexity of the process, our analysis shows that just two dimensionless parameters characterize the spatial and temporal evolution of the protein pattern: a ratio of membrane elasticity to protein stiffness, and the ratio of a hydrodynamic time scale for fluid flow relative to the protein binding rate. A simple phase diagram encompasses the variety of patterns that can arise. PMID:26699430

  1. Datamining protein structure databanks for crystallization patterns of proteins.

    PubMed

    Valafar, Homayoun; Prestegard, James H; Valafar, Faramarz

    2002-12-01

    A study of 345 protein structures selected among 1,500 structures determined by nuclear magnetic resonance (NMR) methods, revealed useful correlations between crystallization properties and several parameters for the studied proteins. NMR methods of structure determination do not require the growth of protein crystals, and hence allow comparison of properties of proteins that have or have not been the subject of crystallographic approaches. One- and two-dimensional statistical analyses of the data confirmed a hypothesized relation between the size of the molecule and its crystallization potential. Furthermore, two-dimensional Bayesian analysis revealed a significant relationship between relative ratio of different secondary structures and the likelihood of success for crystallization trials. The most immediate result is an apparent correlation of crystallization potential with protein size. Further analysis of the data revealed a relationship between the unstructured fraction of proteins and the success of its crystallization. Utilization of Bayesian analysis on the latter correlation resulted in a prediction performance of about 64%, whereas a two-dimensional Bayesian analysis succeeded with a performance of about 75%.

  2. Patterns in protein primary sequences: classification, display and analysis.

    PubMed Central

    Saurugger, P. N.; Metfessel, B. A.

    1991-01-01

    The protein folding code, which is contained in the amino acid chain of a protein, has so far eluded elucidation. However, patterns of hydrophobic residues have previously been identified which show a specificity towards certain secondary structural elements. We are developing an analysis toolkit to find, visualize, and analyze patterns in primary sequences. Preliminary results show that there exist patterns in primary sequences which are useful for predicting the structural class of amino acid chains, performing especially well for the all-alpha helix and all-beta sheet classes. PMID:1807631

  3. Predicting protein function by frequent functional association pattern mining in protein interaction networks.

    PubMed

    Cho, Young-Rae; Zhang, Aidong

    2010-01-01

    Predicting protein function from protein interaction networks has been challenging because of the complexity of functional relationships among proteins. Most previous function prediction methods depend on the neighborhood of or the connected paths to known proteins. However, their accuracy has been limited due to the functional inconsistency of interacting proteins. In this paper, we propose a novel approach for function prediction by identifying frequent patterns of functional associations in a protein interaction network. A set of functions that a protein performs is assigned into the corresponding node as a label. A functional association pattern is then represented as a labeled subgraph. Our frequent labeled subgraph mining algorithm efficiently searches the functional association patterns that occur frequently in the network. It iteratively increases the size of frequent patterns by one node at a time by selective joining, and simplifies the network by a priori pruning. Using the yeast protein interaction network, our algorithm found more than 1400 frequent functional association patterns. The function prediction is performed by matching the subgraph, including the unknown protein, with the frequent patterns analogous to it. By leave-one-out cross validation, we show that our approach has better performance than previous link-based methods in terms of prediction accuracy. The frequent functional association patterns generated in this study might become the foundations of advanced analysis for functional behaviors of proteins in a system level.

  4. Biomimetic Replication of Microscopic Metal-Organic Framework Patterns Using Printed Protein Patterns.

    PubMed

    Liang, Kang; Carbonell, Carlos; Styles, Mark J; Ricco, Raffaele; Cui, Jiwei; Richardson, Joseph J; Maspoch, Daniel; Caruso, Frank; Falcaro, Paolo

    2015-12-02

    It is demonstrated that metal-organic frameworks (MOFs) can be replicated in a biomimetic fashion from protein patterns. Bendable, fluorescent MOF patterns are formed with micrometer resolution under ambient conditions. Furthermore, this technique is used to grow MOF patterns from fingerprint residue in 30 s with high fidelity. This technique is not only relevant for crime-scene investigation, but also for biomedical applications.

  5. Microtubule patterning in the presence of moving motor proteins.

    PubMed

    White, D; de Vries, G; Martin, J; Dawes, A

    2015-10-07

    Cytoskeletal polymers such as microtubules (MTs) interact with motor proteins to form higher-order structures. In vitro experiments have shown that MT patterns such as asters, bundles, and vortices can form under the influence of a single type of dynamic motor protein. MTs also can form anti-parallel bundles, similar to bundles that form the mitotic spindle during cell division, under the influence of two types of moving motors with opposite directionality. Despite the importance of MT structures, their mechanism of formation is not yet understood. We develop an integro-partial differential equation model to describe the dynamic interactions between MTs and moving motor proteins. Our model takes into account motor protein speed, processivity, density, and directionality, as well as MT treadmilling and reorganization due to interactions with motors. Simulation results show that plus-end directed motor proteins can form vortex patterns at low motor density, while minus-end directed motor proteins form aster patterns at similar densities. Also, motor proteins with opposite directionality are able to organize MTs into anti-parallel bundles. Our model is able to provide a quantitative and qualitative description of MT patterning, providing insights into possible mechanisms of spindle formation.

  6. Excimer laser ablation for spatially controlled protein patterns

    NASA Astrophysics Data System (ADS)

    Thissen, Helmut; Hayes, Jason P.; Kingshott, Peter; Johnson, Graham; Harvey, Erol C.; Griesser, Hans J.

    2001-11-01

    Two-dimensional control over the location of proteins on surfaces is desired for a number of applications including diagnostic tests and tissue engineered medical devices. Many of these applications require patterns of specific proteins that allow subsequent two-dimensionally controlled cell attachment. The ideal technique would allow the deposition of specific protein patterns in areas where cell attachment is required, with complete prevention of unspecific protein adsorption in areas where cells are not supposed to attach. In our study, collagen I was used as an example for an extracellular matrix protein known to support the attachment of bovine corneal epithelial cells. An allylamine plasma polymer was deposited on a silicon wafer substrate, followed by grafting of poly(ethylene oxide). Two-dimensional control over the surface chemistry was achieved using a 248 nm excimer laser. Results obtained by XPS and AFM show that the combination of extremely low-fouling surfaces with excimer laser ablation can be used effectively for the production of spatially controlled protein patterns with a resolution of less than 1 micrometers . Furthermore, it was shown that bovine corneal epithelial cell attachment followed exactly the created protein patterns. The presented method is an effective tool for a number of in vitro and in vivo applications.

  7. Patterns of fluorescent protein expression in Scleractinian corals.

    PubMed

    Gruber, David F; Kao, Hung-Teh; Janoschka, Stephen; Tsai, Julia; Pieribone, Vincent A

    2008-10-01

    Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

  8. Geometry sensing by self-organized protein patterns

    PubMed Central

    Schweizer, Jakob; Loose, Martin; Bonny, Mike; Kruse, Karsten; Mönch, Ingolf; Schwille, Petra

    2012-01-01

    In the living cell, proteins are able to organize space much larger than their dimensions. In return, changes of intracellular space can influence biochemical reactions, allowing cells to sense their size and shape. Despite the possibility to reconstitute protein self-organization with only a few purified components, we still lack knowledge of how geometrical boundaries affect spatiotemporal protein patterns. Following a minimal systems approach, we used purified proteins and photolithographically patterned membranes to study the influence of spatial confinement on the self-organization of the Min system, a spatial regulator of bacterial cytokinesis, in vitro. We found that the emerging protein pattern responds even to the lateral, two-dimensional geometry of the membrane such that, as in the three-dimensional cell, Min protein waves travel along the longest axis of the membrane patch. This shows that for spatial sensing the Min system does not need to be enclosed in a three-dimensional compartment. Using a computational model we quantitatively analyzed our experimental findings and identified persistent binding of MinE to the membrane as requirement for the Min system to sense geometry. Our results give insight into the interplay between geometrical confinement and biochemical patterns emerging from a nonlinear reaction–diffusion system. PMID:22949703

  9. Modeling associated protein-DNA pattern discovery with unified scores.

    PubMed

    Chan, Tak-Ming; Lo, Leung-Yau; Sze-To, Ho-Yin; Leung, Kwong-Sak; Xiao, Xinshu; Wong, Man-Hon

    2013-01-01

    Understanding protein-DNA interactions, specifically transcription factor (TF) and transcription factor binding site (TFBS) bindings, is crucial in deciphering gene regulation. The recent associated TF-TFBS pattern discovery combines one-sided motif discovery on both the TF and the TFBS sides. Using sequences only, it identifies the short protein-DNA binding cores available only in high-resolution 3D structures. The discovered patterns lead to promising subtype and disease analysis applications. While the related studies use either association rule mining or existing TFBS annotations, none has proposed any formal unified (both-sided) model to prioritize the top verifiable associated patterns. We propose the unified scores and develop an effective pipeline for associated TF-TFBS pattern discovery. Our stringent instance-level evaluations show that the patterns with the top unified scores match with the binding cores in 3D structures considerably better than the previous works, where up to 90 percent of the top 20 scored patterns are verified. We also introduce extended verification from literature surveys, where the high unified scores correspond to even higher verification percentage. The top scored patterns are confirmed to match the known WRKY binding cores with no available 3D structures and agree well with the top binding affinities of in vivo experiments.

  10. Fourier Analysis of Conservation Patterns in Protein Secondary Structure.

    PubMed

    Palaniappan, Ashok; Jakobsson, Eric

    2017-01-01

    Residue conservation is a common observation in alignments of protein families, underscoring positions important in protein structure and function. Though many methods measure the level of conservation of particular residue positions, currently we do not have a way to study spatial oscillations occurring in protein conservation patterns. It is known that hydrophobicity shows spatial oscillations in proteins, which is characterized by computing the hydrophobic moment of the protein domains. Here, we advance the study of moments of conservation of protein families to know whether there might exist spatial asymmetry in the conservation patterns of regular secondary structures. Analogous to the hydrophobic moment, the conservation moment is defined as the modulus of the Fourier transform of the conservation function of an alignment of related protein, where the conservation function is the vector of conservation values at each column of the alignment. The profile of the conservation moment is useful in ascertaining any periodicity of conservation, which might correlate with the period of the secondary structure. To demonstrate the concept, conservation in the family of potassium ion channel proteins was analyzed using moments. It was shown that the pore helix of the potassium channel showed oscillations in the moment of conservation matching the period of the α-helix. This implied that one side of the pore helix was evolutionarily conserved in contrast to its opposite side. In addition, the method of conservation moments correctly identified the disposition of the voltage sensor of voltage-gated potassium channels to form a 310 helix in the membrane.

  11. Chemicals and heat generate different protein patterns in Acinetobacter calcoaceticus.

    PubMed

    Benndorf, D; Loffhagen, N; Babel, W

    1997-01-01

    The effect of exposing Acinetobacter calcoaceticus 69-V to DNP-stress and heat shock was examined by two-dimensional gel electrophoresis of proteins, which were detected either by autoradiography or by silver staining. Both DNP stress and heat shock led to altered patterns of protein synthesis or concentration. About 10% of the proteins which were synthesized newly or at an increased rate and about 25% of those which were found newly or with an increased concentration after DNP treatment were identified after heat shock, too.

  12. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed Central

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-01-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity. Images PMID:6286971

  13. Isozyme patterns and protein profiles in neuromuscular disorders.

    PubMed

    Edwards, Y H; Tipler, T D; Morgan-Hughes, J A; Neerunjun, J S; Hopkinson, D A

    1982-06-01

    The isozyme patterns of six different enzymes and the polypeptide profiles of soluble proteins have been examined in muscle biopsy specimens from 74 patients with a wide variety of neuromuscular disorders. About half of the samples showed unusual features in at least one, and often several, of the enzymes and proteins tested. The extent of the biochemical abnormalities was roughly proportional to the severity of the disorders. In all cases the unusual isozymes and polypeptide profiles seemed to reflect a reversion to the fetal pattern of gene expression. However, this change appeared to occur in extant muscle and was not dependent on the appearance of new muscle fibres. Among the enzymes, phosphoglycerate mutase followed by creatine kinase appeared to be the most sensitive index of muscle disorder. The extent of the change in the muscle creatine kinase isozyme pattern was not correlated with the levels of serum creatine kinase activity.

  14. A Versatile Method of Patterning Proteins and Cells.

    PubMed

    Shrirao, Anil B; Kung, Frank H; Yip, Derek; Firestein, Bonnie L; Cho, Cheul H; Townes-Anderson, Ellen

    2017-02-26

    Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

  15. Improved method for predicting protein fold patterns with ensemble classifiers.

    PubMed

    Chen, W; Liu, X; Huang, Y; Jiang, Y; Zou, Q; Lin, C

    2012-01-27

    Protein folding is recognized as a critical problem in the field of biophysics in the 21st century. Predicting protein-folding patterns is challenging due to the complex structure of proteins. In an attempt to solve this problem, we employed ensemble classifiers to improve prediction accuracy. In our experiments, 188-dimensional features were extracted based on the composition and physical-chemical property of proteins and 20-dimensional features were selected using a coupled position-specific scoring matrix. Compared with traditional prediction methods, these methods were superior in terms of prediction accuracy. The 188-dimensional feature-based method achieved 71.2% accuracy in five cross-validations. The accuracy rose to 77% when we used a 20-dimensional feature vector. These methods were used on recent data, with 54.2% accuracy. Source codes and dataset, together with web server and software tools for prediction, are available at: http://datamining.xmu.edu.cn/main/~cwc/ProteinPredict.html.

  16. Protein pattern of Xenopus laevis embryos grown in simulated microgravity.

    PubMed

    Tedeschi, Gabriella; Pagliato, Lara; Negroni, Manuela; Montorfano, Gigliola; Corsetto, Paola; Nonnis, Simona; Negri, Armando; Rizzo, Angela Maria

    2011-03-01

    Numerous studies indicate that microgravity affects cell growth and differentiation in many living organisms, and various processes are modified when cells are placed under conditions of weightlessness. However, until now, there is no coherent explanation for these observations, and little information is available concerning the biomolecules involved. Our aim has been to investigate the protein pattern of Xenopus laevis embryos exposed to simulated microgravity during the first 6 days of development. A proteomic approach was applied to compare the protein profiles of Xenopus embryos developed in simulated microgravity and in normal conditions. Attention was focused on embryos that do not present visible malformations in order to investigate if weightlessness has effects at protein level in the absence of macroscopic alterations. The data presented strongly suggest that some of the major components of the cytoskeleton vary in such conditions. Three major findings are described for the first time: (i) the expression of important factors involved in the organization and stabilization of the cytoskeleton, such as Arp (actin-related protein) 3 and stathmin, is heavily affected by microgravity; (ii) the amount of the two major cytoskeletal proteins, actin and tubulin, do not change in such conditions; however, (iii) an increase in the tyrosine nitration of these two proteins can be detected. The data suggest that, in the absence of morphological alterations, simulated microgravity affects the intracellular movement system of cells by altering cytoskeletal proteins heavily involved in the regulation of cytoskeleton remodelling.

  17. Assembly of designed protein scaffolds into monolayers for nanoparticle patterning.

    PubMed

    Mejias, Sara H; Couleaud, Pierre; Casado, Santiago; Granados, Daniel; Garcia, Miguel Angel; Abad, Jose M; Cortajarena, Aitziber L

    2016-05-01

    The controlled assembly of building blocks to achieve new nanostructured materials with defined properties at different length scales through rational design is the basis and future of bottom-up nanofabrication. This work describes the assembly of the idealized protein building block, the consensus tetratricopeptide repeat (CTPR), into monolayers by oriented immobilization of the blocks. The selectivity of thiol-gold interaction for an oriented immobilization has been verified by comparing a non-thiolated protein building block. The physical properties of the CTPR protein thin biomolecular films including topography, thickness, and viscoelasticity, are characterized. Finally, the ability of these scaffolds to act as templates for inorganic nanostructures has been demonstrated by the formation of well-packed gold nanoparticles (GNPs) monolayer patterned by the CTPR monolayer.

  18. Mapping out Min protein patterns in fully confined fluidic chambers

    PubMed Central

    Caspi, Yaron; Dekker, Cees

    2016-01-01

    The bacterial Min protein system provides a major model system for studying reaction-diffusion processes in biology. Here we present the first in vitro study of the Min system in fully confined three-dimensional chambers that are lithography-defined, lipid-bilayer coated and isolated through pressure valves. We identify three typical dynamical behaviors that occur dependent on the geometrical chamber parameters: pole-to-pole oscillations, spiral rotations, and traveling waves. We establish the geometrical selection rules and show that, surprisingly, Min-protein spiral rotations govern the larger part of the geometrical phase diagram. Confinement as well as an elevated temperature reduce the characteristic wavelength of the Min patterns, although even for confined chambers with a bacterial-level viscosity, the patterns retain a ~5 times larger wavelength than in vivo. Our results provide an essential experimental base for modeling of intracellular Min gradients in bacterial cell division as well as, more generally, for understanding pattern formation in reaction-diffusion systems. DOI: http://dx.doi.org/10.7554/eLife.19271.001 PMID:27885986

  19. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    PubMed Central

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  20. Morphology and protein patterns of honey bee drone accessory glands.

    PubMed

    Cruz-Landim, Carminda da; Dallacqua, Rodrigo Pires

    2005-09-30

    We used light and transmission electron microscopy to examine the morphology of the accessory glands of immature and mature adult males of Apis mellifera L. We also made an electrophoretic analysis of the protein content of the mature gland. The glands of the immature male actively secrete a mucous substance that can be seen in the lumen of the gland of the mature male. This secretion stains with mercury bromophenol blue and with periodic acid-Schiff reaction, which stain glyconjugates. The protein content was higher in the lumen secretion than in the gland wall extracts. The electrophoresis patterns of the wall extracts were different from those of the secretion found in the gland lumen.

  1. The E4 protein; structure, function and patterns of expression

    SciTech Connect

    Doorbar, John

    2013-10-15

    }E4, these kinases regulate one of the E1{sup ∧}E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4. - Highlights: • E4 gene products have a modular structure, and are expressed from the E1{sup ∧}E4 spliced mRNA. • E4 proteins are modified during epithelial differentiation by phosphorylation and proteolysis. • The E4 proteins contribute to genome amplification-efficiency and virus synthesis. • E4 proteins are abundantly expressed and may facilitate efficient virus release and transmission. • High-risk E4 proteins are deposited as amyloid fibres and can be used as infection biomarkers.

  2. Fragile X mental retardation protein (FMRP) interacting proteins exhibit different expression patterns during development.

    PubMed

    Bonaccorso, C M; Spatuzza, M; Di Marco, B; Gloria, A; Barrancotto, G; Cupo, A; Musumeci, S A; D'Antoni, S; Bardoni, B; Catania, M V

    2015-05-01

    Fragile X syndrome is caused by the lack of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein involved in mRNA transport and translation. FMRP is a component of mRNA ribonucleoprotein complexes and it can interact with a range of proteins either directly or indirectly, as demonstrated by two-hybrid selection and co-immunoprecipitation, respectively. Most of FMRP-interacting proteins are RNA-binding proteins such as FXR1P, FXR2P and 82-FIP. Interestingly, FMRP can also interact directly with the cytoplasmic proteins CYFIP1 and CYFIP2, which do not bind RNA and link FMRP to the RhoGTPase pathway. The interaction with these different proteins may modulate the functions of FMRP by influencing its affinity to RNA and by affecting the FMRP ability of cytoskeleton remodeling through Rho/Rac GTPases. To better define the relationship of FMRP with its interacting proteins during brain development, we have analyzed the expression pattern of FMRP and its interacting proteins in the cortex, striatum, hippocampus and cerebellum at different ages in wild type (WT) mice. FMRP and FXR2P were strongly expressed during the first week and gradually decreased thereafter, more rapidly in the cerebellum than in the cortex. FXR1P was also expressed early and showed a reduction at later stages of development with a similar developmental pattern in these two regions. CYFIP1 was expressed at all ages and peaked in the third post-natal week. In contrast, CYFIP2 and 82-FIP (only in forebrain regions) were moderately expressed at P3 and gradually increased after P7. In general, the expression pattern of each protein was similar in the regions examined, except for 82-FIP, which exhibited a strong expression at P3 and low levels at later developmental stages in the cerebellum. Our data indicate that FMRP and its interacting proteins have distinct developmental patterns of expression and suggest that FMRP may be preferentially associated to certain proteins in

  3. Concentration Dependent Ion-Protein Interaction Patterns Underlying Protein Oligomerization Behaviours

    NASA Astrophysics Data System (ADS)

    Batoulis, Helena; Schmidt, Thomas H.; Weber, Pascal; Schloetel, Jan-Gero; Kandt, Christian; Lang, Thorsten

    2016-04-01

    Salts and proteins comprise two of the basic molecular components of biological materials. Kosmotropic/chaotropic co-solvation and matching ion water affinities explain basic ionic effects on protein aggregation observed in simple solutions. However, it is unclear how these theories apply to proteins in complex biological environments and what the underlying ionic binding patterns are. Using the positive ion Ca2+ and the negatively charged membrane protein SNAP25, we studied ion effects on protein oligomerization in solution, in native membranes and in molecular dynamics (MD) simulations. We find that concentration-dependent ion-induced protein oligomerization is a fundamental chemico-physical principle applying not only to soluble but also to membrane-anchored proteins in their native environment. Oligomerization is driven by the interaction of Ca2+ ions with the carboxylate groups of aspartate and glutamate. From low up to middle concentrations, salt bridges between Ca2+ ions and two or more protein residues lead to increasingly larger oligomers, while at high concentrations oligomers disperse due to overcharging effects. The insights provide a conceptual framework at the interface of physics, chemistry and biology to explain binding of ions to charged protein surfaces on an atomistic scale, as occurring during protein solubilisation, aggregation and oligomerization both in simple solutions and membrane systems.

  4. Robust regulation of oscillatory Min-protein patterns

    NASA Astrophysics Data System (ADS)

    Halatek, Jacob; Frey, Erwin

    2012-02-01

    Robust spatial patterning was crucial just from the beginning of cellular evolution, and is key to the development of multicellular organisms. In E. Coli, the oscillatory pole-to-pole dynamics of MinCDE proteins functionality prevent improper cell divisions apart from midcell. Min-oscillations are characterized by the remarkable robustness with which spatial patterns dynamically adapt to variations of cell geometry. Moreover, adaption, and therefore proper cell division, is independent of temperature. These observations raise fundamental questions about the underlying core mechanisms, and about the role of spatial cues. With a conceptually novel and universal approach to cellular geometries, we introduce a robust model based on experimental data, consistently explaining the mechanisms underlying pole-to-pole, striped and circular patterns, as well as the observed temperature-dependence. Contrary to prior conjectures, the model predicts that MinD and cardiolipin domains are not colocalized. The key mechanisms are transient sequestration of MinE, and highly canalized transfer of MinD between polar zones. MinD channeling enhances midcell localization and facilitates stripe formation, revealing the potential optimization process from which robust Min-oscillations originally arose.

  5. Protein profile changes during porcine oocyte aging and effects of caffeine on protein expression patterns.

    PubMed

    Jiang, Guang-Jian; Wang, Ke; Miao, De-Qiang; Guo, Lei; Hou, Yi; Schatten, Heide; Sun, Qing-Yuan

    2011-01-01

    It has been shown that oocyte aging critically affects reproduction and development. By using proteomic tools, in the present study, changes in protein profiles during porcine oocyte aging and effects of caffeine on oocyte aging were investigated. By comparing control MII oocytes with aging MII oocytes, we identified 23 proteins that were up-regulated and 3 proteins that were down-regulated during the aging process. In caffeine-treated oocytes, 6 proteins were identified as up-regulated and 12 proteins were identified as down-regulated. A total of 38 differentially expressed proteins grouped into 5 regulation patterns were determined to relate to the aging and anti-aging process. By using the Gene Ontology system, we found that numerous functional gene products involved in metabolism, stress response, reactive oxygen species and cell cycle regulation were differentially expressed during the oocyte aging process, and most of these proteins are for the first time reported in our study, including 2 novel proteins. In addition, several proteins were found to be modified during oocyte aging. These data contribute new information that may be useful for future research on cellular aging and for improvement of oocyte quality.

  6. The E4 protein; structure, function and patterns of expression.

    PubMed

    Doorbar, John

    2013-10-01

    the E1^E4 proteins main functions, the association with the cellular keratin network, and eventually also its cleavage by the protease calpain which allows assembly into amyloid-like fibres and reorganisation of the keratin network. Although the E4 proteins of different HPV types appear divergent at the level of their primary amino acid sequence, they share a recognisable modular organisation and pattern of expression, which may underlie conserved functions and regulation. Assembly into higher-order multimers and suppression of cell proliferation are common to all E4 proteins examined. Although not yet formally demonstrated, a role in virus release and transmission remains a likely function for E4.

  7. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins.

    PubMed

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-03-07

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions.

  8. ceRNA crosstalk stabilizes protein expression and affects the correlation pattern of interacting proteins

    PubMed Central

    Martirosyan, Araks; De Martino, Andrea; Pagnani, Andrea; Marinari, Enzo

    2017-01-01

    Gene expression is a noisy process and several mechanisms, both transcriptional and post-transcriptional, can stabilize protein levels in cells. Much work has focused on the role of miRNAs, showing in particular that miRNA-mediated regulation can buffer expression noise for lowly expressed genes. Here, using in silico simulations and mathematical modeling, we demonstrate that miRNAs can exert a much broader influence on protein levels by orchestrating competition-induced crosstalk between mRNAs. Most notably, we find that miRNA-mediated cross-talk (i) can stabilize protein levels across the full range of gene expression rates, and (ii) modifies the correlation pattern of co-regulated interacting proteins, changing the sign of correlations from negative to positive. The latter feature may constitute a potentially robust signature of the existence of RNA crosstalk induced by endogenous competition for miRNAs in standard cellular conditions. PMID:28266541

  9. Hierarchical protein patterning by meso to molecular scale self-assembly

    NASA Astrophysics Data System (ADS)

    Andersen, Andreas S.; Sutherland, Duncan S.; Ogaki, Ryosuke

    2015-10-01

    Numerous protein patterning methodologies are used extensively for biomedical research and development. We have developed a novel bottom-up protein patterning method using a combination of self-assembly processes in the meso to molecular scale range to allow hierarchical protein patterns to be straightforwardly fabricated with low cost over large areas. As a proof of principle, we patterned vitronectin in various dimensional hierarchies using meso to nanoscale colloids and self-assembled monolayers.

  10. Autophagy and lysosomal related protein expression patterns in human glioblastoma.

    PubMed

    Giatromanolaki, Alexandra; Sivridis, Efthimios; Mitrakas, Achileas; Kalamida, Dimitra; Zois, Christos E; Haider, Syed; Piperidou, Charitomeni; Pappa, Aglaia; Gatter, Kevin C; Harris, Adrian L; Koukourakis, Michael I

    2014-01-01

    Glioblastoma cells are resistant to apoptotic stimuli with autophagic death prevailing under cytotoxic stress. Autophagy interfering agents may represent a new strategy to test in combination with chemo-radiation. We investigated the patterns of expression of autophagy related proteins (LC3A, LC3B, p62, Beclin 1, ULK1 and ULK2) in a series of patients treated with post-operative radiotherapy. Experiments with glioblastoma cell lines (T98 and U87) were also performed to assess autophagic response under conditions simulating the adverse intratumoral environment. Glioblastomas showed cytoplasmic overexpression of autophagic proteins in a varying extent, so that cases could be grouped into low and high expression groups. 10/23, 5/23, 13/23, 5/23, 8/23 and 9/23 cases examined showed extensive expression of LC3A, LC3B, Beclin 1, Ulk 1, Ulk 2 and p62, respectively. Lysosomal markers Cathepsin D and LAMP2a, as well as the lyososomal biogenesis transcription factor TFEB were frequently overexpressed in glioblastomas (10/23, 11/23, and 10/23 cases, respectively). TFEB was directly linked with PTEN, Cathepsin D, HIF1α, LC3B, Beclin 1 and p62 expression. PTEN was also significantly related with LC3B but not LC3A expression, in both immunohistochemistry and gene expression analysis. Confocal microscopy in T98 and U87 cell lines showed distinct identity of LC3A and LC3B autophagosomes. The previously reported stone-like structure (SLS) pattern of LC3 expression was related with prognosis. SLS were inducible in glioblastoma cell lines under exposure to acidic conditions and 2DG mediated glucose antagonism. The present study provides the basis for autophagic characterization of human glioblastoma for further translational studies and targeted therapy trials.

  11. COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways

    SciTech Connect

    Kook, Sung-Ho; Lim, Shin-Saeng; Cho, Eui-Sic; Lee, Young-Hoon; Han, Seong-Kyu; Lee, Kyung-Yeol; Kwon, Jungkee; Hwang, Jae-Won; Bae, Cheol-Hyeon; Seo, Young-Kwon; Lee, Jeong-Chae

    2014-12-12

    Highlights: • COMP-Ang1 induces Tie-2 activation in BMMSCs, but not in primary osteoblasts. • Tie-2 knockdown inhibits COMP-Ang1-stimulated proliferation and osteoblastogenesis. • Tie-2 knockdown prevents COMP-Ang1-induced activation of PI3K/Akt and p38 MAPK. • COMP-Ang1 induces migration of cells via activation of PI3K/Akt and CXCR4 pathways. • COMP-Ang1 stimulates in vivo migration of PDLSCs into a calvarial defect site of rats. - Abstract: Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

  12. Moderate cyclic tensile strain alters the assembly of cartilage extracellular matrix proteins in vitro.

    PubMed

    Bleuel, Judith; Zaucke, Frank; Brüggemann, Gert-Peter; Heilig, Juliane; Wolter, Marie-Louise; Hamann, Nina; Firner, Sara; Niehoff, Anja

    2015-06-01

    Mechanical loading influences the structural and mechanical properties of articular cartilage. The cartilage matrix protein collagen II essentially determines the tensile properties of the tissue and is adapted in response to loading. The collagen II network is stabilized by the collagen II-binding cartilage oligomeric matrix protein (COMP), collagen IX, and matrilin-3. However, the effect of mechanical loading on these extracellular matrix proteins is not yet understood. Therefore, the aim of this study was to investigate if and how chondrocytes assemble the extracellular matrix proteins collagen II, COMP, collagen IX, and matrilin-3 in response to mechanical loading. Primary murine chondrocytes were applied to cyclic tensile strain (6%, 0.5 Hz, 30 min per day at three consecutive days). The localization of collagen II, COMP, collagen IX, and matrilin-3 in loaded and unloaded cells was determined by immunofluorescence staining. The messenger ribo nucleic acid (mRNA) expression levels and synthesis of the proteins were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and western blots. Immunofluorescence staining demonstrated that the pattern of collagen II distribution was altered by loading. In loaded chondrocytes, collagen II containing fibrils appeared thicker and strongly co-stained for COMP and collagen IX, whereas the collagen network from unloaded cells was more diffuse and showed minor costaining. Further, the applied load led to a higher amount of COMP in the matrix, determined by western blot analysis. Our results show that moderate cyclic tensile strain altered the assembly of the extracellular collagen network. However, changes in protein amount were only observed for COMP, but not for collagen II, collagen IX, or matrilin-3. The data suggest that the adaptation to mechanical loading is not always the result of changes in RNA and/or protein expression but might also be the result of changes in matrix assembly and structure.

  13. Patterns of soybean proline-rich protein gene expression.

    PubMed Central

    Wyatt, R E; Nagao, R T; Key, J L

    1992-01-01

    The expression patterns of three members of a gene family that encodes proline-rich proteins in soybean (SbPRPs) were examined using in situ hybridization experiments. In most instances, the expression of SbPRP genes was intense in a limited number of cell types of a particular organ. SbPRP1 RNA was localized in several cell types of soybean hypocotyls, including cells within the phloem and xylem. SbPRP1 expression increased within epidermal cells in the elongating and mature regions of the hypocotyl; expression was detected also in lignified cells surrounding the hilum of mature seeds. SbPRP2 RNA was present in cortical cells and in the vascular tissue of the hypocotyl, especially cells of the phloem. This gene was expressed also in the inner integuments of the mature seed coat. SbPRP3 RNA was localized specifically to the endodermoid layer of cells surrounding the stele in the elongating region of the hypocotyl, as well as in the epidermal cells of leaves and cotyledons. These data show that members of this gene family exhibit cell-specific expression. The members of the SbPRP gene family are expressed in different types of cells and in some cell types that also express the glycine-rich protein or hydroxyproline-rich glycoprotein classes of genes. PMID:1525563

  14. Fabrication of nanometer-sized protein patterns using atomic force microscopy and selective immobilization.

    PubMed Central

    Wadu-Mesthrige, K; Amro, N A; Garno, J C; Xu, S; Liu , G

    2001-01-01

    A new methodology is introduced to produce nanometer-sized protein patterns. The approach includes two main steps, nanopatterning of self-assembled monolayers using atomic force microscopy (AFM)-based nanolithography and subsequent selective immobilization of proteins on the patterned monolayers. The resulting templates and protein patterns are characterized in situ using AFM. Compared with conventional protein fabrication methods, this approach is able to produce smaller patterns with higher spatial precision. In addition, fabrication and characterization are completed in near physiological conditions. The adsorption configuration and bioreactivity of the proteins within the nanopatterns are also studied in situ. PMID:11259301

  15. [The Reading Literacy test for Secondary Education (CompLEC)].

    PubMed

    Llorens Tatay, Ana Cristina; Gil Pelluch, Laura; Vidal-Abarca Gámez, Eduardo; Martínez Giménez, Tomás; Mañá Lloriá, Amelia; Gilabert Pérez, Ramiro

    2011-11-01

    A new test to evaluate reading literacy, the Test of Reading Literacy for Secondary Education (CompLEC) is presented. CompLEC is based on the PISA assessment framework and new definitions of reading literacy. The test, easy to apply and score, assesses the level of reading literacy of children between 11 and 14 years of age in several reading situations (i.e., public, educational, personal and occupational) and with different types of texts (i.e., continuous and non-continuous). The scale has been standardized with a sample of 1,854 students from five different Spanish regions. Empirical results show that CompLEC is a homogeneous, reliable and valid instrument.

  16. COMP-1 promotes competitive advantage of nematode sperm.

    PubMed

    Hansen, Jody M; Chavez, Daniela R; Stanfield, Gillian M

    2015-03-19

    Competition among sperm to fertilize oocytes is a ubiquitous feature of sexual reproduction as well as a profoundly important aspect of sexual selection. However, little is known about the cellular mechanisms sperm use to gain competitive advantage or how these mechanisms are regulated genetically. In this study, we utilize a forward genetic screen in Caenorhabditis elegans to identify a gene, comp-1, whose function is specifically required in competitive contexts. We show that comp-1 functions in sperm to modulate their migration through and localization within the reproductive tract, thereby promoting their access to oocytes. Contrary to previously described models, comp-1 mutant sperm show no defects in size or velocity, thereby defining a novel pathway for preferential usage. Our results indicate not only that sperm functional traits can influence the outcome of sperm competition, but also that these traits can be modulated in a context-dependent manner depending on the presence of competing sperm.

  17. Protein patterning on silicon-based surface using background hydrophobic thin film.

    PubMed

    Lee, Chang-Soo; Lee, Sang-Ho; Park, Sung-Soo; Kim, Yong-Kweon; Kim, Byung-Gee

    2003-04-01

    A new and convenient protein patterning method on silicon-based surface was developed for protein array by spin coating of hydrophobic thin film (CYTOP). Photolithographic lift-off process was used to display two-dimensional patterns of spatially hydrophilic region. The background hydrophobic thin film was used to suppress nonspecific protein binding, and the hydrophilic target protein binding region was chemically modified to introduce aldehyde group after removal of the photoresist layer. The difference in surface energy between the hydrophilic pattern and background hydrophobic film would induce easier covalent binding of proteins onto defined hydrophilic areas having physical and chemical constraints. Below 1 microg/ml of total protein concentration, the CYTOP hydrophobic film effectively suppressed nonspecific binding of the protein. During the process of protein patterning, inherent property of the hydrophobic thin film was not changed judging from static and dynamic contact angle survey. Quantitative analysis of the protein binding was demonstrated by streptavidin-biotin system.

  18. Matrix Gla Protein expression pattern in the early avian embryo.

    PubMed

    Correia, Elizabeth; Conceição, Natércia; Cancela, M Leonor; Belo, José A

    2016-01-01

    MGP (Matrix Gla Protein) is an extracellular matrix vitamin K dependent protein previously identified as a physiological inhibitor of calcification and shown to be well conserved among vertebrates during evolution. MGP is involved in other mechanisms such as TGF-β and BMP activity, and a proposed modulator of cell-matrix interactions. MGP is expressed early in vertebrate development although its role has not been clarified. Previous work in the chicken embryo found MGP localization predominantly in the aorta and aortic valve base, but no data is available earlier in development. Here we examined MGP expression pattern using whole-mount in situ hybridization and histological sectioning during the initial stages of chick development. MGP was first detected at HH10 in the head and in the forming dorsal aorta. At the moment of the onset of blood circulation, MGP was expressed additionally in the venous plexus which will remodel into the vitelline arteries. By E2.25, it is clear that the vitelline arteries are MGP positive. MGP expression progresses centrifugally throughout the area vasculosa of the yolk sac. Between stages HH17 and HH19 MGP is seen in the dorsal aorta, heart, notochord, nephric duct, roof plate, vitelline arteries and in the yolk sac, beneath main arterial branches and in the vicinity of several vessels and venules. MGP expression persists in these areas at least until E4.5. These data suggest that MGP expression could be associated with cell migration and differentiation and to the onset of angiogenesis in the developing chick embryo. This data has biomedical relevance by pointing to the potential use of chick embryo explants to study molecules involved in artery calcification.

  19. Infrared light induced patterning of proteins on ppNIPAM thermoresponsive thin films: a "protein laser printer".

    PubMed

    Cheng, Xuanhong; Yegan Erdem, E; Takeuchi, Shoji; Fujita, Hiroyuki; Ratner, Buddy D; Böhringer, Karl F

    2010-04-21

    Protein micropatterns have applications in fundamental life sciences and clinical medicine. In this work, we present a new technique to create 2-D protein micropatterns by local activation of a thin film of thermoresponsive plasma-deposited poly(N-isopropylacrylamide) (ppNIPAM) using a computer-controlled infrared laser beam. While the whole substrate is exposed to the protein solution, protein deposition happens only at laser-activated locations. A few seconds of laser exposure is all that is required to form a pattern with resolution in the single micrometre range. Successful ligand binding after protein deposition indicates that protein function remains intact after laser-induced adsorption onto ppNIPAM. This rapid, simple technique advances currently available strategies for protein patterning by its potential to pattern proteins in an enclosed environment or onto a 3-D scaffold.

  20. Hierarchical polymer brush nanoarrays: a versatile way to prepare multiscale patterns of proteins.

    PubMed

    Li, Yunfeng; Zhang, Junhu; Liu, Wendong; Li, Daowei; Fang, Liping; Sun, Hongchen; Yang, Bai

    2013-03-01

    This paper presents a versatile way to prepare multiscale and gradient patterns of proteins. The protein patterns are fabricated by conjugating proteins covalently on patterns of polymer brush that are prepared by techniques combining colloidal lithography with photolithography, and two-step colloidal lithography. Taking advantages of this technique, the parameters of protein patterns, such as height, diameters, periods, and distances between two dots, can be arbitrarily tuned. In addition, the protein patterns with varies of architectures, such as microdiscs, microstripes, microrings, microtriangles, microgrids, etc., consisting of protein nanodots, are prepared and the sample size is up to 4 cm(2). The as-prepared patterns of fibronectin can promote the cell adhesion and cell location.

  1. Computation of a diverging Comp-B detonation

    SciTech Connect

    Bukiet, B.G.

    1989-01-01

    The expansion which occurs in diverging detonations weakens the wave and yields pressures and densities below those occurring in planar geometry. We study the problem of a spherically expanding detonation of Comp-B. The effect of varying the order of reaction as well as the rate law parameters (using an Arrhenius burn model) is studied. 14 refs., 3 figs.

  2. Fabrication and characterization of mesoscale protein patterns using atomic force microscopy (AFM)

    NASA Astrophysics Data System (ADS)

    Gao, Pei

    2011-07-01

    A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing. As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It was also demonstrated that the topography of the immobilized protein pattern affects the antibody binding direction. We found that the anti-lysozyme antibodies binding to the edge lysozyme molecules on the half-buried pattern started from the top but the binding on the extruded pattern started from the side because of their different spatial accessibility. In addition, after incubating lysozyme pattern with anti-lysozyme aptamer in buffer solution for enough long time, some fractal-shaped aptamer fibers with 1-6nm high and up to tens of micrometers long were formed by the self-assembling of aptamer molecules on the surface. The aptamer fibers anchor specifically on the edge of protein patterns, which originates from the biospecific recognition between the aptamer and its target protein. Once these edge-bound fibers have formed, they can serve as scaffolds for further assembly processes. We used these aptamer fibers as templates to fabricate palladium and streptavidin nanowires, which anchored on the pattern edges and never cross over or collapse over each other. The aptamer fiber scaffold potentially can lead to an effective means to fabricate and interface nanowires to existing surface patterns. KEYWORDS: Atomic Force Microscopy (AFM), Protein Patterns, Lysozyme, Aptamer

  3. Protein kinase C modulates ventilatory patterning in the developing rat.

    PubMed

    Bandla, H P; Simakajornboon, N; Graff, G R; Gozal, D

    1999-03-01

    Protein kinase C (PKC) mediates important components of signal transduction pathways underlying neuronal excitability and modulates respiratory timing mechanisms in adult rats. To determine ventilatory effects of systemic PKC inhibition during development, whole-body plethysmographic recordings were conducted in 2-3-d (n = 11), 5-6-d (n = 19), 10-12-d (n = 14), and 20-21-d-old (n = 14) rat pups after treatment with vehicle and Ro 32-0432 (100 mg/kg, intraperitoneally). Ro 32-0432 decreased minute ventilation (V E) by 51.0 +/- 5.5% (mean +/- SEM) in youngest pups (p < 0.01) but only 19.1 +/- 6.8% in 20-21-d-old pups (p < 0.01). V E decreases were always due to frequency reductions with tidal volume (VT) remaining unaffected. Respiratory rate decreases primarily resulted from marked expiratory time (TE) prolongations being more pronounced in 2-3-d-old (115.5 +/- 28.9%) compared with 20-21-d old (36.6 +/- 10.9%; p < 0.002 analysis of variance [ANOVA] ). Expression of the PKC isoforms alpha, beta, gamma, delta, iota, and mu was further examined in brainstem and cortex by immunoblotting and revealed different patterns with postnatal age and location. We conclude that endogenous PKC inhibition elicits age-dependent ventilatory reductions which primarily affect timing mechanisms rather than changes in volume drive. This effect on ventilation abates with increasing postnatal age suggesting that the neural substrate mediating overall respiratory output may be more critically dependent on PKC activity in the immature animal.

  4. Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography.

    PubMed

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y; Maynard, Heather D

    2015-03-20

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein, we present a new resist that protects proteins during electron-beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively crosslink to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron-beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high-precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometre and nanometre scale without requiring cleanroom conditions.

  5. Trehalose Glycopolymer Resists Allow Direct Writing of Protein Patterns by Electron-Beam Lithography

    PubMed Central

    Bat, Erhan; Lee, Juneyoung; Lau, Uland Y.; Maynard, Heather D.

    2015-01-01

    Direct-write patterning of multiple proteins on surfaces is of tremendous interest for a myriad of applications. Precise arrangement of different proteins at increasingly smaller dimensions is a fundamental challenge to apply the materials in tissue engineering, diagnostics, proteomics and biosensors. Herein we present a new resist that protects proteins during electron beam exposure and its application in direct-write patterning of multiple proteins. Polymers with pendant trehalose units are shown to effectively cross-link to surfaces as negative resists, while at the same time providing stabilization to proteins during the vacuum and electron beam irradiation steps. In this manner, arbitrary patterns of several different classes of proteins such as enzymes, growth factors and immunoglobulins are realized. Utilizing the high precision alignment capability of electron-beam lithography, surfaces with complex patterns of multiple proteins are successfully generated at the micrometer and nanometer scale without requiring cleanroom conditions. PMID:25791943

  6. Easy fabrication of thin membranes with through holes. Application to protein patterning.

    PubMed

    Masters, Thomas; Engl, Wilfried; Weng, Zhe L; Arasi, Bakya; Gauthier, Nils; Viasnoff, Virgile

    2012-01-01

    Since protein patterning on 2D surfaces has emerged as an important tool in cell biology, the development of easy patterning methods has gained importance in biology labs. In this paper we present a simple, rapid and reliable technique to fabricate thin layers of UV curable polymer with through holes. These membranes are as easy to fabricate as microcontact printing stamps and can be readily used for stencil patterning. We show how this microfabrication scheme allows highly reproducible and highly homogeneous protein patterning with micron sized resolution on surfaces as large as 10 cm(2). Using these stencils, fragile proteins were patterned without loss of function in a fully hydrated state. We further demonstrate how intricate patterns of multiple proteins can be achieved by stacking the stencil membranes. We termed this approach microserigraphy.

  7. BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

    PubMed

    Wei, Qing; La, David; Kihara, Daisuke

    2017-01-01

    Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

  8. THE USE OF EVOLUTIONARY PATTERNS IN PROTEIN ANNOTATION

    PubMed Central

    Wilkins, Angela; Bachman, Ben; Erdin, Serkan; Lichtarge, Olivier

    2012-01-01

    Summary With genomic data skyrocketing, their biological interpretation remains a serious challenge. Diverse computational methods address this problem by pointing to the existence of recurrent patterns among sequence, structure, and function. These patterns emerge naturally from evolutionary variation, natural selection, and divergence—the defining features of biological systems—and they identify molecular events and shapes that underlie specificity of function and allosteric communication. Here we review these methods, and the patterns they identify in case studies and in proteome-wide applications, to infer and rationally redesign function. PMID:22633559

  9. Variants within the COMP and THBS2 genes are not associated with Achilles tendinopathy in a case-control study of South African and Australian populations.

    PubMed

    Saunders, Colleen J; Van Der Merwe, Lize; Cook, Jill; Handley, Christopher J; Collins, Malcolm; September, Alison V

    2014-01-01

    Cartilage oligomeric matrix protein is a structural protein of the extracellular matrix, while thrombospondin-2 is a matricellular protein involved in cell-matrix interactions. Recent studies have shown that genetic variation is a significant risk factor for Achilles tendinopathy, and the genes encoding cartilage oligomeric matrix protein (COMP) and thrombospondin-2 (THBS2) were identified as good candidate genes for association with Achilles tendinopathy. This study aimed to test the association of sequence variants within these candidate genes with the risk of Achilles tendinopathy in participants from South Africa (SA) and Australia (AUS). Three-hundred and forty (133 SA; 207 AUS) control participants with no history of Achilles tendinopathy and 178 (94 SA; 84 AUS) participants clinically diagnosed with Achilles tendinopathy were genotyped for five single nucleotide polymorphisms within the COMP and THBS2 genes in this case-control study. There was no difference in genotype distributions between control and tendinopathy groups for either the THBS2 variants rs9505888, rs6422747 and rs9283850, or the COMP variants rs730079 and rs28494505 in the SA and AUS populations. As the selection of COMP and THBS2 as candidate genes was hypothesis driven, based on biological function, the possibility that other variants within these genes are associated with Achilles tendinopathy cannot be excluded.

  10. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda

    PubMed Central

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-bin; Ye, Jin-zhou; Yang, Man-jun; Jiang, Ming; Peng, Xuan-xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication. PMID:28210241

  11. Outer Membrane Proteins form Specific Patterns in Antibiotic-Resistant Edwardsiella tarda.

    PubMed

    Peng, Bo; Wang, Chao; Li, Hui; Su, Yu-Bin; Ye, Jin-Zhou; Yang, Man-Jun; Jiang, Ming; Peng, Xuan-Xian

    2017-01-01

    Outer membrane proteins of Gram-negative bacteria play key roles in antibiotic resistance. However, it is unknown whether outer membrane proteins that respond to antibiotics behave in a specific manner. The present study specifically investigated the differentially expressed outer membrane proteins of an antibiotic-resistant bacterium, Edwardsiella tarda, a Gram-negative pathogen that can lead to unnecessary mass medication of antimicrobials and consequently resistance development in aquaculture and a spectrum of intestinal and extraintestinal diseases in humans. The comparison of a clinically isolated strain to the laboratory derived kanamycin-, tetracycline-, or chloramphenicol-resistant strains identified their respective outer membrane proteins expression patterns, which are distinct to each other. Similarly, the same approach was utilized to profile the patterns in double antibiotic-resistant bacteria. Surprisingly, one pattern is always dominant over the other as to these three antibiotics; the pattern of chloramphenicol is over tetracycline, which is over kanamycin. This type of pattern was also confirmed in clinically relevant multidrug-resistant bacteria. In addition, the presence of plasmid encoding antibiotic-resistant genes also alters the outer membrane protein profile in a similar manner. Our results demonstrate that bacteria adapt the antibiotic stress through the regulation of outer membrane proteins expression. And more importantly, different outer membrane protein profiles were required to cope with different antibiotics. This type of specific pattern provides the rationale for the development of novel strategy to design outer membrane protein arrays to identify diverse multidrug resistance profiles as biomarkers for clinical medication.

  12. Complex chromatin condensation patterns and nuclear protein transitions during spermiogenesis: examples from mollusks.

    PubMed

    Chiva, M; Saperas, N; Ribes, E

    2011-12-01

    In this paper we review and analyze the chromatin condensation pattern during spermiogenesis in several species of mollusks. Previously, we had described the nuclear protein transitions during spermiogenesis in these species. The results of our study show two types of condensation pattern: simple patterns and complex patterns, with the following general characteristics: (a) When histones (always present in the early spermatid nucleus) are directly replaced by SNBP (sperm nuclear basic proteins) of the protamine type, the spermiogenic chromatin condensation pattern is simple. However, if the replacement is not direct but through intermediate proteins, the condensation pattern is complex. (b) The intermediate proteins found in mollusks are precursor molecules that are processed during spermiogenesis to the final protamine molecules. Some of these final protamines represent proteins with the highest basic amino acid content known to date, which results in the establishment of a very strong electrostatic interaction with DNA. (c) In some instances, the presence of complex patterns of chromatin condensation clearly correlates with the acquisition of specialized forms of the mature sperm nuclei. In contrast, simple condensation patterns always lead to rounded, oval or slightly cylindrical nuclei. (d) All known cases of complex spermiogenic chromatin condensation patterns are restricted to species with specialized sperm cells (introsperm). At the time of writing, we do not know of any report on complex condensation pattern in species with external fertilization and, therefore, with sperm cells of the primitive type (ect-aquasperm). (e) Some of the mollusk an spermiogenic chromatin condensation patterns of the complex type are very similar (almost identical) to those present in other groups of animals. Interestingly, the intermediate proteins involved in these cases can be very different.In this study, we discuss the biological significance of all these features and

  13. Modulation of cell adhesion complexes by surface protein patterns.

    PubMed

    Pesen, Devrim; Haviland, David B

    2009-03-01

    Cell adhesion is an important process in several biological phenomena. To investigate the formation and organization of focal adhesions, we developed a patterning approach based on electron beam lithography. Nanodots (radius <1230 nm) and nanorings (inner radius <320 nm) of fibronectin (FN) were patterned on a K-Casein background. Intracellular vinculin immunofluorescence mirrored the FN nanopatterns. Atomic force microscopy showed that FN nanodots and nanorings organize the immediate cytoskeleton into straight fibrils and diverging fibril bundles, respectively. Our results suggest that a minimum of approximately 40 FN molecules is required for a cell to form a focal adhesion.

  14. Aggrecan- and COMP-loaded poly-(lactic-co-glycolic acid) nanoparticles stimulate chondrogenic differentiation of human mesenchymal stem cells.

    PubMed

    Jeon, Su Yeon; Park, Ji Sun; Yang, Han Na; Woo, Dae Gyun; Park, Keun-Hong

    2014-02-01

    During embryogenesis, specific proteins expressed in cells have key roles in the formation of differentiated cells and tissues. Delivery of specific proteins into specific cells, both in vitro and in vivo, has proved to be exceedingly difficult. In this study, we developed a safe and efficient protein delivery system using encapsulation of proteins into biodegradable poly-(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The PLGA NPs were used to deliver proteins into human mesenchymal stem cells (hMSCs). Fluorescent markers loaded into the PLGA NPs were used to verify the internalization of NPs into hMSCs using FACS analysis and confocal microscopy. With these methods, we demonstrated that the encapsulated model proteins are readily delivered into hMSCs, released from the NP vehicles, and, finally, moved into the cytosols. Using chondrogenesis-related proteins such as aggrecan and cartilage oligomeric matrix protein (COMP), chondrogenic differentiation of hMSCs treated with aggrecan and COMP encapsulated PLGA NPs was clearly observed and caused to differentiate into chondrocytes.

  15. Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks.

    PubMed

    Ramakrishnan, Saminathan; Subramaniam, Sivaraman; Stewart, A Francis; Grundmeier, Guido; Keller, Adrian

    2016-11-16

    DNA origami has become a widely used method for synthesizing well-defined nanostructures with promising applications in various areas of nanotechnology, biophysics, and medicine. Recently, the possibility to transfer the shape of single DNA origami nanostructures into different materials via molecular lithography approaches has received growing interest due to the great structural control provided by the DNA origami technique. Here, we use ordered monolayers of DNA origami nanostructures with internal cavities on mica surfaces as molecular lithography masks for the fabrication of regular protein patterns over large surface areas. Exposure of the masked sample surface to negatively charged proteins results in the directed adsorption of the proteins onto the exposed surface areas in the holes of the mask. By controlling the buffer and adsorption conditions, the protein coverage of the exposed areas can be varied from single proteins to densely packed monolayers. To demonstrate the versatility of this approach, regular nanopatterns of four different proteins are fabricated: the single-strand annealing proteins Redβ and Sak, the iron-storage protein ferritin, and the blood protein bovine serum albumin (BSA). We furthermore demonstrate the desorption of the DNA origami mask after directed protein adsorption, which may enable the fabrication of hierarchical patterns composed of different protein species. Because selectivity in adsorption is achieved by electrostatic interactions between the proteins and the exposed surface areas, this approach may enable also the large-scale patterning of other charged molecular species or even nanoparticles.

  16. Image reversal for direct electron beam patterning of protein coated surfaces.

    PubMed

    Pesen, Devrim; Erlandsson, Anna; Ulfendahl, Mats; Haviland, David B

    2007-11-01

    Electron beam lithography (EBL) is used to create surfaces with protein patterns, which are characterized by immunofluorescence and atomic force microscopies. Both negative and positive image processes are realized by electron beam irradiation of proteins absorbed on a silicon surface, where image reversal is achieved by selectively binding a second species of protein to the electron beam exposed areas on the first protein layer. Biofunctionality at the cellular level was established by culturing cortical cells on patterned lines of fibronectin adsorbed on a bovine serum albumin background for 7 days in culture.

  17. Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

    SciTech Connect

    Valley, Cary T.; Porter, Douglas F.; Qiu, Chen; Campbell, Zachary T.; Tanaka Hall, Traci M.; Wickens, Marvin

    2012-06-28

    mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved 'two-handed' pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.

  18. Protein patterns of black fungi under simulated Mars-like conditions.

    PubMed

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-05-29

    Two species of microcolonial fungi - Cryomyces antarcticus and Knufia perforans - and a species of black yeasts-Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure.

  19. Protein patterns of black fungi under simulated Mars-like conditions

    PubMed Central

    Zakharova, Kristina; Marzban, Gorji; de Vera, Jean-Pierre; Lorek, Andreas; Sterflinger, Katja

    2014-01-01

    Two species of microcolonial fungi – Cryomyces antarcticus and Knufia perforans - and a species of black yeasts–Exophiala jeanselmei - were exposed to thermo-physical Mars-like conditions in the simulation chamber of the German Aerospace Center. In this study the alterations at the protein expression level from various fungi species under Mars-like conditions were analyzed for the first time using 2D gel electrophoresis. Despite of the expectations, the fungi did not express any additional proteins under Mars simulation that could be interpreted as stress induced HSPs. However, up-regulation of some proteins and significant decreasing of protein number were detected within the first 24 hours of the treatment. After 4 and 7 days of the experiment protein spot number was increased again and the protein patterns resemble the protein patterns of biomass from normal conditions. It indicates the recovery of the metabolic activity under Martian environmental conditions after one week of exposure. PMID:24870977

  20. A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels

    NASA Astrophysics Data System (ADS)

    Deforest, Cole A.; Tirrell, David A.

    2015-05-01

    Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.

  1. In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana

    PubMed Central

    Urbanus, Susan L; de Folter, Stefan; Shchennikova, Anna V; Kaufmann, Kerstin; Immink, Richard GH; Angenent, Gerco C

    2009-01-01

    Background MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during

  2. Channel surface patterning of alternating biomimetic protein combinations for enhanced microfluidic tumor cell isolation.

    PubMed

    Launiere, Cari; Gaskill, Marissa; Czaplewski, Gregory; Myung, Ja Hye; Hong, Seungpyo; Eddington, David T

    2012-05-01

    Here, we report a new method for multicomponent protein patterning in a microchannel and also a technique for improving immunoaffinity-based circulating tumor cell (CTC) capture by patterning regions of alternating adhesive proteins using the new method. The first of two proteins, antiepithelial cell adhesion molecule (anti-EpCAM), provides the specificity for CTC capture. The second, E-selectin, increases CTC capture under shear. Patterning regions with and without E-selectin allows captured leukocytes, which also bind E-selectin and are unwanted impurities in CTC isolation, to roll a short distance and detach from the capture surface. This reduces leukocyte capture by up to 82%. The patterning is combined with a leukocyte elution step in which a calcium chelating buffer effectively deactivates E-selectin so that leukocytes may be rinsed away 60% more efficiently than with a buffer containing calcium. The alternating patterning of this biomimetic protein combination, used in conjunction with the elution step, reduces capture of leukocytes while maintaining a high tumor cell capture efficiency that is up to 1.9 times higher than the tumor cell capture efficiency of a surface with only anti-EpCAM. The new patterning technique described here does not require mask alignment and can be used to spatially control the immobilization of any two proteins or protein mixtures inside a sealed microfluidic channel.

  3. Quantity of dietary protein intake, but not pattern of intake, affects net protein balance primarily through differences in protein synthesis in older adults.

    PubMed

    Kim, Il-Young; Schutzler, Scott; Schrader, Amy; Spencer, Horace; Kortebein, Patrick; Deutz, Nicolaas E P; Wolfe, Robert R; Ferrando, Arny A

    2015-01-01

    To examine whole body protein turnover and muscle protein fractional synthesis rate (MPS) following ingestions of protein in mixed meals at two doses of protein and two intake patterns, 20 healthy older adult subjects (52-75 yr) participated in one of four groups in a randomized clinical trial: a level of protein intake of 0.8 g (1RDA) or 1.5 g·kg(-1)·day(-1) (∼2RDA) with uneven (U: 15/20/65%) or even distribution (E: 33/33/33%) patterns of intake for breakfast, lunch, and dinner over the day (1RDA-U, 1RDA-E, 2RDA-U, or 2RDA-E). Subjects were studied with primed continuous infusions of L-[(2)H5]phenylalanine and L-[(2)H2]tyrosine on day 4 following 3 days of diet habituation. Whole body protein kinetics [protein synthesis (PS), breakdown, and net balance (NB)] were expressed as changes from the fasted to the fed states. Positive NB was achieved at both protein levels, but NB was greater in 2RDA vs. 1RDA (94.8 ± 6.0 vs. 58.9 ± 4.9 g protein/750 min; P = 0.0001), without effects of distribution on NB. The greater NB was due to the higher PS with 2RDA vs. 1RDA (15.4 ± 4.8 vs. -18.0 ± 8.4 g protein/750 min; P = 0.0018). Consistent with PS, MPS was greater with 2RDA vs. 1RDA, regardless of distribution patterns. In conclusion, whole body net protein balance was greater with protein intake above recommended dietary allowance (0.8 g protein·kg(-1)·day(-1)) in the context of mixed meals, without demonstrated effects of protein intake pattern, primarily through higher rates of protein synthesis at whole body and muscle levels.

  4. Comparative SDS-page protein patterns of four ascaridid nematodes.

    PubMed

    Ashour, A A; Taha, H A; Mohammad A el-H

    1995-12-01

    In order to investigate the degree of homogeneity and heterogeneity of the ascaridid nematodes. Toxascaris leonina, Parascaris equorum, Toxocara canis and T. vitulorum, protein extracts from adult worms of the four nematodes were resolved into a number of bands. Comparative analysis of dominant bands showed that 13 bands were common among the four species, but certain unique bands were also found in each species including 4 in T. vitulorum, one in T. leonina, two in T. canis, while P. equorum shares both T. canis and T. leonina in most of their bands. Among the four ascaridid studied, T. vitulorum appears to be the most divergent species.

  5. High-quality combinatorial protein libraries using the binary patterning approach.

    PubMed

    Bradley, Luke H

    2014-01-01

    Protein combinatorial libraries have become a platform technology for exploring protein sequence space for novel molecules for use in research, synthetic biology, biotechnology, and medicine. To expedite the isolation of proteins with novel/desired functions using screens and selections, high-quality approaches that generate protein libraries rich in folded and soluble structures are desirable for this goal. The binary patterning approach is a protein library design method that incorporates elements of both rational design and combinatorial diversity to specify the arrangement of polar and nonpolar amino acid residues in the context of a desired, folded tertiary structure template. An overview of the considerations necessary to design and construct binary patterned libraries of de novo and natural proteins is presented.

  6. Fluorescent Biotin Analogues for Microstructure Patterning and Selective Protein Immobilization.

    PubMed

    Krishna, K Vijaya; Ghosh, Subhadip; Sharma, Bikramjit; Singh, Leeju; Mukherjee, Saptarshi; Verma, Sandeep

    2015-11-24

    Benzyl substitution on ureido nitrogens of biotin led to manifestation of aggregation-induced emission, which was studied by steady-state fluorescence, microscopy, and TD-DFT, providing a rationale into the observed photophysical behavior. Besides exhibiting solvatochromism, the biotin derivatives revealed emission peaks centered at ∼430 and 545 nm, which has been attributed to the π-π stacking interactions. Our TD-DFT results also correlate the spectroscopic data and quantify the nature of transitions involved. The isothermal titration calorimetry data substantiates that the binding of the biotin derivatives with avidin are pretty strong. These derivatives on lithographic patterning present a platform for site specific strept(avidin) immobilization, thus opening avenues for potential applications exploiting these interactions. The fluorescent biotin derivatives can thus find applications in cellular biology and imaging.

  7. [Analysis of gingival crevicular fluid. Relation of isoelectric focusing protein patterns to clinical evaluation].

    PubMed

    Aoki, Y; Yoshinaga, E; Tamazawa, O

    1988-12-01

    The purpose of this study was to determine the protein patterns in gingival crevicular fluid relation to the isoelectric focusing protein patterns of GCF and to clinical evaluations. GCF was collected with filter paper from 105 subjects. The probing depth, the gingival index (Löe & Silness) and the plaque index (Silness & Löe) as clinical evaluations The results follow: 1. The main isoelectric focusing protein patterns of GCF were between pH 5.5 and 7.5. In comparison, the GCF and the serum from the same patients showed patterns to similar serum albumin. 2. Between of GCF pH 5.5 and 7.5 the protein patterns that ranged over 60% was pI 5.65, 6.45, 6.55, 6.75 and 7.00. The frequencies of the ranges of protein patterns and clinical evaluation were compared by the X2 test. pI 5.65, 6.45, 6.55 and 6.75 and PD were significant different, as were pI 6.45, 6.55 and 6.75 and GI. But each pI and PIl. were not significantly different.

  8. Extracellular matrix protein patterns guide human chondrocytes adhesion and alignment characterized by vimentin and matrilin-3.

    PubMed

    Pan, Chang-Jiang; Ding, Hong-Yan; Dong, Yun-Xiao

    2013-02-01

    The main purpose of the present study is to investigate the influences of collagen VI (col-VI) patterns on human chondrocytes behaviors. To this end, col-VI stripes with varying width and interstripe spacing are created on polystyrene (PS) surfaces by microcontact printing (μCP). Human chondrocytes are then seeded on these protein patterns and the cell adhesion and alignment are investigated by staining the vimentin and matrilin-3 secreted by seeded chondrocytes. The results indicate that the cells preferentially attach onto the protein areas, rendering cell patterns and the elongated cell shapes. The pattern dimensions can significantly influence cell adhesion, spreading and orientation. The stripe protein patterns can guide cell adhesion and alignment. The cell morphologies can be controlled by carefully designing the pattern shapes and sizes. Our results suggest that the protein patterns can be used to modify biomaterials' surfaces for selective cell-binding and cell alignment. It could provide some cues for the development of novel implantable biomaterials, such as tissue-engineered scaffolds for cartilage replacement, where specific cell alignment is needed.

  9. Protein patterning utilizing region-specific control of wettability by surface modification under atmospheric pressure

    NASA Astrophysics Data System (ADS)

    Lee, Donghee; Kwon, Min-Sung; Hyun, Ji-Chul; Jun, Chang-Duk; Chung, Euiheon; Yang, Sung

    2013-09-01

    Wettability control can be crucial in improving the uniformity of selective protein immobilization in high-density microarrays. In this study, we propose an atmospheric-pressure plasma-enhanced chemical vapor deposition (AP-PECVD)-based method in conjunction with photolithography to implement region-specific control of wettability on Si substrate. The proposed PECVD method under atmospheric pressure condition would be a useful alternative of conventional reactive plasma-based treatments methods requiring vacuum condition for uniform protein patterning. Layers with dissimilar wettability and roughness prepared by AP-PECVD process using tetraethoxysilane (TEOS) or TEOS-O2 as precursors could realize uniform protein patterning in a micrometer-scale.

  10. Global Patterns of Protein Domain Gain and Loss in Superkingdoms

    PubMed Central

    Nasir, Arshan; Kim, Kyung Mo; Caetano-Anollés, Gustavo

    2014-01-01

    Domains are modules within proteins that can fold and function independently and are evolutionarily conserved. Here we compared the usage and distribution of protein domain families in the free-living proteomes of Archaea, Bacteria and Eukarya and reconstructed species phylogenies while tracing the history of domain emergence and loss in proteomes. We show that both gains and losses of domains occurred frequently during proteome evolution. The rate of domain discovery increased approximately linearly in evolutionary time. Remarkably, gains generally outnumbered losses and the gain-to-loss ratios were much higher in akaryotes compared to eukaryotes. Functional annotations of domain families revealed that both Archaea and Bacteria gained and lost metabolic capabilities during the course of evolution while Eukarya acquired a number of diverse molecular functions including those involved in extracellular processes, immunological mechanisms, and cell regulation. Results also highlighted significant contemporary sharing of informational enzymes between Archaea and Eukarya and metabolic enzymes between Bacteria and Eukarya. Finally, the analysis provided useful insights into the evolution of species. The archaeal superkingdom appeared first in evolution by gradual loss of ancestral domains, bacterial lineages were the first to gain superkingdom-specific domains, and eukaryotes (likely) originated when an expanding proto-eukaryotic stem lineage gained organelles through endosymbiosis of already diversified bacterial lineages. The evolutionary dynamics of domain families in proteomes and the increasing number of domain gains is predicted to redefine the persistence strategies of organisms in superkingdoms, influence the make up of molecular functions, and enhance organismal complexity by the generation of new domain architectures. This dynamics highlights ongoing secondary evolutionary adaptations in akaryotic microbes, especially Archaea. PMID:24499935

  11. The EPA CompTox Chemistry Dashboard - an online resource ...

    EPA Pesticide Factsheets

    The U.S. Environmental Protection Agency (EPA) Computational Toxicology Program integrates advances in biology, chemistry, and computer science to help prioritize chemicals for further research based on potential human health risks. This work involves computational and data driven approaches that integrate chemistry, exposure and biological data. As an outcome of these efforts the National Center for Computational Toxicology (NCCT) has measured, assembled and delivered an enormous quantity and diversity of data for the environmental sciences including high-throughput in vitro screening data, in vivo and functional use data, exposure models and chemical databases with associated properties. A series of software applications and databases have been produced over the past decade to deliver these data. Recent work has focused on the development of a new architecture that assembles the resources into a single platform. With a focus on delivering access to Open Data streams, web service integration accessibility and a user-friendly web application the CompTox Dashboard provides access to data associated with ~720,000 chemical substances. These data include research data in the form of bioassay screening data associated with the ToxCast program, experimental and predicted physicochemical properties, product and functional use information and related data of value to environmental scientists. This presentation will provide an overview of the CompTox Dashboard and its va

  12. Expression Patterns of Extracellular Matrix Proteins during Posterior Commissure Development

    PubMed Central

    Stanic, Karen; Saldivia, Natalia; Förstera, Benjamín; Torrejón, Marcela; Montecinos, Hernán; Caprile, Teresa

    2016-01-01

    Extracellular matrix (ECM) molecules are pivotal for central nervous system (CNS) development, facilitating cell migration, axonal growth, myelination, dendritic spine formation, and synaptic plasticity, among other processes. During axon guidance, the ECM not only acts as a permissive or non-permissive substrate for navigating axons, but also modulates the effects of classical guidance cues, such as netrin or Eph/ephrin family members. Despite being highly important, little is known about the expression of ECM molecules during CNS development. Therefore, this study assessed the molecular expression patterns of tenascin, HNK-1, laminin, fibronectin, perlecan, decorin, and osteopontin along chick embryo prosomere 1 during posterior commissure development. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. Located in the dorso-caudal portion of prosomere 1, posterior commissure axons primarily arise from the neurons of basal pretectal nuclei that run dorsally to the roof plate midline, where some turn toward the ipsilateral side. Expressional analysis of ECM molecules in this area these revealed to be highly arranged, and molecule interactions with axon fascicles suggested involvement in processes other than structural support. In particular, tenascin and the HNK-1 epitope extended in ventro-dorsal columns and enclosed axons during navigation to the roof plate. Laminin and osteopontin were expressed in the midline, very close to axons that at this point must decide between extending to the contralateral side or turning to the ipsilateral side. Finally, fibronectin, decorin, and perlecan appeared unrelated to axonal pathfinding in this region and were instead restricted to the external limiting membrane. In summary, the present report provides evidence for an intricate expression of different extracellular molecules that may cooperate in guiding posterior commissure axons. PMID:27733818

  13. Conserved patterns hidden within group A Streptococcus M protein hypervariability recognize human C4b-binding protein

    SciTech Connect

    Buffalo, Cosmo Z.; Bahn-Suh, Adrian J.; Hirakis, Sophia P.; Biswas, Tapan; Amaro, Rommie E.; Nizet, Victor; Ghosh, Partho

    2016-09-05

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ~90%). Such broad recognition is rare, and we discovered a unique mechanism for this through the structure determination of four sequence-diverse M proteins in complexes with C4BP. The structures revealed a uniform and tolerant ‘reading head’ in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies that target the M–C4BP interaction, and also inform a path towards vaccine design.

  14. Proteomic analysis of cow, yak, buffalo, goat and camel milk whey proteins: quantitative differential expression patterns.

    PubMed

    Yang, Yongxin; Bu, Dengpan; Zhao, Xiaowei; Sun, Peng; Wang, Jiaqi; Zhou, Lingyun

    2013-04-05

    To aid in unraveling diverse genetic and biological unknowns, a proteomic approach was used to analyze the whey proteome in cow, yak, buffalo, goat, and camel milk based on the isobaric tag for relative and absolute quantification (iTRAQ) techniques. This analysis is the first to produce proteomic data for the milk from the above-mentioned animal species: 211 proteins have been identified and 113 proteins have been categorized according to molecular function, cellular components, and biological processes based on gene ontology annotation. The results of principal component analysis showed significant differences in proteomic patterns among goat, camel, cow, buffalo, and yak milk. Furthermore, 177 differentially expressed proteins were submitted to advanced hierarchical clustering. The resulting clustering pattern included three major sample clusters: (1) cow, buffalo, and yak milk; (2) goat, cow, buffalo, and yak milk; and (3) camel milk. Certain proteins were chosen as characterization traits for a given species: whey acidic protein and quinone oxidoreductase for camel milk, biglycan for goat milk, uncharacterized protein (Accession Number: F1MK50 ) for yak milk, clusterin for buffalo milk, and primary amine oxidase for cow milk. These results help reveal the quantitative milk whey proteome pattern for analyzed species. This provides information for evaluating adulteration of specific specie milk and may provide potential directions for application of specific milk protein production based on physiological differences among animal species.

  15. Changes in the pattern of protein synthesis during zoospore germination in Blastocladiella emersonii.

    PubMed Central

    Silva, A M; Maia, J C; Juliani, M H

    1987-01-01

    Using two-dimensional gel electrophoresis, we analyzed the pattern of proteins synthesized during Blastocladiella emersonii zoospore germination in an inorganic solution, in both the presence and absence of actinomycin D. During the transition from zoospore to round cells (the first 25 min), essentially no qualitative differences were noticeable, indicating that the earliest stages of germination are entirely preprogrammed with stored RNA. Later in germination (after 25 min), however, changes in the pattern of protein synthesis were found. Some of these proteins (a total of 6 polypeptides) correspond possibly to a selective translation of stored messages, whereas the majority of the changed proteins (22 polypeptides) corresponds to newly synthesized mRNA. Thus, multiple levels of protein synthesis regulation seem to occur during zoospore germination, involving both transcriptional and translational controls. We also analyzed the pattern of protein synthesis during germination in a nutrient medium; synthesis of specific polypeptides occurred during late germination. During early germination posttranslational control was also observed, several labeled proteins from zoospores being specifically degraded or charge modified. Images PMID:3571161

  16. The CompHP Core Competencies Framework for Health Promotion in Europe

    ERIC Educational Resources Information Center

    Barry, Margaret M.; Battel-Kirk, Barbara; Dempsey, Colette

    2012-01-01

    Background: The CompHP Project on Developing Competencies and Professional Standards for Health Promotion in Europe was developed in response to the need for new and changing health promotion competencies to address health challenges. This article presents the process of developing the CompHP Core Competencies Framework for Health Promotion across…

  17. Inverse immunostaining pattern for synthesized versus endocytosed alpha-granule proteins in human bone marrow megakaryocytes.

    PubMed

    de Larouzière, V; Brouland, J P; Souni, F; Drouet, L; Cramer, E

    1998-06-01

    The time of appearance and pattern of expression of several alpha-granule proteins, von Willebrand factor (VWF), fibrinogen and immunoglobulins (Ig) were examined and compared in human bone marrow megakaryocytes (MK) using an immunocytochemical approach. VWF is synthesized by immature MK, whereas it has been shown that fibrinogen is incorporated from the plasma into alpha-granules. The present study was undertaken in order to determine whether there are chronological and morphological differences in the expression of VWF and fibrinogen in vivo in human MK. Seven paraffin-embedded biopsies of normal human bone marrow were labelled with specific antibodies for VWF and for fibrinogen, detected by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. and analysed by immunomorphometry. We found a clear, statistically significant. difference in the labelling pattern of VWF and fibrinogen. The expression of other endocytosed alpha-granule proteins, immunoglobulins G and A, was therefore studied in bone marrow MK from two patients with multiple myeloma, one with monoclonal IgG and one with monoclonal IgA. The immunostaining pattern was similar to that of fibrinogen and different from VWF, and characteristic of endocytosed alpha-granule proteins. This study demonstrates that: (i) immunohistochemical staining of MK alpha-granules proteins distinguishes the peripheral cockade distribution pattern of endocytosed protein from the perinuclear pattern of endogenously synthesized proteins; (ii) VWF is present in human bone marrow MK when fibrinogen is not yet detectable: (iii) VWF synthesis ceases while fibrinogen is still being incorporated: (iv) immunoglobulins can be detected in MK cytoplasm, with a staining pattern resembling that of fibrinogen.

  18. Inference of Hopfield-Potts patterns from covariation in protein families: calculation and statistical error bars

    NASA Astrophysics Data System (ADS)

    Cocco, Simona; Monasson, Rémi; Weigt, Martin

    2013-12-01

    We consider the Hopfield-Potts model for the covariation between residues in protein families recently introduced in Cocco, Monasson, Weigt (2013). The patterns of the model are inferred from the data within a new gauge, more symmetric in the residues. We compute the statistical error bars on the pattern components. Results are illustrated on real data for a response regulator receiver domain (Pfam ID PF00072) family.

  19. Changes in protein patterns and in vivo protein synthesis during senescence of hibiscus petals. [Hibiscus rosa-sinensis

    SciTech Connect

    Woodson, W.R.; Handa, A.K.

    1986-04-01

    Changes in proteins associated with senescence of the flowers of Hibiscus rosa-sinensis was studied using SDS-PAGE. Total extractable protein from petals decreased with senescence. Changes were noted in patterns of proteins from aging petals. Flower opening and senescence was associated with appearance and disappearance of several polypeptides. One new polypeptide with an apparent mw of 41 kd was first seen the day of flower opening and increased to over 9% of the total protein content of senescent petal tissue. Protein synthesis during aging was investigated by following uptake and incorporation of /sup 3/H-leucine into TCA-insoluble fraction of petal discs. Protein synthesis, as evidenced by the percent of label incorporated into the TCA-insoluble fraction, was greatest (32%) the day before flower opening. Senescent petal tissue incorporated 4% of label taken up into protein. Proteins were separated by SDS-PAGE and labelled polypeptides identified by fluorography. In presenescent petal tissue, radioactivity was distributed among several major polypeptides. In senescent tissue, much of the radioactivity was concentrated in the 41 kd polypeptide.

  20. How Surface Heterogeneity Affects Protein Adsorption: Annealing of OTS Patterns and Albumin Adsorption Kinetics*

    PubMed Central

    Hodgkinson, Gerald N.; Hlady, Vladimir

    2009-01-01

    Fluorescence microscopy and intensity histogram analysis techniques were used to monitor spatially-resolved albumin adsorption kinetics to model heterogeneous surfaces on sub-μm scales. Several distinct protein subpopulations were resolved, each represented by a normal distribution of adsorption densities on the adsorbent surface. Histogram analyses provided dynamic information of mean adsorption density, spread in adsorption density, and surface area coverage for each distinct protein subpopulation. A simple adsorption model is proposed in which individual protein binding events are predicted by the summation of multiple protein's surface sub-site interactions with different binding energy sub-sites on adsorbent surfaces. This model is predictive of the albumin adsorption on the patterns produced by one step μ-contact printing (μCP) of octadecyltrichlorosilane (OTS) on glass but fails to describe adsorption once the same patterns are altered by a thermal annealing step. PMID:19746205

  1. Pattern recognition of the secondary structure of proteins (alpha-helix and beta-structure).

    PubMed

    Tohá, J C; Soto, M A; Chinga, H

    1990-09-21

    In this paper, an algorithm for the pattern recognition of secondary structure of proteins is proposed. The procedure simultaneously evaluates the contribution of all the residues of a given peptide to its conformation. By means of the algorithm it is possible to select from a universe of well known proteins the most representative alpha-helix and beta-structure peptides, and to use these peptides, as screening matrices to define the unknown structure of any peptide.

  2. The Kinase Regulator Mob1 Acts as a Patterning Protein for Stentor Morphogenesis

    PubMed Central

    Slabodnick, Mark M.; Ruby, J. Graham; Dunn, Joshua G.; Feldman, Jessica L.; DeRisi, Joseph L.; Marshall, Wallace F.

    2014-01-01

    Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1—with conserved functions in cell division and morphogenesis from plants to humans—as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system. PMID:24823688

  3. The kinase regulator mob1 acts as a patterning protein for stentor morphogenesis.

    PubMed

    Slabodnick, Mark M; Ruby, J Graham; Dunn, Joshua G; Feldman, Jessica L; DeRisi, Joseph L; Marshall, Wallace F

    2014-05-01

    Morphogenesis and pattern formation are vital processes in any organism, whether unicellular or multicellular. But in contrast to the developmental biology of plants and animals, the principles of morphogenesis and pattern formation in single cells remain largely unknown. Although all cells develop patterns, they are most obvious in ciliates; hence, we have turned to a classical unicellular model system, the giant ciliate Stentor coeruleus. Here we show that the RNA interference (RNAi) machinery is conserved in Stentor. Using RNAi, we identify the kinase coactivator Mob1--with conserved functions in cell division and morphogenesis from plants to humans-as an asymmetrically localized patterning protein required for global patterning during development and regeneration in Stentor. Our studies reopen the door for Stentor as a model regeneration system.

  4. Establishing knowledge on the sequence arrangement pattern of nucleated protein folding

    PubMed Central

    Leng, Fei; Xu, Chao; Xia, Xia-Yu; Pan, Xian-Ming

    2017-01-01

    The heat-tolerance mechanisms of (hyper)thermophilic proteins provide a unique opportunity to investigate the unsolved protein folding problem. In an attempt to determine whether the interval between residues in sequence might play a role in determining thermostability, we constructed a sequence interval-dependent value function to calculate the residue pair frequency. Additionally, we identified a new sequence arrangement pattern, where like-charged residues tend to be adjacently assembled, while unlike-charged residues are distributed over longer intervals, using statistical analysis of a large sequence database. This finding indicated that increasing the intervals between unlike-charged residues can increase protein thermostability, with the arrangement patterns of these charged residues serving as thermodynamically favorable nucleation points for protein folding. Additionally, we identified that the residue pairs K-E, R-E, L-V and V-V involving long sequence intervals play important roles involving increased protein thermostability. This work demonstrated a novel approach for considering sequence intervals as keys to understanding protein folding. Our findings of novel relationships between residue arrangement and protein thermostability can be used in industry and academia to aid the design of thermostable proteins. PMID:28273143

  5. Flexible method for fabricating protein patterns on superhydrophobic platforms controlled by magnetic field.

    PubMed

    Wang, Jian; Li, Hao; Zou, Haoyang; Wang, Chenmiao; Zhang, Hao; Mano, João F; Song, Wenlong

    2017-02-28

    Inspired by the rolling of water droplets on lotus leaves, we developed a novel, magnetic field-controlled patterning method for water-soluble proteins and other functional materials on superhydrophobic platforms. This simple method can be used to fabricate biochips and open micro-fluidic devices in a simple way.

  6. Protein synthesis patterns of Paracoccidiodes brasiliensis isolates in stage-specific forms and during cellular differentiation.

    PubMed

    Salem-Izacc, S M; Jesuino, R S; Brito, W A; Pereira, M; Felipe, M S; Soares, C M

    1997-01-01

    In this paper we compared the protein synthesis patterns of Paracoccidioides brasiliensis isolates. The protein profiles were compared for both yeast and mycelial forms and similarity analysis among them was performed by calculating similarity matrices and grouping the isolates in dendrograms. The examined isolates exhibited highly variable cellular morphology at 36 degrees C, when typical yeast cells were expected. On the other hand, at 26 degrees C all the isolates showed mycelial morphology. The analysis of protein synthesis profiles made it possible to cluster the P. brasiliensis isolates into groups that correlated with the morphological data. Interestingly, growth at 36 degrees C strongly decreased the heterogeneity of protein synthesis patterns seen in mycelial isolates. It was possible to cluster the isolates grown at 36 degrees C in three groups based on their two-dimensional protein synthesis analysis. The similarity index observed among the mycelial isolates was lower than that obtained with yeast cells, suggesting a more homogenous gene expression pattern in the host-adapted form than in the saprobic phase.

  7. Inheritance patterns of enzymes and serum proteins of mallard-black duck hybrids

    USGS Publications Warehouse

    Morgan, R.P.; Meritt, D.W.; Block, S.B.; Cole, M.A.; Sulkin, S.T.; Lee, F.B.; Henny, C.J.

    1984-01-01

    From 1974 to 1976, a breeding program was used to produce hybrids of black ducks and mallards for the evaluation of inheritance patterns of serum proteins and serum, liver and muscle enzymes. In addition to the crosses designed to produce hybrids, a series of matings in 1975 and 1976 were designed to evaluate inheritance patterns of a hybrid with either a black duck or mallard. At the F1 level, hybrids were easily distinguished using serum proteins. However, once a hybrid was crossed back to either a mallard or black duck, only 12?23% of the progeny were distinguishable from black ducks or mallards using serum proteins and 23?39% using esterases. Muscle, serum and liver enzymes were similar between the two species.

  8. Inheritance patterns of enzymes and serum proteins of mallard-black duck hybrids

    USGS Publications Warehouse

    Morgan, R.P.; Meritt, D.W.; Block, S.B.; Cole, M.

    1984-01-01

    From 1974 to 1976, a breeding program was used to produce hybrids of black ducks and mallards for the evaluation of inheritance patterns of serum proteins and serum, liver and muscle enzymes. In addition to the crosses designed to produce hybrids, a series of matings in 1975 and 1976 were designed to evaluate inheritance patterns of a hybrid with either a black duck or mallard. At the F1 level, hybrids were easily distinguished using serum proteins. However, once a hybrid was crossed back to either a mallard or black duck, only 12-23% of the progeny were distinguishable from black ducks or mallards using serum proteins and 23-39% using esterases. Muscle, serum and liver enzymes were similar between the two species.

  9. Protein secretory patterns of rat Sertoli and peritubular cells are influenced by culture conditions

    SciTech Connect

    Kierszenbaum, A.L.; Crowell, J.A.; Shabanowitz, R.B.; DePhilip, R.M.; Tres, L.L.

    1986-08-01

    An approach combining two-dimensional gel electrophoresis and autoradiography was used to correlate patterns of secretory proteins in cultures of Sertoli and peritubular cells with those observed in the incubation medium from segments of seminiferous tubules. Sertoli cells in culture and in seminiferous tubules secreted three proteins designated S70 (Mr 72,000-70,000), S45 (Mr 45,000), and S35 (Mr 35,000). Cultured Sertoli and peritubular cells and incubated seminiferous tubules secreted two proteins designated SP1 (Mr 42,000) and SP2 (Mr 50,000). SP1 and S45 have similar Mr but differ from each other in isoelectric point (pI). Cultured peritubular cells secreted a protein designated P40 (Mr 40,000) that was also seen in intact seminiferous tubules but not in seminiferous tubules lacking the peritubular cell wall. However, a large number of high-Mr proteins were observed only in the medium of cultured peritubular cells but not in the incubation medium of intact seminiferous tubules. Culture conditions influence the morphology and patterns of protein secretion of cultured peritubular cells. Peritubular cells that display a flat-stellate shape transition when placed in culture medium free of serum (with or without hormones and growth factors), accumulate various proteins in the medium that are less apparent when these cells are maintained in medium supplemented with serum. Two secretory proteins stimulated by follicle-stimulating hormone (FSH) (designated SCm1 and SCm2) previously found in the medium of cultured Sertoli cells, were also observed in the incubation medium of seminiferous tubular segments stimulated by FSH. Results of this study show that, although cultured Sertoli and peritubular cells synthesize and secrete proteins also observed in segments of incubated seminiferous tubules anther group of proteins lacks seminiferous tubular correlates.

  10. Ser/Thr Motifs in Transmembrane Proteins: Conservation Patterns and Effects on Local Protein Structure and Dynamics

    PubMed Central

    del Val, Coral; White, Stephen H.

    2014-01-01

    We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide. PMID:22836667

  11. Biophysical Models of Protein Evolution: Understanding the Patterns of Evolutionary Sequence Divergence.

    PubMed

    Echave, Julian; Wilke, Claus O

    2017-03-15

    For decades, rates of protein evolution have been interpreted in terms of the vague concept of functional importance. Slowly evolving proteins or sites within proteins were assumed to be more functionally important and thus subject to stronger selection pressure. More recently, biophysical models of protein evolution, which combine evolutionary theory with protein biophysics, have completely revolutionized our view of the forces that shape sequence divergence. Slowly evolving proteins have been found to evolve slowly because of selection against toxic misfolding and misinteractions, linking their rate of evolution primarily to their abundance. Similarly, most slowly evolving sites in proteins are not directly involved in function, but mutating these sites has a large impact on protein structure and stability. In this article, we review the studies in the emerging field of biophysical protein evolution that have shaped our current understanding of sequence divergence patterns. We also propose future research directions to develop this nascent field. Expected final online publication date for the Annual Review of Biophysics Volume 46 is May 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  12. Protein coverage on polymer nanolayers leading to mesenchymal stem cell patterning.

    PubMed

    You, Jungmok; Yoshida, Akihito; Heo, June Seok; Kim, Han-Soo; Kim, Hyun Ok; Tamada, Kaoru; Kim, Eunkyoung

    2011-10-21

    Interactions of gelatin and albumin with a photo-reactive diphenylamino-s-triazine bridged p-phenylene vinylene polymer (DTOPV) were examined by using surface plasmon resonance (SPR) spectroscopy to explore the effect of the polymer structure on protein coverage of DTOPV nanofilms. The SPR data revealed a significant increase of gelatin adsorption on UV-DTOPV nanofilms, while the adsorption of albumin was decreased by UV exposure in the time frame of the experiment. We also found that the selective adsorption of these proteins was highly dependent on the protein concentration; the highest selectivity of protein adsorption was obtained at the lowest concentration (3.5 μg ml(-1)), while no selective adsorption was confirmed at high concentrations (350 and 1000 μg ml(-1)). The selective attachment of mesenchymal stem cells (MSCs) was directly correlated with the selective adsorption of these proteins onto DTOPV nanofilms. The MSCs attachment onto UV-DTOPV films was promoted with only small mass coverage of gelatin, which led to MSC patterning onto the patterned DTOPV nanofilms successfully. The role of cell adhesion proteins that we found in this study will be a clue to elucidate the complex response of biomolecules on functional polymer nanolayers, and contribute to build up biocompatible surfaces on various advanced materials for the sake of cell engineering and medical implants.

  13. Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures

    PubMed Central

    Tesei, Donatella; Marzban, Gorji; Zakharova, Kristina; Isola, Daniela; Selbmann, Laura; Sterflinger, Katja

    2012-01-01

    Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi – Coniosporium perforans, Exophiala jeanselmei – and of the extremophilic fungus – Friedmanniomyces endolithicus – were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum. PMID:22862921

  14. Alteration of protein patterns in black rock inhabiting fungi as a response to different temperatures.

    PubMed

    Tesei, Donatella; Marzban, Gorji; Zakharova, Kristina; Isola, Daniela; Selbmann, Laura; Sterflinger, Katja

    2012-08-01

    Rock inhabiting fungi are among the most stress tolerant organisms on Earth. They are able to cope with different stressors determined by the typical conditions of bare rocks in hot and cold extreme environments. In this study first results of a system biological approach based on two-dimensional protein profiles are presented. Protein patterns of extremotolerant black fungi -Coniosporium perforans, Exophiala jeanselmei - and of the extremophilic fungus -Friedmanniomyces endolithicus - were compared with the cosmopolitan and mesophilic hyphomycete Penicillium chrysogenum in order to follow and determine changes in the expression pattern under different temperatures. The 2D protein gels indicated a temperature dependent qualitative change in all the tested strains. Whereas the reference strain P. chrysogenum expressed the highest number of proteins at 40 °C, thus exhibiting real signs of temperature induced reaction, black fungi, when exposed to temperatures far above their growth optimum, decreased the number of proteins indicating a down-regulation of their metabolism. Temperature of 1 °C led to an increased number of proteins in all of the analysed strains, with the exception of P. chrysogenum. These first results on temperature dependent reactions in rock inhabiting black fungi indicate a rather different strategy to cope with non-optimal temperature than in the mesophilic hyphomycete P. chrysogenum.

  15. Ultrathin coatings from isocyanate terminated star PEG prepolymers: patterning of proteins on the layers.

    PubMed

    Groll, Juergen; Haubensak, Wulf; Ameringer, Thomas; Moeller, Martin

    2005-03-29

    This study presents the easy and fast patterning of low molecular weight molecules that act as binding partners for proteins on Star PEG coatings. These coatings are prepared from isocyanate terminated star shaped prepolymers and form a highly cross-linked network on the substrate in which the stars are connected via urea groups and free amino groups are present. Streptavidin has been patterned on these layers by microcontact printing (muCP) of an amino reactive biotin derivative and consecutive binding of streptavidin to the biotin. Patterns of Ni(2+)-nitriltriacetic acid (NTA) receptors have been prepared by printing amino functional NTA molecules in freshly prepared Star PEG layers that still contain amino reactive isocyanate groups. Complexation of the NTA groups with Ni(II) ions enabled the binding of His-tag enhanced green fluorescent protein (EGFP) in the desired pattern on the substrates. Since the unmodified Star PEG layers prevent unspecific protein adsorption, His-EGFP could selectively be bound to the sample by immersion into crude, nonpurified His-tag EGFP containing cell lysate.

  16. Biased inheritance of the protein PatN frees vegetative cells to initiate patterned heterocyst differentiation.

    PubMed

    Risser, Douglas D; Wong, Francis C Y; Meeks, John C

    2012-09-18

    Heterocysts, cells specialized for nitrogen fixation in certain filamentous cyanobacteria, appear singly in a nonrandom spacing pattern along the chain of vegetative cells. A two-stage, biased initiation and competitive resolution model has been proposed to explain the establishment of this spacing pattern. There is substantial evidence that competitive resolution of a subset of cells initiating differentiation occurs by interactions between a self-enhancing activator protein, HetR, and a diffusible pentapeptide inhibitor PatS-5 (RGSGR). Results presented here show that the absence of a unique membrane protein, PatN, in Nostoc punctiforme strain ATCC 29133 leads to a threefold increase in heterocyst frequency and a fourfold decrease in the vegetative cell interval between heterocysts. A PatN-GFP translational fusion shows a pattern of biased inheritance in daughter vegetative cells of ammonium-grown cultures. Inactivation of another heterocyst patterning gene, patA, is epistatic to inactivation of patN, and transcription of patA increases in a patN-deletion strain, implying that patN may function by modulating levels of patA. The presence of PatN is hypothesized to decrease the competency of a vegetative cell to initiate heterocyst differentiation, and the cellular concentration of PatN is dependent on cell division that results in cells transiently depleted of PatN. We suggest that biased inheritance of cell-fate determinants is a phylogenetic domain-spanning paradigm in the development of biological patterns.

  17. Analysis of evolutionary conservation patterns and their influence on identifying protein functional sites.

    PubMed

    Fang, Chun; Noguchi, Tamotsu; Yamana, Hayato

    2014-10-01

    Evolutionary conservation information included in position-specific scoring matrix (PSSM) has been widely adopted by sequence-based methods for identifying protein functional sites, because all functional sites, whether in ordered or disordered proteins, are found to be conserved at some extent. However, different functional sites have different conservation patterns, some of them are linear contextual, some of them are mingled with highly variable residues, and some others seem to be conserved independently. Every value in PSSMs is calculated independently of each other, without carrying the contextual information of residues in the sequence. Therefore, adopting the direct output of PSSM for prediction fails to consider the relationship between conservation patterns of residues and the distribution of conservation scores in PSSMs. In order to demonstrate the importance of combining PSSMs with the specific conservation patterns of functional sites for prediction, three different PSSM-based methods for identifying three kinds of functional sites have been analyzed. Results suggest that, different PSSM-based methods differ in their capability to identify different patterns of functional sites, and better combining PSSMs with the specific conservation patterns of residues would largely facilitate the prediction.

  18. Identifying known unknowns using the US EPA's CompTox ...

    EPA Pesticide Factsheets

    Chemical features observed using high-resolution mass spectrometry can be tentatively identified using online chemical reference databases by searching molecular formulae and monoisotopic masses and then rank-ordering of the hits using appropriate relevance criteria. The most likely candidate “known unknowns,” which are those chemicals unknown to an investigator but contained within a reference database or literature source, rise to the top of a chemical list when rank-ordered by the number of associated data sources. The U.S. EPA’s CompTox Chemistry Dashboard is a curated and freely available resource for chemistry and computational toxicology research, containing more than 720,000 chemicals of relevance to environmental health science. In this research, the performance of the Dashboard for identifying “known unknowns” was evaluated against that of the online ChemSpider database, one of the primary resources used by mass spectrometrists, using multiple previously studied datasets reported in the peer-reviewed literature totaling 162 chemicals. These chemicals were examined using both applications via molecular formula and monoisotopic mass searches followed by rank-ordering of candidate compounds by associated references or data sources. A greater percentage of chemicals ranked in the top position when using the Dashboard, indicating an advantage of this application over ChemSpider for identifying known unknowns using data source ranking. Addition

  19. Mapping Protein Abundance Patterns in the Brain Using Voxelation Combined with Liquid Chromatography and Mass Spectrometry

    PubMed Central

    Petyuk, Vladislav A.; Qian, Wei-Jun; Smith, Richard D.; Smith, Desmond J.

    2009-01-01

    Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps. PMID:19654045

  20. Patterning protein molecules on poly(ethylene glycol) coated Si(111).

    PubMed

    Jun, Yongseok; Cha, Taewoon; Guo, Athena; Zhu, X-Y

    2004-08-01

    We demonstrate spatially localized immobilization of protein molecules on high-density poly(ethylene glycol) (PEG) coated Si(111). Patterns of HO- and CH3O-terminated PEG regions are formed on silicon surfaces based on soft lithography techniques and an efficient reaction between alcohol functional groups and chlorine-terminated silicon. Activation of the HO-terminated PEG brush is achieved via either partial oxidation to form aldehyde groups or via attachment of efficient leaving groups. Protein molecules are covalently immobilized to these activated regions on the PEG/Si surface.

  1. Mapping protein abundance patterns in the brain using voxelation combined with liquid chromatography and mass spectrometry

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Smith, Richard D.; Smith, Desmond J.

    2010-02-01

    Voxelation creates expression atlases by high-throughput analysis of spatially registered cubes or voxels harvested from the brain. The modality independence of voxelation allows a variety of bioanalytical techniques to be used to map abundance. Protein expression patterns in the brain can be obtained using liquid chromatography (LC) combined with mass spectrometry (MS). Here we describe the methodology of voxelation as it pertains particularly to LC-MS proteomic analysis: sample preparation, instrumental set up and analysis, peptide identification and protein relative abundance quantitation. We also briefly describe some of the advantages, limitations and insights into the brain that can be obtained using combined proteomic and transcriptomic maps

  2. Protein Modification by Deamidation Indicates Variations in Joint Extracellular Matrix Turnover*

    PubMed Central

    Catterall, Jonathan B.; Hsueh, Ming F.; Stabler, Thomas V.; McCudden, Christopher R.; Bolognesi, Michael; Zura, Robert; Jordan, Joanne M.; Renner, Jordan B.; Feng, Sheng; Kraus, Virginia B.

    2012-01-01

    As extracellular proteins age, they undergo and accumulate nonenzymatic post-translational modifications that cannot be repaired. We hypothesized that these could be used to systemically monitor loss of extracellular matrix due to chronic arthritic diseases such as osteoarthritis (OA). To test this, we predicted sites of deamidation in cartilage oligomeric matrix protein (COMP) and confirmed, by mass spectroscopy, the presence of deamidated (Asp64) and native (Asn64) COMP epitopes (mean 0.95% deamidated COMP (D-COMP) relative to native COMP) in cartilage. An Asp64, D-COMP-specific ELISA was developed using a newly created monoclonal antibody 6-1A12. In a joint replacement study, serum D-COMP (p = 0.017), but not total COMP (p = 0.5), declined significantly after replacement demonstrating a joint tissue source for D-COMP. In analyses of 450 participants from the Johnston County Osteoarthritis Project controlled for age, gender, and race, D-COMP was associated with radiographic hip (p < 0.0001) but not knee (p = 0.95) OA severity. In contrast, total COMP was associated with radiographic knee (p < 0.0001) but not hip (p = 0.47) OA severity. D-COMP was higher in soluble proteins extracted from hip cartilage proximal to OA lesions compared with remote from lesions (p = 0.007) or lesional and remote OA knee (p < 0.01) cartilage. Total COMP in cartilage did not vary by joint site or proximity to the lesion. This study demonstrates the presence of D-COMP in articular cartilage and the systemic circulation, and to our knowledge, it is the first biomarker to show specificity for a particular joint site. We believe that enrichment of deamidated epitope in hip OA cartilage indicates a lesser repair response of hip OA compared with knee OA cartilage. PMID:22179616

  3. The effect of venting on cookoff of a melt-castable explosive (Comp-B)

    SciTech Connect

    Hobbs, Michael L.; Kaneshige, Michael J.

    2015-03-01

    Occasionally, our well-controlled cookoff experiments with Comp-B give anomalous results when venting conditions are changed. For example, a vented experiment may take longer to ignite than a sealed experiment. In the current work, we show the effect of venting on thermal ignition of Comp-B. We use Sandia’s Instrumented Thermal Ignition (SITI) experiment with various headspace volumes in both vented and sealed geometries to study ignition of Comp-B. In some of these experiments, we have used a boroscope to observe Comp-B as it melts and reacts. We propose that the mechanism for ignition involves TNT melting, dissolution of RDX, and complex bubbly liquid flow. High pressure inhibits bubble formation and flow is significantly reduced. At low pressure, a vigorous dispersed bubble flow was observed.

  4. The effect of venting on cookoff of a melt-castable explosive (Comp-B)

    DOE PAGES

    Hobbs, Michael L.; Kaneshige, Michael J.

    2015-03-01

    Occasionally, our well-controlled cookoff experiments with Comp-B give anomalous results when venting conditions are changed. For example, a vented experiment may take longer to ignite than a sealed experiment. In the current work, we show the effect of venting on thermal ignition of Comp-B. We use Sandia’s Instrumented Thermal Ignition (SITI) experiment with various headspace volumes in both vented and sealed geometries to study ignition of Comp-B. In some of these experiments, we have used a boroscope to observe Comp-B as it melts and reacts. We propose that the mechanism for ignition involves TNT melting, dissolution of RDX, and complexmore » bubbly liquid flow. High pressure inhibits bubble formation and flow is significantly reduced. At low pressure, a vigorous dispersed bubble flow was observed.« less

  5. The ZIC gene family encodes multi-functional proteins essential for patterning and morphogenesis.

    PubMed

    Houtmeyers, Rob; Souopgui, Jacob; Tejpar, Sabine; Arkell, Ruth

    2013-10-01

    The zinc finger of the cerebellum gene (ZIC) discovered in Drosophila melanogaster (odd-paired) has five homologs in Xenopus, chicken, mice, and humans, and seven in zebrafish. This pattern of gene copy expansion is accompanied by a divergence in gene and protein structure, suggesting that Zic family members share some, but not all, functions. ZIC genes are implicated in neuroectodermal development and neural crest cell induction. All share conserved regions encoding zinc finger domains, however their heterogeneity and specification remain unexplained. In this review, the evolution, structure, and expression patterns of the ZIC homologs are described; specific functions attributable to individual family members are supported. A review of data from functional studies in Xenopus and murine models suggest that ZIC genes encode multifunctional proteins operating in a context-specific manner to drive critical events during embryogenesis. The identification of ZIC mutations in congenital syndromes highlights the relevance of these genes in human development.

  6. MAMP (microbe-associated molecular pattern)-induced changes in plasma membrane-associated proteins.

    PubMed

    Uhlíková, Hana; Solanský, Martin; Hrdinová, Vendula; Šedo, Ondrej; Kašparovský, Tomáš; Hejátko, Jan; Lochman, Jan

    2017-03-01

    Plant plasma membrane associated proteins play significant roles in Microbe-Associated Molecular Pattern (MAMP) mediated defence responses including signal transduction, membrane transport or energetic metabolism. To elucidate the dynamics of proteins associated with plasma membrane in response to cryptogein, a well-known MAMP of defence reaction secreted by the oomycete Phytophthora cryptogea, 2D-Blue Native/SDS gel electrophoresis of plasma membrane fractions was employed. This approach revealed 21 up- or down-regulated protein spots of which 15 were successfully identified as proteins related to transport through plasma membrane, vesicle trafficking, and metabolic enzymes including cytosolic NADP-malic enzyme and glutamine synthetase. Observed changes in proteins were also confirmed on transcriptional level by qRT-PCR analysis. In addition, a significantly decreased accumulation of transcripts observed after employment of a mutant variant of cryptogein Leu41Phe, exhibiting a conspicuous defect in induction of resistance, sustains the contribution of identified proteins in cryptogein-triggered cellular responses. Our data provide further evidence for dynamic MAMP-induced changes in plasma membrane associated proteins.

  7. Automatic classification and pattern discovery in high-throughput protein crystallization trials.

    PubMed

    Cumbaa, Christian; Jurisica, Igor

    2005-01-01

    Conceptually, protein crystallization can be divided into two phases search and optimization. Robotic protein crystallization screening can speed up the search phase, and has a potential to increase process quality. Automated image classification helps to increase throughput and consistently generate objective results. Although the classification accuracy can always be improved, our image analysis system can classify images from 1,536-well plates with high classification accuracy (85%) and ROC score (0.87), as evaluated on 127 human-classified protein screens containing 5,600 crystal images and 189,472 non-crystal images. Data mining can integrate results from high-throughput screens with information about crystallizing conditions, intrinsic protein properties, and results from crystallization optimization. We apply association mining, a data mining approach that identifies frequently occurring patterns among variables and their values. This approach segregates proteins into groups based on how they react in a broad range of conditions, and clusters cocktails to reflect their potential to achieve crystallization. These results may lead to crystallization screen optimization, and reveal associations between protein properties and crystallization conditions. We also postulate that past experience may lead us to the identification of initial conditions favorable to crystallization for novel proteins.

  8. Abundantly and rarely expressed Lhc protein genes exhibit distinct regulation patterns in plants.

    PubMed

    Klimmek, Frank; Sjödin, Andreas; Noutsos, Christos; Leister, Dario; Jansson, Stefan

    2006-03-01

    We have analyzed gene regulation of the Lhc supergene family in poplar (Populus spp.) and Arabidopsis (Arabidopsis thaliana) using digital expression profiling. Multivariate analysis of the tissue-specific, environmental, and developmental Lhc expression patterns in Arabidopsis and poplar was employed to characterize four rarely expressed Lhc genes, Lhca5, Lhca6, Lhcb7, and Lhcb4.3. Those genes have high expression levels under different conditions and in different tissues than the abundantly expressed Lhca1 to 4 and Lhcb1 to 6 genes that code for the 10 major types of higher plant light-harvesting proteins. However, in some of the datasets analyzed, the Lhcb4 and Lhcb6 genes as well as an Arabidopsis gene not present in poplar (Lhcb2.3) exhibited minor differences to the main cooperative Lhc gene expression pattern. The pattern of the rarely expressed Lhc genes was always found to be more similar to that of PsbS and the various light-harvesting-like genes, which might indicate distinct physiological functions for the rarely and abundantly expressed Lhc proteins. The previously undetected Lhcb7 gene encodes a novel plant Lhcb-type protein that possibly contains an additional, fourth, transmembrane N-terminal helix with a highly conserved motif. As the Lhcb4.3 gene seems to be present only in Eurosid species and as its regulation pattern varies significantly from that of Lhcb4.1 and Lhcb4.2, we conclude it to encode a distinct Lhc protein type, Lhcb8.

  9. Discovering co-occurring patterns and their biological significance in protein families

    PubMed Central

    2014-01-01

    Background The large influx of biological sequences poses the importance of identifying and correlating conserved regions in homologous sequences to acquire valuable biological knowledge. These conserved regions contain statistically significant residue associations as sequence patterns. Thus, patterns from two conserved regions co-occurring frequently on the same sequences are inferred to have joint functionality. A method for finding conserved regions in protein families with frequent co-occurrence patterns is proposed. The biological significance of the discovered clusters of conserved regions with co-occurrences patterns can be validated by their three-dimensional closeness of amino acids and the biological functionality found in those regions as supported by published work. Methods Using existing algorithms, we discovered statistically significant amino acid associations as sequence patterns. We then aligned and clustered them into Aligned Pattern Clusters (APCs) corresponding to conserved regions with amino acid conservation and variation. When one APC frequently co-occured with another APC, the two APCs have high co-occurrence. We then clustered APCs with high co-occurrence into what we refer to as Co-occurrence APC Clusters (Co-occurrence Clusters). Results Our results show that for Co-occurrence Clusters, the three-dimensional distance between their amino acids is closer than average amino acid distances. For the Co-occurrence Clusters of the ubiquitin and the cytochrome c families, we observed biological significance among the residing amino acids of the APCs within the same cluster. In ubiquitin, the residues are responsible for ubiquitination as well as conventional and unconventional ubiquitin-bindings. In cytochrome c, amino acids in the first co-occurrence cluster contribute to binding of other proteins in the electron transport chain, and amino acids in the second co-occurrence cluster contribute to the stability of the axial heme ligand

  10. Mathematical Characterization of Protein Sequences Using Patterns as Chemical Group Combinations of Amino Acids.

    PubMed

    Das, Jayanta Kumar; Das, Provas; Ray, Korak Kumar; Choudhury, Pabitra Pal; Jana, Siddhartha Sankar

    2016-01-01

    Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as 'FPKATD' and 'Y/FTNEKL' without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids' pattern in different proteins.

  11. Mathematical Characterization of Protein Sequences Using Patterns as Chemical Group Combinations of Amino Acids

    PubMed Central

    Choudhury, Pabitra Pal; Jana, Siddhartha Sankar

    2016-01-01

    Comparison of amino acid sequence similarity is the fundamental concept behind the protein phylogenetic tree formation. By virtue of this method, we can explain the evolutionary relationships, but further explanations are not possible unless sequences are studied through the chemical nature of individual amino acids. Here we develop a new methodology to characterize the protein sequences on the basis of the chemical nature of the amino acids. We design various algorithms for studying the variation of chemical group transitions and various chemical group combinations as patterns in the protein sequences. The amino acid sequence of conventional myosin II head domain of 14 family members are taken to illustrate this new approach. We find two blocks of maximum length 6 aa as ‘FPKATD’ and ‘Y/FTNEKL’ without repeating the same chemical nature and one block of maximum length 20 aa with the repetition of chemical nature which are common among all 14 members. We also check commonality with another motor protein sub-family kinesin, KIF1A. Based on our analysis we find a common block of length 8 aa both in myosin II and KIF1A. This motif is located in the neck linker region which could be responsible for the generation of mechanical force, enabling us to find the unique blocks which remain chemically conserved across the family. We also validate our methodology with different protein families such as MYOI, Myosin light chain kinase (MLCK) and Rho-associated protein kinase (ROCK), Na+/K+-ATPase and Ca2+-ATPase. Altogether, our studies provide a new methodology for investigating the conserved amino acids’ pattern in different proteins. PMID:27930687

  12. Expression Pattern of Fatty Acid Binding Proteins in Celiac Disease Enteropathy

    PubMed Central

    Bottasso Arias, Natalia M.; García, Marina; Bondar, Constanza; Guzman, Luciana; Redondo, Agustina; Chopita, Nestor; Córsico, Betina; Chirdo, Fernando G.

    2015-01-01

    Celiac disease (CD) is an immune-mediated enteropathy that develops in genetically susceptible individuals following exposure to dietary gluten. Severe changes at the intestinal mucosa observed in untreated CD patients are linked to changes in the level and in the pattern of expression of different genes. Fully differentiated epithelial cells express two isoforms of fatty acid binding proteins (FABPs): intestinal and liver, IFABP and LFABP, respectively. These proteins bind and transport long chain fatty acids and also have other important biological roles in signaling pathways, particularly those related to PPARγ and inflammatory processes. Herein, we analyze the serum levels of IFABP and characterize the expression of both FABPs at protein and mRNA level in small intestinal mucosa in severe enteropathy and normal tissue. As a result, we observed higher levels of circulating IFABP in untreated CD patients compared with controls and patients on gluten-free diet. In duodenal mucosa a differential FABPs expression pattern was observed with a reduction in mRNA levels compared to controls explained by the epithelium loss in severe enteropathy. In conclusion, we report changes in FABPs' expression pattern in severe enteropathy. Consequently, there might be alterations in lipid metabolism and the inflammatory process in the small intestinal mucosa. PMID:26346822

  13. Ethanol and Cancer Induce Similar Changes on Protein Expression Pattern of Human Fibroblast Cell

    PubMed Central

    Zamanian–Azodi, Mona; Rezaei-Tavirani, Mostafa; Rahmati-Rad, Sara; Rezaei Tavirani, Majid

    2016-01-01

    Ethanol has a vast consumption around the world. Many researches confirmed some adverse effect of this component on human health. In addition, recent studies showed significant alteration in both cellular population, and protein profile of human foreskin fibroblast cell line (HFFF2) in the specific dosage of ethanol. Here, the role and interaction of some proteins (characterized by significant alteration in expression due to ethanol effect) analyzed by proteomics and evaluated by considering cancerous case. 2D-electrophoresis findings of comparison of normal fibroblast cells and treated fibroblast with 270 mM dosage of ethanol analyzed by using SameSpots software, R software, and Cytoscape for protein-protein interaction (PPI) investigation. Six proteins with significantly altered expression associated with fundamental properties in a cell identified in ethanol-treated sample. These include AnnexinA5, Heterogeneous nuclear ribonucleoprotein A1, Rho-GDP dissociation inhibitor, Cathepsin L, Cu/Zn-SOD, Rho-GDP dissociation inhibitor, and Serpin peptidase inhibitor. Surprisingly, all these proteins were down-regulated and this pattern is similar to nasopharyngeal carcinoma-associated stromal fibroblast sample. Additionally, protein-protein interaction (PPI) indicates that HNRNPA1, SERPINE1 are hub proteins. Once their expression alters, it can impose vast changes on other molecular function. Based on this approach, ethanol may target same pathways that are related to cancer onset. In addition, some epidemiologic studies proved that ethanol consumption is related to increment of cancer risk. Therefore, more investigation is required in this regard to elicit the feasible relationship. PMID:28228815

  14. From particle self-assembly to functionalized sub-micron protein patterns

    NASA Astrophysics Data System (ADS)

    Blättler, T. M.; Binkert, A.; Zimmermann, M.; Textor, M.; Vörös, J.; Reimhult, E.

    2008-02-01

    Biologically relevant nanopatterns are useful platforms to address fundamental questions, for example, regarding protein-protein and cell-protein interactions. For the creation of nanopatterns, complex and expensive instrumentation is often needed. We present a simple but versatile patterning method using a combination of particle and subsequent molecular self-assembly to produce ordered structures in the micron and sub-micron range. Polystyrene particles were, in a first step, assembled via dip-coating or dried in a drying cell. Silicon wafers and glass slides coated with SiO2 and a top layer of 11 nm of TiO2 were used as substrates. Large hexagonally ordered particle monolayers were formed with high reproducibility. These were subsequently shrunk in a controlled manner by exposure to a O2/N2 plasma and subsequently used as etching masks to transfer the particle pattern onto the substrate, creating TiO2 features in an SiO2 background. After removing the mask the oxide contrast was translated in three simple dip-and-rinse steps into a biochemical contrast of protein-coated features in an inert background. In short, alkane phosphates were first selectively adsorbed to the TiO2 features. Then the SiO2 background was backfilled using poly(L-lysine)-graft-poly(ethylene glycol) and finally streptavidin was adsorbed to the hydrophobic alkane phosphate SAMs, allowing subsequent binding and hybridization of biotinylated DNA.

  15. Plasma protein adsorption patterns on surfaces of Amphotericin B-containing fat emulsions.

    PubMed

    Schmidt, Sven; Müller, Rainer H

    2003-03-18

    Nephrotoxicity of the conventional Amphotericin B formulation Fungizone is the most common side effect in treatment of systemic fungal infections. Lipid formulations of Amphotericin B including fat emulsions showed a reduced nephrotoxicity. In vivo distribution studies of lipid formulations have shown an accumulation of Amphotericin B in liver and spleen, while concentration in the kidneys is reduced. Blood proteins adsorbed onto particles after intravenous administration are regarded as the key factors determining their in vivo fate. Two-dimensional polyacrylamid gel electrophoresis is a powerful tool for analysis of protein adsorption patterns. This paper deals with the question if there is any correlation between proteins adsorbed on surfaces of AmB fat emulsions produced with a new production technique and the potentially organ distribution of this formulation.

  16. Regulation of gene expression in prediapausing embryos of the silkworm, Bombyx mori: pattern of protein synthesis.

    PubMed

    Dorel, C; Coulon, M

    1988-03-01

    Specific qualitative and quantitative changes in protein synthesis occur from the fertilization to the onset of diapause in the silkworm. We have used two-dimensional gel electrophoresis to analyse the patterns of proteins synthesized in prediapausing eggs of Bombyx. This analysis has been carried out with in vivo labelled polypeptides and with proteins synthesized in vitro by RNA isolated at different stages. The oocyte contains an abundant supply of diverse mRNA which are translatable in vitro. A set of proteins with molecular weight range of 68,000 to 74,000 and isoelectric points of 5.85-5.95 (hereafter referred to as No. 30) is specific of the germ-anlage stage. Transcripts encoding the No. 30 proteins are not detectable in oocytes, and inhibition of transcription by actinomycin D indicates that No. 30 mRNA are synthesized de novo. Treating eggs at the germ-anlage stage with 4 N HCl at 46 degrees C prevents diapause and is accompanied by overproduction of No. 30 protein. The induction of No. 30 synthesis is also the main event of the heat shock response. The implications of these findings in relation to early embryonic development and prevention of diapause are discussed.

  17. Evolutionarily Conserved Pattern of Interactions in a Protein Revealed by Local Thermal Expansion Properties.

    PubMed

    Dellarole, Mariano; Caro, Jose A; Roche, Julien; Fossat, Martin; Barthe, Philippe; García-Moreno E, Bertrand; Royer, Catherine A; Roumestand, Christian

    2015-07-29

    The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function.

  18. Assembly of transmembrane helices of simple polytopic membrane proteins from sequence conservation patterns.

    PubMed

    Park, Yungki; Helms, Volkhard

    2006-09-01

    The transmembrane (TM) domains of most membrane proteins consist of helix bundles. The seemingly simple task of TM helix bundle assembly has turned out to be extremely difficult. This is true even for simple TM helix bundle proteins, i.e., those that have the simple form of compact TM helix bundles. Herein, we present a computational method that is capable of generating native-like structural models for simple TM helix bundle proteins having modest numbers of TM helices based on sequence conservation patterns. Thus, the only requirement for our method is the presence of more than 30 homologous sequences for an accurate extraction of sequence conservation patterns. The prediction method first computes a number of representative well-packed conformations for each pair of contacting TM helices, and then a library of tertiary folds is generated by overlaying overlapping TM helices of the representative conformations. This library is scored using sequence conservation patterns, and a subsequent clustering analysis yields five final models. Assuming that neighboring TM helices in the sequence contact each other (but not that TM helices A and G contact each other), the method produced structural models of Calpha atom root-mean-square deviation (CA RMSD) of 3-5 A from corresponding crystal structures for bacteriorhodopsin, halorhodopsin, sensory rhodopsin II, and rhodopsin. In blind predictions, this type of contact knowledge is not available. Mimicking this, predictions were made for the rotor of the V-type Na(+)-adenosine triphosphatase without such knowledge. The CA RMSD between the best model and its crystal structure is only 3.4 A, and its contact accuracy reaches 55%. Furthermore, the model correctly identifies the binding pocket for sodium ion. These results demonstrate that the method can be readily applied to ab initio structure prediction of simple TM helix bundle proteins having modest numbers of TM helices.

  19. Engineering the Pattern of Protein Glycosylation Modulates the Thermostability of a GH11 Xylanase*

    PubMed Central

    Fonseca-Maldonado, Raquel; Vieira, Davi Serradella; Alponti, Juliana Sanchez; Bonneil, Eric; Thibault, Pierre; Ward, Richard John

    2013-01-01

    Protein glycosylation is a common post-translational modification, the effect of which on protein conformational and stability is incompletely understood. Here we have investigated the effects of glycosylation on the thermostability of Bacillus subtilis xylanase A (XynA) expressed in Pichia pastoris. Intact mass analysis of the heterologous wild-type XynA revealed two, three, or four Hex8–16GlcNAc2 modifications involving asparagine residues at positions 20, 25, 141, and 181. Molecular dynamics (MD) simulations of the XynA modified with various combinations of branched Hex9GlcNAc2 at these positions indicated a significant contribution from protein-glycan interactions to the overall energy of the glycoproteins. The effect of glycan content and glycosylation position on protein stability was evaluated by combinatorial mutagenesis of all six potential N-glycosylation sites. The majority of glycosylated enzymes expressed in P. pastoris presented increased thermostability in comparison with their unglycosylated counterparts expressed in Escherichia coli. Steric effects of multiple glycosylation events were apparent, and glycosylation position rather than the number of glycosylation events determined increases in thermostability. The MD simulations also indicated that clustered glycan chains tended to favor less stabilizing glycan-glycan interactions, whereas more dispersed glycosylation patterns favored stabilizing protein-glycan interactions. PMID:23846692

  20. Expression Pattern and Subcellular Localization of the Ovate Protein Family in Rice

    PubMed Central

    Yu, Hui; Jiang, Wenzhu; Liu, Qing; Zhang, Hui; Piao, Mingxin; Chen, Zhengdao; Bian, Mingdi

    2015-01-01

    The Arabidopsis ovate family proteins (AtOFPs) have been shown to function as transcriptional repressors and regulate multiple aspects of plant growth and development. There are 31 genes that encode the full-length OVATE-domain containing proteins in the rice genome. In this study, the gene structure analysis revealed that OsOFPs are intron poor. Phylogenetic analysis suggested that OVATE proteins from rice, Arabidopsis and tomato can be divided into 4 groups (I–IV). Real-time quantitative polymerase chain reaction (RT-qPCR) analysis identified OsOFPs with different tissue-specific expression patterns at all stages of development in the rice plant. Interestingly, nearly half of the total number of OsOFP family was more highly expressed during the seed developmental stage. In addition, seed developmental cis-elements were found in the promoter region of the OsOFPs. Subcellular localization analysis revealed that YFP-OsOFP fusion proteins predominantly localized in the nucleus. Our results suggest that OsOFPs may act as regulatory proteins and play pivotal roles in the growth and development of rice. PMID:25760462

  1. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    PubMed Central

    Wu, Fabai; van Schie, Bas G.C.; Keymer, Juan E.; Dekker, Cees

    2016-01-01

    The boundary of a cell defines the shape and scale for its subcellular organisation. However, the effects of the cell’s spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shape Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2x1x1 to 11x6x1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe, and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale in adaptation to the cell size within a characteristic length range of 3–6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling, and an adaptive range, when and only when facilitated by the three-dimensional confinement of cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood. PMID:26098227

  2. Symmetry and scale orient Min protein patterns in shaped bacterial sculptures

    NASA Astrophysics Data System (ADS)

    Wu, Fabai; van Schie, Bas G. C.; Keymer, Juan E.; Dekker, Cees

    2015-08-01

    The boundary of a cell defines the shape and scale of its subcellular organization. However, the effects of the cell's spatial boundaries as well as the geometry sensing and scale adaptation of intracellular molecular networks remain largely unexplored. Here, we show that living bacterial cells can be ‘sculpted’ into defined shapes, such as squares and rectangles, which are used to explore the spatial adaptation of Min proteins that oscillate pole-to-pole in rod-shaped Escherichia coli to assist cell division. In a wide geometric parameter space, ranging from 2 × 1 × 1 to 11 × 6 × 1 μm3, Min proteins exhibit versatile oscillation patterns, sustaining rotational, longitudinal, diagonal, stripe and even transversal modes. These patterns are found to directly capture the symmetry and scale of the cell boundary, and the Min concentration gradients scale with the cell size within a characteristic length range of 3-6 μm. Numerical simulations reveal that local microscopic Turing kinetics of Min proteins can yield global symmetry selection, gradient scaling and an adaptive range, when and only when facilitated by the three-dimensional confinement of the cell boundary. These findings cannot be explained by previous geometry-sensing models based on the longest distance, membrane area or curvature, and reveal that spatial boundaries can facilitate simple molecular interactions to result in far more versatile functions than previously understood.

  3. Discovery of protein acetylation patterns by deconvolution of peptide isomer mass spectra.

    PubMed

    Abshiru, Nebiyu; Caron-Lizotte, Olivier; Rajan, Roshan Elizabeth; Jamai, Adil; Pomies, Christelle; Verreault, Alain; Thibault, Pierre

    2015-10-15

    Protein post-translational modifications (PTMs) play important roles in the control of various biological processes including protein-protein interactions, epigenetics and cell cycle regulation. Mass spectrometry-based proteomics approaches enable comprehensive identification and quantitation of numerous types of PTMs. However, the analysis of PTMs is complicated by the presence of indistinguishable co-eluting isomeric peptides that result in composite spectra with overlapping features that prevent the identification of individual components. In this study, we present Iso-PeptidAce, a novel software tool that enables deconvolution of composite MS/MS spectra of isomeric peptides based on features associated with their characteristic fragment ion patterns. We benchmark Iso-PeptidAce using dilution series prepared from mixtures of known amounts of synthetic acetylated isomers. We also demonstrate its applicability to different biological problems such as the identification of site-specific acetylation patterns in histones bound to chromatin assembly factor-1 and profiling of histone acetylation in cells treated with different classes of HDAC inhibitors.

  4. Altered Prion Protein Expression Pattern in CSF as a Biomarker for Creutzfeldt-Jakob Disease

    PubMed Central

    Torres, Mauricio; Cartier, Luis; Matamala, José Manuel; Hernández, Nancy; Woehlbier, Ute; Hetz, Claudio

    2012-01-01

    Creutzfeldt-Jakob disease (CJD) is the most frequent human Prion-related disorder (PrD). The detection of 14-3-3 protein in the cerebrospinal fluid (CSF) is used as a molecular diagnostic criterion for patients clinically compatible with CJD. However, there is a pressing need for the identification of new reliable disease biomarkers. The pathological mechanisms leading to accumulation of 14-3-3 protein in CSF are not fully understood, however neuronal loss followed by cell lysis is assumed to cause the increase in 14-3-3 levels, which also occurs in conditions such as brain ischemia. Here we investigated the relation between the levels of 14-3-3 protein, Lactate dehydrogenase (LDH) activity and expression of the prion protein (PrP) in CSF of sporadic and familial CJD cases. Unexpectedly, we found normal levels of LDH activity in CJD cases with moderate levels of 14-3-3 protein. Increased LDH activity was only observed in a percentage of the CSF samples that also exhibited high 14-3-3 levels. Analysis of the PrP expression pattern in CSF revealed a reduction in PrP levels in all CJD cases, as well as marked changes in its glycosylation pattern. PrP present in CSF of CJD cases was sensitive to proteases. The alterations in PrP expression observed in CJD cases were not detected in other pathologies affecting the nervous system, including cases of dementia and tropical spastic paraparesis/HTLV-1 associated myelopathy (HAM/TSP). Time course analysis in several CJD patients revealed that 14-3-3 levels in CSF are dynamic and show a high degree of variability during the end stage of the disease. Post-mortem analysis of brain tissue also indicated that 14-3-3 protein is upregulated in neuronal cells, suggesting that its expression is modulated during the course of the disease. These results suggest that a combined analysis of 14-3-3 and PrP expression pattern in CSF is a reliable biomarker to confirm the clinical diagnosis of CJD patients and follow disease progression

  5. Cartilage Oligomeric Matrix Protein Increases in Photodamaged Skin.

    PubMed

    Kobayashi, Masaki; Kawabata, Keigo; Kusaka-Kikushima, Ayumi; Sugiyama, Yoshinori; Mabuchi, Tomotaka; Takekoshi, Susumu; Miyasaka, Muneo; Ozawa, Akira; Sakai, Shingo

    2016-06-01

    Cartilage oligomeric matrix protein (COMP) is a structural component of cartilage. Recent studies have described COMP as a pathogenic factor that promotes collagen deposition in fibrotic skin disorders such as scleroderma and keloid skin. Although collagen, a major dermis component, is thought to decrease in photoaged skin, recent reports have demonstrated the presence of tightly packed collagen fibrils with a structural resemblance to fibrosis in the papillary dermis of photoaged skin. Here we examined how photoaging damage relates to COMP expression and localization in photoaged skin. In situ hybridization revealed an increase in COMP-mRNA-positive cells with the progress of photoaging in preauricular skin (sun-exposed skin). The signal intensity of immunostaining for COMP increased with photoaging in not only the papillary dermis but also the reticular dermis affected by advancing solar elastosis. Immunoelectron microscopy detected the colocalization of COMP with both elastotic materials and collagen fibrils in photoaged skin. Ultraviolet light A irradiation of human dermal fibroblasts induced COMP expression at both the mRNA and protein levels. Ultraviolet light A-induced COMP expression was inhibited by an anti-transforming growth factor-β antibody or SB431542, an activin receptor-like kinase 5 inhibitor. These results suggest that the transforming growth factor-β-mediated upregulation of COMP expression may contribute to the modulation of dermal extracellular matrix in the photoaging process.

  6. Direct Write Protein Patterns for Multiplexed Cytokine Detection From Live Cells Using Electron Beam Lithography

    PubMed Central

    Lau, Uland Y.; Saxer, Sina S.; Lee, Juneyoung; Bat, Erhan; Maynard, Heather D.

    2016-01-01

    Simultaneous detection of multiple biomarkers, such as extracellular signaling molecules, is a critical aspect in disease profiling and diagnostics. Precise positioning of antibodies on surfaces, especially at the micro- and nano- scale, is important for the improvement of assays, biosensors, and diagnostics on the molecular level, and therefore, the pursuit of device miniaturization for parallel, fast, low-volume assays is a continuing challenge. Here, we describe a multiplexed cytokine immunoassay utilizing electron beam lithography and a trehalose glycopolymer as a resist for the direct writing of antibodies on silicon substrates allowing for micro- and nano-scale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and anti-tumor necrosis factor alpha (TNFα) antibodies were directly patterned. Retention of the specific binding properties of the patterned antibodies was shown by the capture of secreted cytokines from stimulated RAW 264.7 macrophages. A sandwich immunoassay was employed using gold nanoparticles and enhancement with silver for the detection and visualization of bound cytokines to the patterns by localized surface plasmon resonance detected with dark field microscopy. Multiplexing with both IL-6 and TNFα on a single chip was also successfully demonstrated with high specificity and in relevant cell culture conditions and at different times after cell stimulation. The direct fabrication of capture antibody patterns for cytokine detection described here could be useful for biosensing applications. PMID:26679368

  7. Coordinate control of terminal dendrite patterning and dynamics by the membrane protein Raw.

    PubMed

    Lee, Jiae; Peng, Yun; Lin, Wen-Yang; Parrish, Jay Z

    2015-01-01

    The directional flow of information in neurons depends on compartmentalization: dendrites receive inputs whereas axons transmit them. Axons and dendrites likewise contain structurally and functionally distinct subcompartments. Axon/dendrite compartmentalization can be attributed to neuronal polarization, but the developmental origin of subcompartments in axons and dendrites is less well understood. To identify the developmental bases for compartment-specific patterning in dendrites, we screened for mutations that affect discrete dendritic domains in Drosophila sensory neurons. From this screen, we identified mutations that affected distinct aspects of terminal dendrite development with little or no effect on major dendrite patterning. Mutation of one gene, raw, affected multiple aspects of terminal dendrite patterning, suggesting that Raw might coordinate multiple signaling pathways to shape terminal dendrite growth. Consistent with this notion, Raw localizes to branch-points and promotes dendrite stabilization together with the Tricornered (Trc) kinase via effects on cell adhesion. Raw independently influences terminal dendrite elongation through a mechanism that involves modulation of the cytoskeleton, and this pathway is likely to involve the RNA-binding protein Argonaute 1 (AGO1), as raw and AGO1 genetically interact to promote terminal dendrite growth but not adhesion. Thus, Raw defines a potential point of convergence in distinct pathways shaping terminal dendrite patterning.

  8. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography Using Surface Acoustic Waves.

    PubMed

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela H; French, Jarrod B; Huang, Tony Jun

    2015-06-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming, a surface acoustic wave-based method for manipulating and patterning crystals is developed. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and submicrometer-sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but will also make it possible to collect data on samples that were previously intractable.

  9. Precise Manipulation and Patterning of Protein Crystals for Macromolecular Crystallography using Surface Acoustic Waves

    PubMed Central

    Guo, Feng; Zhou, Weijie; Li, Peng; Mao, Zhangming; Yennawar, Neela; French, Jarrod B.; Jun Huang, Tony

    2015-01-01

    Advances in modern X-ray sources and detector technology have made it possible for crystallographers to collect usable data on crystals of only a few micrometers or less in size. Despite these developments, sample handling techniques have significantly lagged behind and often prevent the full realization of current beamline capabilities. In order to address this shortcoming we have developed a surface acoustic wave-based method for manipulating and patterning crystals. This method, which does not damage the fragile protein crystals, can precisely manipulate and pattern micrometer and sub-micrometer sized crystals for data collection and screening. The technique is robust, inexpensive, and easy to implement. This method not only promises to significantly increase efficiency and throughput of both conventional and serial crystallography experiments, but also will make it possible to collect data on samples that were previously intractable. PMID:25641793

  10. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin

    PubMed Central

    Riddle, Nicole C.; Minoda, Aki; Kharchenko, Peter V.; Alekseyenko, Artyom A.; Schwartz, Yuri B.; Tolstorukov, Michael Y.; Gorchakov, Andrey A.; Jaffe, Jacob D.; Kennedy, Cameron; Linder-Basso, Daniela; Peach, Sally E.; Shanower, Gregory; Zheng, Haiyan; Kuroda, Mitzi I.; Pirrotta, Vincenzo; Park, Peter J.; Elgin, Sarah C.R.; Karpen, Gary H.

    2011-01-01

    Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the “silencing” marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin–heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both “activation” marks (e.g., H3K4me3 and H3K36me3) and “silencing” marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin. PMID:21177972

  11. Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin.

    PubMed

    Riddle, Nicole C; Minoda, Aki; Kharchenko, Peter V; Alekseyenko, Artyom A; Schwartz, Yuri B; Tolstorukov, Michael Y; Gorchakov, Andrey A; Jaffe, Jacob D; Kennedy, Cameron; Linder-Basso, Daniela; Peach, Sally E; Shanower, Gregory; Zheng, Haiyan; Kuroda, Mitzi I; Pirrotta, Vincenzo; Park, Peter J; Elgin, Sarah C R; Karpen, Gary H

    2011-02-01

    Eukaryotic genomes are packaged in two basic forms, euchromatin and heterochromatin. We have examined the composition and organization of Drosophila melanogaster heterochromatin in different cell types using ChIP-array analysis of histone modifications and chromosomal proteins. As anticipated, the pericentric heterochromatin and chromosome 4 are on average enriched for the "silencing" marks H3K9me2, H3K9me3, HP1a, and SU(VAR)3-9, and are generally depleted for marks associated with active transcription. The locations of the euchromatin-heterochromatin borders identified by these marks are similar in animal tissues and most cell lines, although the amount of heterochromatin is variable in some cell lines. Combinatorial analysis of chromatin patterns reveals distinct profiles for euchromatin, pericentric heterochromatin, and the 4th chromosome. Both silent and active protein-coding genes in heterochromatin display complex patterns of chromosomal proteins and histone modifications; a majority of the active genes exhibit both "activation" marks (e.g., H3K4me3 and H3K36me3) and "silencing" marks (e.g., H3K9me2 and HP1a). The hallmark of active genes in heterochromatic domains appears to be a loss of H3K9 methylation at the transcription start site. We also observe complex epigenomic profiles of intergenic regions, repeated transposable element (TE) sequences, and genes in the heterochromatic extensions. An unexpectedly large fraction of sequences in the euchromatic chromosome arms exhibits a heterochromatic chromatin signature, which differs in size, position, and impact on gene expression among cell types. We conclude that patterns of heterochromatin/euchromatin packaging show greater complexity and plasticity than anticipated. This comprehensive analysis provides a foundation for future studies of gene activity and chromosomal functions that are influenced by or dependent upon heterochromatin.

  12. Probing thyroglobulin in undiluted human serum based on pattern recognition and competitive adsorption of proteins

    NASA Astrophysics Data System (ADS)

    Wang, Ran; Huang, Shuai; Li, Jing; Chae, Junseok

    2014-10-01

    Thyroglobulin (Tg) is a sensitive indicator of persistent or recurrent differentiated thyroid cancer of follicular cell origin. Detection of Tg in human serum is challenging as bio-receptors, such as anti-Tg, used in immunoassay have relatively weak binding affinity. We engineer sensing surfaces using the competitive adsorption of proteins, termed the Vroman Effect. Coupled with Surface Plasmon Resonance, the "cross-responsive" interactions of Tg on the engineered surfaces produce uniquely distinguishable multiple signature patterns, which are discriminated using Linear Discriminant Analysis. Tg-spiked samples, down to 2 ng/ml Tg in undiluted human serum, are sensitively and selectively discriminated from the control (undiluted human serum).

  13. Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori.

    PubMed

    Gong, Da-Ping; Zhang, Hui-Jie; Zhao, Ping; Lin, Ying; Xia, Qing-You; Xiang, Zhong-Huai

    2007-03-01

    Insect chemosensory proteins (CSPs) as well as odorant-binding proteins (OBPs) have been supposed to transport hydrophobic chemicals to receptors on sensory neurons. Compared with OBPs, CSPs are expressed more broadly in various insect tissues. We performed a genome-wide analysis of the candidate CSP gene family in the silkworm. A total of 20 candidate CSPs, including 3 gene fragments and 2 pseudogenes, were characterized based on their conserved cysteine residues and their similarity to CSPs in other insects. Some of these genes were clustered in the silkworm genome. The gene expression pattern of these candidates was investigated using RT-PCR and microarray, and the results showed that these genes were expressed primarily in mature larvae and the adult moth, suggesting silkworm CSPs may be involved in development. The majority of silkworm CSP genes are expressed broadly in tissues including the antennae, head, thorax, legs, wings, epithelium, testes, ovaries, pheromone glands, wing disks, and compound eyes.

  14. Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors.

    PubMed

    Spitzweck, Bettina; Brankatschk, Marko; Dickson, Barry J

    2010-02-05

    The orthogonal array of axon pathways in the Drosophila CNS is constructed in part under the control of three Robo family axon guidance receptors: Robo1, Robo2 and Robo3. Each of these receptors is responsible for a distinct set of guidance decisions. To determine the molecular basis for these functional specializations, we used homologous recombination to create a series of 9 "robo swap" alleles: expressing each of the three Robo receptors from each of the three robo loci. We demonstrate that the lateral positioning of longitudinal axon pathways relies primarily on differences in gene regulation, not distinct combinations of Robo proteins as previously thought. In contrast, specific features of the Robo1 and Robo2 proteins contribute to their distinct functions in commissure formation. These specializations allow Robo1 to prevent crossing and Robo2 to promote crossing. These data demonstrate how diversification of expression and structure within a single family of guidance receptors can shape complex patterns of neuronal wiring.

  15. Injury, nerve, and wound epidermis related electrophoretic and fluorographic protein patterns in forelimbs of adult newts

    SciTech Connect

    Garling, D.J.; Tassava, R.A.

    1984-08-01

    Polyacrylamide slab gel electrophoresis and (/sup 35/S)methionine fluorography were used to examine proteins in regenerating newt limbs, amputated denervated limbs, unamputated denervated limbs, and separated blastema mesodermal core and wound epidermis. A total of 27 protein electrophoretic bands were obtained from amputated limbs and 24 bands from unamputated limbs. Amputation resulted in the appearance of 4 new bands and the loss of 1 band as compared to unamputated limbs. These 5 banding differences were apparent on stained gels 3 days postamputation and were maintained through 10 weeks postamputation (complete regenerate stage). Only one band in unamputated limbs was always detectable on fluorographs, whereas virtually all of the stainable bands of amputated limbs were visible on fluorographs. Amputation clearly stimulated a marked, generalized increase in the synthesis of limb proteins. The 5 amputation induced changes were equally evident in stained gels of both innervated and denervated limbs. Amputated denervated limbs possessed a full set of fluorographic bands (including the 5 differences) through 18 days postamputation. However, denervation without amputation was not sufficient to alter the stainable banding pattern. Wound epidermis and mesodermal core both displayed the 5 banding differences and had identical banding patterns with the exception of one epidermal specific band. This band was also present in whole limb skin but was absent in unamputated mesodermal limb tissue. This was the only band of unamputated limbs that was consistently detectable by fluorography. It is concluded that amputation induces nerve independent changes in protein synthesis that are common to both mesodermal core and wound epidermis. These changes may represent preparation for cellular proliferation.

  16. Dynamic Pattern of HOXB9 Protein Localization during Oocyte Maturation and Early Embryonic Development in Mammals

    PubMed Central

    Sauvegarde, Caroline; Paul, Delphine; Bridoux, Laure; Jouneau, Alice; Degrelle, Séverine; Hue, Isabelle; Rezsohazy, René; Donnay, Isabelle

    2016-01-01

    Background We previously showed that the homeodomain transcription factor HOXB9 is expressed in mammalian oocytes and early embryos. However, a systematic and exhaustive study of the localization of the HOXB9 protein, and HOX proteins in general, during mammalian early embryonic development has so far never been performed. Results The distribution of HOXB9 proteins in oocytes and the early embryo was characterized by immunofluorescence from the immature oocyte stage to the peri-gastrulation period in both the mouse and the bovine. HOXB9 was detected at all studied stages with a dynamic expression pattern. Its distribution was well conserved between the two species until the blastocyst stage and was mainly nuclear. From that stage on, trophoblastic cells always showed a strong nuclear staining, while the inner cell mass and the derived cell lines showed important dynamic variations both in staining intensity and in intra-cellular localization. Indeed, HOXB9 appeared to be progressively downregulated in epiblast cells and only reappeared after gastrulation had well progressed. The protein was also detected in the primitive endoderm and its derivatives with a distinctive presence in apical vacuoles of mouse visceral endoderm cells. Conclusions Together, these results could suggest the existence of unsuspected functions for HOXB9 during early embryonic development in mammals. PMID:27798681

  17. Sex hormones and expression pattern of cytoskeletal proteins in the rat brain throughout pregnancy.

    PubMed

    González-Arenas, Aliesha; Piña-Medina, Ana Gabriela; González-Flores, Oscar; Galván-Rosas, Agustín; Porfirio Gómora-Arrati; Camacho-Arroyo, Ignacio

    2014-01-01

    Pregnancy involves diverse changes in brain function that implicate a re-organization in neuronal cytoskeleton. In this physiological state, the brain is in contact with several hormones that it has never been exposed, as well as with very high levels of hormones that the brain has been in touch throughout life. Among the latter hormones are progesterone and estradiol which regulate several brain functions, including learning, memory, neuroprotection, and the display of sexual and maternal behavior. These functions involve changes in the structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity is regulated by estradiol and progesterone. We have found that the expression pattern of Microtubule Associated Protein 2, Tau, and Glial Fibrillary Acidic Protein changes in a tissue-specific manner in the brain of the rat throughout gestation and the start of lactation, suggesting that these proteins participate in the plastic changes observed in the brain during pregnancy. This article is part of a Special Issue entitled 'Pregnancy and Steroids'.

  18. Experimental manipulation of compaction of the mouse embryo alters patterns of protein phosphorylation

    SciTech Connect

    Bloom, T. )

    1991-03-01

    Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarisation by which cellular asymmetry is first established. Changes in the pattern of protein phosphorylation have been correlated with this early event of development. In the study reported here, groups of embryos were treated in ways known to affect particular features of compaction and were then labeled with ({sup 32}P)orthophosphate; the phosphoproteins obtained were examined following electrophoresis in one and two dimensions. Four-cell embryos were treated with protein synthesis inhibitors, which advance cell flattening. This treatment resulted in only minor differences from the phosphoprotein profile of untreated four-cell embryos. Inhibition of protein synthesis at the eight-cell stage has little effect on cell flattening or polarisation. However, some phosphoproteins that are observed normally in eight-cell but not in four-cell embryos were no longer detectable if labeling took place in the presence of protein synthesis inhibitors. Eight-cell embryos incubated in phorbol 12-myristate 13-acetate, which disrupts various features of compaction, showed a relative increase in the phosphorylation of a group of phosphoprotein spots associated with the eight-cell but not with the four-cell stage. Embryos incubated in Ca2(+)-free medium, which prevents intercellular flattening and delays polarisation, showed a relative decrease in the phosphorylation of the same group of phosphoprotein spots. The behaviour of these phosphoproteins may therefore be correlated with some of the features of compaction.

  19. Cell patterning via linker-free protein functionalization of an organic conducting polymer (polypyrrole) electrode.

    PubMed

    Bax, Daniel V; Tipa, Roxana S; Kondyurin, Alexey; Higgins, Michael J; Tsoutas, Kostadinos; Gelmi, Amy; Wallace, Gordon G; McKenzie, David R; Weiss, Anthony S; Bilek, Marcela M M

    2012-07-01

    The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of the conducting polymer, polypyrrole, which possesses electrical properties. PIII treatment enabled persistent, covalent binding of the cell adhesive protein, tropoelastin, without employing chemical linking molecules. In contrast tropoelastin was readily eluted from the untreated surface. Through this differential persistence of binding, surface bound tropoelastin supported cell adhesion and spreading on the PIII treated but not the untreated polypyrrole surface. The application of a steel shadow mask during PIII treatment allowed for spatial definition of tropoelastin exclusively to PIII treated regions. The general applicability of this approach to other extracellular matrix proteins was illustrated using collagen I, which displayed similar results to tropoelastin but required extended washing conditions. This approach allowed fine patterning of cell adhesion and spreading to tropoelastin and collagen, specifically on PIII treated polypyrrole regions. We therefore present a methodology to alter the functionality of polypyrrole surfaces, generating surfaces that can spatially control cellular interactions through protein functionalization with the potential for electrical stimulation.

  20. Fabrication of Self-Cleaning, Reusable Titania Templates for Nanometer and Micrometer Scale Protein Patterning.

    PubMed

    Moxey, Mark; Johnson, Alexander; El-Zubir, Osama; Cartron, Michael; Dinachali, Saman Safari; Hunter, C Neil; Saifullah, Mohammad S M; Chong, Karen S L; Leggett, Graham J

    2015-06-23

    The photocatalytic self-cleaning characteristics of titania facilitate the fabrication of reuseable templates for protein nanopatterning. Titania nanostructures were fabricated over square centimeter areas by interferometric lithography (IL) and nanoimprint lithography (NIL). With the use of a Lloyd's mirror two-beam interferometer, self-assembled monolayers of alkylphosphonates adsorbed on the native oxide of a Ti film were patterned by photocatalytic nanolithography. In regions exposed to a maximum in the interferogram, the monolayer was removed by photocatalytic oxidation. In regions exposed to an intensity minimum, the monolayer remained intact. After exposure, the sample was etched in piranha solution to yield Ti nanostructures with widths as small as 30 nm. NIL was performed by using a silicon stamp to imprint a spin-cast film of titanium dioxide resin; after calcination and reactive ion etching, TiO2 nanopillars were formed. For both fabrication techniques, subsequent adsorption of an oligo(ethylene glycol) functionalized trichlorosilane yielded an entirely passive, protein-resistant surface. Near-UV exposure caused removal of this protein-resistant film from the titania regions by photocatalytic degradation, leaving the passivating silane film intact on the silicon dioxide regions. Proteins labeled with fluorescent dyes were adsorbed to the titanium dioxide regions, yielding nanopatterns with bright fluorescence. Subsequent near-UV irradiation of the samples removed the protein from the titanium dioxide nanostructures by photocatalytic degradation facilitating the adsorption of a different protein. The process was repeated multiple times. These simple methods appear to yield durable, reuseable samples that may be of value to laboratories that require nanostructured biological interfaces but do not have access to the infrastructure required for nanofabrication.

  1. Discovery of protein acetylation patterns by deconvolution of peptide isomer mass spectra

    PubMed Central

    Abshiru, Nebiyu; Caron-Lizotte, Olivier; Rajan, Roshan Elizabeth; Jamai, Adil; Pomies, Christelle; Verreault, Alain; Thibault, Pierre

    2015-01-01

    Protein post-translational modifications (PTMs) play important roles in the control of various biological processes including protein–protein interactions, epigenetics and cell cycle regulation. Mass spectrometry-based proteomics approaches enable comprehensive identification and quantitation of numerous types of PTMs. However, the analysis of PTMs is complicated by the presence of indistinguishable co-eluting isomeric peptides that result in composite spectra with overlapping features that prevent the identification of individual components. In this study, we present Iso-PeptidAce, a novel software tool that enables deconvolution of composite MS/MS spectra of isomeric peptides based on features associated with their characteristic fragment ion patterns. We benchmark Iso-PeptidAce using dilution series prepared from mixtures of known amounts of synthetic acetylated isomers. We also demonstrate its applicability to different biological problems such as the identification of site-specific acetylation patterns in histones bound to chromatin assembly factor-1 and profiling of histone acetylation in cells treated with different classes of HDAC inhibitors. PMID:26468920

  2. Rapid Patterning of 1-D Collagenous Topography as an ECM Protein Fibril Platform for Image Cytometry

    PubMed Central

    Xue, Niannan; Li, Xia; Bertulli, Cristina; Li, Zhaoying; Patharagulpong, Atipat; Sadok, Amine; Huang, Yan Yan Shery

    2014-01-01

    Cellular behavior is strongly influenced by the architecture and pattern of its interfacing extracellular matrix (ECM). For an artificial culture system which could eventually benefit the translation of scientific findings into therapeutic development, the system should capture the key characteristics of a physiological microenvironment. At the same time, it should also enable standardized, high throughput data acquisition. Since an ECM is composed of different fibrous proteins, studying cellular interaction with individual fibrils will be of physiological relevance. In this study, we employ near-field electrospinning to create ordered patterns of collagenous fibrils of gelatin, based on an acetic acid and ethyl acetate aqueous co-solvent system. Tunable conformations of micro-fibrils were directly deposited onto soft polymeric substrates in a single step. We observe that global topographical features of straight lines, beads-on-strings, and curls are dictated by solution conductivity; whereas the finer details such as the fiber cross-sectional profile are tuned by solution viscosity. Using these fibril constructs as cellular assays, we study EA.hy926 endothelial cells' response to ROCK inhibition, because of ROCK's key role in the regulation of cell shape. The fibril array was shown to modulate the cellular morphology towards a pre-capillary cord-like phenotype, which was otherwise not observed on a flat 2-D substrate. Further facilitated by quantitative analysis of morphological parameters, the fibril platform also provides better dissection in the cells' response to a H1152 ROCK inhibitor. In conclusion, the near-field electrospun fibril constructs provide a more physiologically-relevant platform compared to a featureless 2-D surface, and simultaneously permit statistical single-cell image cytometry using conventional microscopy systems. The patterning approach described here is also expected to form the basics for depositing other protein fibrils, seen among

  3. A fibrinogen-related protein identified from hepatopancreas of crayfish is a potential pattern recognition receptor.

    PubMed

    Chen, Qiming; Bai, Suhua; Dong, Chaohua

    2016-09-01

    Fibrinogen-related protein (FREP) family is a large group of proteins containing fibrinogen-like (FBG) domain and plays multiple physiological roles in animals. However, their immune functions in crayfish are not fully explored. In the present study, a novel fibrinogen-like protein (designated as PcFBN1) was identified and characterized from hepatopancreas of red swamp crayfish Procambarus clarkii. The cDNA sequence of PcFBN1 contains an open reading frame (ORF) of 1353 bp encoding a protein of 450 amino acids. Sequence and structural analysis indicated that PcFBN1 contains an FBG domain in C-terminal and a putative signal peptide of 19 amino acids in N-terminal. Semi-quantitative PCR revealed that the main expression of PcFBN1 was observed in hepatopancreas and hemocyte. Temporal expression analysis exhibited that PcFBN1 expression could be significantly induced by heat-killed Aeromonas hydrophila. Tissue distribution and temporal change of PcFBN1 suggested that PcFBN1 may be involved in immune responses of red swamp crayfish. Recombinant PcFBN1 protein binds and agglutinates both gram-negative bacteria Escherichia coli and gram-positive bacteria Micrococcus lysodeikticus. Moreover, binding and agglutination is Ca(2+) dependent. Further analysis indicated that PcFBN1 recognizes some acetyl group-containing substance LPS and PGN. RNAi experiment revealed that PcFBN1 is required for bacterial clearance and survival from A. hydrophila infection. Reduction of PcFBN1 expression significantly decreased the survival and enhanced the number of A. hydrophila in the hemolymph. These results indicated that PcFBN1 plays an important role in the innate immunity of red swamp crayfish as a potential pattern recognition receptor.

  4. A Robust and Engineerable Self-Assembling Protein Template for the Synthesis and Patterning of Ordered Nanoparticle Arrays

    NASA Technical Reports Server (NTRS)

    McMillan, R. Andrew; Howard, Jeanie; Zaluzec, Nestor J.; Kagawa, Hiromi K.; Li, Yi-Fen; Paavola, Chad D.; Trent, Jonathan D.

    2004-01-01

    Self-assembling biomolecules that form highly ordered structures have attracted interest as potential alternatives to conventional lithographic processes for patterning materials. Here we introduce a general technique for patterning materials on the nanoscale using genetically modified protein cage structures called chaperonins that self-assemble into crystalline templates. Constrained chemical synthesis of transition metal nanoparticles is specific to templates genetically functionalized with poly-Histidine sequences. These arrays of materials are ordered by the nanoscale structure of the crystallized protein. This system may be easily adapted to pattern a variety of materials given the rapidly growing list of peptide sequences selected by screening for specificity for inorganic materials.

  5. Many Local Pattern Texture Features: Which Is Better for Image-Based Multilabel Human Protein Subcellular Localization Classification?

    PubMed Central

    Xu, Ying-Ying; Shen, Hong-Bin

    2014-01-01

    Human protein subcellular location prediction can provide critical knowledge for understanding a protein's function. Since significant progress has been made on digital microscopy, automated image-based protein subcellular location classification is urgently needed. In this paper, we aim to investigate more representative image features that can be effectively used for dealing with the multilabel subcellular image samples. We prepared a large multilabel immunohistochemistry (IHC) image benchmark from the Human Protein Atlas database and tested the performance of different local texture features, including completed local binary pattern, local tetra pattern, and the standard local binary pattern feature. According to our experimental results from binary relevance multilabel machine learning models, the completed local binary pattern, and local tetra pattern are more discriminative for describing IHC images when compared to the traditional local binary pattern descriptor. The combination of these two novel local pattern features and the conventional global texture features is also studied. The enhanced performance of final binary relevance classification model trained on the combined feature space demonstrates that different features are complementary to each other and thus capable of improving the accuracy of classification. PMID:25050396

  6. Two different immunostaining patterns of beta-amyloid precursor protein (APP) may distinguish traumatic from nontraumatic axonal injury.

    PubMed

    Hayashi, Takahito; Ago, Kazutoshi; Nakamae, Takuma; Higo, Eri; Ogata, Mamoru

    2015-09-01

    Immunostaining for beta-amyloid precursor protein (APP) is recognized as an effective tool for detecting traumatic axonal injury, but it also detects axonal injury due to ischemic or other metabolic causes. Previously, we reported two different patterns of APP staining: labeled axons oriented along with white matter bundles (pattern 1) and labeled axons scattered irregularly (pattern 2) (Hayashi et al. (Leg Med (Tokyo) 11:S171-173, 2009). In this study, we investigated whether these two patterns are consistent with patterns of trauma and hypoxic brain damage, respectively. Sections of the corpus callosum from 44 cases of blunt head injury and equivalent control tissue were immunostained for APP. APP was detected in injured axons such as axonal bulbs and varicose axons in 24 of the 44 cases of head injuries that also survived for three or more hours after injury. In 21 of the 24 APP-positive cases, pattern 1 alone was observed in 14 cases, pattern 2 alone was not observed in any cases, and both patterns 1 and 2 were detected in 7 cases. APP-labeled injured axons were detected in 3 of the 44 control cases, all of which were pattern 2. These results suggest that pattern 1 indicates traumatic axonal injury, while pattern 2 results from hypoxic insult. These patterns may be useful to differentiate between traumatic and nontraumatic axonal injuries.

  7. Different mechanical loading protocols influence serum cartilage oligomeric matrix protein levels in young healthy humans.

    PubMed

    Niehoff, A; Kersting, U G; Helling, S; Dargel, J; Maurer, J; Thevis, M; Brüggemann, G-P

    2010-10-01

    The purpose of the study was to investigate whether a relationship between the loading mode of physical activity and serum cartilage oligomeric matrix protein (COMP) concentration exists and whether the lymphatic system contributes to COMP release into the serum. Serum COMP levels were determined in healthy male subjects before, after and at 18 further time points within 7 h at four separate experimental days with four different loading interventions. The loading intervention included high impact running exercise, slow but deep knee bends, and lymphatic drainage of 30 min duration, respectively, and a resting protocol. The serum COMP levels were measured using a commercially available quantitative enzyme-linked immunosorbent assay. An increase (p < 0.001) in serum COMP concentration was detected immediately after 30 min running exercise. Slow but deep knee bends did not cause any significant changes in serum COMP levels. Lymphatic drainage also had no effect on the serum COMP concentration. After 30 min of complete rest the serum COMP level was significantly (p = 0.008) reduced. The elevation of COMP serum concentration seems to depend on the loading mode of the physical activity and to reflect the extrusion of COMP fragments from the impact loaded articular cartilage or synovial fluid.

  8. Proteomic Analyses Reveal Common Promiscuous Patterns of Cell Surface Proteins on Human Embryonic Stem Cells and Sperms

    PubMed Central

    Gu, Bin; Zhang, Jiarong; Wu, Ying; Zhang, Xinzong; Tan, Zhou; Lin, Yuanji; Huang, Xiao; Chen, Liangbiao; Yao, Kangshou; Zhang, Ming

    2011-01-01

    Background It has long been proposed that early embryos and reproductive organs exhibit similar gene expression profiles. However, whether this similarity is propagated to the protein level remains largely unknown. We have previously characterised the promiscuous expression pattern of cell surface proteins on mouse embryonic stem (mES) cells. As cell surface proteins also play critical functions in human embryonic stem (hES) cells and germ cells, it is important to reveal whether a promiscuous pattern of cell surface proteins also exists for these cells. Methods and Principal Findings Surface proteins of hES cells and human mature sperms (hSperms) were purified by biotin labelling and subjected to proteomic analyses. More than 1000 transmembrane or secreted cell surface proteins were identified on the two cell types, respectively. Proteins from both cell types covered a large variety of functional categories including signal transduction, adhesion and transporting. Moreover, both cell types promiscuously expressed a wide variety of tissue specific surface proteins, and some surface proteins were heterogeneously expressed. Conclusions/Significance Our findings indicate that the promiscuous expression of functional and tissue specific cell surface proteins may be a common pattern in embryonic stem cells and germ cells. The conservation of gene expression patterns between early embryonic cells and reproductive cells is propagated to the protein level. These results have deep implications for the cell surface signature characterisation of pluripotent stem cells and germ cells and may lead the way to a new area of study, i.e., the functional significance of promiscuous gene expression in pluripotent and germ cells. PMID:21559292

  9. 76 FR 61747 - CompONE Services, LTD, Ithaca, NY; Notice of Negative Determination Regarding Application for...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-05

    ... Employment and Training Administration CompONE Services, LTD, Ithaca, NY; Notice of Negative Determination... administrative reconsideration of the negative ] determination regarding workers' eligibility to apply for Trade... York (CompONE Services). The negative determination was issued on August 3, 2011. The...

  10. Strategic Pay Reform: A Student Outcomes-Based Evaluation of Denver's ProComp Teacher Pay Initiative

    ERIC Educational Resources Information Center

    Goldhaber, Dan; Walch, Joe

    2012-01-01

    Denver Public Schools utilizes one of the nation's highest profile alternative teacher compensation systems, and a key element of Denver's Professional Compensation System for Teachers (ProComp) is pay for performance. This study analyzes the student achievement implications of ProComp utilizing matched student- and teacher-level data from 2003 to…

  11. Architectural adaptation and protein expression patterns of Salmonella enterica serovar Enteritidis biofilms under laminar flow conditions.

    PubMed

    Mangalappalli-Illathu, Anil K; Lawrence, John R; Swerhone, George D W; Korber, Darren R

    2008-03-31

    Salmonella enterica serovar Enteritidis is a significant biofilm-forming pathogen. The influence of a 10-fold difference in nutrient laminar flow velocity on the dynamics of Salmonella Enteritidis biofilm formation and protein expression profiles were compared in order to ascertain how flow velocity influenced biofilm structure and function. Low-flow (0.007 cm s(-1)) biofilms consisted of diffusely-arranged microcolonies which grew until merging by approximately 72 h. High-flow (0.07 cm s(-1)) biofilms were significantly thicker (36+/-3 microm (arithmetic mean+/-standard error; n=225) versus 16+/-2 microm for low-flow biofilms at 120 h) and consisted of large bacterial mounds interspersed by water channels. Lectin-binding analysis of biofilm exopolymers revealed a significantly higher (P<0.05) proportion of N-acetylgalactosamine (GalNAc) in low-flow biofilms (55.2%), relative to only 1.2% in high-flow biofilms. Alternatively, the proportions of alpha-L-fucose and N-acetylglucosamine (GlcNAc2)-N-acetylneuraminic acid (NeuNAc) polymer-conjugates were significantly higher (P<0.05) in high-flow biofilms (69.1% and 29.6%, respectively) than low-flow biofilms (33.1% and 11.7%, respectively). Despite an apparent flow rate-based physiologic effect on biofilm structure and exopolymer composition, no major shift in whole-cell protein expression patterns was seen between 168 h-old low-flow and high-flow biofilms, and notably did not include any response involving the stress response proteins, DnaK, SodB, and Tpx. Proteins involved in degradation and energy metabolism (PduA, GapA, GpmA, Pgk, and RpiA), RNA and protein biosynthesis (Tsf, TufA, and RpoZ), cell processes (Crr, MalE, and PtsH), and adaptation (GrcA), and some hypothetical proteins (YcbL and YnaF) became up-regulated in both biofilm systems relative to a 168 h-old planktonic cell control. Our results indicate that Salmonella Enteritidis biofilms altered their structure and extracellular glycoconjugate composition

  12. Distinctive patterns of p53 protein expression and microsatellite instability in human colorectal cancer.

    PubMed

    Nyiraneza, Christine; Jouret-Mourin, Anne; Kartheuser, Alex; Camby, Philippe; Plomteux, Olivier; Detry, Roger; Dahan, Karin; Sempoux, Christine

    2011-12-01

    Although evidence suggests an inverse relationship between microsatellite instability and p53 alterations in colorectal cancer, no study has thoroughly examined the use of p53 immunohistochemistry in phenotyping colorectal cancers. We investigated the value of p53 immunohistochemistry in microsatellite instability-positive colorectal cancers prescreening and attempted to clarify the relationship between DNA mismatch repair system and p53 pathway. In a series of 104 consecutive colorectal cancers, we performed p53 immunohistochemistry, TP53 mutational analysis, DNA mismatch repair system efficiency evaluation (DNA mismatch repair system immunohistochemistry, microsatellite instability status, MLH1/MSH2 germ line, and BRAF, murine double minute 2, and p21 immunohistochemistry. Microsatellite instability high was observed in 25 of 104 colorectal cancers, with DNA mismatch repair system protein loss (24/25) and germ line (8/25) or BRAF mutations (8/25). p53 immunohistochemistry revealed 3 distinct patterns of expression: complete negative immunostaining associated with truncating TP53 mutations (P < .0001), diffuse overexpression associated with missense TP53 mutations (P < .0001), and restricted overexpression characterized by a limited number of homogenously scattered strongly positive tumor cells in 36.5% of colorectal cancers. This latest pattern was associated with wild-type TP53 and microsatellite instability high colorectal cancers (P < .0001) including all Lynch tumors (8/8), but its presence among 22% of DNA mismatch repair system-competent colorectal cancers decreased its positive predictive value (55.2% [95% confidence interval, 45%-65%]). It was also correlated with murine double minute 2 overexpression (P < .0001) and inversely with p21 loss (P = .0002), independently of microsatellite instability status. In conclusion, a restricted pattern of p53 overexpression is preferentially associated with microsatellite instability high phenotype and could

  13. Two dimensional protein patterns of bronchoalveolar lavage fluid from non-smokers, smokers, and subjects exposed to asbestos.

    PubMed Central

    Lindahl, M.; Ekström, T.; Sörensen, J.; Tagesson, C.

    1996-01-01

    BACKGROUND: Bronchoalveolar lavage (BAL) fluid contains a large number of proteins which comprise a potential resource for studying respiratory effects due to occupational and environmental exposures. A study was undertaken to compare protein patterns of BAL fluid from non-smokers, smokers, and subjects exposed to asbestos. METHODS: BAL fluid samples were analysed with two dimensional gel electrophoresis (2-DE). The separated proteins were detected, quantified, and pattern-matched between different individuals with a computerised imaging system designed for evaluations of 2-DE patterns. RESULTS: About 200 different protein spots were detected in each sample of BAL fluid. As is the case with blood plasma, the BAL fluid samples contained large amounts of albumin, transferrin, and immunoglobulins. Higher levels of basic proteins were found in smokers than in non-smokers, while subjects exposed to asbestos had increased amounts of several high molecular weight proteins as well as basic proteins. Lower levels of albumin and higher levels of immunoglobulins were found in smokers than in non-smokers, while higher levels of transferrin were found in asbestos exposed subjects than in unexposed subjects. Moreover, in the group exposed to asbestos differences were found between patients with pleuritis and patients with pleural plaque, and one protein spot was found only in two patients with progressive pleural disease. CONCLUSION: These results suggest that both smokers and asbestos exposed subjects have significant changes in their airway protein expression compared with non-smokers and unexposed subjects. It is inferred that analysis of protein patterns in the BAL fluid with 2-DE may be used to detect and characterise, at a molecular level, respiratory effects due to occupational and environmental exposures. Images PMID:8977605

  14. Joint Factor Analysis of the College Student Experiences Questionnaire and the ACT COMP Objective Exam.

    ERIC Educational Resources Information Center

    Davis, Todd M.; Murrell, Patricia H.

    1990-01-01

    The American College Testing Program's College Outcome Measures Project Objective test (COMP-O) and College Student Experiences Questionnaire (CSEQ) scales for graduating college seniors were analyzed for joint factor structure at a large doctoral-granting institution. No common constructs were found in the two instruments. (Author/MSE)

  15. Schools, Teachers, Students and Computers: A Cross-National Perspective. IEA-Comped Study Stage 2.

    ERIC Educational Resources Information Center

    Pelgrum, W. J., Ed.; And Others

    The Computers in Education (Comped) study was designed as a two-stage survey. The first stage (1987-1990) was aimed at gathering information from a representative sample of schools at elementary, lower secondary and upper secondary level with regard to the state of computer use in education. The survey's focus was on the extent and availability of…

  16. Developing Consensus on the CompHP Professional Standards for Health Promotion in Europe

    ERIC Educational Resources Information Center

    Speller, Viv; Parish, Richard; Davison, Heather; Zilnyk, Anna

    2012-01-01

    Building on the CompHP Core Competencies for health promotion the Professional Standards for Health Promotion have been developed and consulted on across Europe. The standards were formulated to fit within the complexity of professional, occupational and educational standards frameworks in Europe as learning outcome standards with performance…

  17. Teacher Mobility and Financial Incentives: A Descriptive Analysis of Denver's ProComp

    ERIC Educational Resources Information Center

    Fulbeck, Eleanor S.

    2014-01-01

    Extensive teacher mobility can undermine policy efforts to develop a high-quality workforce. In response, policymakers have increasingly championed financial incentives to retain teachers. In 2006, the Denver Public Schools adopted an alternative teacher compensation reform, the Professional Compensation System for Teachers ("ProComp").…

  18. Differentiating disease subtypes by using pathway patterns constructed from gene expressions and protein networks.

    PubMed

    Hung, Fei-Hung; Chiu, Hung-Wen

    2015-01-01

    Gene expression profiles differ in different diseases. Even if diseases are at the same stage, such diseases exhibit different gene expressions, not to mention the different subtypes at a single lesion site. Distinguishing different disease subtypes at a single lesion site is difficult. In early cases, subtypes were initially distinguished by doctors. Subsequently, further differences were found through pathological experiments. For example, a brain tumor can be classified according to its origin, its cell-type origin, or the tumor site. Because of the advancements in bioinformatics and the techniques for accumulating gene expressions, researchers can use gene expression data to classify disease subtypes. Because the operation of a biopathway is closely related to the disease mechanism, the application of gene expression profiles for clustering disease subtypes is insufficient. In this study, we collected gene expression data of healthy and four myelodysplastic syndrome subtypes and applied a method that integrated protein-protein interaction and gene expression data to identify different patterns of disease subtypes. We hope it is efficient for the classification of disease subtypes in adventure.

  19. Stem fasciation in cacti and succulent species--tissue anatomy, protein pattern and RAPD polymorphisms.

    PubMed

    El-Banna, A N; El-Nady, M F; Dewir, Y H; El-Mahrouk, M E

    2013-09-01

    Fasciated and normal stem segments of Opuntia microdasys, Opuntia cylindrica, Huernia primulina and Euphorbia lactea were collected from the same plant and compared for their anatomy, water relations and genetic variations. Anatomical differences in terms of thickness of cuticle, vascular bundle, xylem and phloem were analyzed in both normal and fasciated stems. The mucilage cells were higher in the fasciated form of Opuntia microdasys than that in the normal form. Water status in terms of total water content (TWC), water deficit and relative water content (RWC) was influenced by fasciation. Genetic variations were tested in normal and fasciated stems using randomly amplified polymorphic DNA (RAPD) fingerprints and SDS-PAGE of soluble protein extracts. SDS-PAGE protein and RAPD analysis confirmed that normal and fasciated tissues were genetically different. Polymerase chain reaction (PCR) yielded different polymorphic banding patterns that were unique to each primer and distinguishable over all samples. The PCR results of normal and fasciated samples were significantly different in cases of primers P1, P2 and P3. These results indicate that occurrence of fasciation in Opuntia microdasys, Opuntia cylindrica, Huernia primulina and Euphorbia lactea is an epigenetic mutation of tissues.

  20. Heterogeneous patterns on block copolymer thin film via solvent annealing: Effect on protein adsorption

    NASA Astrophysics Data System (ADS)

    Shen, Lei; Zhu, Jintao; Liang, Haojun

    2015-03-01

    Heterogeneous patterns consisting of nanometer-scaled hydrophobic/hydrophilic domains were generated by self-assembly of poly(styrene)-block-poly(2-hydroxyethyl methacrylate) (PS-b-PHEMA) block copolymer thin film. The effect of the heterogeneity of the polymer film surface on the nonspecific adsorption of the protein human plasma fibrinogen (FBN, 5.0 × 5.0 × 47.5 nm3) was investigated. The kinetics of the FBN adsorption varies from a single-component Langmuir model on homogeneous hydrophilic PHEMA to a two-stage spreading relaxation model on homogeneous hydrophobic PS surface. On a heterogeneous PS-b-PHEMA surface with majority PS part, the initial FBN adsorption rate remains the same as that on the homogeneous PS surface. However, hydrophilic PHEMA microdomains on the heterogeneous surface slow down the second spreading stage of the FBN adsorption process, leading to a surface excess of adsorbed FBN molecules less than the presumed one simply calculated as adsorption onto multiple domains. Importantly, when the PS-b-PHEMA surface is annealed to form minority domelike PS domains (diameter: ˜50-100 nm) surrounded by a majority PHEMA matrix, such surface morphology proves to be strongly protein-repulsive. These interesting findings can be attributed to the enhancement of the spread FBN molecule in a mobile state by the heterogeneity of polymer film surface before irreversible adsorption occurs.

  1. Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis

    PubMed Central

    Ghosh, Ratna; de Campos, Marília K. F.; Huang, Jin; Huh, Seong K.; Orlowski, Adam; Yang, Yuan; Tripathi, Ashutosh; Nile, Aaron; Lee, Hsin-Chieh; Dynowski, Marek; Schäfer, Helen; Róg, Tomasz; Lete, Marta G.; Ahyayauch, Hasna; Alonso, Alicia; Vattulainen, Ilpo; Igumenova, Tatyana I.; Schaaf, Gabriel; Bankaitis, Vytas A.

    2015-01-01

    Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P2 binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen. PMID:25739452

  2. Using phylogenomic patterns and gene ontology to identify proteins of importance in plant evolution.

    PubMed

    Cibrián-Jaramillo, Angélica; De la Torre-Bárcena, Jose E; Lee, Ernest K; Katari, Manpreet S; Little, Damon P; Stevenson, Dennis W; Martienssen, Rob; Coruzzi, Gloria M; DeSalle, Rob

    2010-07-12

    We use measures of congruence on a combined expressed sequenced tag genome phylogeny to identify proteins that have potential significance in the evolution of seed plants. Relevant proteins are identified based on the direction of partitioned branch and hidden support on the hypothesis obtained on a 16-species tree, constructed from 2,557 concatenated orthologous genes. We provide a general method for detecting genes or groups of genes that may be under selection in directions that are in agreement with the phylogenetic pattern. Gene partitioning methods and estimates of the degree and direction of support of individual gene partitions to the overall data set are used. Using this approach, we correlate positive branch support of specific genes for key branches in the seed plant phylogeny. In addition to basic metabolic functions, such as photosynthesis or hormones, genes involved in posttranscriptional regulation by small RNAs were significantly overrepresented in key nodes of the phylogeny of seed plants. Two genes in our matrix are of critical importance as they are involved in RNA-dependent regulation, essential during embryo and leaf development. These are Argonaute and the RNA-dependent RNA polymerase 6 found to be overrepresented in the angiosperm clade. We use these genes as examples of our phylogenomics approach and show that identifying partitions or genes in this way provides a platform to explain some of the more interesting organismal differences among species, and in particular, in the evolution of plants.

  3. Decreased expression of Fyn protein and disbalanced alternative splicing patterns in platelets from patients with schizophrenia.

    PubMed

    Hattori, Kotaro; Fukuzako, Hiroshi; Hashiguchi, Tomo; Hamada, Shun; Murata, Yoji; Isosaka, Tomoko; Yuasa, Shigeki; Yagi, Takeshi

    2009-07-30

    Fyn, a Src-family kinase, is highly expressed in brain tissue and blood cells. In the mouse brain, Fyn participates in brain development, synaptic transmission through the phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunits, and the regulation of emotional behavior. Recently, we found that Fyn is required for the signal transduction in striatal neurons that is initiated by haloperidol, an antipsychotic drug. To determine whether Fyn abnormalities are present in patients with schizophrenia, we analyzed Fyn expression in platelet samples from 110 patients with schizophrenia, 75 of the patients' first-degree relatives, and 130 control subjects. A Western blot analysis revealed significantly lower levels of Fyn protein among the patients with schizophrenia and their relatives, compared with the level in the control group. At the mRNA level, the splicing patterns of fyn were altered in the patients and their relatives; specifically, the ratio of fynDelta7, in which exon 7 is absent, was elevated. An expression study in HEK293T cells revealed that FynDelta7 had a dominant-negative effect on the phosphorylation of Fyn's substrate. These results suggest novel deficits in Fyn function, manifested as the downregulation of Fyn protein or the altered transcription of the fyn gene, in patients with schizophrenia.

  4. Multiplex protein pattern unmixing using a non-linear variable-weighted support vector machine as optimized by a particle swarm optimization algorithm.

    PubMed

    Yang, Qin; Zou, Hong-Yan; Zhang, Yan; Tang, Li-Juan; Shen, Guo-Li; Jiang, Jian-Hui; Yu, Ru-Qin

    2016-01-15

    Most of the proteins locate more than one organelle in a cell. Unmixing the localization patterns of proteins is critical for understanding the protein functions and other vital cellular processes. Herein, non-linear machine learning technique is proposed for the first time upon protein pattern unmixing. Variable-weighted support vector machine (VW-SVM) is a demonstrated robust modeling technique with flexible and rational variable selection. As optimized by a global stochastic optimization technique, particle swarm optimization (PSO) algorithm, it makes VW-SVM to be an adaptive parameter-free method for automated unmixing of protein subcellular patterns. Results obtained by pattern unmixing of a set of fluorescence microscope images of cells indicate VW-SVM as optimized by PSO is able to extract useful pattern features by optimally rescaling each variable for non-linear SVM modeling, consequently leading to improved performances in multiplex protein pattern unmixing compared with conventional SVM and other exiting pattern unmixing methods.

  5. Chromosome-wise Protein Interaction Patterns and Their Impact on Functional Implications of Large-Scale Genomic Aberrations.

    PubMed

    Kirk, Isa Kristina; Weinhold, Nils; Belling, Kirstine; Skakkebæk, Niels Erik; Jensen, Thomas Skøt; Leffers, Henrik; Juul, Anders; Brunak, Søren

    2017-03-22

    Gene copy-number changes influence phenotypes through gene-dosage alteration and subsequent changes of protein complex stoichiometry. Human trisomies where gene copy numbers are increased uniformly over entire chromosomes provide generic cases for studying these relationships. In most trisomies, gene and protein level alterations have fatal consequences. We used genome-wide protein-protein interaction data to identify chromosome-specific patterns of protein interactions. We found that some chromosomes encode proteins that interact infrequently with each other, chromosome 21 in particular. We combined the protein interaction data with transcriptome data from human brain tissue to investigate how this pattern of global interactions may affect cellular function. We identified highly connected proteins that also had coordinated gene expression. These proteins were associated with important neurological functions affecting the characteristic phenotypes for Down syndrome and have previously been validated in mouse knockout experiments. Our approach is general and applicable to other gene-dosage changes, such as arm-level amplifications in cancer.

  6. Electrophoretic protein patterns and numerical analysis of Candida albicans from the oral cavities of healthy children.

    PubMed

    Boriollo, Marcelo Fabiano Gomes; Rosa, Edvaldo Antonio Ribeiro; Bernardo, Wagner Luis de Carvalho; Gonçalves, Reginaldo Bruno; Höfling, José Francisco

    2003-01-01

    dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < or = SD < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.

  7. Terminal sequence importance of de novo proteins from binary-patterned library: stable artificial proteins with 11- or 12-amino acid alphabet.

    PubMed

    Okura, Hiromichi; Takahashi, Tsuyoshi; Mihara, Hisakazu

    2012-06-01

    Successful approaches of de novo protein design suggest a great potential to create novel structural folds and to understand natural rules of protein folding. For these purposes, smaller and simpler de novo proteins have been developed. Here, we constructed smaller proteins by removing the terminal sequences from stable de novo vTAJ proteins and compared stabilities between mutant and original proteins. vTAJ proteins were screened from an α3β3 binary-patterned library which was designed with polar/ nonpolar periodicities of α-helix and β-sheet. vTAJ proteins have the additional terminal sequences due to the method of constructing the genetically repeated library sequences. By removing the parts of the sequences, we successfully obtained the stable smaller de novo protein mutants with fewer amino acid alphabets than the originals. However, these mutants showed the differences on ANS binding properties and stabilities against denaturant and pH change. The terminal sequences, which were designed just as flexible linkers not as secondary structure units, sufficiently affected these physicochemical details. This study showed implications for adjusting protein stabilities by designing N- and C-terminal sequences.

  8. Patterns of Protein Evolution in Cytochrome c Oxidase 1 (COI) from the Class Arachnida

    PubMed Central

    Young, Monica R; Hebert, Paul D. N.

    2015-01-01

    Because sequence information is now available for the 648bp barcode region of cytochrome c oxidase 1 (COI) from more than 400,000 animal species, this gene segment can be used to probe patterns of mitochondrial evolution. The present study examines levels of amino acid substitution and the frequency of indels in COI from 4177 species of arachnids, including representatives from all 16 orders and 43% of its families (267/625). It examines divergences at three taxonomic levels—among members of each order to an outgroup, among families in each order and among BINs, a species proxy, in each family. Order Distances vary fourfold (0.10–0.39), while the mean of the Family Distances for the ten orders ranges fivefold (0.07–0.35). BIN Distances show great variation, ranging from 0.01 or less in 12 families to more than 0.25 in eight families. Patterns of amino acid substitution in COI are generally congruent with previously reported variation in nucleotide substitution rates in arachnids, but provide some new insights, such as clear rate acceleration in the Opiliones. By revealing a strong association between elevated rates of nucleotide and amino acid substitution, this study builds evidence for the selective importance of the rate variation among arachnid lineages. Moreover, it establishes that groups whose COI genes have elevated levels of amino acid substitution also regularly possess indels, a dramatic form of protein reconfiguration. Overall, this study suggests that the mitochondrial genome of some arachnid groups is dynamic with high rates of amino acid substitution and frequent indels, while it is ‘locked down’ in others. Dynamic genomes are most prevalent in arachnids with short generation times, but the possible impact of breeding system deserves investigation since many of the rapidly evolving lineages reproduce by haplodiploidy, a mode of reproduction absent in ‘locked down’ taxa. PMID:26308206

  9. Cartilage oligomeric matrix protein and its binding partners in the cartilage extracellular matrix: interaction, regulation and role in chondrogenesis.

    PubMed

    Acharya, Chitrangada; Yik, Jasper H N; Kishore, Ashleen; Van Dinh, Victoria; Di Cesare, Paul E; Haudenschild, Dominik R

    2014-07-01

    Thrombospondins (TSPs) are widely known as a family of five calcium-binding matricellular proteins. While these proteins belong to the same family, they are encoded by different genes, regulate different cellular functions and are localized to specific regions of the body. TSP-5 or Cartilage Oligomeric Matrix Protein (COMP) is the only TSP that has been associated with skeletal disorders in humans, including pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). The pentameric structure of COMP, the evidence that it interacts with multiple cellular proteins, and the recent reports of COMP acting as a 'lattice' to present growth factors to cells, inspired this review of COMP and its interacting partners. In our review, we have compiled the interactions of COMP with other proteins in the cartilage extracellular matrix and summarized their importance in maintaining the structural integrity of cartilage as well as in regulating cellular functions.

  10. NMR spin relaxation in proteins: The patterns of motion that dissipate power to the bath

    NASA Astrophysics Data System (ADS)

    Shapiro, Yury E.; Meirovitch, Eva

    2014-04-01

    We developed in recent years the two-body coupled-rotator slowly relaxing local structure (SRLS) approach for the analysis of NMR relaxation in proteins. The two bodies/rotators are the protein (diffusion tensor D1) and the spin-bearing probe, e.g., the 15N-1H bond (diffusion tensor, D2), coupled by a local potential (u). A Smoluchowski equation is solved to yield the generic time correlation functions (TCFs), which are sums of weighted exponentials (eigenmodes). By Fourier transformation one obtains the generic spectral density functions (SDFs) which underlie the experimental relaxation parameters. The typical paradigm is to characterize structural dynamics in terms of the best-fit values of D1, D2, and u. Additional approaches we pursued employ the SRLS TCFs, SDFs, or eigenmodes as descriptors. In this study we develop yet another perspective. We consider the SDF as function of the angular velocity associated with the fluctuating fields underlying NMR relaxation. A parameter called j-fraction, which represents the relative contribution of eigenmode, i, to a given value of the SDF function at a specific frequency, ω, is defined. j-fraction profiles of the dominant eigenmodes are derived for 0 ≤ ω ≤ 1012 rad/s. They reveal which patterns of motion actuate power dissipation at given ω-values, what are their rates, and what is their relative contribution. Simulations are carried out to determine the effect of timescale separation, D1/D2, axial potential strength, and local diffusion axiality. For D1/D2 ≤ 0.01 and strong local potential of 15 kBT, power is dissipated by global diffusion, renormalized (by the strong potential) local diffusion, and probe diffusion on the surface of a cone (to be called cone diffusion). For D1/D2 = 0.1, power is dissipated by mixed eigenmodes largely of a global-diffusion-type or cone-diffusion-type, and a nearly bare renormalized-local-diffusion eigenmode. For D1/D2 > 0.1, most eigenmodes are of a mixed type. The analysis is

  11. Phenols content and 2-D electrophoresis protein pattern: a promising tool to monitor Posidonia meadows health state

    PubMed Central

    Migliore, Luciana; Rotini, Alice; Randazzo, Davide; Albanese, Nadia N; Giallongo, Agata

    2007-01-01

    Background The endemic seagrass Posidonia oceanica (L.) Delile colonizes soft bottoms producing highly productive meadows that play a crucial role in coastal ecosystems dynamics. Human activities and natural events are responsible for a widespread meadows regression; to date the identification of "diagnostic" tools to monitor conservation status is a critical issue. In this study the feasibility of a novel tool to evaluate ecological impacts on Posidonia meadows has been tested. Quantification of a putative stress indicator, i.e. phenols content, has been coupled to 2-D electrophoretic protein analysis of rhizome samples. Results The overall expression pattern from Posidonia rhizome was determined using a preliminary proteomic approach, 437 protein spots were characterized by pI and molecular weight. We found that protein expression differs in samples belonging to sites with high or low phenols: 22 unique protein spots are peculiar of "low phenols" and 27 other spots characterize "high phenols" samples. Conclusion Posidonia showed phenols variations within the meadow, that probably reflect the heterogeneity of environmental pressures. In addition, comparison of the 2-D electrophoresis patterns allowed to highlight qualitative protein expression differences in response to these pressures. These differences may account for changes in metabolic/physiological pathways as adaptation to stress. A combined approach, based on phenols content determination and 2-D electrophoresis protein pattern, seems a promising tool to monitor Posidonia meadows health state. PMID:17663776

  12. Patterns of sequence conservation in the S-Layer proteins and related sequences in Clostridium difficile.

    PubMed

    Calabi, Emanuela; Fairweather, Neil

    2002-07-01

    Clostridium difficile is the etiological agent of antibiotic-associated diarrhea. Among the factors that may play a role in infection are S-layer proteins (SLPs). Previous work has shown these to consist mainly of two components, resulting from the cleavage of a precursor encoded by the slpA gene. The high-molecular-weight (MW) subunit is related both to amidases from B. subtilis and to at least another 28 gene products in C. difficile strain 630. To gain insight into the functions of the SLPs and related proteins, we have further investigated the pattern of variability both at the slpA locus and at six nearby paralogs. Sequencing of the slpA gene from an S-layer group II strain and a variant S-layer group strain confirms a high degree of divergence in the low-MW SLP, which may result from diversifying selection. A highly conserved motif, however, is found at the C terminus in all low-MW subunits and may be essential for SlpA precursor cleavage. In strain 167, a variant cleavage product is present, suggesting a secondary processing site. Southern blotting analysis shows slpA-like open reading frames (ORFs) 2 to 7 to be conserved in all nine strains tested, with one exception: ORF2, which encodes a 66-kDa polypeptide coextracted at low pH with the main SLPs in strain 630, may be partially deleted in strain 167. Polymorphism within the slpA-ORF7 cluster may be more pronounced in the region proximal to the slpA gene. Unexpectedly, a high-MW subunit probe cross hybridizes to sequences outside the slpA locus, which appear to vary in number in different strains.

  13. Conserved patterns hidden within group A Streptococcus M protein hypervariability are responsible for recognition of human C4b-binding protein

    PubMed Central

    Buffalo, Cosmo Z.; Bahn-Suh, Adrian J.; Hirakis, Sophia P.; Biswas, Tapan; Amaro, Rommie E.; Nizet, Victor; Ghosh, Partho

    2016-01-01

    No vaccine exists against group A Streptococcus (GAS), a leading cause of worldwide morbidity and mortality. A severe hurdle is the hypervariability of its major antigen, the M protein, with >200 different M types known. Neutralizing antibodies typically recognize M protein hypervariable regions (HVRs) and confer narrow protection. In stark contrast, human C4b-binding protein (C4BP), which is recruited to the GAS surface to block phagocytic killing, interacts with a remarkably large number of M protein HVRs (apparently ~90%). Such broad recognition is rare, and we discovered a unique mechanism for this through structure determination of four sequence-diverse M proteins in complex with C4BP. The structures revealed a uniform and tolerant ‘reading head’ in C4BP, which detected conserved sequence patterns hidden within hypervariability. Our results open up possibilities for rational therapies targeting the M-C4BP interaction, and also inform a path towards vaccine design. PMID:27595425

  14. Patterns and mechanisms of ancestral histone protein inheritance in budding yeast.

    PubMed

    Radman-Livaja, Marta; Verzijlbergen, Kitty F; Weiner, Assaf; van Welsem, Tibor; Friedman, Nir; Rando, Oliver J; van Leeuwen, Fred

    2011-06-01

    Replicating chromatin involves disruption of histone-DNA contacts and subsequent reassembly of maternal histones on the new daughter genomes. In bulk, maternal histones are randomly segregated to the two daughters, but little is known about the fine details of this process: do maternal histones re-assemble at preferred locations or close to their original loci? Here, we use a recently developed method for swapping epitope tags to measure the disposition of ancestral histone H3 across the yeast genome over six generations. We find that ancestral H3 is preferentially retained at the 5' ends of most genes, with strongest retention at long, poorly transcribed genes. We recapitulate these observations with a quantitative model in which the majority of maternal histones are reincorporated within 400 bp of their pre-replication locus during replication, with replication-independent replacement and transcription-related retrograde nucleosome movement shaping the resulting distributions of ancestral histones. We find a key role for Topoisomerase I in retrograde histone movement during transcription, and we find that loss of Chromatin Assembly Factor-1 affects replication-independent turnover. Together, these results show that specific loci are enriched for histone proteins first synthesized several generations beforehand, and that maternal histones re-associate close to their original locations on daughter genomes after replication. Our findings further suggest that accumulation of ancestral histones could play a role in shaping histone modification patterns.

  15. On-chip parylene-C microstencil for simple-to-use patterning of proteins and cells on polydimethylsiloxane.

    PubMed

    Lee, Donghee; Yang, Sung

    2013-04-10

    Polydimethylsiloxane (PDMS) is widely used as a substrate in miniaturized devices, given its suitability for execution of biological and chemical assays. Here, we present a patterning approach for PDMS, which uses an on-chip Parylene-C microstencil to pattern proteins and cells. To implement the on-chip Parylene-C microstencil, we applied SiOx-like nanoparticle layers using atmospheric-pressure plasma-enhanced chemical vapor deposition (AP-PECVD) of tetraethyl orthosilicate (TEOS) mixed with oxygen. The complete removal of Parylene-C from PDMS following application of SiOx-like nanoparticle layers was demonstrated by various surface characterization analysis, including optical transparency, surface morphology, chemical composition, and peel-off force. Furthermore, the effects of the number of AP-PECVD treatments were investigated. Our approach overcomes the tendency of Parylene-C to peel off incompletely from PDMS, which has limited its use with PDMS to date. The on-chip Parylene-C microstencil approach that is based on this Parylene-C peel-off process on PDMS can pattern proteins with 2-μm resolution and cells at single-cell resolution with a vacancy ratio as small as 10%. This provides superior user-friendliness and a greater degree of geometrical freedom than previously described approaches that require meticulous care in handling of stencil. Thus, this patterning method could be applied in various research fields to pattern proteins or cells on the flexible PDMS substrate.

  16. The semantics of Chemical Markup Language (CML) for computational chemistry : CompChem

    PubMed Central

    2012-01-01

    This paper introduces a subdomain chemistry format for storing computational chemistry data called CompChem. It has been developed based on the design, concepts and methodologies of Chemical Markup Language (CML) by adding computational chemistry semantics on top of the CML Schema. The format allows a wide range of ab initio quantum chemistry calculations of individual molecules to be stored. These calculations include, for example, single point energy calculation, molecular geometry optimization, and vibrational frequency analysis. The paper also describes the supporting infrastructure, such as processing software, dictionaries, validation tools and database repositories. In addition, some of the challenges and difficulties in developing common computational chemistry dictionaries are discussed. The uses of CompChem are illustrated by two practical applications. PMID:22870956

  17. User's Guide to PreComp (Pre-Processor for Computing Composite Blade Properties)

    SciTech Connect

    Bir, G. S.

    2006-01-01

    PreComp (Pre-processor for computing Composite blade structural properties) was developed to compute the stiffness and inertial properties of a composite blade. The code may also be used to compute the structural properties of a metallic blade by treating it as a special case of an isotropic composite material. This guide provides step-by-step instructions on how to prepare input files (specify blade external geometry and internal structural layup of composite laminates), how to execute the code, and how to interpret the output properties. PreComp performs extensive checks for completeness, range, and viability of input data; these are also discussed in this manual. The code runs fast, usually in a fraction of a second, and requires only a modest knowledge of the composites and laminates schedule typically used in blades.

  18. On-line process analysis innovation: DiComp (tm) shunting dielectric sensor technology

    NASA Technical Reports Server (NTRS)

    Davis, Craig R.; Waldman, Frank A.

    1993-01-01

    The DiComp Shunting Dielectric Sensor (SDS) is a new patent-pending technology developed under the Small Business Innovation Research Program (SBIR) for NASA's Kennedy Space Center. The incorporation of a shunt electrode into a conventional fringing field dielectric sensor makes the SDS uniquely sensitive to changes in material dielectric properties in the KHz to MHz range which were previously detectable only at GHz measurement frequencies. The initial NASA application of the SDS for Nutrient Delivery Control has demonstrated SDS capabilities for thickness and concentration measurement of Hoagland nutrient solutions. The commercial introduction of DiComp SDS technology for concentration and percent solids measurements in dispersions, emulsions and solutions represents a new technology for process measurements for liquids in a variety of industries.

  19. The semantics of Chemical Markup Language (CML) for computational chemistry : CompChem.

    PubMed

    Phadungsukanan, Weerapong; Kraft, Markus; Townsend, Joe A; Murray-Rust, Peter

    2012-08-07

    : This paper introduces a subdomain chemistry format for storing computational chemistry data called CompChem. It has been developed based on the design, concepts and methodologies of Chemical Markup Language (CML) by adding computational chemistry semantics on top of the CML Schema. The format allows a wide range of ab initio quantum chemistry calculations of individual molecules to be stored. These calculations include, for example, single point energy calculation, molecular geometry optimization, and vibrational frequency analysis. The paper also describes the supporting infrastructure, such as processing software, dictionaries, validation tools and database repositories. In addition, some of the challenges and difficulties in developing common computational chemistry dictionaries are discussed. The uses of CompChem are illustrated by two practical applications.

  20. Protein patterns of brush-border fragments in congenital lactose malabsorption and in specific hypolactasia of the adult.

    PubMed

    Freiburghaus, A U; Schmitz, J; Schindler, M; Rotthauwe, H W; Kuitunen, P; Launiala, K; Hadorn, B

    1976-05-06

    Brush-border membrane proteins of the small-bowel mucosa were separated on polyacrylamide gels from intestinal biopsy specimens obtained from four children with congenital lactose malabsorption and from two adults with specific hypolactasia. In three patients with the congenital type of lactase deficiency the protein band corresponding to brush-border lactase was reduced in intensity, but was never completely absent. No difference in gel patterns was detected when this pattern in congenital deficiency was compared to that obtained from the two patients with adult-type selective hypolactasia. In one patient with congenital lactose malabsorption the protein band corresponding to lactase activity was not detectable. The findings suggest that the mechanisms leading to low lactase activity in the congenital and adult forms of lactose intolerance are similar.

  1. Electrophoretic pattern and distribution of cytoskeletal proteins in flat-epitheloid and stellate process-bearing astrocytes in primary culture.

    PubMed

    Ciesielski-Treska, J; Ulrich, G; Mensch, C; Aunis, D

    1984-01-01

    One- and two-dimensional electrophoresis patterns and distribution of major cytoskeletal proteins were studied in primary astrocytes with either flat-epitheloid or stellate appearance. No major differences in the electrophoretic patterns of actin, tubulin, glial fibrillary acidic protein (GFAP) and vimentin were detected between flat-epitheloid and stellate process-bearing astrocytes produced by the exposure of cultures to dibutyryl cyclic AMP (dBcAMP). However the morphological changes of astrocytes were accompanied by marked changes in the quantitative distribution of cytoskeletal proteins. The most prominent change was a large and specific decrease in the amount of actin, detected by [(35)S]methionine incorporation, densitometric scanning of one-dimensional gels and DNase inhibition assay. In stellate astrocytes produced by a 4 day treatment with dibutyryl cyclic AMP, the amount of actin decreased by 50%. This decrease was not apparently related to the depolymerization of actin.

  2. util_2comp: Planck-based two-component dust model utilities

    NASA Astrophysics Data System (ADS)

    Meisner, Aaron

    2014-11-01

    The util_2comp software utilities generate predictions of far-infrared Galactic dust emission and reddening based on a two-component dust emission model fit to Planck HFI, DIRBE and IRAS data from 100 GHz to 3000 GHz. These predictions and the associated dust temperature map have angular resolution of 6.1 arcminutes and are available over the entire sky. Implementations in IDL and Python are included.

  3. A Request for the Conference and Symposia Grant from COMP Division of American Chemical Society

    DTIC Science & Technology

    2015-03-02

    34Modeling and Simulations of Electrochemical Interfaces and Materials for Energy Storage" symposium within the Computers in Chemistry (COMP) Division of...Laboratory organized the "Modeling and Simulations of Electrochemical Interfaces and Materials for Energy Storage" symposium within the Computers in...Emilio Esposito, Scott Wildman Monday, August 11, 2014 Oral Session Modeling and Simulations of Electrochemical Interfaces and Materials for Energy

  4. The CoMP-S Instrument at the Lomnický Peak Observatory: Status Report

    NASA Astrophysics Data System (ADS)

    Kučera, A.; Ambróz, J.; Gömöry, P.; Habaj, P.; Kavka, J.; Kozák, M.; Schwartz, P.; Rybák, J.; Tomczyk, S.; Sewell, S.; Aumiller, P.; Summers, R.; Watt, A.

    2016-04-01

    The Coronal Multi-channel Polarimeter for Slovakia (CoMP-S) has been installed at the high-altitude Lomnicky Peak Observatory of the Astronomical Institute of SAS (2633 m a.s.l.) in 2011. The instrument was designed and manufactured by HAO/NCAR (Boulder, USA) with a tunable Lyot filter and polarimeter for visible and near IR spectral regions. This instrument is proposed for coronagraphic observations of magnetic and velocity fields in the solar corona and in prominences. A fundamental upgrade of this instrument has been prepared with pair of cameras sensitive in the near IR spectral region in a new camera module. This upgrade is being incorporated to the instrument in course of the year 2014. In this contribution the technical parameters of the final configuration of the CoMP-S instrument containing four cameras, covering both visible and near IR spectral regions, are described. We also present a potential of the CoMP-S instrument for coronagraphic spectro-polarimetric observations of the solar corona and prominences with a capability for sequential measurements of the spectral profiles of all prominent emission lines in spectral region from 500 to 1100 nm.

  5. Comp Plan: A computer program to generate dose and radiobiological metrics from dose-volume histogram files

    SciTech Connect

    Holloway, Lois Charlotte; Miller, Julie-Anne; Kumar, Shivani; Whelan, Brendan M.; Vinod, Shalini K.

    2012-10-01

    Treatment planning studies often require the calculation of a large number of dose and radiobiological metrics. To streamline these calculations, a computer program called Comp Plan was developed using MATLAB. Comp Plan calculates common metrics, including equivalent uniform dose, tumor control probability, and normal tissue complication probability from dose-volume histogram data. The dose and radiobiological metrics can be calculated for the original data or for an adjusted fraction size using the linear quadratic model. A homogeneous boost dose can be added to a given structure if desired. The final output is written to an Excel file in a format convenient for further statistical analysis. Comp Plan was verified by independent calculations. A lung treatment planning study comparing 45 plans for 7 structures using up to 6 metrics for each structure was successfully analyzed within approximately 5 minutes with Comp Plan. The code is freely available from the authors on request.

  6. Using contrast patterns between true complexes and random subgraphs in PPI networks to predict unknown protein complexes

    PubMed Central

    Liu, Quanzhong; Song, Jiangning; Li, Jinyan

    2016-01-01

    Most protein complex detection methods utilize unsupervised techniques to cluster densely connected nodes in a protein-protein interaction (PPI) network, in spite of the fact that many true complexes are not dense subgraphs. Supervised methods have been proposed recently, but they do not answer why a group of proteins are predicted as a complex, and they have not investigated how to detect new complexes of one species by training the model on the PPI data of another species. We propose a novel supervised method to address these issues. The key idea is to discover emerging patterns (EPs), a type of contrast pattern, which can clearly distinguish true complexes from random subgraphs in a PPI network. An integrative score of EPs is defined to measure how likely a subgraph of proteins can form a complex. New complexes thus can grow from our seed proteins by iteratively updating this score. The performance of our method is tested on eight benchmark PPI datasets and compared with seven unsupervised methods, two supervised and one semi-supervised methods under five standards to assess the quality of the predicted complexes. The results show that in most cases our method achieved a better performance, sometimes significantly. PMID:26868667

  7. Evaluation of stereo-array isotope labeling (SAIL) patterns for automated structural analysis of proteins with CYANA.

    PubMed

    Ikeya, Teppei; Terauchi, Tsutomu; Güntert, Peter; Kainosho, Masatsune

    2006-07-01

    Recently we have developed the stereo-array isotope labeling (SAIL) technique to overcome the conventional molecular size limitation in NMR protein structure determination by employing complete stereo- and regiospecific patterns of stable isotopes. SAIL sharpens signals and simplifies spectra without the loss of requisite structural information, thus making large classes of proteins newly accessible to detailed solution structure determination. The automated structure calculation program CYANA can efficiently analyze SAIL-NOESY spectra and calculate structures without manual analysis. Nevertheless, the original SAIL method might not be capable of determining the structures of proteins larger than 50 kDa or membrane proteins, for which the spectra are characterized by many broadened and overlapped peaks. Here we have carried out simulations of new SAIL patterns optimized for minimal relaxation and overlap, to evaluate the combined use of SAIL and CYANA for solving the structures of larger proteins and membrane proteins. The modified approach reduces the number of peaks to nearly half of that observed with uniform labeling, while still yielding well-defined structures and is expected to enable NMR structure determinations of these challenging systems.

  8. FoxP2 protein levels regulate cell morphology changes and migration patterns in the vertebrate developing telencephalon.

    PubMed

    Garcia-Calero, Elena; Botella-Lopez, Arancha; Bahamonde, Olga; Perez-Balaguer, Ariadna; Martinez, Salvador

    2016-07-01

    In the mammalian telencephalon, part of the progenitor cells transition from multipolar to bipolar morphology as they invade the mantle zone. This associates with changing patterns of radial migration. However, the molecules implicated in these morphology transitions are not well known. In the present work, we analyzed the function of FoxP2 protein in this process during telencephalic development in vertebrates. We analyzed the expression of FoxP2 protein and its relation with cell morphology and migratory patterns in mouse and chicken developing striatum. We observed FoxP2 protein expressed in a gradient from the subventricular zone to the mantle layer in mice embryos. In the FoxP2 low domain cells showed multipolar migration. In the striatal mantle layer where FoxP2 protein expression is higher, cells showed locomoting migration and bipolar morphology. In contrast, FoxP2 showed a high and homogenous expression pattern in chicken striatum, thus bipolar morphology predominated. Elevation of FoxP2 in the striatal subventricular zone by in utero electroporation promoted bipolar morphology and impaired multipolar radial migration. In mouse cerebral cortex we obtained similar results. FoxP2 promotes transition from multipolar to bipolar morphology by means of gradiental expression in mouse striatum and cortex. Together these results indicate a role of FoxP2 differential expression in cell morphology control of the vertebrate telencephalon.

  9. NMR spin relaxation in proteins: The patterns of motion that dissipate power to the bath

    SciTech Connect

    Shapiro, Yury E. E-mail: yuryeshapiro@gmail.com; Meirovitch, Eva E-mail: yuryeshapiro@gmail.com

    2014-04-21

    We developed in recent years the two-body coupled-rotator slowly relaxing local structure (SRLS) approach for the analysis of NMR relaxation in proteins. The two bodies/rotators are the protein (diffusion tensor D{sub 1}) and the spin-bearing probe, e.g., the {sup 15}N−{sup 1}H bond (diffusion tensor, D{sub 2}), coupled by a local potential (u). A Smoluchowski equation is solved to yield the generic time correlation functions (TCFs), which are sums of weighted exponentials (eigenmodes). By Fourier transformation one obtains the generic spectral density functions (SDFs) which underlie the experimental relaxation parameters. The typical paradigm is to characterize structural dynamics in terms of the best-fit values of D{sub 1}, D{sub 2}, and u. Additional approaches we pursued employ the SRLS TCFs, SDFs, or eigenmodes as descriptors. In this study we develop yet another perspective. We consider the SDF as function of the angular velocity associated with the fluctuating fields underlying NMR relaxation. A parameter called j-fraction, which represents the relative contribution of eigenmode, i, to a given value of the SDF function at a specific frequency, ω, is defined. j-fraction profiles of the dominant eigenmodes are derived for 0 ≤ ω ≤ 10{sup 12} rad/s. They reveal which patterns of motion actuate power dissipation at given ω-values, what are their rates, and what is their relative contribution. Simulations are carried out to determine the effect of timescale separation, D{sub 1}/D{sub 2}, axial potential strength, and local diffusion axiality. For D{sub 1}/D{sub 2} ≤ 0.01 and strong local potential of 15 k{sub B}T, power is dissipated by global diffusion, renormalized (by the strong potential) local diffusion, and probe diffusion on the surface of a cone (to be called cone diffusion). For D{sub 1}/D{sub 2} = 0.1, power is dissipated by mixed eigenmodes largely of a global-diffusion-type or cone-diffusion-type, and a nearly bare renormalized

  10. Interaction of gold and silver nanoparticles with human plasma: Analysis of protein corona reveals specific binding patterns.

    PubMed

    Lai, Wenjia; Wang, Qingsong; Li, Lumeng; Hu, Zhiyuan; Chen, Jiankui; Fang, Qiaojun

    2017-04-01

    Determining how nanomaterials interact with plasma will assist in understanding their effects on the biological system. This work presents a systematic study of the protein corona formed from human plasma on 20nm silver and gold nanoparticles with three different surface modifications, including positive and negative surface charges. The results show that all nanoparticles, even those with positive surface modifications, acquire negative charges after interacting with plasma. Approximately 300 proteins are identified on the coronas, while 99 are commonly found on each nanomaterial. The 20 most abundant proteins account for over 80% of the total proteins abundance. Remarkably, the surface charge and core of the nanoparticles, as well as the isoelectric point of the plasma proteins, are found to play significant roles in determining the nanoparticle coronas. Albumin and globulins are present at levels of less than 2% on these nanoparticle coronas. Fibrinogen, which presents in the plasma but not in the serum, preferably binds to negatively charged gold nanoparticles. These observations demonstrate the specific plasma protein binding pattern of silver and gold nanoparticles, as well as the importance of the surface charge and core in determining the protein corona compositions. The potential downstream biological impacts of the corona proteins were also investigated.

  11. Effect of water salinity on total protein and electrophoretic pattern of serum proteins of grass carp, Ctenopharyngodon idella

    PubMed Central

    Peyghan, Rahim; Khadjeh, Gholam Hosain; Enayati, Ala

    2014-01-01

    In this study the effects of water salinity on serum total protein and its components in grass carp were investigated. The aim of this study was to determine the effect of salinity tolerance of fish on total serum protein level and its components as an indicator of liver and kidney activity. One hundred and twenty grass carp were divided into four groups, randomly. The first three groups were reared in concentration of 4, 8 and 12 g L-1 of salt solution, respectively, and the fourth group was reared in freshwater and served as control. After 3 weeks, blood samples were collected and after harvesting the blood serum, serum total protein and protein components were measured with Biuret and electrophoresis methods, respectively. Results showed that mean value of serum total protein in the control and three salinities groups were 2.75, 3.28, 2.90 and 3.13 g dL-1, respectively. Five fractions of serum protein were electrophoretically observed as: albumin (Alb), alpha-1 globulin (α1-glu), alpha-2 globulin (α2-glu), beta globulin (β-glu) and gamma globulin (γ-glu). There were not any significant differences between the average mean of serum total protein of experimental and control groups (p > 0.05). However, Alb, α1-glu and β-glu levels in the experimental groups were significantly higher than in the control group (p < 0.05). The average of α2-glu and γ-glu revealed no significant difference between the experimental groups (p > 0.05). In conclusion, our results showed that increasing water salinity could have a significant effect on Alb, α1-glu and β-glu levels but not on total serum protein in grass carp. PMID:25568723

  12. Distribution patterns of small-molecule ligands in the protein universe and implications for origin of life and drug discovery

    PubMed Central

    Ji, Hong-Fang; Kong, De-Xin; Shen, Liang; Chen, Ling-Ling; Ma, Bin-Guang; Zhang, Hong-Yu

    2007-01-01

    Background Extant life depends greatly on the binding of small molecules (such as ligands) with macromolecules (such as proteins), and one ligand can bind multiple proteins. However, little is known about the global patterns of ligand-protein mapping. Results By examining 2,186 well-defined small-molecule ligands and thousands of protein domains derived from a database of druggable binding sites, we show that a few ligands bind tens of protein domains or folds, whereas most ligands bind only one, which indicates that ligand-protein mapping follows a power law. Through assigning the protein-binding orders (early or late) for bio-ligands, we demonstrate that the preferential attachment principle still holds for the power-law relation between ligands and proteins. We also found that polar molecular surface area, H-bond acceptor counts, H-bond donor counts and partition coefficient are potential factors to discriminate ligands from ordinary molecules and to differentiate super ligands (shared by three or more folds) from others. Conclusion These findings have significant implications for evolution and drug discovery. First, the chronology of ligand-protein binding can be inferred by the power-law feature of ligand-protein mapping. Some nucleotide-containing ligands, such as ATP, ADP, GDP, NAD, FAD, dihydro-nicotinamide-adenine-dinucleotide phosphate (NDP), nicotinamide-adenine-dinucleotide phosphate (NAP), flavin mononucleotide (FMN) and AMP, are found to be the earliest cofactors bound to proteins, agreeing with the current understanding of evolutionary history. Second, the finding that about 30% of ligands are shared by two or more domains will help with drug discovery, such as in finding new functions from old drugs, developing promiscuous drugs and depending more on natural products. PMID:17727706

  13. Dietary Protein Intake and Distribution Patterns of Well-Trained Dutch Athletes.

    PubMed

    Gillen, Jenna B; Trommelen, Jorn; Wardenaar, Floris C; Brinkmans, Naomi Y J; Versteegen, Joline J; Jonvik, Kristin L; Kapp, Christoph; de Vries, Jeanne; van den Borne, Joost J G C; Gibala, Martin J; van Loon, Luc J C

    2016-10-06

    Dietary protein intake should be optimized in all athletes to ensure proper recovery and enhance the skeletal muscle adaptive response to exercise training. In addition to total protein intake, the use of specific protein-containing food sources and the distribution of protein throughout the day are relevant for optimizing protein intake in athletes. In the present study, we examined the daily intake and distribution of various protein-containing food sources in a large cohort of strength, endurance and team-sport athletes. Well-trained male (n=327) and female (n=226) athletes completed multiple web-based 24-h dietary recalls over a 2-4 wk period. Total energy intake, the contribution of animal- and plant-based proteins to daily protein intake, and protein intake at six eating moments were determined. Daily protein intake averaged 108±33 and 90±24 g in men and women, respectively, which corresponded to relative intakes of 1.5±0.4 and 1.4±0.4 g/kg. Dietary protein intake was correlated with total energy intake in strength (r=0.71, p<0.001), endurance (r=0.79, p<0.001) and team-sport (r=0.77, p<0.001) athletes. Animal and plant-based sources of protein intake was 57% and 43%, respectively. The distribution of protein intake was 19% (19±8 g) at breakfast, 24% (25±13 g) at lunch and 38% (38±15 g) at dinner. Protein intake was below the recommended 20 g for 58% of athletes at breakfast, 36% at lunch and 8% at dinner. In summary, this survey of athletes revealed they habitually consume > 1.2 g protein/kg/d, but the distribution throughout the day may be suboptimal to maximize the skeletal muscle adaptive response to training.

  14. Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration.

    PubMed

    Li, Aiqing; Benoit, Joshua B; Lopez-Martinez, Giancarlo; Elnitsky, Michael A; Lee, Richard E; Denlinger, David L

    2009-05-01

    Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2-DE and nanoscale capillary LC/MS/MS. Twenty-four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration-regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.

  15. Microcystin-Bound Protein Patterns in Different Cultures of Microcystis aeruginosa and Field Samples

    PubMed Central

    Wei, Nian; Hu, Lili; Song, Lirong; Gan, Nanqin

    2016-01-01

    Micocystin (MC) exists in Microcystis cells in two different forms, free and protein-bound. We examined the dynamic change in extracellular free MCs, intracellular free MCs and protein-bound MCs in both batch cultures and semi-continuous cultures, using high performance liquid chromatography and Western blot. The results showed that the free MC per cell remained constant, while the quantity of protein-bound MCs increased with the growth of Microcystis cells in both kinds of culture. Significant changes in the dominant MC-bound proteins occurred in the late exponential growth phase of batch cultures, while the dominant MC-bound proteins in semi-continuous cultures remained the same. In field samples collected at different months in Lake Taihu, the dominant MC-bound proteins were shown to be similar, but the amount of protein-bound MC varied and correlated with the intracellular MC content. We identified MC-bound proteins by two-dimensional electrophoresis immunoblots and mass spectrometry. The 60 kDa chaperonin GroEL was a prominent MC-bound protein. Three essential glycolytic enzymes and ATP synthase alpha subunit were also major targets of MC-binding, which might contribute to sustained growth in semi-continuous culture. Our results indicate that protein-bound MC may be important for sustaining growth and adaptation of Microcystis sp. PMID:27754336

  16. Travelling lipid domains in a dynamic model for protein-induced pattern formation in biomembranes

    NASA Astrophysics Data System (ADS)

    John, Karin; Bär, Markus

    2005-06-01

    Cell membranes are composed of a mixture of lipids. Many biological processes require the formation of spatial domains in the lipid distribution of the plasma membrane. We have developed a mathematical model that describes the dynamic spatial distribution of acidic lipids in response to the presence of GMC proteins and regulating enzymes. The model encompasses diffusion of lipids and GMC proteins, electrostatic attraction between acidic lipids and GMC proteins as well as the kinetics of membrane attachment/detachment of GMC proteins. If the lipid-protein interaction is strong enough, phase separation occurs in the membrane as a result of free energy minimization and protein/lipid domains are formed. The picture is changed if a constant activity of enzymes is included into the model. We chose the myristoyl-electrostatic switch as a regulatory module. It consists of a protein kinase C that phosphorylates and removes the GMC proteins from the membrane and a phosphatase that dephosphorylates the proteins and enables them to rebind to the membrane. For sufficiently high enzymatic activity, the phase separation is replaced by travelling domains of acidic lipids and proteins. The latter active process is typical for nonequilibrium systems. It allows for a faster restructuring and polarization of the membrane since it acts on a larger length scale than the passive phase separation. The travelling domains can be pinned by spatial gradients in the activity; thus the membrane is able to detect spatial clues and can adapt its polarity dynamically to changes in the environment.

  17. Patterns of indirect protein interactions suggest a spatial organization to metabolism.

    PubMed

    Pérez-Bercoff, Åsa; McLysaght, Aoife; Conant, Gavin C

    2011-11-01

    It has long been believed that cells organize their cytoplasm so as to efficiently channel metabolites between sequential enzymes. This metabolic channeling has the potential to yield higher metabolic fluxes as well as better regulatory control over metabolism. One mechanism for achieving such channeling is to ensure that sequential enzymes in a pathway are physically close to each other in the cell. We present evidence that indirect protein interactions between related enzymes represent a global mechanism for achieving metabolic channeling; the intuition being that protein interactions between enzymes and non-enzymatic mediator proteins are a powerful means of physically associating enzymes in a modular fashion. By analyzing the metabolic and protein-protein interactions networks of Escherichia coli, yeast and humans, we are able to show that all three species have many more indirect protein interactions linking enzymes that share metabolites than would be expected by chance. Moreover, these interactions are distributed non-randomly in the metabolic network. Our analyses in yeast and E. coli show that reactions possessing such interactions also show higher flux than do those lacking them. On the basis of these observations, we suggest that an important role of protein interactions with mediator proteins is to contribute to the spatial organization of the cell. This hypothesis is supported by the fact that these mediator proteins are also enriched with annotations related to signal transduction, a system where scaffolding proteins are known to limit cross-talk by controlling spatial localization.

  18. Ultrasound Technologies for the Spatial Patterning of Cells and Extracellular Matrix Proteins and the Vascularization of Engineered Tissue

    NASA Astrophysics Data System (ADS)

    Garvin, Kelley A.

    Technological advancements in the field of tissue engineering could save the lives of thousands of organ transplant patients who die each year while waiting for donor organs. Currently, two of the primary challenges preventing tissue engineers from developing functional replacement tissues and organs are the need to recreate complex cell and extracellular microenvironments and to vascularize the tissue to maintain cell viability and function. Ultrasound is a form of mechanical energy that can noninvasively and nondestructively interact with tissues at the cell and protein level. In this thesis, novel ultrasound-based technologies were developed for the spatial patterning of cells and extracellular matrix proteins and the vascularization of three-dimensional engineered tissue constructs. Acoustic radiation forces associated with ultrasound standing wave fields were utilized to noninvasively control the spatial organization of cells and cell-bound extracellular matrix proteins within collagen-based engineered tissue. Additionally, ultrasound induced thermal mechanisms were exploited to site-specifically pattern various extracellular matrix collagen microstructures within a single engineered tissue construct. Finally, ultrasound standing wave field technology was used to promote the rapid and extensive vascularization of three-dimensional tissue constructs. As such, the ultrasound technologies developed in these studies have the potential to provide the field of tissue engineering with novel strategies to spatially pattern cells and extracellular matrix components and to vascularize engineered tissue, and thus, could advance the fabrication of functional replacement tissues and organs in the field of tissue engineering.

  19. Contrasting evolutionary patterns of spore coat proteins in two Bacillus species groups are linked to a difference in cellular structure

    PubMed Central

    2013-01-01

    Background The Bacillus subtilis-group and the Bacillus cereus-group are two well-studied groups of species in the genus Bacillus. Bacteria in this genus can produce a highly resistant cell type, the spore, which is encased in a complex protective protein shell called the coat. Spores in the B. cereus-group contain an additional outer layer, the exosporium, which encircles the coat. The coat in B. subtilis spores possesses inner and outer layers. The aim of this study is to investigate whether differences in the spore structures influenced the divergence of the coat protein genes during the evolution of these two Bacillus species groups. Results We designed and implemented a computational framework to compare the evolutionary histories of coat proteins. We curated a list of B. subtilis coat proteins and identified their orthologs in 11 Bacillus species based on phylogenetic congruence. Phylogenetic profiles of these coat proteins show that they can be divided into conserved and labile ones. Coat proteins comprising the B. subtilis inner coat are significantly more conserved than those comprising the outer coat. We then performed genome-wide comparisons of the nonsynonymous/synonymous substitution rate ratio, dN/dS, and found contrasting patterns: Coat proteins have significantly higher dN/dS in the B. subtilis-group genomes, but not in the B. cereus-group genomes. We further corroborated this contrast by examining changes of dN/dS within gene trees, and found that some coat protein gene trees have significantly different dN/dS between the B subtilis-clade and the B. cereus-clade. Conclusions Coat proteins in the B. subtilis- and B. cereus-group species are under contrasting selective pressures. We speculate that the absence of the exosporium in the B. subtilis spore coat effectively lifted a structural constraint that has led to relaxed negative selection pressure on the outer coat. PMID:24283940

  20. Nuclear levels and patterns of histone H3 modification and HP1 proteins after inhibition of histone deacetylases.

    PubMed

    Bártová, Eva; Pacherník, Jirí; Harnicarová, Andrea; Kovarík, Ales; Kovaríková, Martina; Hofmanová, Jirina; Skalníková, Magdalena; Kozubek, Michal; Kozubek, Stanislav

    2005-11-01

    The effects of the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and sodium butyrate (NaBt) were studied in A549, HT29 and FHC human cell lines. Global histone hyperacetylation, leading to decondensation of interphase chromatin, was characterized by an increase in H3(K9) and H3(K4) dimethylation and H3(K9) acetylation. The levels of all isoforms of heterochromatin protein, HP1, were reduced after HDAC inhibition. The observed changes in the protein levels were accompanied by changes in their interphase patterns. In control cells, H3(K9) acetylation and H3(K4) dimethylation were substantially reduced to a thin layer at the nuclear periphery, whereas TSA and NaBt caused the peripheral regions to become intensely acetylated at H3(K9) and dimethylated at H3(K4). The dispersed pattern of H3(K9) dimethylation was stable even at the nuclear periphery of HDACi-treated cells. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment. We conclude that inhibition of histone deacetylases caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications.

  1. SHH Protein Variance in the Limb Bud Is Constrained by Feedback Regulation and Correlates with Altered Digit Patterning

    PubMed Central

    Zhang, Rui; Lee, Chanmi; Lawson, Lisa Y.; Svete, Lillian J.; McIntyre, Lauren M.; Harfe, Brian D.

    2017-01-01

    mRNA variance has been proposed to play key roles in normal development, population fitness, adaptability, and disease. While variance in gene expression levels may be beneficial for certain cellular processes, for example in a cell’s ability to respond to external stimuli, variance may be detrimental for the development of some organs. In the bilaterally symmetric vertebrate limb buds, the amount of Sonic Hedgehog (SHH) protein present at specific stages of development is essential to ensure proper patterning of this structure. To our surprise, we found that SHH protein variance is present during the first 10 hr of limb development. The variance is virtually eliminated after the first 10 hr of limb development. By examining mutant animals, we determined that the ability of the limb bud apical ectodermal ridge (AER) to respond to SHH protein was required for reducing SHH variance during limb formation. One consequence of the failure to eliminate variance in SHH protein was the presence of polydactyly and an increase in digit length. These data suggest a potential novel mechanism in which alterations in SHH variance during evolution may have driven changes in limb patterning and digit length. PMID:28131983

  2. C-H…pi interactions in proteins: prevalence, pattern of occurrence, residue propensities, location, and contribution to protein stability.

    PubMed

    Kumar, Manjeet; Balaji, Petety V

    2014-02-01

    C-H…pi interactions are a class of non-covalent interactions found in different molecular systems including organic crystals, proteins and nucleic acids. High-resolution protein structures have been analyzed in the present study to delineate various aspects of C-H…pi interactions. Additionally, to determine the extent to which redundancy of a database biases the outcome, two datasets differing from each other in the level of redundancy have been analyzed. On average, only one out of six {with C-H(Aro) group} or eight {with C-H(Ali) group} residues in a protein participate as C-H group donors. Neither the frequency of occurrence in proteins nor the number of C-H groups present in it is correlated to the propensity of an amino acid to participate in C-H…pi interactions. Most of the residues that participate in C-H…pi interactions are solvent-shielded. Solvent shielded nature of most of the C-H…pi interactions and prevalence of intra- as well as inter-secondary structural element C-H…pi interactions suggest that the contribution of these interactions to the enthalpy of folded form will be significant. The separation in the primary structure between donor and acceptor residues is found to be correlated to secondary structure type. Other insights obtained from this study include the presence of networks of C-H…pi interactions spanning multiple secondary structural elements. To our knowledge this has not been reported so far. A substantial number of residues involved in C-H…pi interactions are found in catalytic and ligand binding sites suggesting their possible role in maintaining active site geometry. No significant differences of C-H…pi interactions in the two datasets are found for any of the parameters/features analyzed.

  3. Pineapple translation factor SUI1 and ribosomal protein L36 promoters drive constitutive transgene expression patterns in Arabidopsis thaliana.

    PubMed

    Koia, Jonni; Moyle, Richard; Hendry, Caroline; Lim, Lionel; Botella, José Ramón

    2013-03-01

    The availability of a variety of promoter sequences is necessary for the genetic engineering of plants, in basic research studies and for the development of transgenic crops. In this study, the promoter and 5' untranslated regions of the evolutionally conserved protein translation factor SUI1 gene and ribosomal protein L36 gene were isolated from pineapple and sequenced. Each promoter was translationally fused to the GUS reporter gene and transformed into the heterologous plant system Arabidopsis thaliana. Both the pineapple SUI1 and L36 promoters drove GUS expression in all tissues of Arabidopsis at levels comparable to the CaMV35S promoter. Transient assays determined that the pineapple SUI1 promoter also drove GUS expression in a variety of climacteric and non-climacteric fruit species. Thus the pineapple SUI1 and L36 promoters demonstrate the potential for using translation factor and ribosomal protein genes as a source of promoter sequences that can drive constitutive transgene expression patterns.

  4. CapsidMaps: protein-protein interaction pattern discovery platform for the structural analysis of virus capsids using Google Maps.

    PubMed

    Carrillo-Tripp, Mauricio; Montiel-García, Daniel Jorge; Brooks, Charles L; Reddy, Vijay S

    2015-04-01

    Structural analysis and visualization of protein-protein interactions is a challenging task since it is difficult to appreciate easily the extent of all contacts made by the residues forming the interfaces. In the case of viruses, structural analysis becomes even more demanding because several interfaces coexist and, in most cases, these are formed by hundreds of contacting residues that belong to multiple interacting coat proteins. CapsidMaps is an interactive analysis and visualization tool that is designed to benefit the structural virology community. Developed as an improved extension of the φ-ψ Explorer, here we describe the details of its design and implementation. We present results of analysis of a spherical virus to showcase the features and utility of the new tool. CapsidMaps also facilitates the comparison of quaternary interactions between two spherical virus particles by computing a similarity (S)-score. The tool can also be used to identify residues that are solvent exposed and in the process of locating antigenic epitope regions as well as residues forming the inside surface of the capsid that interact with the nucleic acid genome. CapsidMaps is part of the VIPERdb Science Gateway, and is freely available as a web-based and cross-browser compliant application at http://viperdb.scripps.edu.

  5. Salt stress-induced protein pattern associated with photosynthetic parameters and andrographolide content in Andrographis paniculata Nees.

    PubMed

    Talei, Daryush; Valdiani, Alireza; Maziah, Mahmood; Sagineedu, Sreenivasa Rao; Abiri, Rambod

    2015-01-01

    Andrographis paniculata is a multifunctional medicinal plant and a potent source of bioactive compounds. Impact of environmental stresses such as salinity on protein diversification, as well as the consequent changes in the photosynthetic parameters and andrographolide content (AG) of the herb, has not yet been thoroughly investigated. The present study showed that the salinity affects the protein pattern, and subsequently, it decreased the photosynthetic parameters, protein content, total dry weight, and total crude extract. Exceptionally, the AG content was increased (p ≤ 0.01). Moreover, it was noticed that the salinity at 12 dS m(-1) led to the maximum increase in AG content in all accessions. Interestingly, the leaf protein analysis revealed that the two polymorphic protein bands as low- and medium-sized of 17 and 45 kDa acted as the activator agents for the photosynthetic parameters and AG content. Protein sequencing and proteomic analysis can be conducted based on the present findings in the future.

  6. Identifying known unknowns using the US EPA's CompTox Chemistry Dashboard.

    PubMed

    McEachran, Andrew D; Sobus, Jon R; Williams, Antony J

    2017-03-01

    Chemical features observed using high-resolution mass spectrometry can be tentatively identified using online chemical reference databases by searching molecular formulae and monoisotopic masses and then rank-ordering of the hits using appropriate relevance criteria. The most likely candidate "known unknowns," which are those chemicals unknown to an investigator but contained within a reference database or literature source, rise to the top of a chemical list when rank-ordered by the number of associated data sources. The U.S. EPA's CompTox Chemistry Dashboard is a curated and freely available resource for chemistry and computational toxicology research, containing more than 720,000 chemicals of relevance to environmental health science. In this research, the performance of the Dashboard for identifying known unknowns was evaluated against that of the online ChemSpider database, one of the primary resources used by mass spectrometrists, using multiple previously studied datasets reported in the peer-reviewed literature totaling 162 chemicals. These chemicals were examined using both applications via molecular formula and monoisotopic mass searches followed by rank-ordering of candidate compounds by associated references or data sources. A greater percentage of chemicals ranked in the top position when using the Dashboard, indicating an advantage of this application over ChemSpider for identifying known unknowns using data source ranking. Additional approaches are being developed for inclusion into a non-targeted analysis workflow as part of the CompTox Chemistry Dashboard. This work shows the potential for use of the Dashboard in exposure assessment and risk decision-making through significant improvements in non-targeted chemical identification. Graphical abstract Identifying known unknowns in the US EPA's CompTox Chemistry Dashboard from molecular formula and monoisotopic mass inputs.

  7. RNA-protein distance patterns in ribosomes reveal the mechanism of translational attenuation.

    PubMed

    Yu, DongMei; Zhang, Chao; Qin, PeiWu; Cornish, Peter V; Xu, Dong

    2014-11-01

    Elucidating protein translational regulation is crucial for understanding cellular function and drug development. A key molecule in protein translation is ribosome, which is a super-molecular complex extensively studied for more than a half century. The structure and dynamics of ribosome complexes were resolved recently thanks to the development of X-ray crystallography, Cryo-EM, and single molecule biophysics. Current studies of the ribosome have shown multiple functional states, each with a unique conformation. In this study, we analyzed the RNA-protein distances of ribosome (2.5 MDa) complexes and compared these changes among different ribosome complexes. We found that the RNA-protein distance is significantly correlated with the ribosomal functional state. Thus, the analysis of RNA-protein binding distances at important functional sites can distinguish ribosomal functional states and help understand ribosome functions. In particular, the mechanism of translational attenuation by nascent peptides and antibiotics was revealed by the conformational changes of local functional sites.

  8. Progesterone-dependent sexual behavior and protein patterns in the ventromedial hypothalamus of the adult female rat

    SciTech Connect

    Montemayor, M.E.; Roy, E.J.; Giometti, C.S.; Taylor, J.

    1994-09-01

    Controversy exists concerning mechanisms by which progesterone exerts central nervous system effects on behavior. Progesterone may affect behavior by genomic regulation of protein synthesis. Alternatively, it may work through non-genomic mechanisms, consistent with its short latency to act. Recent work suggests that progesterone may elicit its effects on sexual behavior by more than one mechanism in a tissue specific manner. In the present study, we have examined whether progesterone facilitation of sexual behavior is correlated with modification of protein synthesis patterns in the ventromedial hypothalamus (VMH). Ovariectomized rats were divided into three groups: estradiol (4 ug/ka at 0 and 18 hrs), estradiol (at 0 and 18 hrs) plus progesterone (2 mg/kg at 37 hrs), and vehicle only. {sup 35}S-labeled cysteine and methionine were bilaterally infused into the VMH at 37 hrs (the time of progesterone administration). Following 4 hrs of infusion, animals were tested for sexual behavior and sacrificed. Newly synthesized VMH proteins were separated by two dimensional gel electrophoresis followed by fluorography. Analysis of approximately 660 spots/fluorogram in two independent replications indicated that no protein was completely induced or lost as a result of being treated with progesterone. The abundances of several proteins were significantly altered in response to progesterone treatment in each replication; however, none were changed in abundance in both replications. These findings present no evidence that progesterone causes detectable alterations in VIMH protein patterns between 10-100 kDa in the 4.8-6.7 apparent pI range.

  9. Dissolution Rate, Weathering Mechanics, and Friability of TNT, Comp B, Tritonal, and Octol

    DTIC Science & Technology

    2010-02-01

    instrumentation (Figure 77). We selected five Comp B chunks from a LO detonation (LO 5), a piece of C4 whose history is not known from Demolition...the explosive pieces and the camera were somewhat protected from birds and wolves . The disadvantage was that the tripod might have protected the...military ranges: Camp Edwards, Massachusetts, USA . Environmental Pollution, 129(1):13–21. Dionne, B.C., D.P. Rounbehler, E.K. Achter, J.R. Hobbs and D.H

  10. Ovocalyxin-36 is a pattern recognition protein in chicken eggshell membranes.

    PubMed

    Cordeiro, Cristianne M M; Esmaili, Hamed; Ansah, George; Hincke, Maxwell T

    2013-01-01

    The avian eggshell membranes are essential elements in the fabrication of the calcified shell as a defense against bacterial penetration. Ovocalyxin-36 (OCX-36) is an abundant avian eggshell membrane protein, which shares protein sequence homology to bactericidal permeability-increasing protein (BPI), lipopolysaccharide-binding protein (LBP) and palate, lung and nasal epithelium clone (PLUNC) proteins. We have developed an efficient method to extract OCX-36 from chicken eggshell membranes for purification with cation and anion exchange chromatographies. Purified OCX-36 protein exhibited lipopolysaccharide (LPS) binding activity and bound lipopolysaccharide (LPS) from Escherichia coli O111:B4 in a dose-dependent manner. OCX-36 showed inhibitory activity against growth of Staphylococcus aureus ATCC 6538. OCX-36 single nucleotide polymorphisms (SNPs) were verified at cDNA 211 position and the corresponding proteins proline-71 (Pro-71) or serine-71 (Ser-71) were purified from eggs collected from genotyped hens. A significant difference between Pro-71 and Ser-71 OCX-36 for S. aureus lipoteichoic acid (LTA) binding activity was detected. The current study is a starting point to understand the innate immune role that OCX-36 may play in protection against bacterial invasion of both embryonated eggs (relevant to avian reproductive success) and unfertilized table eggs (relevant to food safety).

  11. Enhanced protein adsorption and patterning on nanostructured latex-coated paper.

    PubMed

    Juvonen, Helka; Määttänen, Anni; Ihalainen, Petri; Viitala, Tapani; Sarfraz, Jawad; Peltonen, Jouko

    2014-06-01

    Specific interactions of extracellular matrix proteins with cells and their adhesion to the substrate are important for cell growth. A nanopatterned latex-coated paper substrate previously shown to be an excellent substrate for cell adhesion and 2D growth was studied for directed immobilization of proteins. The nanostructured latex surface was formed by short-wavelength IR irradiation of a two-component latex coating consisting of a hydrophilic film-forming styrene butadiene acrylonitrile copolymer and hydrophobic polystyrene particles. The hydrophobic regions of the IR-treated latex coating showed strong adhesion of bovine serum albumin (cell repelling protein), fibronectin (cell adhesive protein) and streptavidin. Opposite to the IR-treated surface, fibronectin and streptavidin had a poor affinity toward the untreated pristine latex coating. Detailed characterization of the physicochemical surface properties of the latex-coated substrates revealed that the observed differences in protein affinity were mainly due to the presence or absence of the protein repelling polar and charged surface groups. The protein adsorption was assisted by hydrophobic (dehydration) interactions.

  12. G protein-coupled receptors show unusual patterns of intrinsic unfolding.

    PubMed

    Jaakola, Veli-Pekka; Prilusky, Jaime; Sussman, Joel L; Goldman, Adrian

    2005-02-01

    Intrinsically unstructured proteins (IUPs) or IUP-like regions often play key roles in controlling processes ranging from transcription to the cell cycle. In silico such proteins can be identified by their sequence properties; they have low hydrophobicity and high net charge. In this study, we applied the FoldIndex (http://bioportal.weizmann.ac.il/fldbin/findex) program to analyze human G protein-coupled receptors and compared them with membrane proteins of known structure and with IUPs. We show that human G protein-coupled receptor (GPCR) extramembranous domains include long (>50 residues) disordered segments, unlike membrane proteins of known structure. The predicted disorder occurred primarily in the N-terminal, C-terminal and third intracellular domain regions: 55, 69 and 56% of the human GPCRs were disordered in these regions, respectively. This increased flexibility may therefore be critical for GPCR function. Surprisingly, however, the kinds of residues used in GPCR unstructured regions were different than in hitherto-identified IUPs. The GPCR third intracellular loop domains contain very high percentages of Arg, Lys and His residues, especially Arg, but the percentage of Glu, Asp and Pro is no higher than in folded proteins. We propose that this has structural and functional consequences.

  13. Different zinc sensitivity of Brassica organs is accompanied by distinct responses in protein nitration level and pattern.

    PubMed

    Feigl, Gábor; Kolbert, Zsuzsanna; Lehotai, Nóra; Molnár, Árpád; Ördög, Attila; Bordé, Ádám; Laskay, Gábor; Erdei, László

    2016-03-01

    Zinc is an essential microelement, but its excess exerts toxic effects in plants. Heavy metal stress can alter the metabolism of reactive oxygen (ROS) and nitrogen species (RNS) leading to oxidative and nitrosative damages; although the participation of these processes in Zn toxicity and tolerance is not yet known. Therefore this study aimed to evaluate the zinc tolerance of Brassica organs and the putative correspondence of it with protein nitration as a relevant marker for nitrosative stress. Both examined Brassica species (B. juncea and B. napus) proved to be moderate Zn accumulators; however B. napus accumulated more from this metal in its organs. The zinc-induced damages (growth diminution, altered morphology, necrosis, chlorosis, and the decrease of photosynthetic activity) were slighter in the shoot system of B. napus than in B. juncea. The relative zinc tolerance of B. napus shoot was accompanied by moderate changes of the nitration pattern. In contrast, the root system of B. napus suffered more severe damages (growth reduction, altered morphology, viability loss) and slighter increase in nitration level compared to B. juncea. Based on these, the organs of Brassica species reacted differentially to excess zinc, since in the shoot system modification of the nitration pattern occurred (with newly appeared nitrated protein bands), while in the roots, a general increment in the nitroproteome could be observed (the intensification of the same protein bands being present in the control samples). It can be assumed that the significant alteration of nitration pattern is coupled with enhanced zinc sensitivity of the Brassica shoot system and the general intensification of protein nitration in the roots is attached to relative zinc endurance.

  14. Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

    SciTech Connect

    Vogel, Lotte K. . E-mail: vogel@imbg.ku.dk; Larsen, Jakob E.; Hansen, Martin; Truffer, Renato

    2005-05-13

    Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity of secretion is determined by conformational sensitive sorting signals.

  15. A component based noise correction method (CompCor) for BOLD and perfusion based fMRI.

    PubMed

    Behzadi, Yashar; Restom, Khaled; Liau, Joy; Liu, Thomas T

    2007-08-01

    A component based method (CompCor) for the reduction of noise in both blood oxygenation level-dependent (BOLD) and perfusion-based functional magnetic resonance imaging (fMRI) data is presented. In the proposed method, significant principal components are derived from noise regions-of-interest (ROI) in which the time series data are unlikely to be modulated by neural activity. These components are then included as nuisance parameters within general linear models for BOLD and perfusion-based fMRI time series data. Two approaches for the determination of the noise ROI are considered. The first method uses high-resolution anatomical data to define a region of interest composed primarily of white matter and cerebrospinal fluid, while the second method defines a region based upon the temporal standard deviation of the time series data. With the application of CompCor, the temporal standard deviation of resting-state perfusion and BOLD data in gray matter regions was significantly reduced as compared to either no correction or the application of a previously described retrospective image based correction scheme (RETROICOR). For both functional perfusion and BOLD data, the application of CompCor significantly increased the number of activated voxels as compared to no correction. In addition, for functional BOLD data, there were significantly more activated voxels detected with CompCor as compared to RETROICOR. In comparison to RETROICOR, CompCor has the advantage of not requiring external monitoring of physiological fluctuations.

  16. A novel competence gene, comP, is essential for natural transformation of Acinetobacter sp. strain BD413.

    PubMed Central

    Porstendörfer, D; Drotschmann, U; Averhoff, B

    1997-01-01

    Acinetobacter sp. strain BD413 (= ATCC 33305), a nutritionally versatile bacterium, has an extremely efficient natural transformation system. Here we describe the generation of eight transformation-affected mutants of Acinetobacter sp. strain BD413 by insertional mutagenesis. These mutants were found by Southern blot analysis and complementation studies to result from single nptII marker insertions at different chromosomal loci. DNA binding and uptake studies with one mutant, T205, revealed that the transformation deficiency of this mutant results from a complete lack of DNA binding and, therefore, uptake activity. A novel competence gene essential for natural transformation, named comP, was cloned by complementation of mutant T205. The nucleotide sequence of comP was determined, and its deduced 15-kDa polypeptide displays significant similarities to type IV pilins. Analysis of the ultrastructure of a transformation-deficient comP mutant and the transformation-competent wild-type strain revealed that both are covered with bundle-forming thin fimbriae (3 to 4 nm in diameter) and individual thick fimbriae (6 nm in diameter). These results provide evidence that the pilinlike ComP is unrelated to the piluslike structures of strain BD413. Taking all data into account, we propose that ComP functions as a major subunit of an organelle acting as a channel or pore mediating DNA binding and/or uptake in Acinetobacter sp. strain BD413. PMID:9361398

  17. Molecular identification and expression patterns of odorant binding protein and chemosensory protein genes in Athetis lepigone (Lepidoptera: Noctuidae)

    PubMed Central

    Zhu, Xiu-Yun; Ma, Ji-Fang; Dong, Zhi-Ping; Xu, Ji-Wei; Kang, Ke

    2017-01-01

    The olfaction system of insects plays an important role in mediating various physiological behaviors, including locating hosts, avoiding predators, and recognizing mates and oviposition sites. Therefore, some key genes in the system present valuable opportunities as targets for developing novel green pesticides. Athetis lepigone, a noctuid moth can feed on more than 30 different host plants making it a serious polyphagous pest worldwide, and it has become one of the major maize pests in northern China since 2011. However, there are no reports on effective and environmentally friendly pesticides for the control of this pest. In this study, we identified 28 genes encoding putative odorant binding proteins (OBPs) and 20 chemosensory protein (CSPs) genes based on our previous A. lepigone transcriptomic data. A tissue expression investigation and phylogenetic analysis were conducted in an effort to postulate the functions of these genes. Our results show that nearly half (46.4%) of the AlOBPs exhibited antennae-biased expression while many of the AlCSPs were highly abundant in non-antennal tissues. These results will aid in exploring the chemosensory mechanisms of A. lepigone and developing environmentally friendly pesticides against this pest in the future. PMID:28382236

  18. Effects of light on protein patterns in gravitropically stimulated root caps of corn

    NASA Technical Reports Server (NTRS)

    Feldman, L. J.; Gildow, V.

    1984-01-01

    In certain cultivars of corn (Zea mays var. Merit), light stimulates gravitropic bending of the root by influencing events in the root cap. In this paper, we report on changes in root cap proteins which occur as a result of the light treatment and single out specific proteins as potentially having a role in mediating the gravitropic response. For this work, we have used root caps maintained aseptically in culture media supplemented with auxin. If auxin is deleted from the culture medium, the protein profiles observed following illumination differ from that seen in caps provided light while in auxin-supplemented media. We also report that several of the proteins for which synthesis is stimulated by light appear to turn over rapidly, usually within 0.5 hour of formation.

  19. Identification of Methanococcus Jannaschii Proteins in 2-D Gel Electrophoresis Patterns by Mass Spectrometry

    DOE R&D Accomplishments Database

    Liang, X.

    1998-06-10

    The genome of Methanococcus jannaschii has been sequenced completely and has been found to contain approximately 1,770 predicted protein-coding regions. When these coding regions are expressed and how their expression is regulated, however, remain open questions. In this work, mass spectrometry was combined with two-dimensional gel electrophoresis to identify which proteins the genes produce under different growth conditions, and thus investigate the regulation of genes responsible for functions characteristic of this thermophilic representative of the methanogenic Archaea.

  20. Aquaporin inhibition changes protein phosphorylation pattern following sperm motility activation in fish.

    PubMed

    Zilli, Loredana; Beirão, José; Schiavone, Roberta; Herraez, Maria Paz; Cabrita, Elsa; Storelli, Carlo; Vilella, Sebastiano

    2011-09-01

    Our previous studies demonstrated that osmolality is the key signal in sperm motility activation in Sparus aurata spermatozoa. In particular, we have proposed that the hyper-osmotic shock triggers water efflux from spermatozoa via aquaporins. This water efflux determines the cell volume reduction and, in turn, the rise in the intracellular concentration of ions. This increase could lead to the activation of adenylyl cyclase and of the cAMP-signaling pathway, causing the phosphorylation of sperm proteins and then the initiation of sperm motility. This study confirms the important role of sea bream AQPs (Aqp1a and Aqp10b) in the beginning of sperm motility. In fact, when these proteins are inhibited by HgCl(2), the phosphorylation of some proteins (174 kDa protein of head; 147, 97 and 33 kDa proteins of flagella), following the hyper-osmotic shock, was inhibited (totally or partially). However, our results also suggest that more than one transduction pathways could be activated when sea bream spermatozoa were ejaculated in seawater, since numerous proteins showed an HgCl(2)(AQPs)-independent phosphorylation state after motility activation. The role played by each different signal transduction pathways need to be clarified.

  1. Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins

    PubMed Central

    Kudryashova, Elena; Koneru, Pratibha C.; Kvaratskhelia, Mamuka; Strömstedt, Adam A.; Lu, Wuyuan; Kudryashov, Dmitri S.

    2016-01-01

    Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation, and angiogenesis, disintegrating bacterial membranes, and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural “anti-chaperones”, i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV, and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not β-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage, and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins – the intrinsically low thermodynamic protein stability. PMID:27581352

  2. TUNABLE TENSOR VOTING FOR REGULARIZING PUNCTATE PATTERNS OFMEMBRANE-BOUND PROTEIN SIGNALS

    SciTech Connect

    Loss, Leandro; Bebis, George; Parvin, Bahram

    2009-04-29

    Membrane-bound protein, expressed in the basal-lateral region, is heterogeneous and an important endpoint for understanding biological processes. At the optical resolution, membrane-bound protein can be visualized as being diffused (e.g., E-cadherin), punctate (e.g., connexin), or simultaneously diffused and punctate as a result of sample preparation or conditioning. Furthermore, there is a significant amount of heterogeneity as a result of technical and biological variations. This paper aims at enhancing membrane-bound proteins that are expressed between epithelial cells so that quantitative analysis can be enabled on a cell-by-cell basis. We propose a method to detect and enhance membrane-bound protein signal from noisy images. More precisely, we build upon the tensor voting framework in order to produce an efficient method to detect and refine perceptually interesting linear structures in images. The novelty of the proposed method is in its iterative tuning of the tensor voting fields, which allows the concentration of the votes only over areas of interest. The method is shown to produce high quality enhancements of membrane-bound protein signals with combined punctate and diffused characteristics. Experimental results demonstrate the benefits of using tunable tensor voting for enhancing and differentiating cell-cell adhesion mediated by integral cell membrane protein.

  3. Determination of the electrophoretic pattern of somatic and excretory-secretory proteins of Ligula intestinalis parasite in spirlin (Alburnoides bipunctatus).

    PubMed

    Youssefi, M R; Hosseinifard, S M; Halimi, M; Kordafshari, S

    2012-12-01

    Ligula intestinalis parasite is a cestodes that causes remarkable damages to fish. It is also of prime importance in economic and hygienic aspects. SDS-PAGE and western blotting are the methods that can be used to determine the electerophoretic pattern of somatic and excretory-secretory proteins of parasites. In this study, after obtaining the plerocercoidal stage of this parasite from the spirlin (Alburnoides bipunctatus), its somatic proteins were prepared using ultrasonicae, and excretory-secretory proteins were prepared using the PBS solution. After protein assay, which included using the Bradford method and then SDS-PAGE on these two antigenic solutions, 5 protein bands of 26, 33, 38, 58, 70kDa in somatic antigens, and 7 bands of 25, 28, 33, 43, 49, 60, 70kDa in excretory-secretory antigens were observed. After western blotting on both antigens and adding the primary antibody (the sera of infected fish) and then the secondary antibody (Rabbit Anti-fish Polyclonal Antibody Conjugated from Abnova Corporation) no band was seen in excretory-secretory antigen. And only in the 55kDa band of somatic antigen, a positive response, in comparison of fish positive serum was observed.

  4. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    PubMed Central

    Shi, Tujin; Niepel, Mario; McDermott, Jason E.; Gao, Yuqian; Nicora, Carrie D.; Chrisler, William B.; Markillie, Lye M.; Petyuk, Vladislav A.; Smith, Richard D.; Rodland, Karin D.; Sorger, Peter K.; Qian, Wei-Jun; Wiley, H. Steven

    2016-01-01

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components—16 core proteins and 10 feedback regulators—of the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling. PMID:27405981

  5. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway.

    PubMed

    Shi, Tujin; Niepel, Mario; McDermott, Jason E; Gao, Yuqian; Nicora, Carrie D; Chrisler, William B; Markillie, Lye M; Petyuk, Vladislav A; Smith, Richard D; Rodland, Karin D; Sorger, Peter K; Qian, Wei-Jun; Wiley, H Steven

    2016-07-12

    Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components-16 core proteins and 10 feedback regulators-of the epidermal growth factor receptor (EGFR)-mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling.

  6. Dorsal–Ventral patterning: Crescent is a dorsally secreted Frizzled-related protein that competitively inhibits Tolloid proteases

    PubMed Central

    Ploper, Diego; Lee, Hojoon X.; De Robertis, Edward M.

    2011-01-01

    In Xenopus, dorsal–ventral (D–V) patterning can self-regulate after embryo bisection. This is mediated by an extracellular network of proteins secreted by the dorsal and ventral centers of the gastrula. Different proteins of similar activity can be secreted at these two poles, but under opposite transcriptional control. Here we show that Crescent, a dorsal protein, can compensate for the loss of Sizzled, a ventral protein. Crescent is a secreted Frizzled-Related Protein (sFRP) known to regulate Wnt8 and Wnt11 activity. We now find that Crescent also regulates the BMP pathway. Crescent expression was increased by the BMP antagonist Chordin and repressed by BMP4, while the opposite was true for Sizzled. Crescent knock-down increased the expression of BMP target genes, and synergized with Sizzled morpholinos. Thus, Crescent loss-of-function is compensated by increased expression of its ventral counterpart Sizzled. Crescent overexpression dorsalized whole embryos but not ventral half-embryos, indicating that Crescent requires a dorsal component to exert its anti-BMP activity. Crescent protein lost its dorsalizing activity in Chordin-depleted embryos. When co-injected, Crescent and Chordin proteins greatly synergized in the dorsalization of Xenopus embryos. The molecular mechanism of these phenotypes is explained by the ability of Crescent to inhibit Tolloid metalloproteinases, which normally degrade Chordin. Enzyme kinetic studies showed that Crescent was a competitive inhibitor of Tolloid activity, which bound to Tolloid/BMP1 with a KD of 11 nM. In sum, Crescent is a new component of the D–V pathway, which functions as the dorsal counterpart of Sizzled, through the regulation of chordinases of the Tolloid family. PMID:21295563

  7. Comprehensive characterization of expression patterns of protein 4.1 family members in mouse adrenal gland: implications for functions.

    PubMed

    Wang, Hua; Liu, Congrong; Debnath, Gargi; Baines, Anthony J; Conboy, John G; Mohandas, Narla; An, Xiuli

    2010-10-01

    The members of the protein 4.1 family, 4.1R, 4.1G, 4.1N, and 4.1B, are encoded by four genes, all of which undergo complex alternative splicing. It is well established that 4.1R, the prototypical member of the family, serves as an adapter that links the spectrin-actin based cytoskeleton to the plasma membrane in red cells. It is required for mechanical resilience of the membrane, and it ensures the cell surface accumulation of selected membrane proteins. However, the function of 4.1 proteins outside erythrocytes remains under-explored, especially in endocrine tissues. Transcripts of all 4.1 homologs have previously been documented to be abundantly expressed in adrenal gland. In order to begin to decipher the function of 4.1 proteins in adrenal gland, we performed a detailed characterization of the expression pattern of various 4.1 proteins and their cellular localization. We show that 4.1R (~80 and ~135 kDa) splice forms are expressed on the membrane of all cells, while a ~160 kDa 4.1G splice form is distributed in the cytoplasm and the membrane of zona glomerulosa and of medullary cells. Two 4.1N splice forms, ~135 and ~95 kDa, are present in the peri-nuclear region of both zona glomerulosa and medullary cells, while a single ~130 kDa 4.1B splice form, is detected in all layers of adrenal gland in both the cytoplasm and the membrane. The characterization of distinct splice forms of various 4.1 proteins with diverse cellular and sub-cellular localization indicates multiple functions for this family of proteins in endocrine functions of adrenal gland.

  8. Gene expression patterns, and protein metabolic and histological analyses for muscle development in Peking duck.

    PubMed

    Zhang, Rong-Ping; Liu, He-He; Li, Qing-Qing; Wang, Yan; Liu, Jun-Ying; Hu, Ji-Wei; Yan, Xi-Ping; Gou, Hua; Li, Liang; Wang, Ji-Wen

    2014-12-01

    In this study, we aimed to use duck breast muscle and leg muscle, the 2 main productive muscle organs, as a model to elucidate the molecular mechanism controlling how the 2 muscles have different deposition capabilities, and to analyze the mechanisms facilitating duck muscle development posthatching. Peking duck breast muscle and leg muscle were collected 3, 7, and 16 wk posthatching. The morphology of the myofibers was observed by paraffin sectioning the muscles. The expression of genes involved in protein metabolism [mammalian target of rapamycin (mTOR), RPS6-p70-protein kinase (S6K), forkhead box O1 (FoxO1), muscle RING finger 1 (MuRF1), and atrogin-1 (MAFbx)] was detected using real-time quantitative PCR and Western blot assays, and the results indicated that breast muscle had a stronger capacity for both protein synthesis and protein degradation compared with leg muscle. Satellite cell frequency declined during muscle development in both tissues, and the expression of Pax3/7, satellite cell marker genes, was not significantly different between breast muscle and leg muscle. No notable apoptosis was observed in either tissue. The results of this study suggest that protein metabolism signaling is the main reason promoting duck skeletal muscle mass gain.

  9. Competing Lipid-Protein and Protein-Protein Interactions Determine Clustering and Gating Patterns in the Potassium Channel from Streptomyces lividans (KcsA)*

    PubMed Central

    Molina, M. Luisa; Giudici, A. Marcela; Poveda, José A.; Fernández-Ballester, Gregorio; Montoya, Estefanía; Renart, M. Lourdes; Fernández, Asia M.; Encinar, José A.; Riquelme, Gloria; Morales, Andrés; González-Ros, José M.

    2015-01-01

    There is increasing evidence to support the notion that membrane proteins, instead of being isolated components floating in a fluid lipid environment, can be assembled into supramolecular complexes that take part in a variety of cooperative cellular functions. The interplay between lipid-protein and protein-protein interactions is expected to be a determinant factor in the assembly and dynamics of such membrane complexes. Here we report on a role of anionic phospholipids in determining the extent of clustering of KcsA, a model potassium channel. Assembly/disassembly of channel clusters occurs, at least partly, as a consequence of competing lipid-protein and protein-protein interactions at nonannular lipid binding sites on the channel surface and brings about profound changes in the gating properties of the channel. Our results suggest that these latter effects of anionic lipids are mediated via the Trp67–Glu71–Asp80 inactivation triad within the channel structure and its bearing on the selectivity filter. PMID:26336105

  10. The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom.

    PubMed Central

    Calvete, J. J.; Moreno-Murciano, M. P.; Sanz, L.; Jürgens, M.; Schrader, M.; Raida, M.; Benjamin, D. C.; Fox, J. W.

    2000-01-01

    The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed. PMID:10933502

  11. Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns.

    PubMed

    Bauer, F; Urdaci, M; Aigle, M; Crouzet, M

    1993-08-01

    Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. Here we report results concerning one of these mutants, rvs167, which shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism.

  12. Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns.

    PubMed Central

    Bauer, F; Urdaci, M; Aigle, M; Crouzet, M

    1993-01-01

    Mutations in genes necessary for survival in stationary phase were isolated to understand the ability of wild-type Saccharomyces cerevisiae to remain viable during prolonged periods of nutritional deprivation. Here we report results concerning one of these mutants, rvs167, which shows reduced viability and abnormal cell morphology upon carbon and nitrogen starvation. The mutant exhibits the same response when cells are grown in high salt concentrations and other unfavorable growth conditions. The RVS167 gene product displays significant homology with the Rvs161 protein and contains a SH3 domain at the C-terminal end. Abnormal actin distribution is associated with the mutant phenotype. In addition, while the budding pattern of haploid strains remains axial in standard growth conditions, the budding pattern of diploid mutant strains is random. The gene RVS167 therefore could be implicated in cytoskeletal reorganization in response to environmental stresses and could act in the budding site selection mechanism. Images PMID:8336735

  13. ToF-SIMS Analysis of Adsorbed Proteins: Principal Component Analysis of the Primary Ion Species Effect on the Protein Fragmentation Patterns.

    PubMed

    Muramoto, Shin; Graham, Daniel J; Wagner, Matthew S; Lee, Tae Geol; Moon, Dae Won; Castner, David G

    2011-12-15

    In time-of-flight secondary ion mass spectrometry (ToF-SIMS), the choice of primary ion used for analysis can influence the resulting mass spectrum. This is because different primary ion types can produce different fragmentation pathways. In this study, analysis of single-component protein monolayers were performed using monatomic, tri-atomic, and polyatomic primary ion sources. Eight primary ions (Cs(+), Au(+), Au(3) (+), Bi(+), Bi(3) (+), Bi(3) (++), C(60) (+)) were used to examine to the low mass (m/z < 200) fragmentation patterns from five different proteins (bovine serum albumin, bovine serum fibrinogen, bovine immunoglobulin G and chicken egg white lysozyme) adsorbed onto mica surfaces. Principal component analysis (PCA) processing of the ToF-SIMS data showed that variation in peak intensity caused by the primary ions was greater than differences in protein composition. The spectra generated by Cs(+), Au(+) and Bi(+) primary ions were similar, but the spectra generated by monatomic, tri-atomic and polyatomic primary ion ions varied significantly. C(60) primary ions increased fragmentation of the adsorbed proteins in the m/z < 200 region, resulting in more intense low m/z peaks. Thus, comparison of data obtained by one primary ion species with that obtained by another primary ion species should be done with caution. However, for the spectra generated using a given primary ion beam, discrimination between the spectra of different proteins followed similar trends. Therefore, a PCA model of proteins created with a given ion source should only be applied to datasets obtained using the same ion source. The type of information obtained from PCA depended on the peak set used. When only amino acid peaks were used, PCA was able to identify the relationship between proteins by their amino acid composition. When all peaks from m/z 12-200 were used, PCA separated proteins based on a ratio of C(4)H(8)N(+) to K(+) peak intensities. This ratio correlated with the thickness

  14. Chelating Surfaces for Native State Proteins Patterning: The Human Serum Albumin Case.

    PubMed

    Giamblanco, Nicoletta; Tuccitto, Nunzio; Zappalà, Gabriella; Sfuncia, Gianfranco; Licciardello, Antonino; Marletta, Giovanni

    2015-10-21

    The paper reports a new "soft" surface functionalization strategy, based on a highly selective ion metal chelation process. The proposed stepwise methodology implies at first the construction of a monolayer of terpyridine-based thiol (Tpy), whose highly packed structuring has been followed in situ by using quartz crystal microbalance (QCM-D) measurements, showing that the monolayers consist of about 2.7 × 10(14) Tpy/cm(2). Then, the tridentate sites of the each Tpy moiety are employed to partially chelate divalent metal ions, providing an effective platform to anchoring proteins by completing the metal ion coordination with an available site on the protein of interest. We report the case study of the application of the process to the HSA immobilization onto various surfaces, including Tpy-Fe(II) and Tpy-Cu(II) complexes, as well as hydrophilic bare gold substrates and hydrophobic self-assembled Tpy-based monolayers. It is shown that the chelation interaction between Tpy-Cu(II) complexes and HSA produces the highest and most robust HSA immobilization, with an adsorbed mass at the steady state of ∼800 ng/cm(2), with respect to an average adsorption of ∼350 ng/cm(2) for the other surfaces. Furthermore, Cu(II)-chelated surfaces seem to promote a sort of protein "soft" landing, preventing the ubiquitous surface-induced major unfolding and transmitting an orientation information to the protein, owing to the highly specific symmetry coordination of the Tpy-Cu(II)-protein complex. Indeed, the interaction with a specific monoclonal antiboby (anti-HSA) indicated the lack of a significant protein denaturation, while a massive reorientation/denaturation process was found for all the remaining surfaces, including the Tpy-Fe(II) complex. Finally, the metal-ion-dependent HSA immobilization selectivity has been exploited to obtain micropatterned surfaces, based on the strikingly different strength of interaction and stability observed for Fe(II) and Cu(II) complexes.

  15. Rapid photochemical surface patterning of proteins in thiol-ene based microfluidic devices.

    PubMed

    Lafleur, Josiane P; Kwapiszewski, Radoslaw; Jensen, Thomas G; Kutter, Jörg P

    2013-02-21

    The suitable optical properties of thiol-ene polymers combined with the ease of modifying their surface for the attachment of recognition molecules make them ideal candidates in many biochip applications. This paper reports the rapid one-step photochemical surface patterning of biomolecules in microfluidic thiol-ene chips. This work focuses on thiol-ene substrates featuring an excess of thiol groups at their surface. The thiol-ene stoichiometric composition can be varied to precisely control the number of surface thiol groups available for surface modification up to an average surface density of 136 ± 17 SH nm(-2). Biotin alkyne was patterned directly inside thiol-ene microchannels prior to conjugation with fluorescently labelled streptavidin. The surface bound conjugates were detected by evanescent wave-induced fluorescence (EWIF), demonstrating the success of the grafting procedure and its potential for biochip applications.

  16. Laboratory-scale protein striping system for patterning biomolecules onto paper-based immunochromatographic test strips.

    PubMed

    Nash, Michael A; Hoffman, John M; Stevens, Dean Y; Hoffman, Allan S; Stayton, Patrick S; Yager, Paul

    2010-09-07

    A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.

  17. Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

    SciTech Connect

    Shi, T.; Niepel, M.; McDermott, J. E.; Gao, Y.; Nicora, C. D.; Chrisler, W. B.; Markillie, L. M.; Petyuk, V. A.; Smith, R. D.; Rodland, K. D.; Sorger, P. K.; Qian, W. -J.; Wiley, H. S.

    2016-07-12

    It is not known whether cancer cells generally show quantitative differences in the expression of signaling pathway proteins that could dysregulate signal transduction. To explore this issue, we first defined the primary components of the EGF-MAPK pathway in normal human mammary epithelial cells, identifying 16 core proteins and 10 feedback regulators. We then quantified their absolute abundance across a panel of normal and cancer cell lines. We found that core pathway proteins were expressed at very similar levels across all cell types. In contrast, the EGFR and transcriptionally controlled feedback regulators were expressed at highly variable levels. The absolute abundance of most core pathway proteins was between 50,000- 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower levels (2,000-5,000 per cell). MAPK signaling showed saturation in all cells between 3,000-10,000 occupied EGFR, consistent with the idea that low adaptor levels limit signaling. Our results suggest that the core MAPK pathway is essentially invariant across different cell types, with cell- specific differences in signaling likely due to variable levels of feedback regulators. The low abundance of adaptors relative to the EGFR could be responsible for previous observation of saturable signaling, endocytosis, and high affinity EGFR.

  18. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

    PubMed

    Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.

  19. Strategy for allosteric analysis based on protein-patterned stationary phase in microfluidic chip.

    PubMed

    Bi, Hongyan; Weng, Xuexiang; Qu, Haiyun; Kong, Jilie; Yang, Pengyuan; Liu, Baohong

    2005-01-01

    An effective method is presented for the on-chip analysis of chiral interactions with a successful depression of nonspecific adsorption. The alumina gel-derived protein network on poly(methyl methacrylate) (PMMA) microchannel was explored to form a protein-stationary phase and then used to carry out electrophoresis for fast enantioseparation coupled with electrochemical detection. On the basis of the chemical modification of a synthesized copolymer containing silane-functionalized scaffold, alumina sol-gel could react readily with the silane groups and form steady microstructure on the chip surface achieving the encapsulation of functional biomolecules. Compared with the native PMMA microchannels, the modified surfaces exhibited much better wettability, more stable and enhanced electroosmotic mobility, and less nonspecific adsorption. The water contact angle and EOF of alumina-gel-derived PMMA substrate were 22 degrees and 4.3 x 10(-4) cm(2) V(-1) s(-1), compared to those of 73 degrees and 1.9 x 10(-4) cm(2) V(-1) s(-1) from the untreated one, respectively. Bovine serum albumin, acting as a target protein, could be stably and homogeneously immobilized in the modified PMMA microchannel to fabricate a protein-stationary phase. Under a mild condition, D- and L-tryptophan were efficiently separated with a resolution of 1.57. The as-prepared microchip can perform chiral separations within short time, indicating that the general protocol has the potential to provide a platform for high throughput screening of enantiomer candidates such as those biochemical drugs with protein targets and the research of receptor interactions.

  20. The Evolution and Expression Pattern of Human Overlapping lncRNA and Protein-coding Gene Pairs

    PubMed Central

    Ning, Qianqian; Li, Yixue; Wang, Zhen; Zhou, Songwen; Sun, Hong; Yu, Guangjun

    2017-01-01

    Long non-coding RNA overlapping with protein-coding gene (lncRNA-coding pair) is a special type of overlapping genes. Protein-coding overlapping genes have been well studied and increasing attention has been paid to lncRNAs. By studying lncRNA-coding pairs in human genome, we showed that lncRNA-coding pairs were more likely to be generated by overprinting and retaining genes in lncRNA-coding pairs were given higher priority than non-overlapping genes. Besides, the preference of overlapping configurations preserved during evolution was based on the origin of lncRNA-coding pairs. Further investigations showed that lncRNAs promoting the splicing of their embedded protein-coding partners was a unilateral interaction, but the existence of overlapping partners improving the gene expression was bidirectional and the effect was decreased with the increased evolutionary age of genes. Additionally, the expression of lncRNA-coding pairs showed an overall positive correlation and the expression correlation was associated with their overlapping configurations, local genomic environment and evolutionary age of genes. Comparison of the expression correlation of lncRNA-coding pairs between normal and cancer samples found that the lineage-specific pairs including old protein-coding genes may play an important role in tumorigenesis. This work presents a systematically comprehensive understanding of the evolution and the expression pattern of human lncRNA-coding pairs. PMID:28344339

  1. Pyruvate dehydrogenase complex: mRNA and protein expression patterns of E1α subunit genes in human spermatogenesis.

    PubMed

    Pinheiro, Ana; Silva, Maria João; Graça, Inês; Silva, Joaquina; Sá, Rosália; Sousa, Mário; Barros, Alberto; Tavares de Almeida, Isabel; Rivera, Isabel

    2012-09-10

    During spermatogenesis, germ cells undergo a complex process of cell differentiation and morphological restructuring, which depends on the coordinated expression of different genes. Some vital examples are those involved in cell energy metabolism, namely the genes encoding the E1α subunit of pyruvate dehydrogenase complex: the somatic PDHA1 (X-linked) and the testis-specific PDHA2 (autosomal). There are no data related to the study at the RNA and protein levels of PDHA genes during human spermatogenesis. The present study aimed to describe the mRNA and protein expression patterns of the human PDHA genes during spermatogenesis. Expression profiles of the PDHA1 and PDHA2 genes were characterized using different human tissues and cells. Diploid and haploid germ cells fractions were obtained from testis tissues. The mRNA profiles were analyzed by quantitative RT-PCR, whereas the protein profiles were evaluated by immunohistochemistry, western blotting and two-dimensional electrophoresis. Expression of the PDHA1 gene was found in all somatic cells, whereas expression of PDHA2 gene was restricted to germ cells. The switch from X-linked to autosomic gene expression occurred in spermatocytes. Data suggest the activation of PDHA2 gene expression is most probably a mechanism to ensure the continued expression of the protein, thus allowing germ cell viability and functionality.

  2. Two secretory protein genes in Chironomus tentans have arisen by gene duplication and exhibit different developmental expression patterns.

    PubMed

    Galli, J; Wieslander, L

    1993-05-20

    The salivary gland cells in the dipteran Chironomus tentans produce approximately 15 different secretory proteins, with relative molecular masses ranging between 1 x 10(4) and 1 x 10(6). Together these proteins form two types of extra corporal tubes, a larval protective housing and feeding tube or a pupation tube. The developmental change in tube formation is accompanied by a switch in production from one combination of secretory proteins to another. Here we characterize two genes, the sp38-40.A and B genes, which encode secretory proteins with relative molecular masses of 38,000 to 40,000. The two genes are located 346 base-pairs apart in the same orientation and have presumably arisen by gene duplication as the result of an illegitimate recombination event. Both genes contain two regions with cysteine codons, surrounded by regions with short repeats coding for proline and charged amino acid residues. The two genes and alleles of the genes differ in their number of repeats. This structure resembles the structure of the Balbiani ring (BR) genes, which encode the four largest salivary gland secretory proteins. The sp38-40.A and B genes are therefore likely to belong to a BR multigene family containing all or most of the 15 salivary gland secretory protein genes. The expression of the sp38-40.A and B genes are different: the A gene is expressed throughout the larval fourth instar but considerably less in the prepupal stage, while the B gene shows the opposite expression pattern. The developmental regulation of the expression of the two genes has therefore diverged after the gene duplication event.

  3. Different BCR/Abl protein suppression patterns as a converging trait of chronic myeloid leukemia cell adaptation to energy restriction.

    PubMed

    Bono, Silvia; Lulli, Matteo; D'Agostino, Vito Giuseppe; Di Gesualdo, Federico; Loffredo, Rosa; Cipolleschi, Maria Grazia; Provenzani, Alessandro; Rovida, Elisabetta; Dello Sbarba, Persio

    2016-12-20

    BCR/Abl protein drives the onset and progression of Chronic Myeloid Leukemia (CML). We previously showed that BCR/Abl protein is suppressed in low oxygen, where viable cells retain stem cell potential. This study addressed the regulation of BCR/Abl protein expression under oxygen or glucose shortage, characteristic of the in vivo environment where cells resistant to tyrosine kinase inhibitors (TKi) persist. We investigated, at transcriptional, translational and post-translational level, the mechanisms involved in BCR/Abl suppression in K562 and KCL22 CML cells. BCR/abl mRNA steady-state analysis and ChIP-qPCR on BCR promoter revealed that BCR/abl transcriptional activity is reduced in K562 cells under oxygen shortage. The SUnSET assay showed an overall reduction of protein synthesis under oxygen/glucose shortage in both cell lines. However, only low oxygen decreased polysome-associated BCR/abl mRNA significantly in KCL22 cells, suggesting a decreased BCR/Abl translation. The proteasome inhibitor MG132 or the pan-caspase inhibitor z-VAD-fmk extended BCR/Abl expression under oxygen/glucose shortage in K562 cells. Glucose shortage induced autophagy-dependent BCR/Abl protein degradation in KCL22 cells. Overall, our results showed that energy restriction induces different cell-specific BCR/Abl protein suppression patterns, which represent a converging route to TKi-resistance of CML cells. Thus, the interference with BCR/Abl expression in environment-adapted CML cells may become a useful implement to current therapy.

  4. DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning.

    PubMed

    Rodriguez, Kevin; Perales, Mariano; Snipes, Stephen; Yadav, Ram Kishor; Diaz-Mendoza, Mercedes; Reddy, G Venugopala

    2016-10-11

    The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.

  5. Localization and expression pattern of amelotin, odontogenic ameloblast-associated protein and follicular dendritic cell-secreted protein in the junctional epithelium of inflamed gingiva.

    PubMed

    Nakayama, Yohei; Kobayashi, Ryoki; Matsui, Sari; Matsumura, Hiroyoshi; Iwai, Yasunobu; Noda, Keisuke; Yamazaki, Mizuho; Kurita-Ochiai, Tomoko; Yoshimura, Atsutoshi; Shinomura, Tamayuki; Ganss, Bernhard; Ogata, Yorimasa

    2016-11-02

    The purpose of this study is to elucidate the localization of amelotin (AMTN), odontogenic ameloblast-associated protein (ODAM) and follicular dendritic cell-secreted protein (FDC-SP) at the junctional epithelium (JE) in Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans infected mice and inflamed and non-inflamed human gingiva. We performed immunostaining to determine the localization and expression pattern of AMTN, ODAM and FDC-SP. AMTN, ODAM and FDC-SP in A. actinomycetemcomitans infected mice did not change dramatically compared with non-infected mice. AMTN and FDC-SP expressions were observed stronger in P. gingivalis infected mice at early stage. However, at the following stage, the coronal part of the AMTN expression disappeared from the JE, and FDC-SP expression decreased due to severe inflammation by P. gingivalis. ODAM expressed internal and external basal lamina, and the expression increased not only at early stage but also at the following stage in the inflammatory JE induced by P. gingivalis. In the human gingival tissues, AMTN was detected at the surface of the sulcular epithelium and JE in the non-inflamed and inflamed gingiva, and the localization did not change the process of inflammation. ODAM and FDC-SP were more widely detected at the sulcular epithelium and JE in the non-inflamed gingiva. In the inflamed gingiva, localization of ODAM and FDC-SP was spread into the gingival epithelium, compared to AMTN. These studies demonstrated that the expression pattern of AMTN, ODAM and FDC-SP at the JE were changed during inflammation process and these three proteins might play an important role in the resistance to inflammation.

  6. Expression pattern in retinal photoreceptors of POMGnT1, a protein involved in muscle-eye-brain disease

    PubMed Central

    Uribe, Mary Luz; Haro, Carmen; Campello, Laura; Cruces, Jesús; Martín-Nieto, José

    2016-01-01

    Purpose The POMGNT1 gene, encoding protein O-linked-mannose β-1,2-N-acetylglucosaminyltransferase 1, is associated with muscle-eye-brain disease (MEB) and other dystroglycanopathies. This gene’s lack of function or expression causes hypoglycosylation of α-dystroglycan (α-DG) in the muscle and the central nervous system, including the brain and the retina. The ocular symptoms of patients with MEB include retinal degeneration and detachment, glaucoma, and abnormal electroretinogram. Nevertheless, the POMGnT1 expression pattern in the healthy mammalian retina has not yet been investigated. In this work, we address the expression of the POMGNT1 gene in the healthy retina of a variety of mammals and characterize the distribution pattern of this gene in the adult mouse retina and the 661W photoreceptor cell line. Methods Using reverse transcription (RT)–PCR and immunoblotting, we studied POMGNT1 expression at the mRNA and protein levels in various mammalian species, from rodents to humans. Immunofluorescence confocal microscopy analyses were performed to characterize the distribution profile of its protein product in mouse retinal sections and in 661W cultured cells. The intranuclear distribution of POMT1 and POMT2, the two enzymes preceding POMGnT1 in the α-DG O-mannosyl glycosylation pathway, was also analyzed. Results POMGNT1 mRNA and its encoded protein were expressed in the neural retina of all mammals studied. POMGnT1 was located in the cytoplasmic fraction in the mouse retina and concentrated in the myoid portion of the photoreceptor inner segments, where the protein colocalized with GM130, a Golgi complex marker. The presence of POMGnT1 in the Golgi complex was also evident in 661W cells. However, and in contrast to retinal tissue, POMGnT1 additionally accumulated in the nucleus of the 661W photoreceptors. Colocalization was found within this organelle between POMGnT1 and POMT1/2, the latter associated with euchromatic regions of the nucleus. Conclusions

  7. Persistent dominant follicle alters pattern of oviductal secretory proteins from cows at estrus.

    PubMed

    Binelli, M; Hampton, J; Buhi, W C; Thatcher, W W

    1999-07-01

    The experimental objective was to compare synthesis of oviductal secretory proteins of dairy cows bearing a persistent dominant follicle (PDF) versus a fresh dominant follicle (FDF) at estrus. On Day 7 after synchronized estrus (Day 0), cows received an intravaginal progesterone device and injection of prostaglandin F2alpha (PGF2alpha). On Day 9, cows received an injection of a GnRH agonist (FDF group; n = 3) or received no injection (PDF group, n = 3). On Day 16, all cows received PGF2alpha, and progesterone devices were removed. At slaughter on Day 18 or Day 19, oviducts ipsilateral and contralateral to the dominant follicle were divided into infundibulum, ampulla, and isthmus regions. Explants from oviductal regions were cultured in minimal essential medium supplemented with [3H]leucine for 24 h. Two-dimensional fluorographs of proteins in conditioned media were analyzed by densitometry. Rate of incorporation of [3H]leucine into macromolecules was greater in the infundibulum, ampulla, and isthmus of FDF cows (p < 0.01). Overall, intensities of radiolabeled secretory protein (P) 2 and P13 were greater for FDF than for PDF. In the ampulla, P14 was more intense for FDF while P7 was more intense for PDF. Abundance of P1 in the isthmus was greater for PDF cows. Across regions, P5, P6, P8, P9, and P11 were more intense for PDF than for FDF in the ipsilateral side. In the contralateral side, P19 was more intense for PDF than for FDF, whereas P6, P8, P9, and P11 were more intense for FDF. Differences in biosynthetic activity and in secreted oviductal proteins from cows bearing a PDF may contribute to the decrease in fertility associated with a PDF.

  8. Distinct Patterns of Gene and Protein Expression Elicited by Organophosphorus Pesticides in Caenorhabditis elegans

    DTIC Science & Technology

    2009-01-01

    alterations in the expression of a number of genes and proteins involved in cell death. Neuronal death in response to OP exposure in C . elegans is...level of free acetylcholine. At face value, the evidence argues against the occurrence of necrosis. C . elegans has six aspartyl protease genes (asp-1...axon damage [2]. There are two genes in the C . elegans genome homologous to the vertebrate secondary OP target, NTE (ZK370.4 and M110.7; [27] and

  9. Target pattern recognition by complement proteins of the classical and alternative pathways.

    PubMed

    Kang, Yu-Hoi; Tan, Lee Aun; Carroll, Maria V; Gentle, Madeleine E; Sim, Robert B

    2009-01-01

    The complement system is a major component of the innate defence of animals against invading microorganisms, and is also essential for the recognition and clearance of damaged or structurally-altered host cells or macromolecules. The system is activated by three different pathways, each of which responds, using different recognition molecules, to a very wide range of activators. The recognition protein of the complement classical pathway, C1q is described in detail here, with comparisons to the alternative pathway.

  10. Multiplexed protein profiling after aneurysmal subarachnoid hemorrhage: characterization of differential expression patterns in cerebral vasospasm.

    PubMed

    Walcott, Brian P; Patel, Anoop P; Stapleton, Christopher J; Trivedi, Rikin A; Young, Adam M H; Ogilvy, Christopher S

    2014-12-01

    Cerebral vasospasm is a major contributor to delayed morbidity following aneurysmal subarachnoid hemorrhage. We sought to evaluate differential plasma protein levels across time in patients with aneurysmal subarachnoid hemorrhage to identify potential biomarkers and to better understand the pathogenesis of cerebral vasospasm. Nine female patients with aneurysmal subarachnoid hemorrhage underwent serial analysis of 239 different serum protein levels using quantitative, multiplexed immunoassays (DiscoveryMAP 250+ v2.0, Myriad RBM, Austin, TX, USA) on post-hemorrhage days 0 and 5. A repeated measures analysis of variance determined that mean protein concentration decreased significantly in patients who developed vasospasm versus those who did not for alpha-2-macroglobulin (F [1.00,7.00]=16.33, p=0.005), angiogenin (F [1.00,7.00]=7.65, p=0.028), apolipoprotein A-IV (F [1.00,7.00]=6.308, p=0.040), granulocyte colony-stimulating factor (F [1.00,7.00]=9.08, p=0.020), macrophage-stimulating protein (F [1.00,7.00]=24.21, p=0.002), tetranectin (F [1.00,7.00]=5.46, p<0.039), vascular endothelial growth factor receptor 3 (F [1.00,7.00]=6.94, p=0.034), and significantly increased for vitronectin (F [1.00,7.00]=5.79, p=0.047). These biomarkers may be of value in detecting cerebral vasospasm, possibly aiding in the identification of patients at high-risk prior to neurological deterioration.

  11. Global Alignment of Pairwise Protein Interaction Networks for Maximal Common Conserved Patterns

    DOE PAGES

    Tian, Wenhong; Samatova, Nagiza F.

    2013-01-01

    A number of tools for the alignment of protein-protein interaction (PPI) networks have laid the foundation for PPI network analysis. Most of alignment tools focus on finding conserved interaction regions across the PPI networks through either local or global mapping of similar sequences. Researchers are still trying to improve the speed, scalability, and accuracy of network alignment. In view of this, we introduce a connected-components based fast algorithm, HopeMap, for network alignment. Observing that the size of true orthologs across species is small comparing to the total number of proteins in all species, we take a different approach basedmore » on a precompiled list of homologs identified by KO terms. Applying this approach to S. cerevisiae (yeast) and D. melanogaster (fly), E. coli K12 and S. typhimurium , E. coli K12 and C. crescenttus , we analyze all clusters identified in the alignment. The results are evaluated through up-to-date known gene annotations, gene ontology (GO), and KEGG ortholog groups (KO). Comparing to existing tools, our approach is fast with linear computational cost, highly accurate in terms of KO and GO terms specificity and sensitivity, and can be extended to multiple alignments easily.« less

  12. Adaptive expression pattern of different proteins involved in cellular calcium homeostasis in denervated rat vas deferens.

    PubMed

    Quintas, Luis Eduardo M; Cunha, Valéria M N; Scaramello, Christianne B V; da Silva, Cláudia L M; Caricati-Neto, Afonso; Lafayette, Simone S L; Jurkiewicz, Aron; Noël, François

    2005-11-21

    The activity and protein expression of plasma membrane and sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPases and ryanodine receptors were investigated in surgically denervated rat vas deferens. The function of thapsigargin-sensitive but not thapsigargin-resistant (Ca2+-Mg2+)ATPase (from sarco(endo)plasmic reticulum and plasma membrane, respectively), evidenced by enzyme activity and Ca2+ uptake experiments, was significantly depressed by 30-50% when compared to innervated vas. Western blots showed that such reduction in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase performance was accompanied by a decrement of similar magnitude in sarco(endo)plasmic reticulum (Ca2+-Mg2+)ATPase type 2 protein expression, without any significant change in plasma membrane (Ca2+-Mg2+)ATPase expression. Finally, [3H]ryanodine binding revealed that the density of ryanodine binding sites was reduced by 45% after denervation without modification in affinity. The present findings demonstrate that sarco(endo)plasmic reticulum proteins involved in intracellular calcium homeostasis are clearly down-regulated and brings further evidence of a modified calcium translocation in denervated rat vas deferens.

  13. Epidemiology of virulence-associated plasmids and outer membrane protein patterns within seven common Salmonella serotypes.

    PubMed

    Helmuth, R; Stephan, R; Bunge, C; Hoog, B; Steinbeck, A; Bulling, E

    1985-04-01

    Antibiotic-sensitive Salmonella isolates belonging to seven common serotypes and originating from 29 different countries from all continents were investigated for their plasmid DNA content (337 isolates) and their outer membrane protein profiles (216 isolates). Of the S. typhimurium, S. enteritidis, S. dublin, and S. choleraesuis isolates, 90% or more carried a serotype-specific plasmid. The molecular sizes of the plasmids were 60 megadaltons (Md) for S. typhimurium, 37 Md for S. enteritidis, 56 Md for S. dublin, and 30 Md for S. choleraesuis. The outer membrane protein profiles were homogeneous within each of the seven serotypes, except that a minority of S. enteritidis and S. dublin strains were lacking one major outer membrane protein. Virulence studies were performed with 39 representative strains by measuring the 50% lethal doses (LD50S) after oral infection of mice. The LD50 values obtained for plasmid-positive strains of S. typhimurium, S. enteritidis, and S. dublin were up to 10(6)-fold lower than the values obtained for the plasmid-free strains of the same serotype. Only the plasmid-positive strains could invade the livers of orally infected mice, and only they were resistant to the bactericidal activity of 90% guinea pig serum. Strains of S. infantis were generally plasmid free, whereas S. panama and S. heidelberg isolates carried heterogeneous plasmid populations. The virulence properties of the latter three serotypes could not be correlated with the predominant plasmids found in these strains.

  14. Old and new findings on lipopolysaccharide-binding protein: a soluble pattern-recognition molecule.

    PubMed

    Schumann, Ralf R

    2011-08-01

    LBP [LPS (lipopolysaccharide)-binding protein] was discovered approximately 25 years ago. Since then, substantial progress has been made towards our understanding of its function in health and disease. Furthermore, the discovery of a large protein family sharing functional and structural attributes has helped in our knowledge. Still, key questions are unresolved, and here an overview on the old and new findings on LBP is given. LBP is an acute-phase protein of the liver, but is also synthesized in other cells of the organism. While LBP is named after the ability to bind to LPS of Gram-negative bacteria, it also can recognize other bacterial compounds, such as lipopeptides. It has been shown that LBP is needed to combat infections; however, the main mechanism of action is still not clear. New findings on natural genetic variations of LBP leading to functional consequences may help in further elucidating the mechanism of LBP and its role in innate immunity and disease.

  15. Dynamic and quasi-static measurements of PBXN-5 and comp-B explosives

    SciTech Connect

    Brown, Geoffrey W; Ten Cate, James A; Deluca, Racci; Rae, Philip J; Todd, Steven N

    2009-03-12

    We have measured dynamic and quasi-static mechanical properties of PBXN-5 and Comp-B explosive materials to provide input data for modeling efforts. Dynamic measurements included acoustic and split-Hopkinson pressure bar tests. Quasi-static testing was done in compression on a load frame. Hopkinson bar and quasistatic testing was done at five temperatures from -50{sup o}C to 50{sup o}C. Our results were dominated by the low density of the samples and showed up as low acoustic velocities and lower strengths, as compared to other materials of the same or similar formulations. The effects seem to be consistent with the high porosity of the materials. The data do provide useful input to models that include density as a parameter and suggest caution when using measurements of ideal materials to predict behavior of damaged materials.

  16. Development-related expression patterns of protein-coding and miRNA genes involved in porcine muscle growth.

    PubMed

    Wang, F J; Jin, L; Guo, Y Q; Liu, R; He, M N; Li, M Z; Li, X W

    2014-11-27

    Muscle growth and development is associated with remarkable changes in protein-coding and microRNA (miRNA) gene expression. To determine the expression patterns of genes and miRNAs related to muscle growth and development, we measured the expression levels of 25 protein-coding and 16 miRNA genes in skeletal and cardiac muscles throughout 5 developmental stages by quantitative reverse transcription-polymerase chain reaction. The Short Time-Series Expression Miner (STEM) software clustering results showed that growth-related genes were downregulated at all developmental stages in both the psoas major and longissimus dorsi muscles, indicating their involvement in early developmental stages. Furthermore, genes related to muscle atrophy, such as forkhead box 1 and muscle ring finger, showed unregulated expression with increasing age, suggesting a decrease in protein synthesis during the later stages of skeletal muscle development. We found that development of the cardiac muscle was a complex process in which growth-related genes were highly expressed during embryonic development, but they did not show uniform postnatal expression patterns. Moreover, the expression level of miR-499, which enhances the expression of the β-myosin heavy chain, was significantly different in the psoas major and longissimus dorsi muscles, suggesting the involvement of miR-499 in the determination of skeletal muscle fiber types. We also performed correlation analyses of messenger RNA and miRNA expression. We found negative relationships between miR-486 and forkhead box 1, and miR-133a and serum response factor at all developmental stages, suggesting that forkhead box 1 and serum response factor are potential targets of miR-486 and miR-133a, respectively.

  17. Pattern of seroreactivity against feline foamy virus proteins in domestic cats from Germany.

    PubMed

    Bleiholder, Anne; Mühle, Michael; Hechler, Torsten; Bevins, Sarah; vandeWoude, Sue; Denner, Joachim; Löchelt, Martin

    2011-10-15

    The prevalence of feline foamy virus (FFV, spumaretrovirinae) in naturally infected domestic cats ranges between 30 and 80% FFV positive animals depending on age, sex and geographical region analyzed. Two serotypes have been reported for FFV designated FUV7-like and F17/951-like. Serotype-specific neutralization has been shown to correlate with sequence divergence in the surface (SU) domain of the envelope protein (Env). We analyzed a serum collection of 262 domestic cat sera from Germany using a GST-capture ELISA setup screening for Gag and Bet specific antibodies and identified 39% FFV positive animals. Due to the heterogeneity of the serological samples, cut-offs for Gag and Bet reactivity had to be experimentally determined since application of calculated cut-off values yielded some false-positive results; the new cut-off values turned out to be also fully applicable to a previous study. Using the already established FUV7 ElpSU antigen and the newly cloned and produced F17/951 ElpSU antigen, both consisting of the corresponding ectodomains of the envelope leader protein (Elp) and SU protein, we aimed at the detection of Env-specific antibodies and discrimination between the two known FFV serotypes within the diagnostic FFV ELISA. We validated the ElpSU antigens using cat reference sera of known serotype and screened with this assay domestic cat sera from Germany. Use of the FUV7- and F17/951 ElpSU antigens in ELISA resulted in the detection of Env-specific antibodies in both cat reference sera and sera from domestic cats in Germany, but failed to allow serotyping at the same time.

  18. DNA methylation patterns of protein coding genes and long noncoding RNAs in female schizophrenic patients.

    PubMed

    Liao, Qi; Wang, Yunliang; Cheng, Jia; Dai, Dongjun; Zhou, Xingyu; Zhang, Yuzheng; Gao, Shugui; Duan, Shiwei

    2015-02-01

    Schizophrenia (SCZ) is a complex mental disorder contributed by both genetic and epigenetic factors. Long noncoding RNAs (lncRNAs) was recently found playing an important regulatory role in mental disorders. However, little was known about the DNA methylation of lncRNAs, although numerous SCZ studies have been performed on genetic polymorphisms or epigenetic marks in protein coding genes. We presented a comprehensive genome wide DNA methylation study of both protein coding genes and lncRNAs in female patients with paranoid and undifferentiated SCZ. Using the methyl-CpG binding domain (MBD) protein-enriched genome sequencing (MBD-seq), 8,163 and 764 peaks were identified in paranoid and undifferentiated SCZ, respectively (p < 1 × 10-5). Gene ontology analysis showed that the hypermethylated regions were enriched in the genes related to neuron system and brain for both paranoid and undifferentiated SCZ (p < 0.05). Among these peaks, 121 peaks were located in gene promoter regions that might affect gene expression and influence the SCZ related pathways. Interestingly, DNA methylation of 136 and 23 known lncRNAs in Refseq database were identified in paranoid and undifferentiated SCZ, respectively. In addition, ∼20% of intergenic peaks annotated based on Refseq genes were overlapped with lncRNAs in UCSC and gencode databases. In order to show the results well for most biological researchers, we created an online database to display and visualize the information of DNA methyation peaks in both types of SCZ (http://www.bioinfo.org/scz/scz.htm). Our results showed that the aberrant DNA methylation of lncRNAs might be another important epigenetic factor for SCZ.

  19. Expression patterns of Wnt signaling component, secreted frizzled‑related protein 3 in astrocytoma and glioblastoma.

    PubMed

    Pećina-Šlaus, Nives; Kafka, Anja; Varošanec, Ana Maria; Marković, Leon; Krsnik, Željka; Njirić, Niko; Mrak, Goran

    2016-05-01

    Secreted frizzled-related protein 3 (SFRP3) is a member of the family of soluble proteins, which modulate the Wnt signaling cascade. Novel research has identified aberrant expression of SFRPs in different types of cancer. In the present study the expression intensities and localizations of the SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. The results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Tumors were classified into four malignancy grades according to the World Health Organization guidelines. Moderate (P=0.014) and strong (P=0.028) nuclear expression levels were significantly different in pilocytic (grade I) and diffuse (grade II) astrocytomas demonstrating higher expression values, as compared with anaplastic astrocytoma (grade III) and glioblastoma (grade IV). When the sample was divided into two groups, the moderate and high cytoplasmic expression levels were observed to be significantly higher in glioblastomas than in the group comprising astrocytoma II and III. Furthermore, the results indicated that high grade tumors were associated with lower values of moderate (P=0.002) and strong (P=0.018) nuclear expression in comparison to low grade tumors. Analysis of cytoplasmic staining demonstrated that strong cytoplasmic expression was significantly higher in the astrocytoma III and IV group than in the astrocytoma I and II group (P=0.048). Furthermore, lower grade astrocytomas exhibited reduced membranous SFRP3 staining when compared with higher grade astrocytomas and this difference was statistically significant (P=0.036). The present results demonstrated that SFRP3 protein expression levels were decreased in the nucleus in higher grade astrocytoma (indicating the expected behavior of an antagonist of Wnt signaling), whereas when the SFRP3 was located in the

  20. Expression patterns of Wnt signaling component, secreted frizzled-related protein 3 in astrocytoma and glioblastoma

    PubMed Central

    PEĆINA-ŠLAUS, NIVES; KAFKA, ANJA; VAROŠANEC, ANA MARIA; MARKOVIĆ, LEON; KRSNIK, ŽELJKA; NJIRIĆ, NIKO; MRAK, GORAN

    2016-01-01

    Secreted frizzled-related protein 3 (SFRP3) is a member of the family of soluble proteins, which modulate the Wnt signaling cascade. Novel research has identified aberrant expression of SFRPs in different types of cancer. In the present study the expression intensities and localizations of the SFRP3 protein across different histopathological grades of astrocytic brain tumors were investigated by immunohistochemistry, digital scanning and image analysis. The results demonstrated that the differences between expression levels and malignancy grades were statistically significant. Tumors were classified into four malignancy grades according to the World Health Organization guidelines. Moderate (P=0.014) and strong (P=0.028) nuclear expression levels were significantly different in pilocytic (grade I) and diffuse (grade II) astrocytomas demonstrating higher expression values, as compared with anaplastic astrocytoma (grade III) and glioblastoma (grade IV). When the sample was divided into two groups, the moderate and high cytoplasmic expression levels were observed to be significantly higher in glioblastomas than in the group comprising astrocytoma II and III. Furthermore, the results indicated that high grade tumors were associated with lower values of moderate (P=0.002) and strong (P=0.018) nuclear expression in comparison to low grade tumors. Analysis of cytoplasmic staining demonstrated that strong cytoplasmic expression was significantly higher in the astrocytoma III and IV group than in the astrocytoma I and II group (P=0.048). Furthermore, lower grade astrocytomas exhibited reduced membranous SFRP3 staining when compared with higher grade astrocytomas and this difference was statistically significant (P=0.036). The present results demonstrated that SFRP3 protein expression levels were decreased in the nucleus in higher grade astrocytoma (indicating the expected behavior of an antagonist of Wnt signaling), whereas when the SFRP3 was located in the cytoplasm an

  1. Patterns of proteins synthesized in the R15 neuron of Aplysia. Temporal studies and evidence for processing

    PubMed Central

    1976-01-01

    The time-course of changes in the pattern of newly synthesized proteins in the R15 neuron of the parietovisceral ganglion of Aplysia californica has been studied at 14 degrees C. 5% polyacrylamide gels containing sodium dodecyl sulfate (SDS) have been used to separate newly synthesized (leucine-labeled) proteins from the neuron. We have demonstrated that the pattern of newly synthesized proteins from the R15 neuron does not change significantly if 5-h pulses of labeled leucine are given during the first 72 h of in vitro incubation of the excised ganglion. However, the level of leucine incorporation begins to decline somewhere between 17 and 43 h after the ganglion is isolated; at 43 and 69 h the levels of incorporation fell to 29 and 10% of the initial level, respectively. A number of conclusions have been drawn from the use of a sequential, double-label type of experiment in the same cell. There is processing of SDS-soluble, 12,000-dalton (12k) material to 6,000-9,000-dalton (6-9k) material. These materials are the two major peaks on gels after long labeling periods and together account for about 35% of all newly synthesized proteins. After synthesis of 12k material, there is a gradual disappearance of 12k (half-life about 8 h) and simultaneous appearance of 6-9k material on the gels, as the postsynthesis "chase" period of ganglia incubation is increased. The processing of 12k to 6-9k material occurs even in the presence of anisomycin, a protein syntehsis inhibitor, during the chase period. While the rate of 12k to 6-9k conversion can vary from cell to cell, it appears to remain consistent within, and is characteristic of, any individual R15. We detect no circadian rhythm in either the rate of 12k synthesis or the rate of 12k to 6-9k processing with 5-h label periods. These results are discussed in relation to the roles of 12k and 6-9k material in the R15 neuron. PMID:932671

  2. Patterned polymer nanowire arrays as an effective protein immobilizer for biosensing and HIV detection

    NASA Astrophysics Data System (ADS)

    Shen, Yue; Liu, Yingyi; Zhu, Guang; Fang, Hao; Huang, Yunhui; Jiang, Xingyu; Wang, Zhong L.

    2012-12-01

    We report an array of polymeric nanowires for effectively immobilizing biomolecules on biochips owing to the large surface area. The nanowires were fabricated in predesigned patterns using an inductively coupled plasma (ICP) etching process. Microfluidic biochips integrated using the substrates with arrays of nanowires and polydimethylsiloxane channels have been demonstrated to be effective for detecting antigens, and a detection limit of antigens at 0.2 μg mL-1 has been achieved, which is improved by a factor of 50 compared to that based on flat substrates without the nanowires. In addition, the high sensitivity for clinical detection of human immunodeficiency virus (HIV) antibody has also been demonstrated, showing a 20 times enhancement in fluorescent signal intensity between the samples with positive and negative HIV.

  3. Skeletal muscle regeneration on protein-grafted and microchannel-patterned scaffold for hypopharyngeal tissue engineering.

    PubMed

    Shen, Zhisen; Guo, Shanshan; Ye, Dong; Chen, Jingjing; Kang, Cheng; Qiu, Shejie; Lu, Dakai; Li, Qun; Xu, Kunjie; Lv, Jingjing; Zhu, Yabin

    2013-01-01

    In the field of tissue engineering, polymeric materials with high biocompatibility like polylactic acid and polyglycolic acid have been widely used for fabricating living constructs. For hypopharynx tissue engineering, skeletal muscle is one important functional part of the whole organ, which assembles the unidirectionally aligned myotubes. In this study, a polyurethane (PU) scaffold with microchannel patterns was used to provide aligning guidance for the seeded human myoblasts. Due to the low hydrophilicity of PU, the scaffold was grafted with silk fibroin (PU-SF) or gelatin (PU-Gel) to improve its cell adhesion properties. Scaffolds were observed to degrade slowly over time, and their mechanical properties and hydrophilicities were improved through the surface grafting. Also, the myoblasts seeded on PU-SF had the higher proliferative rate and better differentiation compared with those on the control or PU-Gel. Our results demonstrate that polyurethane scaffolds seeded with myoblasts hold promise to guide hypopharynx muscle regeneration.

  4. Skeletal Muscle Regeneration on Protein-Grafted and Microchannel-Patterned Scaffold for Hypopharyngeal Tissue Engineering

    PubMed Central

    Shen, Zhisen; Guo, Shanshan; Ye, Dong; Chen, Jingjing; Kang, Cheng; Qiu, Shejie; Lu, Dakai; Li, Qun; Xu, Kunjie; Lv, Jingjing

    2013-01-01

    In the field of tissue engineering, polymeric materials with high biocompatibility like polylactic acid and polyglycolic acid have been widely used for fabricating living constructs. For hypopharynx tissue engineering, skeletal muscle is one important functional part of the whole organ, which assembles the unidirectionally aligned myotubes. In this study, a polyurethane (PU) scaffold with microchannel patterns was used to provide aligning guidance for the seeded human myoblasts. Due to the low hydrophilicity of PU, the scaffold was grafted with silk fibroin (PU-SF) or gelatin (PU-Gel) to improve its cell adhesion properties. Scaffolds were observed to degrade slowly over time, and their mechanical properties and hydrophilicities were improved through the surface grafting. Also, the myoblasts seeded on PU-SF had the higher proliferative rate and better differentiation compared with those on the control or PU-Gel. Our results demonstrate that polyurethane scaffolds seeded with myoblasts hold promise to guide hypopharynx muscle regeneration. PMID:24175281

  5. Expression patterns and binding properties of three pheromone binding proteins in the diamondback moth, Plutella xyllotella.

    PubMed

    Sun, Mengjing; Liu, Yang; Wang, Guirong

    2013-01-01

    Pheromone binding proteins (PBPs) play a key role in transporting hydrophobic sex pheromone components emitted by con-specific female across aqueous sensillar lymph to the surface of olfactory receptor neurons. A number of PBPs have been cloned, however, details of their function are still largely unknown. Here three pheromone binding protein genes in the diamondback moth, Plutella xyllotella were cloned. The three PxylPBP genes are not only expressed in chemosensory tissues but also expressed in female reproductive organs and male legs. To better understand the functions of PxylPBPs in the initial steps of pheromone recognition, three PxylPBPs were expressed in Escherichia coli and the ligand-binding specificities of purified recombinant PBPs were investigated. Fluorescence binding assays indicate that three PxylPBPs not only robustly bound all four sex pheromone components but also significantly bound pheromone analogs with at least one double bond, while weakly bound tested plant volatiles. Although pheromone analogs bound PBPs, they could not elicit the moth's electrophysiological response. These experiments provide evidence that PxylPBPs have limited selectivity of pheromone components and analogs and some downstream components such as odor receptors might be involved in selectivity and specificity of pheromone perception in P. xyllotella.

  6. DNA Origami Reorganizes upon Interaction with Graphite: Implications for High-Resolution DNA Directed Protein Patterning

    PubMed Central

    Rahman, Masudur; Neff, David; Green, Nathaniel; Norton, Michael L.

    2016-01-01

    Although there is a long history of the study of the interaction of DNA with carbon surfaces, limited information exists regarding the interaction of complex DNA-based nanostructures with the important material graphite, which is closely related to graphene. In view of the capacity of DNA to direct the assembly of proteins and optical and electronic nanoparticles, the potential for combining DNA-based materials with graphite, which is an ultra-flat, conductive carbon substrate, requires evaluation. A series of imaging studies utilizing Atomic Force Microscopy has been applied in order to provide a unified picture of this important interaction of structured DNA and graphite. For the test structure examined, we observe a rapid destabilization of the complex DNA origami structure, consistent with a strong interaction of single-stranded DNA with the carbon surface. This destabilizing interaction can be obscured by an intentional or unintentional primary intervening layer of single-stranded DNA. Because the interaction of origami with graphite is not completely dissociative, and because the frustrated, expanded structure is relatively stable over time in solution, it is demonstrated that organized structures of pairs of the model protein streptavidin can be produced on carbon surfaces using DNA origami as the directing material.

  7. Changes of CSF-protein pattern in children with acute lymphoblastic leukemia during prophylactic CNS therapy (Berlin protocol)

    SciTech Connect

    Siemes, H.; Rating, D.; Siegert, M.; Hanefeld, F.; Mueller, S.; Gadner, H.; Riehm, H.

    1980-01-01

    The cerebral spinal fluid (CSF)-protein profiles of ten children with previously untreated acute lymphoblastic leukemia (ALL) were investigated by agarose gel electrophoresis. The profiles were determined at diagnosis and during the fifth to eighth week of treatment when preventive therapy for central nervous system (CNS) leukemia (skull irradiation, intrathecal methotrexate (ithMTX) was administered. The profiles were compared with those obtained from a control group of 67 children and those from 42 patients with acute aseptic meningitis. The data from the latter group demonstrated the CSF-protein pattern of partial blood-CSF barrier (B-CSF-B) breakdown. The children with ALL showed no or only minor signs of a B-CSF-B impairment at diagnosis and after four weeks of systemic treatment. However, CSF changes indicative of a lesion of the B-CSF-B increased in all children continuously during CNS prophylaxis. The protein profile at the end of combined chemotherapy and radiotherapy was very similar to that in patients with acute aseptic meningitis. These observations point to neurotoxic side effects on the CNS barrier system with the combination of cranial radiation and ithMTX. A striking finding was restricted heterogeneity of gamma-globulin, observed in the CSF of nine out of the ten children with ALL before or during treatment. The significance of this abnormality is unknown.

  8. Differential expression patterns among heat-shock protein genes and thermal responses in the whitefly Bemisia tabaci (MEAM 1).

    PubMed

    Díaz, Fernando; Orobio, Rony F; Chavarriaga, Paul; Toro-Perea, Nelson

    2015-08-01

    There is convincing evidence that heat-shock proteins (HSP) are upregulated by stress conditions in insects; however, the relative contribution of each HSP gene to the heat-shock response remains unclear. Here we considered the whitefly Bemisia tabaci (MEAM 1), a phloem feeder and invasive species whose molecular stress response is an important mechanism for overcoming heat stress. We assessed the expression of the hsp23, 40, 70 and 90 genes at the mRNA level when submitted to heat shocks of 40 and 44°C/1h (control at 25°C). For this, we evaluated a set of available and suitable reference genes in order to perform data normalization using the real-time polymerase chain reaction (qRT-PCR) technique, and then confirmed the production of HSP70 protein based on Western blot. Results were compared with the hardening capacity of B. tabaci, measured by fitness components as a response to heat shocks, using 40°C as the induction temperature. Three of the four genes (hsp23, 70 and 90) were upregulated by heat stress at mRNA, showing differential expression patterns. Hsp70 expression was confirmed at the protein level. Hardening significantly increased fitness following heat stress, suggesting that HSPs may contribute to hardening capacity in B. tabaci. Potential role of each gene in the heat-shock response for whiteflies is discussed.

  9. The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns.

    PubMed

    Loose, Martin; Mitchison, Timothy J

    2014-01-01

    Bacterial cytokinesis is commonly initiated by the Z-ring, a cytoskeletal structure that assembles at the site of division. Its primary component is FtsZ, a tubulin superfamily GTPase, which is recruited to the membrane by the actin-related protein FtsA. Both proteins are required for the formation of the Z-ring, but if and how they influence each other's assembly dynamics is not known. Here, we reconstituted FtsA-dependent recruitment of FtsZ polymers to supported membranes, where both proteins self-organize into complex patterns, such as fast-moving filament bundles and chirally rotating rings. Using fluorescence microscopy and biochemical perturbations, we found that these large-scale rearrangements of FtsZ emerge from its polymerization dynamics and a dual, antagonistic role of FtsA: recruitment of FtsZ filaments to the membrane and negative regulation of FtsZ organization. Our findings provide a model for the initial steps of bacterial cell division and illustrate how dynamic polymers can self-organize into large-scale structures.

  10. The Drosophila Retinoblastoma Binding Protein 6 Family Member Has Two Isoforms and Is Potentially Involved in Embryonic Patterning

    PubMed Central

    Hull, Rodney; Oosthuysen, Brent; Cajee, Umar-Faruq; Mokgohloa, Lehlogonolo; Nweke, Ekene; Antunes, Ricardo Jorge; Coetzer, Theresa H. T.; Ntwasa, Monde

    2015-01-01

    The human retinoblastoma binding protein 6 (RBBP6) is implicated in esophageal, lung, hepatocellular and colon cancers. Furthermore, RBBP6 was identified as a strong marker for colon cancer prognosis and as a predisposing factor in familial myeloproliferative neoplasms. Functionally, the mammalian protein interacts with p53 and enhances the activity of Mdm2, the prototypical negative regulator of p53. However, since RBBP6 (known as PACT in mice) exists in multiple isoforms and pact−/− mice exhibit a more severe phenotype than mdm2−/− mutants, it must possess some Mdm2-independent functions. The function of the invertebrate homologue is poorly understood. This is complicated by the absence of the Mdm2 gene in both Drosophila and Caenorhabditis elegans. We have experimentally identified the promoter region of Snama, the Drosophila homologue, analyzed potential transcription factor binding sites and confirmed the existence of an additional isoform. Using band shift and co-immunoprecipitation assays combined with mass spectrometry, we found evidence that this gene may be regulated by, amongst others, DREF, which regulates hundreds of genes related to cell proliferation. The potential transcription factors for Snama fall into distinct functional groups, including anteroposterior embryonic patterning and nucleic acid metabolism. Significantly, previous work in mice shows that pact−/− induces an anteroposterior phenotype in embryos when rescued by simultaneous deletion of p53. Taken together, these observations indicate the significance of RBBP6 proteins in carcinogenesis and in developmental defects. PMID:25955646

  11. Just What Does the ACT Assessment and ACT/COMP Measure Anyway? AIR 1992 Annual Forum Paper.

    ERIC Educational Resources Information Center

    Phillippi, Raymond H.

    A study was done of the relationship between the American College Testing (ACT) Assessment and the ACT/COMP (College Outcomes Measures Program) test and general intellectual ability of college students. The subjects for the study were 133 undergraduates, mostly freshmen, in Introductory Psychology at the University of Tennessee, Knoxville. The…

  12. Structure Identification Using High Resolution Mass Spectrometry Data and the EPA’s CompTox Chemistry Dashboard (EAS)

    EPA Science Inventory

    The iCSS CompTox Dashboard is a publicly accessible dashboard provided by the National Center for Computation Toxicology at the US-EPA. It serves a number of purposes, including providing a chemistry database underpinning many of our public-facing projects (e.g. ToxCast and ExpoC...

  13. eComp at the University of New Mexico: Emphasizing Twenty-First Century Literacies in an Online Composition Program

    ERIC Educational Resources Information Center

    Bourelle, Tiffany; Bourelle, Andrew

    2015-01-01

    With distance education on the rise, a new program at the University of New Mexico provides an innovative way to teach first-year composition in a fully online format. The program, called eComp (short for Electronic Composition), insists that instructors receive formal and educational training before working in the model. In addition, the…

  14. Intravitreal AAV2.COMP-Ang1 Prevents Neurovascular Degeneration in a Murine Model of Diabetic Retinopathy

    PubMed Central

    Cahoon, Judd M.; Rai, Ruju R.; Carroll, Lara S.; Uehara, Hironori; Zhang, Xiaohui; Medina, Reinhold J.; Das, Subtrata K.; Muddana, Santosh K.; Olson, Paul R.; Nielson, Spencer; Walker, Kortnie; Flood, Maggie M.; Messenger, Wyatt B.; Archer, Bonnie J.; Barabas, Peter; Krizaj, David; Gibson, Christopher C.; Li, Dean Y.; Koh, Gou Y.; Gao, Guangping; Stitt, Alan W.

    2015-01-01

    Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the U.S. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here, we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable with nondiabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR. PMID:26340930

  15. The SBASE protein domain library, release 2.0: a collection of annotated protein sequence segments.

    PubMed Central

    Pongor, S; Skerl, V; Cserzö, M; Hátsági, Z; Simon, G; Bevilacqua, V

    1993-01-01

    SBASE 2.0 is the second release of SBASE, a collection of annotated protein domain sequences. SBASE entries represent various structural, functional, ligand-binding and topogenic segments of proteins [Pongor, S. et al. (1993) Prot. Eng., in press]. This release contains 34,518 entries provided with standardized names and it is cross-referenced to the major protein and nucleic acid databanks as well as to the PROSITE catalog of protein sequence patterns [Bairoch, A. (1992) Nucl. Acids Res., 20 suppl, 2013-2018]. SBASE can be used for establishing domain homologies using different database-search tools such as FASTA [Lipman and Pearson (1985) Science, 227, 1436-1441], FASTDB [Brutlag et al. (1990) Comp. Appl. Biosci., 6, 237-245] or BLAST3 [Altschul and Lipman (1990) Proc. Natl. Acad. Sci. USA, 87, 5509-5513] which is especially useful in the case of loosely defined domain types for which efficient consensus patterns can not be established. SBASE 2.0 and a set of search and retrieval tools are freely available on request to the authors or by anonymous 'ftp' file transfer from mean value of ftp.icgeb.trieste.it. PMID:8332532

  16. Proteomic analyses of human cytomegalovirus strain AD169 derivatives reveal highly conserved patterns of viral and cellular proteins in infected fibroblasts.

    PubMed

    Reyda, Sabine; Büscher, Nicole; Tenzer, Stefan; Plachter, Bodo

    2014-01-07

    Human cytomegalovirus (HCMV) particle morphogenesis in infected cells is an orchestrated process that eventually results in the release of enveloped virions. Proteomic analysis has been employed to reveal the complexity in the protein composition of these extracellular particles. Only limited information is however available regarding the proteome of infected cells preceding the release of HCMV virions. We used quantitative mass spectrometry to address the pattern of viral and cellular proteins in cells, infected with derivatives of the AD169 laboratory strain. Our analyses revealed a remarkable conservation in the patterns of viral and of abundant cellular proteins in cells, infected for 2 hours, 2 days, or 4 days. Most viral proteins increased in abundance as the infection progressed over time. Of the proteins that were reliably detectable by mass spectrometry, only IE1 (pUL123), pTRS1, and pIRS1 were downregulated at 4 days after infection. In addition, little variation of viral proteins in the virions of the different viruses was detectable, independent of the expression of the major tegument protein pp65. Taken together these data suggest that there is little variation in the expression program of viral and cellular proteins in cells infected with related HCMVs, resulting in a conserved pattern of viral proteins ultimately associated with extracellular virions.

  17. Microscopic pattern of ice crystal growth in the presence of thermal hysteresis proteins

    SciTech Connect

    Coger, R.; Rubinsky, B. . Dept. of Mechanical Engineering); Fletcher, G. )

    1994-08-01

    This study examines the effect of thermal hysteresis proteins (THPs) from the winter flounder (Psuedopleuronectes americanus) on the ice-water interface morphology during freezing of aqueous solutions. Experiments were performed using a directional solidification stage, and the development of the two-phase interface was observed through a microscope and recorded by a video system. Unusual ice crystal morphologies were observed, including faceted ice crystal growth along the (1100) crystal plane; spicular or needlelike growth in the (1010) direction; and growth parallel to the c-axis, (0001), consisting of incorporated liquid inclusions bounded by hexagonal prism faces. The observed crystallographic structures can be explained as an effect of the interaction between the THPs and the primary prism faces of ice crystals. This results in an increase in the Gibbs free energy of these planes, followed by ice growth into the supercooled liquid adjacent to these faces.

  18. Active machine learning-driven experimentation to determine compound effects on protein patterns.

    PubMed

    Naik, Armaghan W; Kangas, Joshua D; Sullivan, Devin P; Murphy, Robert F

    2016-02-03

    High throughput screening determines the effects of many conditions on a given biological target. Currently, to estimate the effects of those conditions on other targets requires either strong modeling assumptions (e.g. similarities among targets) or separate screens. Ideally, data-driven experimentation could be used to learn accurate models for many conditions and targets without doing all possible experiments. We have previously described an active machine learning algorithm that can iteratively choose small sets of experiments to learn models of multiple effects. We now show that, with no prior knowledge and with liquid handling robotics and automated microscopy under its control, this learner accurately learned the effects of 48 chemical compounds on the subcellular localization of 48 proteins while performing only 29% of all possible experiments. The results represent the first practical demonstration of the utility of active learning-driven biological experimentation in which the set of possible phenotypes is unknown in advance.

  19. Neurofilament protein defines regional patterns of cortical organization in the macaque monkey visual system: a quantitative immunohistochemical analysis

    NASA Technical Reports Server (NTRS)

    Hof, P. R.; Morrison, J. H.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    Visual function in monkeys is subserved at the cortical level by a large number of areas defined by their specific physiological properties and connectivity patterns. For most of these cortical fields, a precise index of their degree of anatomical specialization has not yet been defined, although many regional patterns have been described using Nissl or myelin stains. In the present study, an attempt has been made to elucidate the regional characteristics, and to varying degrees boundaries, of several visual cortical areas in the macaque monkey using an antibody to neurofilament protein (SMI32). This antibody labels a subset of pyramidal neurons with highly specific regional and laminar distribution patterns in the cerebral cortex. Based on the staining patterns and regional quantitative analysis, as many as 28 cortical fields were reliably identified. Each field had a homogeneous distribution of labeled neurons, except area V1, where increases in layer IVB cell and in Meynert cell counts paralleled the increase in the degree of eccentricity in the visual field representation. Within the occipitotemporal pathway, areas V3 and V4 and fields in the inferior temporal cortex were characterized by a distinct population of neurofilament-rich neurons in layers II-IIIa, whereas areas located in the parietal cortex and part of the occipitoparietal pathway had a consistent population of large labeled neurons in layer Va. The mediotemporal areas MT and MST displayed a distinct population of densely labeled neurons in layer VI. Quantitative analysis of the laminar distribution of the labeled neurons demonstrated that the visual cortical areas could be grouped in four hierarchical levels based on the ratio of neuron counts between infragranular and supragranular layers, with the first (areas V1, V2, V3, and V3A) and third (temporal and parietal regions) levels characterized by low ratios and the second (areas MT, MST, and V4) and fourth (frontal regions) levels characterized by

  20. G-protein-coupled receptors and localized signaling in the primary cilium during ventral neural tube patterning.

    PubMed

    Hwang, Sun-Hee; Mukhopadhyay, Saikat

    2015-01-01

    The primary cilium is critical in sonic hedgehog (Shh)-dependent ventral patterning of the vertebrate neural tube. Most mutants that cause disruption of the cilium result in decreased Shh signaling in the neural tube. In contrast, mutations in the intraflagellar complex A (IFT-A) and the tubby family protein, Tulp3, result in increased Shh signaling in the neural tube. Proteomic analysis of Tulp3-binding proteins first pointed to the role of the IFT-A complex in trafficking Tulp3 into the cilia. Tulp3 directs trafficking of rhodopsin family G-protein-coupled receptors (GPCRs) to the cilia, suggesting the role of a GPCR in mediating the paradoxical effects of the Tulp3/IFT-A complex in causing increased Shh signaling. Gpr161 has recently been identified as a Tulp3/IFT-A-regulated GPCR that localizes to the primary cilium. A null knock-out mouse model of Gpr161 phenocopies Tulp3 and IFT-A mutants, and causes increased Shh signaling throughout the neural tube. In the absence of Shh, the bifunctional Gli transcription factors are proteolytically processed into repressor forms in a protein kinase A (PKA) -dependent and cilium-dependent manner. Gpr161 activity results in increased cAMP levels in a Gαs -coupled manner, and determines processing of Gli3. Shh signaling also results in removal of Gpr161 from the cilia, suggesting that Gpr161 functions in a positive feedback loop in the Shh pathway. As PKA-null and Gαs mutant embryos also exhibit increased Shh signaling in the neural tube, Gpr161 is a strong candidate for a GPCR that regulates ciliary cAMP levels, and activates PKA in close proximity to the cilia.

  1. Homology modeling, molecular dynamics, and docking studies of pattern-recognition transmembrane protein-lipopolysaccharide and β-1,3 glucan-binding protein from Fenneropenaeus indicus.

    PubMed

    Sivakamavalli, Jeyachandran; Tripathi, Sunil Kumar; Singh, Sanjeev Kumar; Vaseeharan, Baskaralingam

    2015-01-01

    Lipopolysaccharide and β-1,3 glucan-binding protein (LGBP) is a family of pattern-recognition transmembrane proteins (PRPs) which plays a vital role in the immune mechanism of crustaceans in adverse conditions. Fenneropenaeus indicus LGBP-deduced amino acid has conserved potential recognition motif for β-1,3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp) cell adhesion sites for the activation of innate defense mechanism. In order to understand the stimulating activity of β-1,3 glucan (β-glucan) and its interaction with LGBP, a 3D model of LGBP is generated. Molecular docking is performed with this model, and the results indicate Arg71 with strong hydrogen bond from RGD domain of LGBP. Moreover, from the docking studies, we also suggest that Arg34, Lys68, Val135, and Ala146 in LGBP are important amino acid residues in binding as they have strong bonding interaction in the active site of LGBP. In our in vitro studies, yeast agglutination results suggest that shrimp F. indicus LGBP possesses sugar binding and recognition sites in its structure, which is responsible for agglutination reaction. Our results were synchronized with the already reported evidence both in vivo and in vitro experiments. This investigation may be valuable for further experimental investigation in the synthesis of novel immunomodulator.

  2. cDNA cloning of heat shock protein 90 gene and protein expression pattern in response to heavy metal exposure and thermal stress in planarian Dugesia japonica.

    PubMed

    Ma, Ke-Xue; Chen, Guang-Wen; Liu, De-Zeng

    2012-06-01

    Heat shock protein 90 (HSP90) is an abundant and highly conserved molecular chaperone, playing important roles in multiple cellular stress responses. The full-length cDNA of planarian Dugesia japonica Hsp90 (designated DjHsp90) was firstly cloned using rapid amplification of cDNA ends (RACE) techniques. It is 2,354 bp, including an open reading frame (ORF) of 2,148 bp encoding a polypeptide of 715 amino acids with all five HSP90 family signatures. We sequenced the ORF sequences from genomic DNA, and found only one intron (48 bp) existed in Djhsp90 gene structure. We used western blot and immunohistochemistry to analyze the expression pattern of DjHsp90 in response to heavy metal exposure and thermal stress at the protein level. Our results show that low doses of heavy metals and elevated culture temperature induced, but high doses of heavy metals and severe heat shock inhibited DjHsp90 expression. In response to heavy metals and thermal stress, DjHsp90-positive cells only appeared in the parenchymal tissue under epidermis cells along the bilateral from head to tail. These positive cells are presumably sensor cells that can detect external environment changes. Our work provides basic data for the study of stress responses in planarians.

  3. Directed immobilization of protein-coated nanospheres to nanometer-scale patterns fabricated by electron beam lithography of poly(ethylene glycol) self-assembled monolayers.

    PubMed

    Rundqvist, Jonas; Hoh, Jan H; Haviland, David B

    2006-05-23

    Controlling the spatial organization of biomolecules on solid supports with high resolution is important for a wide range of scientific and technological problems. Here we report a study of electron beam lithography (EBL) patterning of a self-assembled monolayer (SAM) of the amide-containing poly(ethylene glycol) (PEG) thiol CH(3)O(CH(2)CH(2)O)(17)NHCO(CH(2))(2)SH on Au and demonstrate the patterning of biomolecular features with dimensions approaching 40 nm. The electron dose dependence of feature size and pattern resolution is studied in detail by atomic force microscopy (AFM), which reveals two distinct patterning mechanisms. At low doses, the pattern formation occurs by SAM ablation in a self-developing process where the feature size is directly dose-dependent. At higher doses, electron beam-induced deposition of material, so-called contamination writing, is seen in the ablated areas of the SAM. The balance between these two mechanisms is shown to depend on the geometry of the pattern. The patterned SAMs were backfilled with fluorescent 40-nm spheres coated with NeutrAvidin. These protein-coated spheres adhered to exposed areas in the SAM with high selectivity. This direct writing approach for patterning bioactive surfaces is a fast and efficient way to produce patterns with a resolution approaching that of single proteins.

  4. Protein kinase C acts as a molecular detector of firing patterns to mediate sensory gating in Aplysia.

    PubMed

    Wan, Qin; Jiang, Xue-Ying; Negroiu, Andreea M; Lu, Shao-Gang; McKay, Kimberly S; Abrams, Thomas W

    2012-07-08

    Habituation of a behavioral response to a repetitive stimulus enables animals to ignore irrelevant stimuli and focus on behaviorally important events. In Aplysia, habituation is mediated by rapid depression of sensory synapses, which could leave an animal unresponsive to important repetitive stimuli, making it vulnerable to injury. We identified a form of plasticity that prevents synaptic depression depending on the precise stimulus strength. Burst-dependent protection from depression is initiated by trains of 2-4 action potentials and is distinct from previously described forms of synaptic enhancement. The blockade of depression is mediated by presynaptic Ca2+ influx and protein kinase C (PKC) and requires localization of PKC via a PDZ domain interaction with Aplysia PICK1. During protection from depression, PKC acts as a highly sensitive detector of the precise pattern of sensory neuron firing. Behaviorally, burst-dependent protection reduces habituation, enabling animals to maintain responsiveness to stimuli that are functionally important.

  5. Photo-patterned free-standing hydrogel microarrays for massively parallel protein analysis

    NASA Astrophysics Data System (ADS)

    Duncombe, Todd A.; Herr, Amy E.

    2015-03-01

    Microfluidic technologies have largely been realized within enclosed microchannels. While powerful, a principle limitation of closed-channel microfluidics is the difficulty for sample extraction and downstream processing. To address this limitation and expand the utility of microfluidic analytical separation tools, we developed an openchannel hydrogel architecture for rapid protein analysis. Designed for compatibility with slab-gel polyacrylamide gel electrophoresis (PAGE) reagents and instruments, we detail the development of free-standing polyacrylamide gel (fsPAG) microstructures supporting electrophoretic performance rivalling that of microfluidic platforms. Owing to its open architecture - the platform can be easily interfaced with automated robotic controllers and downstream processing (e.g., sample spotters, immunological probing, mass spectroscopy). The fsPAG devices are directly photopatterened atop of and covalently attached to planar polymer or glass surfaces. Due to the fast < 1 hr design-prototype-test cycle - significantly faster than mold based fabrication techniques - rapid prototyping devices with fsPAG microstructures provides researchers a powerful tool for developing custom analytical assays. Leveraging the rapid prototyping benefits - we up-scale from a unit separation to an array of 96 concurrent fsPAGE assays in 10 min run time driven by one electrode pair. The fsPAGE platform is uniquely well-suited for massively parallelized proteomics, a major unrealized goal from bioanalytical technology.

  6. Multiple haplotype-resolved genomes reveal population patterns of gene and protein diplotypes.

    PubMed

    Hoehe, Margret R; Church, George M; Lehrach, Hans; Kroslak, Thomas; Palczewski, Stefanie; Nowick, Katja; Schulz, Sabrina; Suk, Eun-Kyung; Huebsch, Thomas

    2014-11-26

    To fully understand human biology and link genotype to phenotype, the phase of DNA variants must be known. Here we present a comprehensive analysis of haplotype-resolved genomes to assess the nature and variation of haplotypes and their pairs, diplotypes, in European population samples. We use a set of 14 haplotype-resolved genomes generated by fosmid clone-based sequencing, complemented and expanded by up to 372 statistically resolved genomes from the 1000 Genomes Project. We find immense diversity of both haploid and diploid gene forms, up to 4.1 and 3.9 million corresponding to 249 and 235 per gene on average. Less than 15% of autosomal genes have a predominant form. We describe a 'common diplotypic proteome', a set of 4,269 genes encoding two different proteins in over 30% of genomes. We show moreover an abundance of cis configurations of mutations in the 386 genomes with an average cis/trans ratio of 60:40, and distinguishable classes of cis- versus trans-abundant genes. This work identifies key features characterizing the diplotypic nature of human genomes and provides a conceptual and analytical framework, rich resources and novel hypotheses on the functional importance of diploidy.

  7. Tunable two dimensional protein patterns through self-assembly nanosphere template

    NASA Astrophysics Data System (ADS)

    Li, Zhishi; Ruan, Weidong; Shen, Shanshan; Wang, Haiyang; Guo, Zhinan; Xue, Xiangxin; Mao, Zhu; Ji, Wei; Wang, Xu; Song, Wei; Zhao, Bing

    2012-10-01

    By the aim of constructing surfaces for multi-component and multifunctional bioassay, a microsphere lithography technique was employed to control the surface morphology. Two kinds of protein molecules (antibodies) were used as building blocks. As a result, dual-component biocompatible surfaces with alternate immunoglobulin micropatterns were fabricated. The employed antibodies included human Immunoglobulin G (IgG) and rabbit IgG, which composed nanometer scale surface arrays on the surfaces. The antibodies were identified specially by immunoreactions with labeled antigens of fluorescein isothiocyanate (FITC)-antihuman IgG and tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC)-antirabbit IgG. The immune responses were confirmed by confocal fluorescence (FL) microscopy. A study on the sensitivity and quantification was done by using surface-enhanced resonance Raman scattering (SERRS) spectroscopy. The obtained SERRS spectra showed satisfactory resolution in the multi-component detection objects. No interference was observed from inner- or interactions of detecting molecules. The detection limits for both of the antigens reached to as low as 1 ng/mL, which was comparable to FL method. Meanwhile, a good linear relationship between SERRS peak intensity and the logarithm of antigens' concentrations (from 1 ng/mL to 1 mg/mL) were observed. The results demonstrated that SERRS is a very promising detection technique for multi-component immunoassay, and has great potential applications in biotechnology and biochemistry.

  8. Specificity patterns indicate that auxin exporters and receptors are the same proteins.

    PubMed

    Hössel, D; Schmeiser, C; Hertel, R

    2005-01-01

    A study of transport and action of synthetic auxin analogues can help to identify transporters and receptors of this plant hormone. Both aspects--transportability and action on growth--were tested with 2-naphthoxyacetic acid (2-NOA) and compared across several plant species. 2-NOA stimulates elongation effectively at low concentrations in petioles of the gymnosperm Ginkgo biloba L., in hypocotyls or internodes of the dicot legumes, mung bean (Vigna mungo L.) and pea (Pisum sativum L.), in cotyledons of onion (Allium cepa L.) and in leaf bases of chive (Allium schoenoprasum L.), the latter two of the monocot order Asparagales. In contrast, elongation of coleoptile segments of maize (Zea mays L.) is poorly responsive to 2-NOA. Significant auxin-like transport of 2-NOA was observed in segments of mung bean hypocotyls, pea internodes, and chive leaf bases, but not in segments of the grass coleoptiles. Thus, for the two assays, elongation and polar transportability, the same difference in ligand specificity was observed between the grass and all other species assayed. This finding supports the hypothesis that a common protein mediates auxin efflux as well as auxin action on elongation.

  9. Patterns of Nucleotide Substitution in Mitochondrial Protein Coding Genes of Vertebrates

    PubMed Central

    Kumar, S.

    1996-01-01

    Maximum likelihood methods were used to study the differences in substitution rates among the four nucleotides and among different nucleotide sites in mitochondrial protein-coding genes of vertebrates. In the 1st+2nd codon position data, the frequency of nucleotide G is negatively correlated with evolutionary rates of genes, substitution rates vary substantially among sites, and the transition/transversion rate bias (R) is two to five times larger than that expected at random. Generally, largest transition biases and greatest differences in substitution rates among sites are found in the highly conserved genes. The 3rd positions in placental mammal genes exhibit strong nucleotide composition biases and the transitional rates exceed transversional rates by one to two orders of magnitude. Tamura-Nei and Hasegawa-Kishino-Yano models with gamma distributed variable rates among sites (gamma parameter, α) adequately describe the nucleotide substitution process in 1st+2nd position data. In these data, ignoring differences in substitution rates among sites leads to largest biases while estimating substitution rates. Kimura's two-parameter model with variable-rates among sites performs satisfactorily in likelihood estimation of R, α, and overall amount of evolution for 1st+2nd position data. It can also be used to estimate pairwise distances with appropriate values of α for a majority of genes. PMID:8722802

  10. Active machine learning-driven experimentation to determine compound effects on protein patterns

    PubMed Central

    Naik, Armaghan W; Kangas, Joshua D; Sullivan, Devin P; Murphy, Robert F

    2016-01-01

    High throughput screening determines the effects of many conditions on a given biological target. Currently, to estimate the effects of those conditions on other targets requires either strong modeling assumptions (e.g. similarities among targets) or separate screens. Ideally, data-driven experimentation could be used to learn accurate models for many conditions and targets without doing all possible experiments. We have previously described an active machine learning algorithm that can iteratively choose small sets of experiments to learn models of multiple effects. We now show that, with no prior knowledge and with liquid handling robotics and automated microscopy under its control, this learner accurately learned the effects of 48 chemical compounds on the subcellular localization of 48 proteins while performing only 29% of all possible experiments. The results represent the first practical demonstration of the utility of active learning-driven biological experimentation in which the set of possible phenotypes is unknown in advance. DOI: http://dx.doi.org/10.7554/eLife.10047.001 PMID:26840049

  11. Sex-specific basal and hypoglycemic patterns of in vivo caudal dorsal vagal complex astrocyte glycogen metabolic enzyme protein expression.

    PubMed

    Tamrakar, Pratistha; Shrestha, Prem; Briski, Karen P

    2014-10-24

    Astrocytes contribute to neurometabolic stability through uptake, catabolism, and storage of glucose. These cells maintain the major brain glycogen reservoir, which is a critical fuel supply to neurons during glucose deficiency and increased brain activity. We used a combinatory approach incorporating immunocytochemistry, laser microdissection, and Western blotting to investigate the hypothesis of divergent expression of key enzymes regulating glycogen metabolism and glycolysis during in vivo normo- and/or hypoglycemia in male versus female hindbrain astrocytes. Glycogen synthase (GS) and glycogen phosphorylase (GP) levels were both enhanced in dorsal vagal complex astrocytes from vehicle-injected female versus male controls, with incremental increase in GS exceeding GP. Insulin-induced hypoglycemia (IIH) diminished GS and increased glycogen synthase kinase-3-beta (GSK3β) expression in both sexes, but decreased phosphoprotein phosphatase-1 (PP1) levels only in males. Astrocyte GP content was elevated by IIH in male, but not female rats. Data reveal sex-dependent sensitivity of these enzyme proteins to lactate as caudal hindbrain repletion of this energy substrate fully or incompletely reversed hypoglycemic inhibition of GS and prevented hypoglycemic augmentation of GSK3β and GP in females and males, respectively. Sex dimorphic patterns of glycogen branching and debranching enzyme protein expression were also observed. Levels of the rate-limiting glycolytic enzyme, phosphofructokinase, were unaffected by IIH with or without lactate repletion. Current data demonstrating sex-dependent basal and hypoglycemic patterns of hindbrain astrocyte glycogen metabolic enzyme expression imply that glycogen volume and turnover during glucose sufficiency and shortage may vary accordingly.

  12. Gene expression patterns in the black blowfly (Phormia regina) as revealed by two-dimensional electrophoresis of proteins. I. Developmental stage-specific and sex-specific differences

    SciTech Connect

    Harrison, H.H. ); Joslyn, D.J. )

    1991-12-01

    The black blowfly, Phormia regina, has been implicated in human myiasis and as a contact vector of viral and bacterial diseases present in carrion to which female flies are attracted for egg deposition. Inbred strains of P. regina are an excellent model system for studying gene expression in the developmental stages of such holometabolous dipteran parasites. However, information regarding gene and protein expression patterns in regina is limited. The authors used ISO-DALT high-resolution, two-dimensional electrophoresis with solver staining to establish fundamental protein maps for examination of the stage-specific gene expression patterns in the 615 most abundant proteins of the eggs, first- and third-instar larvae, pupae, and male and female adults. They also used a differential extraction technique to identify the major cuticular proteins of the adults. The results show 48 clearly identifiable stage-specific and sex-specific proteins.

  13. Quantitative expression patterns of peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) protein in mice

    SciTech Connect

    Girroir, Elizabeth E.; Hollingshead, Holly E.; He Pengfei; Zhu Bokai; Perdew, Gary H.; Peters, Jeffrey M.

    2008-07-04

    The expression patterns of PPAR{beta}/{delta} have been described, but the majority of these data are based on mRNA data. To date, there are no reports that have quantitatively examined the expression of PPAR{beta}/{delta} protein in mouse tissues. In the present study, a highly specific PPAR{beta}/{delta} antibody was developed, characterized, and used to examine tissue expression patterns of PPAR{beta}/{delta}. As compared to commercially available anti-PPAR{beta}/{delta} antibodies, one of six polyclonal anti-PPAR{beta}/{delta} antibodies developed was significantly more effective for immunoprecipitation of in vitro-translated PPAR{beta}/{delta}. This antibody was used for quantitative Western blot analysis using radioactive detection methods. Expression of PPAR{beta}/{delta} was highest in colon, small intestine, liver, and keratinocytes as compared to other tissues including heart, spleen, skeletal muscle, lung, brain, and thymus. Interestingly, PPAR{beta}/{delta} expression was localized in the nucleus and RXR{alpha} can be co-immunoprecipitated with nuclear PPAR{beta}/{delta}. Results from these studies demonstrate that PPAR{beta}/{delta} expression is highest in intestinal epithelium, liver, and keratinocytes, consistent with significant biological roles in these tissues.

  14. Intraflagellar Transport Protein 172 is essential for primary cilia formation and plays a vital role in patterning the mammalian brain

    PubMed Central

    Gorivodsky, Marat; Mukhopadhyay, Mahua; Wilsch-Braeuninger, Michaela; Phillips, Matthew; Teufel, Andreas; Kim, Changmee; Malik, Nasir; Huttner, Wieland; Westphal, Heiner

    2008-01-01

    IFT172, also known as Selective Lim-domain Binding protein (SLB), is a component of the Intraflagellar Transport (IFT) complex. In order to evaluate the biological role of the Ift172 gene, we generated a loss-of-function mutation in the mouse. The resulting Slb mutant embryos die between E12.5–13.0, and exhibit severe cranio-facial malformations, failure to close the cranial neural tube, holoprosencephaly, heart edema and extensive hemorrhages. Cilia outgrowth in cells of the neuroepithelium is initiated but the axonemes are severely truncated and do not contain visible microtubules. Morphological and molecular analyses revealed a global brain-patterning defect along the dorsal-ventral (DV) and anterior-posterior (AP) axes. We demonstrate that Ift172 gene function is required for early regulation of Fgf8 at the midbrain-hindbrain boundary and maintenance of the isthmic organizer. In addition, Ift172 is required for proper function of the embryonic node, the early embryonic organizer and for formation of the head organizing center (the anterior mesendoderm, or AME). We propose a model suggesting that forebrain and mid-hindbrain growth and AP patterning depends on the early function of Ift172 at gastrulation. Our data suggest that the formation and function of the node and AME in the mouse embryo relies on an indispensable role of Ift172 in cilia morphogenesis and cilia-mediated signaling. PMID:18930042

  15. Growth pattern switch of renal cells and expression of cell cycle related proteins at the early stage of diabetic nephropathy

    SciTech Connect

    Zhang Yanling; Shi Yonghong; Liu Yaling; Dong Hui; Liu, Maodong; Li Ying; Duan Huijun

    2007-11-09

    Renal hypertrophy, partly due to cell proliferation and hypertrophy, has been found correlated to renal function deterioration in diabetes mellitus. We screened the up-regulated cell cycle related genes to investigate cell growth and the expression of cell cycle regulating proteins at the early stage of diabetic nephropathy using STZ-induced diabetic rats. Cyclin E, CDK{sub 2} and P{sup 27} were found significantly up-regulated in diabetic kidney. Increased cell proliferation in the kidney was seen at day 3, peaked at day 5, and returned to normal level at day 30. Cyclin E and CDK{sub 2} expression also peeked at day 5 and P{sup 27} activity peaked at day 14. These findings indicate that a hyperplastic growth period of renal cells is followed by a hypertrophic growth period at the early stage of diabetes. The growth pattern switch may be regulated by cell cycle regulating proteins, Cyclin E, CDK{sub 2}, and P{sup 27}.

  16. Uncoupling Protein 2 and 4 Expression Pattern during Stem Cell Differentiation Provides New Insight into Their Putative Function

    PubMed Central

    Rupprecht, Anne; Sittner, Dana; Smorodchenko, Alina; Hilse, Karolina E.; Goyn, Justus; Moldzio, Rudolf; Seiler, Andrea E. M.; Bräuer, Anja U.; Pohl, Elena E.

    2014-01-01

    Apart from the first family member, uncoupling protein 1 (UCP1), the functions of other UCPs (UCP2-UCP5) are still unknown. In analyzing our own results and those previously published by others, we have assumed that UCP's cellular expression pattern coincides with a specific cell metabolism and changes if the latter is altered. To verify this hypothesis, we analyzed the expression of UCP1-5 in mouse embryonic stem cells before and after their differentiation to neurons. We have shown that only UCP2 is present in undifferentiated stem cells and it disappears simultaneously with the initiation of neuronal differentiation. In contrast, UCP4 is simultaneously up-regulated together with typical neuronal marker proteins TUJ-1 and NeuN during mESC differentiation in vitro as well as during murine brain development in vivo. Notably, several tested cell lines express UCP2, but not UCP4. In line with this finding, neuroblastoma cells that display metabolic features of tumor cells express UCP2, but not UCP4. UCP2's occurrence in cancer, immunological and stem cells indicates that UCP2 is present in cells with highly proliferative potential, which have a glycolytic type of metabolism as a common feature, whereas UCP4 is strongly associated with non-proliferative highly differentiated neuronal cells. PMID:24523901

  17. Divide and Conquer Approach to Contact Map Overlap Problem Using 2D-Pattern Mining of Protein Contact Networks.

    PubMed

    Koneru, Suvarna Vani; Bhavani, Durga S

    2015-01-01

    A novel approach to Contact Map Overlap (CMO) problem is proposed using the two dimensional clusters present in the contact maps. Each protein is represented as a set of the non-trivial clusters of contacts extracted from its contact map. The approach involves finding matching regions between the two contact maps using approximate 2D-pattern matching algorithm and dynamic programming technique. These matched pairs of small contact maps are submitted in parallel to a fast heuristic CMO algorithm. The approach facilitates parallelization at this level since all the pairs of contact maps can be submitted to the algorithm in parallel. Then, a merge algorithm is used in order to obtain the overall alignment. As a proof of concept, MSVNS, a heuristic CMO algorithm is used for global as well as local alignment. The divide and conquer approach is evaluated for two benchmark data sets that of Skolnick and Ding et al. It is interesting to note that along with achieving saving of time, better overlap is also obtained for certain protein folds.

  18. [Cloning and expression pattern of a zinc finger protein gene ShSAP1 in Saccharum officinarum].

    PubMed

    Li, Xiaojun; Cai, Wenwei; Zhang, Shuzhen; Xu, Liping; Chen, Ping; Wang, Jungang

    2011-06-01

    In plants, proteins with A20/AN1 zinc finger domain are involved in stress responses, named as "Stress Associated Protein" (SAP) gene family. Based on Expressed Sequence Tag (EST) sequences information in Badila Saccharum officinarum mature related cDNA library, we cloned an SAP gene from sugarcane full length cDNA library, named ShSAP1 (GenBank: Accession No. HM991960). To characterize ShSAP1, we analyzed its genome structure and expression pattern. Southern blot analysis showed ShSAP1 was present as one or two copy in the genome of Badila. Comparison of ShSAP1 1 008 bp full length cDNA with a genomic frangment (2 241 bp) generated by PCR amplification and sequencing, revealed the presence of two introns (202 bp and 1 052 bp) located in the 5'UTR region. Semiquantitative RT-PCR analysis found ShSAP1 expressed in leaves, roots and stalk in mature sugarcane. Compared with immature stems, ShSAP1 expressed higher in mature stalk. ShSAP1 was induced by different types of treatments, such as salt (200 mmol/L NaCl), drought (10% PEG 6 000), GA3 (200 mg/L), ABA (100 micromol/L) and ET (1 mmol/L) during sugarcane seedling stage. These results indicated that ShSAP1 may function in sugarcane maturation and abiotic stress response processes.

  19. PROCOV: maximum likelihood estimation of protein phylogeny under covarion models and site-specific covarion pattern analysis

    PubMed Central

    Wang, Huai-Chun; Susko, Edward; Roger, Andrew J

    2009-01-01

    Background The covarion hypothesis of molecular evolution holds that selective pressures on a given amino acid or nucleotide site are dependent on the identity of other sites in the molecule that change throughout time, resulting in changes of evolutionary rates of sites along the branches of a phylogenetic tree. At the sequence level, covarion-like evolution at a site manifests as conservation of nucleotide or amino acid states among some homologs where the states are not conserved in other homologs (or groups of homologs). Covarion-like evolution has been shown to relate to changes in functions at sites in different clades, and, if ignored, can adversely affect the accuracy of phylogenetic inference. Results PROCOV (protein covarion analysis) is a software tool that implements a number of previously proposed covarion models of protein evolution for phylogenetic inference in a maximum likelihood framework. Several algorithmic and implementation improvements in this tool over previous versions make computationally expensive tree searches with covarion models more efficient and analyses of large phylogenomic data sets tractable. PROCOV can be used to identify covarion sites by comparing the site likelihoods under the covarion process to the corresponding site likelihoods under a rates-across-sites (RAS) process. Those sites with the greatest log-likelihood difference between a 'covarion' and an RAS process were found to be of functional or structural significance in a dataset of bacterial and eukaryotic elongation factors. Conclusion Covarion models implemented in PROCOV may be especially useful for phylogenetic estimation when ancient divergences between sequences have occurred and rates of evolution at sites are likely to have changed over the tree. It can also be used to study lineage-specific functional shifts in protein families that result in changes in the patterns of site variability among subtrees. PMID:19737395

  20. Effects of dietary protein and fermentable fiber on nitrogen excretion patterns and plasma urea in grower pigs.

    PubMed

    Zervas, S; Zijlstra, R T

    2002-12-01

    Effects of dietary protein concentration (high, 18.5; low, 15.7%) and fermentable fiber (control; soyhulls, SH; and sugar beet pulp, SBP) on N excretion patterns and plasma urea were tested in a 2 x 3 factorial arrangement. The objectives were: 1) to determine if reduced dietary protein together with fermentable fiber would reduce urinary N excretion further than a single diet manipulation, 2) to determine if effects of diet manipulations were similar between pigs with restricted and free access of feed, and 3) to further develop predictions of urinary N excretion using plasma urea. Diets were formulated to 3.30 Mcal digestible energy (DE)/kg and 2.4 g of digestible lysine per Mcal DE, and supplemented with lysine, methionine, tryptophan, threonine, isoleucine, leucine, or valine to ensure meeting an ideal AA profile. Pigs (30.5 +/- 3 kg; n = 36) were housed in metabolism crates with restricted access to feed (3 x 110 kcal DE/kg BW(0.75)) from d 1 to 18, and free access from d 19 to 26. Feces and urine were collected from d 15 to 18 and d 23 to 26, and blood was sampled on d 17 and 25. With restricted access to feed, urinary N was reduced 28% and N retention was reduced 12% for the low- compared to high-protein diet (P < 0.01; as g/d). Fecal N was increased 4% units for SH and 6.5% units for SBP (P < 0.01; as % of N intake) and urinary N was reduced 5% units for SH (P < 0.10) and 9% units for SBP (P < 0.05) compared to the control. With free access to feed, urinary N was reduced 27% (P < 0.05; as g/d) and N retention was reduced 7% (P < 0.10) for the low- compared to high-protein diet. Fecal N was increased 5% units for SH and 9% units for SBP (P < 0.001; as % of N intake), and urinary N was reduced 9% units for SH and 10% units for SBP (P < 0.01) compared to the control. For either restricted or free access to feed, fermentable fiber did not affect N retention (P > 0.10). A protein x fiber interaction was not observed for urinary N excretion (P > 0.10), indicating

  1. Effects of monocular deprivation on the spatial pattern of visually induced expression of c-Fos protein.

    PubMed

    Nakadate, K; Imamura, K; Watanabe, Y

    2012-01-27

    We studied the pattern of expression of a protein product (c-Fos) of immediate-early gene (IEG) in the visual cortex of rats and mice. The basal expression of c-Fos was very low and visual exposure revealed a large number of c-Fos immunopositive cells in the visual cortex. We found that monocular deprivation during the sensitive period of ocular dominance (OD) plasticity significantly changed both the amount and pattern of c-Fos expression upon monocular stimulation of either eye. The number of immunopositive cells in layer IV of binocular subfields of the primary visual cortex (Oc1B) ipsilateral to the stimulated eye was found to be the most sensitive index of the effects of monocular deprivation during the sensitive period, that is, opened eye stimulation induced significantly larger numbers of c-Fos immunopositive cells, whereas closed eye stimulation induced significantly smaller numbers compared with those induced by monocular stimulation in control animals. In the lateral geniculate nucleus and superior colliculus, the pattern of expression of c-Fos following monocular stimulation was not affected by preceding monocular deprivation. Monocular deprivation imposed after the sensitive period did not affect the pattern of induction of c-Fos. Notably, in age-matched old animals that had been raised in total darkness and then experienced monocular deprivation, the distribution and numbers of c-Fos-expressing cells in visual cortex exhibited the same alterations as found in young animals during the sensitive period. These findings suggest that the present activity mapping method using c-Fos as a molecular marker is useful for examining the activity-dependent regulation of cortical plasticity, and provides an alternative method to conventional electrophysiological recording. This method is particularly powerful when applied to knockout or transgenic mice in which sampling biases in electrophysiological recording have been considered inevitable. Furthermore, these

  2. Nutritional Status and Daytime Pattern of Protein Intake on Match, Post-Match, Rest and Training Days in Senior Professional and Youth Elite Soccer Players.

    PubMed

    Bettonviel A, E O; Brinkmans N, Y J; Russcher, Kris; Wardenaar, Floris C; Witard, Oliver C

    2016-06-01

    The nutritional status of elite soccer players across match, postmatch, training and rest days has not been defined. Recent evidence suggests the pattern of dietary protein intake impacts the daytime turnover of muscle proteins and, as such, influences muscle recovery. We assessed the nutritional status and daytime pattern of protein intake in senior professional and elite youth soccer players and compared findings against published recommendations. Fourteen senior professional (SP) and 15 youth elite (YP) soccer players from the Dutch premier division completed nutritional assessments using a 24-hr web-based recall method. Recall days consisted of a match, postmatch, rest, and training day. Daily energy intake over the 4-day period was similar between SP (2988 ± 583 kcal/day) and YP (2938 ± 465 kcal/day; p = .800). Carbohydrate intake over the combined 4-day period was lower in SP (4.7 ± 0.7 g·kg-1 BM·day-1) vs. YP (6.0 ± 1.5 g·kg-1 BM·day-1, p = .006) and SP failed to meet recommended carbohydrate intakes on match and training days. Conversely, recommended protein intakes were met for SP (1.9 ± 0.3 g·kg-1 BM·day-1) and YP (1.7 ± 0.4 g·kg-1 BM·day-1), with no differences between groups (p = .286). Accordingly, both groups met or exceeded recommended daily protein intakes on individual match, postmatch, rest and training days. A similar "balanced" daytime pattern of protein intake was observed in SP and YP. To conclude, SP increased protein intake on match and training days to a greater extent than YP, however at the expense of carbohydrate intake. The daytime distribution of protein intake for YP and SP aligned with current recommendations of a balanced protein meal pattern.

  3. Developmental pattern of the neuronal intermediate filament inaa in the zebrafish retina.

    PubMed

    Liao, Meng-Lin; Peng, Wei-Hau; Kan, Daphne; Chien, Chung-Liang

    2016-12-15

    α-Internexin is a member of the neuronal intermediate filament (nIF) protein family, which also includes peripherin and neurofilament (NF) triplet proteins. Previous studies found that expression of α-internexin precedes that of the NF triplet proteins in mammals and suggested that α-internexin plays a key role in the neuronal cytoskeleton network during development. In this study, we aimed to analyze the expression patterns and function of internexin neuronal intermediate filament protein-alpha a (inaa), the encoding gene of which is a homolog of the mammalian α-internexin, during retinal development in zebrafish. Via in vitro and in vivo studies, we demonstrated that zebrafish inaa is an α-internexin homolog that shares characteristics with nIFs. An immunohistochemical analysis of zebrafish revealed that inaa was distributed dynamically in the developing retina. It was widely localized in retinal neuroepithelial cells at 1 day postfertilization (dpf), and was mainly found in the ganglion cell layer (GCL) and inner part of the inner nuclear layer (INL) from 3-9 dpf; after 14 dpf, it was restricted to the outer nuclear layer (ONL). Moreover, we demonstrated for the first time that inaa acted distinctively from the cytoskeletal scaffold of zebrafish cone photoreceptors during development. In conclusion, we demonstrated the morphological features of a novel nIF, inaa, and illustrated its developmental expression pattern in the zebrafish retina. J. Comp. Neurol. 524:3810-3826, 2016. © 2016 Wiley Periodicals, Inc.

  4. Ambulation speed and corresponding mechanics are associated with changes in serum cartilage oligomeric matrix protein.

    PubMed

    Denning, W Matt; Becker Pardo, Michael; Winward, Jason G; Hunter, Iain; Ridge, Sarah; Hopkins, J Ty; Reese, C Shane; Parcell, Allen C; Seeley, Matthew K

    2016-02-01

    Because serum cartilage oligomeric matrix protein (COMP) has been used to reflect articular cartilage condition, we aimed to identify walking and running mechanics that are associated with changes in serum COMP. Eighteen subjects (9 male, 9 female; age=23 ± 2 yrs.; mass=68.3 ± 9.6 kg; height=1.70 ± 0.08 m) completed 4000 steps on an instrumented treadmill on three separate days. Each day corresponded to a different ambulation speed: slow (preferred walking speed), medium (+50% of slow), and fast (+100% of slow). Synchronized ground reaction force and video data were collected to evaluate walking mechanics. Blood samples were collected pre-, post-, 30-minute post-, and 60-minute post-ambulation to determine serum COMP concentration at these times. Serum COMP increased 29%, 18%, and 5% immediately post ambulation for the fast, medium, and slow sessions (p<0.01). When the speeds were pooled, peak ankle inversion, knee extension, knee abduction, hip flexion, hip extension, and hip abduction moment, and knee flexion angle at impact explained 61.4% of total variance in COMP concentration change (p<0.001). These results indicate that (1) certain joint mechanics are associated with acute change in serum COMP due to ambulation, and (2) increased ambulation speed increases serum COMP concentration.

  5. Rapid diagnosis of acute promyelocytic leukemia by analyzing the immunocytochemical pattern of the PML protein with the monoclonal antibody PG-M3.

    PubMed

    Villamor, N; Costa, D; Aymerich, M; Esteve, J; Carrió, A; Rozman, M; Aguilar, J L; Falini, B; Montserrat, E; Campo, E; Colomer, D

    2000-11-01

    The fusion protein, promyelocytic leukemia-retinoic acid receptor (PML-RAR)alpha, generated by the t(15;17) translocation has an abnormal cellular distribution with colocalization of RARalpha and PML proteins. We analyzed the immunostaining pattern of PML protein using the PG-M3 monoclonal antibody directed against the amino terminal portion of PML (retained in wild-type PML and PML-RARalpha fusion protein) in the diagnosis of acute promyelocytic leukemia (APL). In addition, we compared this test with other methods for detecting the PML-RARalpha fusion gene. A normal immunostaining pattern was observed in nonmyeloid disorders and in 78 of 111 acute myeloid leukemias (AMLs). A microgranular pattern was observed in 25 AMLs, all corresponding to APL. These results were concordant with the reverse transcriptase-polymerase chain reaction results for PML-RARalpha fusion gene. Only 1 case positive for the PML-RARalpha transcript showed a normal protein pattern by immunocytochemistry. PML immunostaining was helpful to rapidly differentiate 7 cases with borderline characteristics and to obtain the diagnosis in 2 cases with scarce material. The effectiveness and low cost of this technique support its routine use as a first-line procedure in the differential diagnosis of AML.

  6. Prediction-for-CompAction: navigation in social environments using generalized cognitive maps.

    PubMed

    Villacorta-Atienza, Jose A; Calvo, Carlos; Makarov, Valeri A

    2015-06-01

    The ultimate navigation efficiency of mobile robots in human environments will depend on how we will appraise them: merely as impersonal machines or as human-like agents. In the latter case, an agent may take advantage of the cooperative collision avoidance, given that it possesses recursive cognition, i.e., the agent's decisions depend on the decisions made by humans that in turn depend on the agent's decisions. To deal with this high-level cognitive skill, we propose a neural network architecture implementing Prediction-for-CompAction paradigm. The network predicts possible human-agent collisions and compacts the time dimension by projecting a given dynamic situation into a static map. Thereby emerging compact cognitive map can be readily used as a "dynamic GPS" for planning actions or mental evaluation of the convenience of cooperation in a given context. We provide numerical evidence that cooperation yields additional room for more efficient navigation in cluttered pedestrian flows, and the agent can choose path to the target significantly shorter than a robot treated by humans as a functional machine. Moreover, the navigation safety, i.e., the chances to avoid accidental collisions, increases under cooperation. Remarkably, these benefits yield no additional load to the mean society effort. Thus, the proposed strategy is socially compliant, and the humanoid agent can behave as "one of us."

  7. A novel deleterious mutation in the COMP gene that causes pseudoachondroplasia

    PubMed Central

    Luo, Huaichao; Yu, Sisi; Lin, Ying; Guo, Qi; Ma, Rongchuan; Ye, Zimeng; Di, Yanan; Li, Ning; Miao, Yuanying; Zhou, Yu; Li, Yuanfeng; Yang, Jiyun; Yang, Zhenglin

    2016-01-01

    Pseudoachondroplasia (PSACH) is a rare and severe genetic disease; therefore, an accurate molecular diagnosis is essential for appropriate disease treatment and family planning. Currently, the diagnosis of PSACH is based mainly on family history, physical examination and radiographic evaluation. Genetic studies of patients with PSACH in Chinese populations have been very limited. With the application of next-generation sequencing (NGS), a comprehensive molecular diagnosis of PSACH is now possible. The purpose of this study was to perform comprehensive NGS-based molecular diagnoses for patients with PSACH in China. We investigated the molecular genetics of one suspected PSACH family in this study. The DNA sample from the proband was sequenced using a custom capture panel that included 249 bone disease genes. Variant calls were filtered and annotated using an in-house automated pipeline. Then, we confirmed the variants by Sanger sequencing in three family members. After co-segregation analysis, the variant, c.1160_1162del of the COMP gene, was identified as a novel mutation responsible for this spontaneous form of PSACH. PMID:27330822

  8. ALE3D Model Predictions and Experimental Analysis of the Cookoff Response of Comp B*

    SciTech Connect

    Maienschein, J L; McClelland, M A; Wardell, J F; Reaugh, J E; Nichols, A L; Tran, T D

    2003-11-24

    ALE3D simulations are presented for the thermal explosion of Comp B (RDX,TNT) in a Scaled Thermal Explosion Experiment (STEX). Candidate models and numerical strategies are being tested using the ALE3D code which simulates the coupled thermal, mechanical, and chemical behavior during heating, ignition, and explosion. The mechanical behavior of the solid constituents is represented by a Steinberg-Guinan model while polynomial and gamma-law expressions are used for the equation of state of the solid and gas species, respectively. A gamma-law model is employed for the air in gaps, and a mixed material model is used for the interface between air and explosive. A three-step chemical kinetics model is used for each of the RDX and TNT reaction sequences during the heating and ignition phases, and a pressure-dependent deflagration model is employed during the rapid expansion. Parameters for the three-step kinetics model are specified using measurements of the One-Dimensional-Time-to-Explosion (ODTX), while measurements for burn rate are employed to determine parameters in the burn front model. We compare model predictions to measurements for temperature fields, ignition temperature, and tube wall strain during the heating, ignition, and explosive phases.

  9. Colonization Pattern of the Biocontrol Strain Pseudomonas chlororaphis MA 342 on Barley Seeds Visualized by Using Green Fluorescent Protein

    PubMed Central

    Tombolini, Riccardo; van der Gaag, Dirk Jan; Gerhardson, Berndt; Jansson, Janet K.

    1999-01-01

    Pseudomonas chlororaphis MA 342 is a potent biocontrol agent that can be used against several seed-borne diseases of cereal crops, including net blotch of barley caused by the fungus Drechslera teres. In this study, strain MA 342 was tagged with the gfp gene (encoding the green fluorescent protein) in order to study the fate of cells after seed inoculation. The gfp-tagged strain, MA 342G2, had the same biocontrol efficacy as the wild type when it was applied at high cell concentrations to seeds but was less effective at lower cell concentrations. By comparing cell counts determined by microscopy to the number of CFU, we found that the number of culturable cells was significantly lower than the total number of bacteria on seeds which were inoculated and dried for 20 h. Confocal microscopy and epifluorescence stereomicroscopy were used to determine the pattern of MA 342G2 colonization and cell aggregation on barley seeds. Immediately after inoculation of seeds, bacteria were found mainly under the seed glume, and there was no particular aggregation pattern. However, after the seeds were sown, irregularly distributed areas of bacterial aggregation were found, which reflected epiphytic colonization of glume cells. There was a trend towards bacterial aggregation near the embryo but never within the embryo. Bacterial aggregates were regularly found in the groove of each seed formed by the base of the coleoptile and the scutellum. Based on these results, we suggest that MA 342 colocalizes with the pathogen D. teres, which facilitates the action of the fungistatic compound(s) produced by this strain. PMID:10427065

  10. Molecular Interactions of the Min Protein System Reproduce Spatiotemporal Patterning in Growing and Dividing Escherichia coli Cells

    PubMed Central

    Walsh, James C.; Angstmann, Christopher N.; Duggin, Iain G.; Curmi, Paul M. G.

    2015-01-01

    Oscillations of the Min protein system are involved in the correct midcell placement of the divisome during Escherichia coli cell division. Based on molecular interactions of the Min system, we formulated a mathematical model that reproduces Min patterning during cell growth and division. Specifically, the increase in the residence time of MinD attached to the membrane as its own concentration increases, is accounted for by dimerisation of membrane-bound MinD and its interaction with MinE. Simulation of this system generates unparalleled correlation between the waveshape of experimental and theoretical MinD distributions, suggesting that the dominant interactions of the physical system have been successfully incorporated into the model. For cells where MinD is fully-labelled with GFP, the model reproduces the stationary localization of MinD-GFP for short cells, followed by oscillations from pole to pole in larger cells, and the transition to the symmetric distribution during cell filamentation. Cells containing a secondary, GFP-labelled MinD display a contrasting pattern. The model is able to account for these differences, including temporary midcell localization just prior to division, by increasing the rate constant controlling MinD ATPase and heterotetramer dissociation. For both experimental conditions, the model can explain how cell division results in an equal distribution of MinD and MinE in the two daughter cells, and accounts for the temperature dependence of the period of Min oscillations. Thus, we show that while other interactions may be present, they are not needed to reproduce the main characteristics of the Min system in vivo. PMID:26018614

  11. The mouse ileal lipid-binding protein gene: a model for studying axial patterning during gut morphogenesis

    PubMed Central

    1994-01-01

    Normal, chimeric-transgenic, and transgenic mice have been used to study the axial patterns of ileal lipid-binding protein gene (Ilbp) expression during and after completion of gut morphogenesis. Ilbp is initially activated in enterocytes in bidirectional wave that expands proximally in the ileum and distally to the colon during late gestation and the first postnatal week. This activation occurs at the same time that a wave of cytodifferentiation of the gut endoderm is completing its unidirectional journey from duodenum to colon. The subsequent contraction of Ilbp's expression domain, followed by its reexpansion from the distal to proximal ileum, coincides with a critical period in gut morphogenesis (postnatal days 7-28) when its proliferative units (crypts) form, establish their final stem cell hierarchy, and then multiply through fission. The wave of reactivation is characterized by changing patterns of Ilbp expression: (a) at the proximal most boundary of the wave, villi contain a mixed population of scattered ileal lipid- binding protein (ILBP)-positive and ILBP-negative enterocytes derived from the same monoclonal crypt; (b) somewhat more distally, villi contain vertical coherent stripes of wholly ILBP-positive enterocytes derived from monoclonal crypts and adjacent, wholly ILBP-negative stripes of enterocytes emanating from other monoclonal crypts; and (c) more distally, all the enterocytes on a villus support Ilbp expression. Functional mapping studies of Ilbp's promoter in transgenic mice indicate that nucleotides -145 to +48 contain cis-acting elements sufficient to produce an appropriately directed distal-to-proximal wave of Ilbp activation in the ileum, to maintain an appropriate axial distribution of monophenotypic wholly reporter-positive villi in the distal portion of the ileum, as well as striped and speckled villi in the proximal portion of its expression domain, and to correctly support reporter production in villus-associated ileal enterocytes

  12. Ribosomal acidic phosphoproteins P1 and P2 are not required for cell viability but regulate the pattern of protein expression in Saccharomyces cerevisiae.

    PubMed Central

    Remacha, M; Jimenez-Diaz, A; Bermejo, B; Rodriguez-Gabriel, M A; Guarinos, E; Ballesta, J P

    1995-01-01

    Saccharomyces cerevisiae strains with either three inactivated genes (triple disruptants) or four inactivated genes (quadruple disruptants) encoding the four acidic ribosomal phosphoproteins, YP1 alpha, YP1 beta, YP2 alpha, and YP2 beta, present in this species have been obtained. Ribosomes from the triple disruptants and, obviously, those from the quadruple strain do not have bound P proteins. All disrupted strains are viable; however, they show a cold-sensitive phenotype, growing very poorly at 23 degrees C. Cell extracts from the quadruple-disruptant strain are about 30% as active as the control in protein synthesis assays and are stimulated by the addition of free acidic P proteins. Strains lacking acidic proteins do not have a higher suppressor activity than the parental strains, and cell extracts derived from the quadruple disruptant do not show a higher degree of misreading, indicating that the absence of acidic proteins does not affect the accuracy of the ribosomes. However, the patterns of protein expressed in the cells as well as in the cell-free protein system are affected by the absence of P proteins from the particles; a wild-type pattern is restored upon addition of exogenous P proteins to the cell extract. In addition, strains carrying P-protein-deficient ribosomes are unable to sporulate but recover this capacity upon transformation with one of the missing genes. These results indicate that acidic proteins are not an absolute requirement for protein synthesis but regulate the activity of the 60S subunit, affecting the translation of certain mRNAs differently. PMID:7651393

  13. Patterns of Expression in the Matrix Proteins Responsible for Nucleation and Growth of Aragonite Crystals in Flat Pearls of Pinctada fucata

    PubMed Central

    Xiang, Liang; Su, Jingtan; Zheng, Guilan; Liang, Jian; Zhang, Guiyou; Wang, Hongzhong; Xie, Liping; Zhang, Rongqing

    2013-01-01

    The initial growth of the nacreous layer is crucial for comprehending the formation of nacreous aragonite. A flat pearl method in the presence of the inner-shell film was conducted to evaluate the role of matrix proteins in the initial stages of nacre biomineralization in vivo. We examined the crystals deposited on a substrate and the expression patterns of the matrix proteins in the mantle facing the substrate. In this study, the aragonite crystals nucleated on the surface at 5 days in the inner-shell film system. In the film-free system, the calcite crystals nucleated at 5 days, a new organic film covered the calcite, and the aragonite nucleated at 10 days. This meant that the nacre lamellae appeared in the inner-shell film system 5 days earlier than that in the film-free system, timing that was consistent with the maximum level of matrix proteins during the first 20 days. In addition, matrix proteins (Nacrein, MSI60, N19, N16 and Pif80) had similar expression patterns in controlling the sequential morphologies of the nacre growth in the inner-film system, while these proteins in the film-free system also had similar patterns of expression. These results suggest that matrix proteins regulate aragonite nucleation and growth with the inner-shell film in vivo. PMID:23776687

  14. Activation of extrasynaptic, but not synaptic, NMDA receptors modifies amyloid precursor protein expression pattern and increases amyloid-ß production.

    PubMed

    Bordji, Karim; Becerril-Ortega, Javier; Nicole, Olivier; Buisson, Alain

    2010-11-24

    Calcium is a key mediator controlling essential neuronal functions depending on electrical activity. Altered neuronal calcium homeostasis affects metabolism of amyloid precursor protein (APP), leading to increased production of β-amyloid (Aβ), and contributing to the initiation of Alzheimer's disease (AD). A linkage between excessive glutamate receptor activation and neuronal Aβ release was established, and recent reports suggest that synaptic and extrasynaptic NMDA receptor (NMDAR) activation may have distinct consequences in plasticity, gene regulation, and neuronal death. Here, we report for the first time that prolonged activation of extrasynaptic NMDAR, but not synaptic NMDAR, dramatically increased the neuronal production of Aβ. This effect was preceded by a shift from APP695 to Kunitz protease inhibitory domain (KPI) containing APPs (KPI-APPs), isoforms exhibiting an important amyloidogenic potential. Conversely, after synaptic NMDAR activation, we failed to detect any KPI-APP expression and neuronal Aβ production was not modified. Calcium imaging data showed that intracellular calcium concentration after extrasynaptic NMDAR stimulation was lower than after synaptic activation. This suggests distinct signaling pathways for each pool of receptors. We found that modification of neuronal APP expression pattern triggered by extrasynaptic NMDAR activation was regulated at an alternative splicing level involving calcium-/calmodulin-dependent protein kinase IV, but overall APP expression remained identical. Finally, memantine dose-dependently inhibited extrasynaptic NMDAR-induced KPI-APPs expression as well as neuronal Aβ release. Altogether, these data suggest that a chronic activation of extrasynaptic NMDAR promotes amyloidogenic KPI-APP expression leading to neuronal Aβ release, representing a causal risk factor for developing AD.

  15. Mobility and Core-Protein Binding Patterns of Disordered C-Terminal Tails in β-Tubulin Isotypes.

    PubMed

    Laurin, Yoann; Eyer, Joel; Robert, Charles H; Prevost, Chantal; Sacquin-Mora, Sophie

    2017-03-28

    Although they play a significant part in the regulation of microtubule structure, dynamics, and function, the disordered C-terminal tails of tubulin remain invisible to experimental structural methods and do not appear in the crystallographic structures that are currently available in the Protein Data Bank. Interestingly, these tails concentrate most of the sequence variability between tubulin isotypes and are the sites of the principal post-translational modifications undergone by this protein. Using homology modeling, we developed two complete models for the human αI/βI- and αI/βIII-tubulin isotypes that include their C-terminal tails. We then investigated the conformational variability of the two β-tails using long time-scale classical molecular dynamics simulations that revealed similar features, notably the unexpected presence of common anchoring regions on the surface of the tuulin dimer, but also distinctive mobility or interaction patterns, some of which could be related to the tail lengths and charge distributions. We also observed in our simulations that the C-terminal tail from the βI isotype, but not the βIII isotype, formed contacts in the putative binding site of a recently discovered peptide that disrupts microtubule formation in glioma cells. Hindering the binding site in the βI isotype would be consistent with this peptide's preferential disruption of microtubule formation in glioma, whose cells overexpress βIII, compared to normal glial cells. While these observations need to be confirmed with more intensive sampling, our study opens new perspectives for the development of isotype-specific chemotherapy drugs.

  16. Anisakis simplex allergy: a murine model of anaphylaxis induced by parasitic proteins displays a mixed Th1/Th2 pattern

    PubMed Central

    Baeza, M L; Conejero, L; Higaki, Y; Martín, E; Pérez, C; Infante, S; Rubio, M; Zubeldia, J M

    2005-01-01

    The study of the singular hypersensitivity reactions to Anisakis simplex (A.s) proteins, may help us to undestand many of the unknown immune interactions between helmiths infections and allergy. We have developed a murine model of allergy to A. simplex, that mimics human A. simplex allergy to study the specific aspects of anaphylaxis induced by parasites. Male C3H/HeJ mice were intraperitoneally sensitized to A. simplex. Mice were then intravenous or orally challenged with A. simplex. Antigen-specific immunoglobulins, polyclonal IgE, anaphylactic symptoms, plasma histamine levels and cytokine profiles were determined. Comparative IgE immunoblot analyses were also performed. Specific IgE, IgG1 and IgG2a were detected in sensitized mice since week 3. Polyclonal IgE raised and peaked with different kinetics. Intravenous A. simplex challenge produced anaphylaxis in mice, accompanied by plasma histamine release. Oral A. simplex challenge in similarly sensitized mice did not caused symptoms nor histamine release. Numerous A. simplex allergens were recognized by sensitized mouse sera, some of them similar to human serum. The A. simplex stimulated splenocytes released IL-10, IFN-γ, IL-4, IL-13 and IL-5. We describe a new animal model of anaphylaxis. It exhibits characteristics of type I hypersensitivity reactions to Anisakis simplex similar to those observed in allergic humans. Different responses to i.v. or oral A. simplex challenges emerged, which did not reflect a window tolerization period. The cytokine profile developed (mixed Th1/Th2 pattern) differed from the observed in classical models of anaphylaxis or allergy to food antigens. This model may permit to investigate the peculiar allergic reactions to parasitic proteins. PMID:16297154

  17. Transcriptional analysis of in vitro expression patterns of Chlamydophila abortus polymorphic outer membrane proteins during the chlamydial developmental cycle

    PubMed Central

    Wheelhouse, Nicholas; Aitchison, Kevin; Spalding, Lucy; Livingstone, Morag; Longbottom, David

    2009-01-01

    Chlamydophila abortus is the aetiological agent of ovine enzootic abortion. Sequencing, annotation and comparative analysis of the genome of C. abortus strain S26/3 has revealed variation in the loci encoding the polymorphic membrane proteins (Pmps). These Pmps resemble autotransporter proteins of the type V secretion system, suggesting an important role in chlamydial pathogenesis. The purpose of this study was to characterise the transcriptional expression patterns of this family during the developmental cycle of C. abortus. McCoy cells were infected with C. abortus and analysed for pmp mRNA expression over a 72 h period. Few pmp transcripts were detected in the early stages of the developmental cycle. Peak expression occurred at 48 h post-infection (p.i.) other than for pmp5E, where it was observed at 24 h p.i. Overall, expression of pmps 5E, 18D and 10G were found to be 40 to 100-fold higher than the lowest expressing pmps (6H, 13G and 15G) at 24 h p.i., while pmps 18D and 17G were 14 to 16-fold higher than the lowest (11G, 14G and 15G) at 48 h. Levels of expression for all the other pmp genes were below one copy per genome at any time point. The expression of all the pmps reduced to near base-line levels by 60 h p.i. These results demonstrate that pmp expression in C. abortus is mid to late cycle, consistent with conversion of the reticulate body to the elementary body. The low level of pmp transcription may be indicative of heterogeneity in expression, suggesting a possible role for some of the Pmps in antigenic variation and chlamydial pathogenesis. PMID:19454212

  18. MERISTEM-DEFECTIVE, an RS domain protein, is required for the correct meristem patterning and function in Arabidopsis.

    PubMed

    Casson, Stuart A; Topping, Jennifer F; Lindsey, Keith

    2009-03-01

    Plant growth and development is dependent on the specification and maintenance of pools of stem cells found in the meristems. Mutations in the Arabidopsis MERISTEM-DEFECTIVE (MDF) gene lead to a loss of stem cell and meristematic activity in the root and vegetative shoot. MDF encodes a putative RS domain protein with a predicted role in transcription or RNA processing control. mdf mutants exhibit decreased levels of PINFORMED2 (PIN2) and PIN4 mRNAs, which is associated with a reduction in PIN:GFP levels, and with a defective auxin maximum in the basal region of the developing mdf embryo and seedling root meristem. Seedling roots also exhibit reduced PLETHORA (PLT), SCARECROW and SHORTROOT gene expression, a loss of stem cell activity, terminal differentiation of the root meristem and defective cell patterning. MDF expression is not defective in the bodenlos, pin1 or eir1/pin2 auxin mutants, and is not modulated by exogenous auxin. plt1 plt2 double mutants have unaffected levels of MDF RNA, indicating that MDF acts upstream of PIN and PLT gene expression. Differentiation of the shoot stem cell pool also occurs in mdf mutants, associated with a reduced WUSCHEL (WUS) expression domain and expanded CLAVATA3 (CLV3) domain. Overexpression of MDF leads to the activation of markers of embryonic identity and ectopic meristem activity in vegetative tissues. These results demonstrate a requirement for the MDF-dependent pathway in regulating PIN/PLT- and WUS/CLV-mediated meristem activity.

  19. [Effect of freezing and cooking on the texture and electrophoretic pattern of the proteins of octopus arms (Octopus vulgaris)].

    PubMed

    Reyes, Genara; Nirchio, Mauro; Bello, Rafael; Borderías, Javier

    2014-09-01

    Texture is the most valuable feature in cephalopods. Factors that mainly affect the texture of octopus are: freezing, scalding and cooking. The aim of this study was to assess the effect of freezing, scalding and length of cooking time on the texture and electrophoretic pattern of proteins of octopus arms. Octopuses were trapped near Margarita Island and carried with ice to the laboratory where they were packed and subjected to: a) freezing at -27 degrees C or at -20 degrees C b) scalding c) cooking for 25 min, 35 min or 45 min. Shear force was determined by Kramer cell on strips of octopus arms. SDS-PAGE was done according to the Laemmli method with 12% polyacrilamide gels. A sensory evaluation of the preference of texture was carried out using a hedonic scale of 7-points and a non-trained panel. Octopus texture was not affected by freezing temperature or scalding. Frozen octopus was softer after cooking than fresh. The longer the cooking time was, the softer the octopus was. Myosin heavy chain (MHC) was not significantly affected by scalding or cooking; however large aggregates heavier than MHC, new bands and loss of resolution of the bands appeared. Myosin and paramyosin bands were more affected by freezing prior to cooking.

  20. KinasePhos 2.0: a web server for identifying protein kinase-specific phosphorylation sites based on sequences and coupling patterns.

    PubMed

    Wong, Yung-Hao; Lee, Tzong-Yi; Liang, Han-Kuen; Huang, Chia-Mao; Wang, Ting-Yuan; Yang, Yi-Huan; Chu, Chia-Huei; Huang, Hsien-Da; Ko, Ming-Tat; Hwang, Jenn-Kang

    2007-07-01

    Due to the importance of protein phosphorylation in cellular control, many researches are undertaken to predict the kinase-specific phosphorylation sites. Referred to our previous work, KinasePhos 1.0, incorporated profile hidden Markov model (HMM) with flanking residues of the kinase-specific phosphorylation sites. Herein, a new web server, KinasePhos 2.0, incorporates support vector machines (SVM) with the protein sequence profile and protein coupling pattern, which is a novel feature used for identifying phosphorylation sites. The coupling pattern [XdZ] denotes the amino acid coupling-pattern of amino acid types X and Z that are separated by d amino acids. The differences or quotients of coupling strength C(XdZ) between the positive set of phosphorylation sites and the background set of whole protein sequences from Swiss-Prot are computed to determine the number of coupling patterns for training SVM models. After the evaluation based on k-fold cross-validation and Jackknife cross-validation, the average predictive accuracy of phosphorylated serine, threonine, tyrosine and histidine are 90, 93, 88 and 93%, respectively. KinasePhos 2.0 performs better than other tools previously developed. The proposed web server is freely available at http://KinasePhos2.mbc.nctu.edu.tw/.

  1. RPL39L is an example of a recently evolved ribosomal protein paralog that shows highly specific tissue expression patterns and is upregulated in ESCs and HCC tumors.

    PubMed

    Wong, Queenie Wing-Lei; Li, Jia; Ng, Sheng Rong; Lim, Seng Gee; Yang, Henry; Vardy, Leah A

    2014-01-01

    Ribosomal proteins (RPs) have been shown to be able to impart selectivity on the translating ribosome implicating them in gene expression control. Many ribosomal proteins are highly conserved and recently a number of ribosomal protein paralogs have been described in mammals. We examined the expression pattern of RPs in differentiating mouse Embryonic Stem Cells (ESCs), paying particular attention to the RP paralogs. We find the RP paralog Rpl39l is highly expressed in ESC and its expression strongly correlates with hepatocellular carcinoma tumor (HCC) samples with high tumor grading and alpha-fetoprotein level giving it diagnostic potential. We further screen the expression pattern of all RPs and their paralogs across 22 different tissues. We find that the more recently evolved RP paralogs show a much greater level of tissue-specific expression. We propose that these RP paralogs evolved more recently to provide a greater level of gene expression control to higher eukaryotes.

  2. The respective roles of polar/nonpolar binary patterns and amino acid composition in protein regular secondary structures explored exhaustively using hydrophobic cluster analysis.

    PubMed

    Rebehmed, Joseph; Quintus, Flavien; Mornon, Jean-Paul; Callebaut, Isabelle

    2016-05-01

    Several studies have highlighted the leading role of the sequence periodicity of polar and nonpolar amino acids (binary patterns) in the formation of regular secondary structures (RSS). However, these were based on the analysis of only a few simple cases, with no direct mean to correlate binary patterns with the limits of RSS. Here, HCA-derived hydrophobic clusters (HC) which are conditioned binary patterns whose positions fit well those of RSS, were considered. All the HC types, defined by unique binary patterns, which were commonly observed in three-dimensional (3D) structures of globular domains, were analyzed. The 180 HC types with preferences for either α-helices or β-strands distinctly contain basic binary units typical of these RSS. Therefore a general trend supporting the "binary pattern preference" assumption was observed. HC for which observed RSS are in disagreement with their expected behavior (discordant HC) were also examined. They were separated in HC types with moderate preferences for RSS, having "weak" binary patterns and versatile RSS and HC types with high preferences for RSS, having "strong" binary patterns and then displaying nonpolar amino acids at the protein surface. It was shown that in both cases, discordant HC could be distinguished from concordant ones by well-differentiated amino acid compositions. The obtained results could, thus, help to complement the currently available methods for the accurate prediction of secondary structures in proteins from the only information of a single amino acid sequence. This can be especially useful for characterizing orphan sequences and for assisting protein engineering and design.

  3. Bayesian Proteoform Modeling Improves Protein Quantification of Global Proteomic Measurements

    SciTech Connect

    Webb-Robertson, Bobbie-Jo M.; Matzke, Melissa M.; Datta, Susmita; Payne, Samuel H.; Kang, Jiyun; Bramer, Lisa M.; Nicora, Carrie D.; Shukla, Anil K.; Metz, Thomas O.; Rodland, Karin D.; Smith, Richard D.; Tardiff, Mark F.; McDermott, Jason E.; Pounds, Joel G.; Waters, Katrina M.

    2014-12-01

    As the capability of mass spectrometry-based proteomics has matured, tens of thousands of peptides can be measured simultaneously, which has the benefit of offering a systems view of protein expression. However, a major challenge is that with an increase in throughput, protein quantification estimation from the native measured peptides has become a computational task. A limitation to existing computationally-driven protein quantification methods is that most ignore protein variation, such as alternate splicing of the RNA transcript and post-translational modifications or other possible proteoforms, which will affect a significant fraction of the proteome. The consequence of this assumption is that statistical inference at the protein level, and consequently downstream analyses, such as network and pathway modeling, have only limited power for biomarker discovery. Here, we describe a Bayesian model (BP-Quant) that uses statistically derived peptides signatures to identify peptides that are outside the dominant pattern, or the existence of multiple over-expressed patterns to improve relative protein abundance estimates. It is a research-driven approach that utilizes the objectives of the experiment, defined in the context of a standard statistical hypothesis, to identify a set of peptides exhibiting similar statistical behavior relating to a protein. This approach infers that changes in relative protein abundance can be used as a surrogate for changes in function, without necessarily taking into account the effect of differential post-translational modifications, processing, or splicing in altering protein function. We verify the approach using a dilution study from mouse plasma samples and demonstrate that BP-Quant achieves similar accuracy as the current state-of-the-art methods at proteoform identification with significantly better specificity. BP-Quant is available as a MatLab ® and R packages at https://github.com/PNNL-Comp-Mass-Spec/BP-Quant.

  4. Consumption of a healthy dietary pattern results in significant reductions in C-reactive protein levels in adults: a meta-analysis.

    PubMed

    Neale, E P; Batterham, M J; Tapsell, L C

    2016-05-01

    Consumption of healthy dietary patterns has been associated with reduced risk of cardiovascular disease and metabolic syndrome. Dietary intervention targets disease prevention, so studies increasingly use biomarkers of underlying inflammation and metabolic syndrome progression to examine the diet-health relationship. The extent to which these biomarkers contribute to the body of evidence on healthy dietary patterns is unknown. The aim of this meta-analysis was to determine the effect of healthy dietary patterns on biomarkers associated with adiposity, insulin resistance, and inflammation in adults. A systematic search of Scopus, PubMed, Web of Science, and Cochrane Central Register of Controlled Trials (all years to April 2015) was conducted. Inclusion criteria were randomized controlled trials; effects of dietary patterns assessed on C-reactive protein (CRP), total adiponectin, high-molecular-weight adiponectin, tumor necrosis factor-α, adiponectin:leptin, resistin, or retinol binding protein 4. Random effects meta-analyses were conducted to assess the weighted mean differences in change or final mean values for each outcome. Seventeen studies were included in the review. These reflected research on dietary patterns associated with the Mediterranean diet, Nordic diet, Tibetan diet, and the Dietary Approaches to Stop Hypertension diet. Consumption of a healthy dietary pattern was associated with significant reductions in CRP (weighted mean difference, -0.75 [-1.16, -0.35]; P = .0003). Non-significant changes were found for all other biomarkers. This analysis found evidence for favorable effects of healthy dietary patterns on CRP, with limited evidence for other biomarkers. Future research should include additional randomized controlled trials incorporating a greater range of dietary patterns and biomarkers.

  5. Distinct von Hippel-Lindau gene and hypoxia-regulated alterations in gene and protein expression patterns of renal cell carcinoma and their effects on metabolism.

    PubMed

    Leisz, Sandra; Schulz, Kristin; Erb, Susanne; Oefner, Peter; Dettmer, Katja; Mougiakakos, Dimitrios; Wang, Ena; Marincola, Francesco M; Stehle, Franziska; Seliger, Barbara

    2015-05-10

    During the last decade the knowledge about the molecular mechanisms of the cellular adaption to hypoxia and the function of the "von Hippel Lindau" (VHL) protein in renal cell carcinoma (RCC) has increased, but there exists little information about the overlap and differences in gene/protein expression of both processes. Therefore the aim of this study was to dissect VHL- and hypoxia-regulated alterations in the metabolism of human RCC using ome-based strategies. The effect of the VHL- and hypoxia-regulated altered gene/protein expression pattern on the cellular metabolism was analyzed by determination of glucose uptake, lactate secretion, extracellular pH, lactate dehydrogenase activity, amino acid content and ATP levels. By employing VHL-/VHL(+) RCC cells cultured under normoxic and hypoxic conditions, VHL-dependent, HIF-dependent as well as VHL-/HIF-independent alterations in the gene and protein expression patterns were identified and further validated in other RCC cell lines. The genes/proteins differentially expressed under these distinct conditions were mainly involved in the cellular metabolism, which was accompanied by an altered metabolism as well as changes in the abundance of amino acids in VHL-deficient cells. In conclusion, the study reveals similarities, but also differences in the genes and proteins controlled by VHL functionality and hypoxia thereby demonstrating differences in the metabolic switch of RCC under these conditions.

  6. Different uranium distribution patterns in cytosolic protein pool of zebrafish gills after chronic and acute waterborne exposures.

    PubMed

    Bucher, Guillaume; Mounicou, Sandra; Simon, Olivier; Floriani, Magali; Lobinski, Ryszard; Frelon, Sandrine

    2014-09-01

    The toxicity of uranium (U) to aquatic organisms depends notably on its compartmentalization in organs, tissues, cells as well as on its distribution among biomolecules. In order to contribute to the understanding of U accumulation and associated toxicity mechanisms in case of waterborne exposure, this study focused on U fate in the gills epithelia, uptake pathway, of the fish model Danio rerio (zebrafish). U distribution among cytosolic biomolecules was investigated after no addition (0μgL(-)(1) (c0) for 3 and 30d), chronic (20μgL(-)(1) (c20) for 30d) and acute (20μgL(-)(1) (c20) and 250μgL(-)(1) (c250) for 3d) exposures to depleted U. Cytosolic U accounted for an average of 24-32% of gills burden for c20 and c250, respectively. Size Exclusion Chromatography (SEC) coupled with Inductively Coupled Plasma-Sector Field Mass Spectrometry (ICP-SFMS) allowed identification of ecotoxicologically relevant U-containing fractions among cytosolic biomolecules as a function of exposure conditions. In c0 and c20 samples, most U (ca.80%) was found in the Low Molecular Weight fraction (LMW, <18kDa), often considered as a detoxifying fraction. In c250 exposed fish, U was equally distributed between LMW (40%) and High Molecular Weight (HMW, 150-670kDa; 40%) fractions, the latter including sensitive metalloproteins. Uranium-biomolecules were co-eluted with endogenous essential metal (Fe, Cu and Zn) species, however, no major influence on their cytosolic concentration and distribution pattern among cytosolic proteins was found.

  7. Cloning and expression pattern of heat shock protein genes from the endoparasitoid wasp, Pteromalus puparum in response to environmental stresses.

    PubMed

    Wang, Huan; Li, Kai; Zhu, Jia-Ying; Fang, Qi; Ye, Gong-Yin; Wang, Huan; Li, Kai; Zhu, Jia-Ying

    2012-04-01

    Six heat shock protein (HSP) genes from five HSP families in the parasitoid, Pteromalus puparum, were evaluated for their response to temperature (-15 ~ 3°C , and 30 ~ 42°C for 1 h), heavy metals (0.5 ~ 5 mM Cd(2+) and Cu(2+) for 24 h and 60 h), and starvation (24 h). Compared with other insect HSPs, all conserved motifs are found in P. puparum HSPs, and they are very similar to those of the recently sequenced ectoparasitoid Nasonia vitripennis. The temporal gene expression patterns indicated that these six HSP genes were all heat-inducible, of which hsp40 was the most inducible. The temperatures for maximal HSP induction at high and low temperature zone were 36 or 39°C and -3°C, respectively. In the hot zone, all HSP genes have the same initial temperature (33°C) for up-regulation. Low concentrations of Cd(2+) for a short-term promoted the expression of all HSP genes, but not high concentrations or long-term treatments. Cu(2+) stress for 24 h increased expression of nearly all HSP. Four HSP genes changed after starvation. We infer that all six HSP genes are sensitive to heat. This may help understand the absence of P. puparum during the summer and winter. The expression profiles of six HSP genes in P. puparum under heavy metal stress indicates that HSP is a short-term response to cellular distress or injury induced by Cd(2+) and Cu(2+).

  8. Pattern of HER-2 Gene Amplification and Protein Expression in Benign, Borderline, and Malignant Ovarian Serous and Mucinous Neoplasms.

    PubMed

    Mohammed, Rabab A A; Makboul, Rania; Elsers, Dalia A H; Elsaba, Tarek M A M; Thalab, Abeer M A B; Shaaban, Omar M

    2016-06-15

    Amplification of HER-2 gene and overexpression of HER-2 receptor play a significant role in the progression of a number of malignancies such as breast cancer. Trastuzumab (anti-HER-2 therapeutic agent) has been used successfully in treatment of breast cancer. The aim of this study was to assess the pattern of HER-2 gene amplification and of HER-2 receptor expression in a spectrum of serous and mucinous ovarian tumors to determine whether HER-2 is altered in these neoplasms similar to that occurring in breast cancer. Formalin-fixed paraffin-embedded microarray tissue sections from 212 specimens were stained with HER-2 antibody using immunohistochemistry and with anti-HER-2 DNA probe using chromogenic in situ hybridization. Specimens consisted of 65 benign tumors (50 serous and 15 mucinous), 26 borderline (13 serous and 13 mucinous), 73 malignant (53 serous carcinoma and 20 mucinous carcinoma), 18 metastatic deposits (13 serous and 5 mucinous), in addition to 30 normal tissues (16 ovarian surface and 14 normal fallopian tube). HER-2 protein-positive expression was not detected in the normal or the benign tissues. Borderline neoplasms showed positive staining, but no overexpression. HER-2 overexpression was seen only in 4 carcinoma specimens: 1/53 (1.8%) primary serous carcinomas and 3/20 (15%) primary mucinous carcinomas. HER-2 gene amplification was seen in 4 specimens: 2 primary mucinous carcinomas and 2 malignant deposits of these 2 mucinous carcinomas. In conclusion, alteration of HER-2 was not detected in ovarian serous neoplasms; however, in mucinous carcinoma, HER-2 amplification and overexpression occur more frequently.

  9. Anemia plus hypoproteinemia in dogs; various proteins in diet show various patterns in blood protein production; beef muscle,. egg, lactalbumin, fibrin, viscera, and supplements.

    PubMed

    WHIPPLE, G H; ROBSCHEIT-ROBBINS, F S

    1951-09-01

    Dogs with sustained anemia plus hypoproteinemia due to bleeding and a continuing low protein or protein-free diet containing abundant iron have been used in the present work to test food proteins and supplements as to their See PDF for Structure capacity to produce new hemoglobin and plasma proteins. The reserve stores of blood protein-producing materials are thus largely depleted in such animals and sustained levels of 6 to 8 gm. per cent hemoglobin and 4 to 5 gm. per cent plasma protein can be maintained for considerable periods of time. The stimulus of double depletion drives the body to use all protein building materials with the utmost conservation. This represents a severe biological test for food and body proteins and its assay value must have significance. Measured by this biological test in these experiments, casein stands well up among the best food proteins. The ratio of plasma protein to hemoglobin is about 40 to 50 per cent, which emphasizes the fact that these dogs produce on most diets about 2 gm. hemoglobin to 1 gm. plasma protein. The reason for this preference for hemoglobin production is obscure. The mass of circulating hemoglobin is greater even in this degree of anemia and the life cycle of hemoglobin is much longer than that of the plasma protein. Egg protein, egg albumin, and lactalbumin all favor the production of more plasma protein and less hemoglobin as compared with casein. The plasma protein to hemoglobin ratio is increased, sometimes above 100 per cent. Supplements to the above proteins of casein digests or several amino acids may return the response toward that which is standard for casein. Histidine as a supplement to egg protein increases the total blood protein output and brings the ratio of plasma protein to hemoglobin toward that of casein. Beef muscle goes to the other extreme and favors new hemoglobin production up to 4 gm. hemoglobin to 1 gm. plasma protein-a ratio of 25 per cent. The total amounts of new blood proteins are

  10. Arabidopsis actin capping protein (AtCP) subunits have different expression patterns, and downregulation of AtCPB confers increased thermotolerance of Arabidopsis after heat shock stress.

    PubMed

    Wang, Jue; Qian, Dong; Fan, Tingting; Jia, Honglei; An, Lizhe; Xiang, Yun

    2012-09-01

    As a heterodimer actin-binding protein, capping protein is composed of α and β subunits, and can stabilize the actin filament cytoskeleton by binding to F-actin ends to inhibit G-actin addition or loss from that end. Until now, studies on plant capping protein have focused on biochemical functions in vitro, and so the expression patterns and physiological functions of actin capping protein in Arabidopsis (AtCP) are poorly understood. In the present study, real-time quantitative PCR and Western blot analysis showed that although AtCP α and β subunits (i.e. AtCPA and AtCPB) were expressed in various tissues, their expression patterns were significantly different. GUS staining further indicated they were present in different parts of the same organs. We also demonstrated that the expression levels of both subunits were induced by heat shock stress. However, only the atcpβ-mutant showed enhanced thermotolerance, and confocal microscopy showed that the actin filaments of the atcpβ-mutant were much more complete than that in the wild-type and the atcpα-mutant after heat treatment at 45 °C for 40 and 45 min. In conclusion, these results demonstrated that AtCPA and AtCPB showed distinct expression patterns in vivo, and that downregulation of AtCPB conferred increased plant thermotolerance after heat shock stress.

  11. The UNUSUAL FLORAL ORGANS gene of Arabidopsis thaliana is an F-box protein required for normal patterning and growth in the floral meristem.

    PubMed

    Samach, A; Klenz, J E; Kohalmi, S E; Risseeuw, E; Haughn, G W; Crosby, W L

    1999-11-01

    Genetic and molecular studies have suggested that the UNUSUAL FLORAL ORGANS (UFO) gene, from Arabidopsis thaliana, is expressed in all shoot apical meristems, and is involved in the regulation of a complex set of developmental events during floral development, including floral meristem and floral organ identity. Results from in situ hybridization using genes expressed early in floral development as probes indicate that UFO controls growth of young floral primordia. Transgenic constructs were used to provide evidence that UFO regulates floral organ identity by activating or maintaining transcription of the class B organ-identity gene APETALA 3, but not PISTILLATA. In an attempt to understand the biochemical mode of action of the UFO gene product, we show here that UFO is an F-box protein that interacts with Arabidopsis SKP1-like proteins, both in the yeast two-hybrid system and in vitro. In yeast and other organisms both F-box proteins and SKP1 homologues are subunits of specific ubiquitin E3 enzyme complexes that target specific proteins for degradation. The protein selected for degradation by the complex is specified by the F-box proteins. It is therefore possible that the role of UFO is to target for degradation specific proteins controlling normal growth patterns in the floral primordia, as well as proteins that negatively regulate APETALA 3 transcription.

  12. Disruption of patterns of immunoreactive glial fibrillary acidic protein processes in the Cebus Apella striate cortex following loss of visual input.

    PubMed

    Colombo, J A; Yáñez, A; Lipina, S

    1999-01-01

    Long, interlaminar, astroglial processes and its patterned organization in the striate cortex of adult primates was previously described. Loss of visual input following bilateral retinal detachment and degeneration in an adult Cebus apella monkey resulted three months later in reduction of interlaminar processes immunoreactive to Glial Fibrillary Acid Protein antibody, loss of the honeycomb-like pattern normally present in tangential sections, and loss of high density patches of terminal segments of those processes in the opercular striate. These results further indicate the highly interactive nature of neuron-glial cerebral cortex architecture, and the dynamic regulation of astroglial interlaminar processes.

  13. Effects of motor patterns on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue

    NASA Technical Reports Server (NTRS)

    Pevzner, L. Z.; Venkov, L.; Cheresharov, L.

    1980-01-01

    Albino rats were kept for a year under conditions of daily motor load or constant hypokinesia. An increase in motor activity results in a rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesia but does not change under the motor load pattern. Motor load causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

  14. Exposures of Sus scrofa to a TASER(®) conducted electrical weapon: no effects on 2-dimensional gel electrophoresis patterns of plasma proteins.

    PubMed

    Jauchem, James R; Cerna, Cesario Z; Lim, Tiffany Y; Seaman, Ronald L

    2014-12-01

    In an earlier study, we found significant changes in red-blood-cell, leukocyte, and platelet counts, and in red-blood-cell membrane proteins, following exposures of anesthetized pigs to a conducted electrical weapon. In the current study, we examined potential changes in plasma proteins [analyzed via two-dimensional gel electrophoresis (2-DGE)] following two 30 s exposures of anesthetized pigs (Sus scrofa) to a TASER (®) C2 conducted electrical weapon. Patterns of proteins, separated by 2-DGE, were consistent and reproducible between animals and between times of sampling. We determined that the blood plasma collection, handling, storage, and processing techniques we used are suitable for swine blood. There were no statistically significant changes in plasma proteins following the conducted-electrical-weapon exposures. Overall gel patterns of fibrinogen were similar to results of other studies of both pigs and humans (in control settings, not exposed to conducted electrical weapons). The lack of significant changes in plasma proteins may be added to the body of evidence regarding relative safety of TASER C2 device exposures.

  15. Protein feeding pattern, casein feeding, or milk-soluble protein feeding did not change the evolution of body composition during a short-term weight loss program.

    PubMed

    Adechian, Solange; Balage, Michèle; Remond, Didier; Migné, Carole; Quignard-Boulangé, Annie; Marset-Baglieri, Agnès; Rousset, Sylvie; Boirie, Yves; Gaudichon, Claire; Dardevet, Dominique; Mosoni, Laurent

    2012-10-15

    Studies have shown that timing of protein intake, leucine content, and speed of digestion significantly affect postprandial protein utilization. Our aim was to determine if one can spare lean body mass during energy restriction by varying the quality and the timing of protein intake. Obese volunteers followed a 6-wk restricted energy diet. Four groups were compared: casein pulse, casein spread, milk-soluble protein (MSP, = whey) pulse, and MSP spread (n = 10-11 per group). In casein groups, caseins were the only protein source; it was MSP in MSP groups. Proteins were distributed in four meals per day in the proportion 8:80:4:8% in the pulse groups; it was 25:25:25:25% in the spread groups. We measured weight, body composition, nitrogen balance, 3-methylhistidine excretion, perception of hunger, plasma parameters, adipose tissue metabolism, and whole body protein metabolism. Volunteers lost 7.5 ± 0.4 kg of weight, 5.1 ± 0.2 kg of fat, and 2.2 ± 0.2 kg of lean mass, with no difference between groups. In adipose tissue, cell size and mRNA expression of various genes were reduced with no difference between groups. Hunger perception was also never different between groups. In the last week, due to a higher inhibition of protein degradation and despite a lower stimulation of protein synthesis, postprandial balance between whole body protein synthesis and degradation was better with caseins than with MSP. It seems likely that the positive effect of caseins on protein balance occurred only at the end of the experiment.

  16. Electrophoresis-tutor: an image-based personal computer program that teaches clinical interpretation of protein electrophoresis patterns of serum, urine, and cerebrospinal fluid.

    PubMed

    Astion, M L; Rank, J; Wener, M H; Torvik, P; Schneider, J B; Killingsworth, L M

    1995-09-01

    High-resolution protein electrophoresis of serum, urine, and cerebrospinal fluid (CSF) can aid in the diagnosis of multiple myeloma, amyloidosis, macroglobulinemia, multiple sclerosis, and other diseases. Electrophoresis-Tutor is a personal computer program based on approximately 150 digital images that teaches the clinical interpretation of agarose gel electrophoretic patterns. The program is divided into the following sections: introduction, CSF, serum, urine, review of disease states, program navigator, and final exam. The CSF section describes normal and abnormal CSF findings with emphasis on oligoclonal banding, as seen in the CSF of patients with multiple sclerosis. The serum section emphasizes monoclonal gammopathy patterns but also has detailed descriptions of inflammation, liver disease, protein-losing disorders, genetic deficiencies, and other patterns. Monoclonal gammopathy is described in the context of specific associated clinical conditions (e.g., myeloma, amyloidosis). For each monoclonal gammopathy example, results of standard electrophoresis, densitometry, and immunofixation are presented. The review of disease states uses animation to illustrate the development and remission of a variety of pathological patterns. The program navigator allows the user to jump quickly to any place in the program. The optional exam contains 20 questions, and detailed feedback is given after each question. Electrophoresis-Tutor can be used as a stand-alone teaching tool, a companion to traditional instruction, or a reference source.

  17. Detection of Cartilage Oligomeric Matrix Protein Using a Quartz Crystal Microbalance

    PubMed Central

    Wang, Shih-Han; Shen, Chi-Yen; Weng, Ting-Chan; Lin, Pin-Hsuan; Yang, Jia-Jyun; Chen, I-Fen; Kuo, Shyh-Ming; Chang, Shwu-Jen; Tu, Yuan-Kun; Kao, Yu-Hsien; Hung, Chih-Hsin

    2010-01-01

    Current methods for diagnosing early stage osteoarthritis (OA) based on the magnetic resonance imaging and enzyme-linked immunosorbent assay methods are specific, but require specialized laboratory facilities and highly trained personal to obtain a definitive result. In this work, a user friendly and non-invasive quartz crystal microbalance (QCM) immunosensor method has been developed to detect Cartilage Oligomeric Matrix Protein (COMP) for early stage OA diagnosis. This QCM immunosensor was fabricated to immobilize COMP antibodies utilizing the self-assembled monolayer technique. The surface properties of the immunosensor were characterized by its FTIR and electrochemical impedance spectra (EIS). The feasibility study was based on urine samples obtained from 41 volunteers. Experiments were carried out in a flow system and the reproducibility of the electrodes was evaluated by the impedance measured by EIS. Its potential dynamically monitored the immunoreaction processes and could increase the efficiency and sensitivity of COMP detection in laboratory-cultured preparations and clinical samples. The frequency responses of the QCM immunosensor changed from 6 kHz when testing 50 ng/mL COMP concentration. The linear regression equation of frequency shift and COMP concentration was determined as: y = 0.0872 x + 1.2138 (R2 = 0.9957). The COMP in urine was also determined by both QCM and EIS for comparison. A highly sensitive, user friendly and cost effective analytical method for the early stage OA diagnosis has thus been successfully developed. PMID:22163547

  18. Temporal patterns of cardiac performance and genes encoding heat shock proteins and metabolic sensors of an intertidal limpet Cellana toreuma during sublethal heat stress.

    PubMed

    Zhang, Shu; Han, Guo-dong; Dong, Yun-wei

    2014-04-01

    Intertidal invertebrates develop effective physiological adaptations to cope with the rapidly changing thermal environment in the intertidal zone. In the present study, the temporal patterns of heart rate, protein carbonyl groups, and genes encoding heat shock proteins (hsp70 and hsp90) and metabolic sensors (ampkα, ampkβ and sirt1) were measured to study the effect of sublethal heat stress on the cardiac function, oxidative stress, heat shock response and cellular metabolism of an intertidal limpet Cellana toreuma. All the physiological parameters are sensitive to temperature and duration of heat stress. Spearman correlation analysis revealed that the correlations between heart rate and levels of heat shock proteins mRNA and metabolic sensors mRNA were statistically significant. These results further suggest that cardiac function plays crucial roles in cellular energy metabolism and heat shock responses. The significant increase of protein carbonyl groups at 34°C after 4h exposure indicated that the failure of cardiac function and the increase of anaerobic metabolism partly leads to the increase of protein carbonyl groups. Generally, the physiological responses to heat stress are sensitive to temperature and are energy-consumptive, as indicated by the upregulation of metabolic sensors mRNA. However, the upregulation of heat shock proteins and metabolic sensors at the post-transcriptional level and related functions need to be confirmed in further experiments.

  19. Differences in serum protein 2D gel electrophoresis patterns of Przewalski's (Mongolian wild horse) and thoroughbred horses.

    PubMed

    Barsuren, Enkhbolor; Namkhai, Bandi; Kong, Hong Sik

    2015-04-01

    The objective of this study was to assess differences in serum protein expression profiles of Przewalski's (Mongolian wild horse) and thoroughbred horses using proteome analysis. The serum proteins were separated by two-dimensional electrophoresis (2-DE) and five different gene products were identified. Proteins represented by the five spots were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS)/MS technology. The identities of all proteins were deduced based on their similarity to proteins in the human plasma protein database. Three proteins (a haptoglobin-2 alpha glycoprotein and two haptoglobin-2beta glycoproteins with different accession numbers) were downregulated in Przewalski's horse sera compared to thoroughbred horse sera. Moreover, two proteins (tetraspanin-18 and pM5) were upregulated in Przewalski's horses compared to thoroughbred horses. Haptoglobin-2 alpha and haptoglobin-2beta may serve as candidate molecules in future studies of inflammation, coagulation, immune modulation and pro-oxidant and antioxidant activity with consequential effects on the entire metabolism of the horse.

  20. Molecular characterization, phylogenetic analysis and expression patterns of five protein arginine methyltransferase genes of channel catfish, Ictalurus punctatus (Rafinesque)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMT), has recently emerged as an important modification in the regulation of gene expression. In this communication, we identified and characterized the channel catfish orthologs to human PRMT 1, 3, 4 and 5, and PRMT4 ...

  1. compMS2Miner: An Automatable Metabolite Identification, Visualization, and Data-Sharing R Package for High-Resolution LC-MS Data Sets.

    PubMed

    Edmands, William M B; Petrick, Lauren; Barupal, Dinesh K; Scalbert, Augustin; Wilson, Mark J; Wickliffe, Jeffrey K; Rappaport, Stephen M

    2017-04-04

    A long-standing challenge of untargeted metabolomic profiling by ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) is efficient transition from unknown mass spectral features to confident metabolite annotations. The compMS(2)Miner (Comprehensive MS(2) Miner) package was developed in the R language to facilitate rapid, comprehensive feature annotation using a peak-picker-output and MS(2) data files as inputs. The number of MS(2) spectra that can be collected during a metabolomic profiling experiment far outweigh the amount of time required for pain-staking manual interpretation; therefore, a degree of software workflow autonomy is required for broad-scale metabolite annotation. CompMS(2)Miner integrates many useful tools in a single workflow for metabolite annotation and also provides a means to overview the MS(2) data with a Web application GUI compMS(2)Explorer (Comprehensive MS(2) Explorer) that also facilitates data-sharing and transparency. The automatable compMS(2)Miner workflow consists of the following steps: (i) matching unknown MS(1) features to precursor MS(2) scans, (ii) filtration of spectral noise (dynamic noise filter), (iii) generation of composite mass spectra by multiple similar spectrum signal summation and redundant/contaminant spectra removal, (iv) interpretation of possible fragment ion substructure using an internal database, (v) annotation of unknowns with chemical and spectral databases with prediction of mammalian biotransformation metabolites, wrapper functions for in silico fragmentation software, nearest neighbor chemical similarity scoring, random forest based retention time prediction, text-mining based false positive removal/true positive ranking, chemical taxonomic prediction and differential evolution based global annotation score optimization, and (vi) network graph visualizations, data curation, and sharing are made possible via the compMS(2)Explorer application. Metabolite identities and

  2. Elucidating the evolutionary history and expression patterns of nucleoside phosphorylase paralogs (vegetative storage proteins) in Populus and the plant kingdom

    PubMed Central

    2013-01-01

    Background Nucleoside phosphorylases (NPs) have been extensively investigated in human and bacterial systems for their role in metabolic nucleotide salvaging and links to oncogenesis. In plants, NP-like proteins have not been comprehensively studied, likely because there is no evidence of a metabolic function in nucleoside salvage. However, in the forest trees genus Populus a family of NP-like proteins function as an important ecophysiological adaptation for inter- and intra-seasonal nitrogen storage and cycling. Results We conducted phylogenetic analyses to determine the distribution and evolution of NP-like proteins in plants. These analyses revealed two major clusters of NP-like proteins in plants. Group I proteins were encoded by genes across a wide range of plant taxa while proteins encoded by Group II genes were dominated by species belonging to the order Malpighiales and included the Populus Bark Storage Protein (BSP) and WIN4-like proteins. Additionally, we evaluated the NP-like genes in Populus by examining the transcript abundance of the 13 NP-like genes found in the Populus genome in various tissues of plants exposed to long-day (LD) and short-day (SD) photoperiods. We found that all 13 of the Populus NP-like genes belonging to either Group I or II are expressed in various tissues in both LD and SD conditions. Tests of natural selection and expression evolution analysis of the Populus genes suggests that divergence in gene expression may have occurred recently during the evolution of Populus, which supports the adaptive maintenance models. Lastly, in silico analysis of cis-regulatory elements in the promoters of the 13 NP-like genes in Populus revealed common regulatory elements known to be involved in light regulation, stress/pathogenesis and phytohormone responses. Conclusion In Populus, the evolution of the NP-like protein and gene family has been shaped by duplication events and natural selection. Expression data suggest that previously

  3. Salinity stress in roots of contrasting barley genotypes reveals time-distinct and genotype-specific patterns for defined proteins.

    PubMed

    Witzel, Katja; Matros, Andrea; Strickert, Marc; Kaspar, Stephanie; Peukert, Manuela; Mühling, Karl H; Börner, Andreas; Mock, Hans-Peter

    2014-02-01

    Soil salinity is one of the most severe abiotic stress factors threatening agriculture worldwide. Hence, particular interest exists in unraveling mechanisms leading to salt tolerance and improved crop plant performance on saline soils. Barley is considered to be one of the most salinity-tolerant crops, but varying levels of tolerance are well characterized. A proteomic analysis of the roots of two contrasting cultivars (cv. Steptoe and cv. Morex) is presented. Young plants were exposed to a period of 1, 4, 7, or 10 d at 0, 100, or 150 mM NaCl. The root proteome was analyzed based on two-dimensional gel electrophoresis. A number of cultivar-specific and salinity stress-responsive proteins were identified. Mass spectrometry-based identification was successful for 74 proteins, and a hierarchical clustering analysis grouped these into five clusters based on similarity of expression profile. The rank product method was applied to statistically access the early and late responses, and this delivered a number of new candidate proteins underlying salinity tolerance in barley. Among these were some germin-like proteins, some pathogenesis-related proteins, and numerous as-yet uncharacterized proteins. Notably, proteins involved in detoxification pathways and terpenoid biosynthesis were detected as early responsive to salinity and may function as a means of modulating growth-regulating mechanisms and membrane stability via fine tuning of phytohormone and secondary metabolism in the root.

  4. Different expression patterns of renal Na(+)/K(+)-ATPase α-isoform-like proteins between tilapia and milkfish following salinity challenges.

    PubMed

    Yang, Wen-Kai; Chung, Chang-Hung; Cheng, Hui Chen; Tang, Cheng-Hao; Lee, Tsung-Han

    2016-12-01

    Euryhaline teleosts can survive in a broad range of salinity via alteration of the molecular mechanisms in certain osmoregulatory organs, including in the gill and kidney. Among these mechanisms, Na(+)/K(+)-ATPase (NKA) plays a crucial role in triggering ion-transporting systems. The switch of NKA isoforms in euryhaline fish gills substantially contributes to salinity adaptation. However, there is little information about switches in the kidneys of euryhaline teleosts. Therefore, the responses of the renal NKA α-isoform protein switch to salinity challenge in euryhaline tilapia (Oreochromis mossambicus) and milkfish (Chanos chanos) with different salinity preferences were examined and compared in this study. Immunohistochemical staining in tilapia kidneys revealed the localization of NKA in renal tubules rather than in the glomeruli, similar to our previous findings in milkfish kidneys. Protein abundance in the renal NKA pan α-subunit-like, α1-, and α3-isoform-like proteins in seawater-acclimated tilapia was significantly higher than in the freshwater group, whereas the α2-isoform-like protein exhibited the opposite pattern of expression. In the milkfish, higher protein abundance in the renal NKA pan α-subunit-like and α1-isoform-like proteins was found in freshwater-acclimated fish, whereas no difference was found in the protein abundance of α2- and α3-isoform-like proteins between groups. These findings suggested that switches for renal NKA α-isoforms, especially the α1-isoform, were involved in renal osmoregulatory mechanisms of euryhaline teleosts. Moreover, differences in regulatory responses of the renal NKA α-subunit to salinity acclimation between tilapia and milkfish revealed that divergent mechanisms for maintaining osmotic balance might be employed by euryhaline teleosts with different salinity preferences.

  5. Distribution of the SynDIG4/proline-rich transmembrane protein 1 in rat brain.

    PubMed

    Kirk, Lyndsey M; Ti, Shu W; Bishop, Hannah I; Orozco-Llamas, Mayra; Pham, Michelle; Trimmer, James S; Díaz, Elva

    2016-08-01

    The modulation of AMPA receptor (AMPAR) content at synapses is thought to be an underlying molecular mechanism of memory and learning. AMPAR content at synapses is highly plastic and is regulated by numerous AMPAR accessory transmembrane proteins such as TARPs, cornichons, and CKAMPs. SynDIG (synapse differentiation-induced gene) defines a family of four genes (SynDIG1-4) expressed in distinct and overlapping patterns in the brain. SynDIG1 was previously identified as a novel transmembrane AMPAR-associated protein that regulates synaptic strength. The related protein SynDIG4 [also known as Prrt1 (proline-rich transmembrane protein 1)] has recently been identified as a component of AMPAR complexes. In this study, we show that SynDIG1 and SynDIG4 have distinct yet overlapping patterns of expression in the central nervous system, with SynDIG4 having especially prominent expression in the hippocampus and particularly within CA1. In contrast to SynDIG1 and other traditional AMPAR auxiliary subunits, SynDIG4 is de-enriched at the postsynaptic density and colocalizes with extrasynaptic GluA1 puncta in primary dissociated neuron culture. These results indicate that, although SynDIG4 shares sequence similarity with SynDIG1, it might act through a unique mechanism as an auxiliary factor for extrasynaptic GluA1-containing AMPARs. J. Comp. Neurol. 524:2266-2280, 2016. © 2015 Wiley Periodicals, Inc.

  6. Proteins.

    ERIC Educational Resources Information Center

    Doolittle, Russell F.

    1985-01-01

    Examines proteins which give rise to structure and, by virtue of selective binding to other molecules, make genes. Binding sites, amino acids, protein evolution, and molecular paleontology are discussed. Work with encoding segments of deoxyribonucleic acid (exons) and noncoding stretches (introns) provides new information for hypotheses. (DH)

  7. Differentiation of species in the Bacillus brevis group and the Bacillus aneurinolyticus group based on the electrophoretic whole-cell protein pattern.

    PubMed

    Shida, O; Takagi, H; Kadowaki, K; Yano, H; Komagata, K

    1996-07-01

    Ninety strains of eleven Bacillus species in the Bacillus brevis group and the Bacillus aneurinolyticus group were compared with the electrophoretic whole-cell protein pattern. The strains were separated into two clusters at the similarity of 55%. One cluster (cluster 1) was consisted of strains from the B. brevis group, and another cluster (cluster 2) was composed of strains from the B. aneurinolyticus group. The cluster 1 was separated into eight subclusters. Out of eight subclusters, seven subclusters contain strains from B. brevis, B. laterosporus, B. agri, B. reuszeri, B. choshinensis, B. formosus, and B. borstelensis. Another subcluster was further separated into two related clusters, which corresponded to B. centrosporus and B. parabrevis, and they were fused at the similarity of 76%. Cluster 2 was separated into two subclusters, which corresponded to B. aneurinolyticus and B. migulanus. The above eleven species showed characteristic patterns distinct from one another, and this correlated well with the published DNA relatedness data. The comparison of the electrophoretic whole-cell protein pattern proved to be useful for evaluation of taxonomic relationships among these taxa.

  8. Alteration of consciousness via diverse photo-acoustic stimulatory patterns. Phenomenology and effect on salivary flow rate, alpha-amylase and total protein levels.

    PubMed

    Beck, Anita; Fábián, Gábor; Fejérdy, Pál; Krause, Wolf-Rainer; Hermann, Péter; Módos, Károly; Varga, Gábor; Fábián, Tibor Károly

    2015-12-01

    Long-term photo-acoustic stimulation is used for the induction of altered states of consciousness for both therapeutic and experimental purposes. Long-term photo-acoustic stimulation also leads to changes in the composition of saliva which have a key contribution to the efficiency of this technique in easing mucosal symptoms of oral psychosomatic patients. The aim of this study is to find out whether there is any cumulative effect of repeated stimulation and whether there are any detectable differences between diverse stimulatory patterns of long lasting photo-acoustic stimulation on the phenomenology of the appearing trance state and on salivary secretion. There was significant cumulative effect in relation with the appearance of day dreaming as phenomenological parameter, and in relation with protein output and amylase/protein ratio as salivary parameter. Pattern specific effect was detectable in relation with salivary flow rate only. Although our results clearly indicate the existence of certain cumulative and stimulation-pattern specific effects of repeated photo-acoustic stimulation, the absolute values of all these effects were relatively small in this study. Therefore, in spite of their theoretical importance there are no direct clinical consequences of these findings. However, our data do not exclude at all the possibility that repeated stimulation with other stimulatory parameters may lead to more pronounced effects. Further studies are needed to make clear conclusion in this respect.

  9. X-ray absorption spectroscopy of an investigational anticancer gallium(III) drug: interaction with serum proteins, elemental distribution pattern, and coordination of the compound in tissue.

    PubMed

    Hummer, Alfred A; Bartel, Caroline; Arion, Vladimir B; Jakupec, Michael A; Meyer-Klaucke, Wolfram; Geraki, Tina; Quinn, Paul D; Mijovilovich, Ana; Keppler, Bernhard K; Rompel, Annette

    2012-06-14

    Tris(8-quinolinolato)gallium(III) (1, KP46) is a very promising investigational anticancer drug. Its interaction with serum proteins, elemental distribution, and coordination in tissue were investigated with X-ray absorption (XAS) methods. Model compounds with mixed O, N, and/or S donor atoms are reported. The coordination and structure of 1 in cell culture medium (minimum essential medium, MEM) and fetal calf serum (FCS) were probed by XANES and EXAFS. The interaction of 1 with the serum proteins apotransferrin (apoTf) and human serum albumin (HSA) was addressed as well. By application of micro-XAS to tissue samples from mice treated with 1, the gallium distribution pattern was analyzed and compared to those of physiological trace elements. The complex 1 turned out to be very stable under physiological conditions, in cell culture media and in tissue samples. The coordination environment of the metal center remains intact in the presence of apoTf and HSA. The gallium distribution pattern in tumor and liver tissue revealed high similarities to the distribution patterns of Zn and Fe, minor similarities to Cu and Ni, and no similarity to Ca.

  10. Fungal Endopolygalacturonases Are Recognized as Microbe-Associated Molecular Patterns by the Arabidopsis Receptor-Like Protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES11[W

    PubMed Central

    Zhang, Lisha; Kars, Ilona; Essenstam, Bert; Liebrand, Thomas W.H.; Wagemakers, Lia; Elberse, Joyce; Tagkalaki, Panagiota; Tjoitang, Devlin; van den Ackerveken, Guido; van Kan, Jan A.L.

    2014-01-01

    Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis. PMID:24259685

  11. Fungal endopolygalacturonases are recognized as microbe-associated molecular patterns by the arabidopsis receptor-like protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1.

    PubMed

    Zhang, Lisha; Kars, Ilona; Essenstam, Bert; Liebrand, Thomas W H; Wagemakers, Lia; Elberse, Joyce; Tagkalaki, Panagiota; Tjoitang, Devlin; van den Ackerveken, Guido; van Kan, Jan A L

    2014-01-01

    Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.

  12. Effects of disruption of the nucleotide pattern in CRID element and Kozak sequence of interferon β on mRNA stability and protein production.

    PubMed

    Kay, Maryam; Hojati, Zohreh; Heidari, Maryam; Bazi, Zahra; Korbekandi, Hassan

    2015-01-01

    Interferon β (IFNβ) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFNβ production by introducing mutations in the coding region of IFNβ, while its amino acid sequence is intact. Two recombinant vectors IFNβ(K) and IFNβ(K+CRID )were designed by site-directed mutagenesis. The IFNβ(K) and IFNβ(K+CRID) have two substitutions in Kozak sequence and four substitutions in CRID sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was also performed for both of them. They were transiently transfected to CHO-dhfr(-) cell line using Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was determined by real time PCR and ELISA. The results of this study indicate that the amount of IFNβ protein produced by CHO cells containing IFNβ(K) has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβ(K+CRID) construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that disruption of AT pattern in CRID element increase RNA and protein production, improve IFNβ mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.

  13. Distribution pattern(s) of sperm protein at 22 kDa (SP22) on fresh, cooled and frozen/thawed equine spermatozoa and expression of SP22 in tissues from the testes and epididymides of normal stallions.

    PubMed

    Miller, L M J; Woodward, E M; Campos, J R; Squires, E L; Troedsson, M H T

    2015-04-01

    The objectives of this study were to (i) verify localization of SP22 on fresh, cooled, and frozen/thawed equine spermatozoa and to (ii) determine SP22 mRNA and protein expression in equine testicular and epididymal tissues. Immunocytochemistry and Western blots were performed on the spermatozoa samples. Northern blots and Western blots were performed on the tissue samples. The immunocytochemistry revealed the presence of SP22 in all samples tested. The fresh spermatozoa stained predominantly over the equatorial segment as did the samples cooled for 1 and 2 days. The samples cooled for 3 days, and the frozen/thawed samples had an increased proportion of no staining. The Western blots revealed SP22 was present on all semen samples tested. The Northern blot of the tissues revealed a 1.0 kb mRNA transcript present in each of the tissues, and the Western blot revealed the presence of SP22 in each of the tissues. As expected, SP22 was found to be altered on cooled and frozen/thawed spermatozoa. Our results suggest that the equatorial pattern is the normal pattern in spermatozoa, while a complete loss of SP22 from the surface of spermatozoa seems to be the staining pattern indicating the most extreme abnormality with scattered staining of the head indicating intermediate damage.

  14. The Tissue-Specific RNA Binding Protein T-STAR Controls Regional Splicing Patterns of Neurexin Pre-mRNAs in the Brain

    PubMed Central

    Ehrmann, Ingrid; Dalgliesh, Caroline; Liu, Yilei; Danilenko, Marina; Crosier, Moira; Overman, Lynn; Arthur, Helen M.; Lindsay, Susan; Clowry, Gavin J.; Venables, Julian P.; Fort, Philippe; Elliott, David J.

    2013-01-01

    The RNA binding protein T-STAR was created following a gene triplication 520–610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns

  15. The tissue-specific RNA binding protein T-STAR controls regional splicing patterns of neurexin pre-mRNAs in the brain.

    PubMed

    Ehrmann, Ingrid; Dalgliesh, Caroline; Liu, Yilei; Danilenko, Marina; Crosier, Moira; Overman, Lynn; Arthur, Helen M; Lindsay, Susan; Clowry, Gavin J; Venables, Julian P; Fort, Philippe; Elliott, David J

    2013-04-01

    The RNA binding protein T-STAR was created following a gene triplication 520-610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of

  16. Yolk proteins in the male reproductive system of the fruit fly Drosophila melanogaster: spatial and temporal patterns of expression.

    PubMed

    Majewska, Magdalena M; Suszczynska, Agnieszka; Kotwica-Rolinska, Joanna; Czerwik, Tomasz; Paterczyk, Bohdan; Polanska, Marta A; Bernatowicz, Piotr; Bebas, Piotr

    2014-04-01

    In insects, spermatozoa develop in the testes as clones of single spermatogonia covered by specialized somatic cyst cells (cc). Upon completion of spermatogenesis, spermatozoa are released to the vas deferens, while the cc remain in the testes and die. In the fruit fly Drosophila melanogaster, the released spermatozoa first reach the seminal vesicles (SV), the organ where post-testicular maturation begins. Here, we demonstrate the temporal (restricted to the evening and early night hours) accumulation of membranous vesicles containing proteins in the SV lumen of D. melanogaster. When SV vesicles were isolated from the semen and co-incubated with testis-derived spermatozoa in vitro, their contents bound to the spermatozoa along their tails. The proteins of the SV vesicles were then characterized using 2-D electrophoresis. We identified a prominent protein spot of around 45-47 kDa, which disappears from the SV vesicles in the night, i.e. shortly after they appear in the SV lumen. Sequencing of peptides derived from this spot by mass spectrometry revealed identity with three yolk proteins (YP1-3). This unexpected result was confirmed by western blotting, which demonstrated that SV vesicles contain proteins that are immunoreactive with an antibody against D. melanogaster YP1-3. The expression of all yp genes was shown to be a unique feature of testis tissues. Using RNA probes we found that their transcripts localize exclusively to the cc that cover fully developed spermatozoa in the distal part of each testis. Temporally, the expression of yp genes was found to be restricted to a short period during the day and is followed by the evening accumulation of YP proteins in the cc. Immunohistochemical staining confirmed that cc are the source of SV vesicles containing YPs that are released into the SV lumen. These vesicles interact with spermatozoa and as a result, YPs become extrinsic proteins of the sperm membrane. Thus, we describe for the first time the expression of

  17. Response of heat shock protein genes of the oriental fruit moth under diapause and thermal stress reveals multiple patterns dependent on the nature of stress exposure.

    PubMed

    Zhang, Bo; Peng, Yu; Zheng, Jincheng; Liang, Lina; Hoffmann, Ary A; Ma, Chun-Sen

    2016-07-01

    Heat shock protein gene (Hsp) families are thought to be important in thermal adaptation, but their expression patterns under various thermal stresses have still been poorly characterized outside of model systems. We have therefore characterized Hsp genes and their stress responses in the oriental fruit moth (OFM), Grapholita molesta, a widespread global orchard pest, and compared patterns of expression in this species to that of other insects. Genes from four Hsp families showed variable expression levels among tissues and developmental stages. Members of the Hsp40, 70, and 90 families were highly expressed under short exposures to heat and cold. Expression of Hsp40, 70, and Hsc70 family members increased in OFM undergoing diapause, while Hsp90 was downregulated. We found that there was strong sequence conservation of members of large Hsp families (Hsp40, Hsp60, Hsp70, Hsc70) across taxa, but this was not always matched by conservation of expression patterns. When the large Hsps as well as small Hsps from OFM were compared under acute and ramping heat stress, two groups of sHsps expression patterns were apparent, depending on whether expression increased or decreased immediately after stress exposure. These results highlight potential differences in conservation of function as opposed to sequence in this gene family and also point to Hsp genes potentially useful as bioindicators of diapause and thermal stress in OFM.

  18. Changes in electrophoretic patterns of soluble and membrane proteins during cold acclimation of red clover seedlings. [Trifolium pratense

    SciTech Connect

    Wolfraim, L.; Dhindsa, R.S.

    1987-04-01

    Two-week old seedlings of a cold-hardy cultivar of red clover (Trifolium pratense cv. Florex) were grown under 12 h photoperiod and day/night temperatures of 25/20C. They were cold-acclimated at 4C for 0, 7 and 14 days, and slowly frozen to -8C for 4h. They were then allowed to thaw gradually to room temperature. Survival was estimated 3 days after thawing. Acclimation for 0, 7 and 14 days resulted in 2, 55 and 64% survival respectively. Seedlings, acclimated for 0, 3, 7, 10, 14 and 20 days were labeled in vivo with (/sup 35/S)Met for 4h. Soluble and membrane proteins were prepared and analyzed by SDS-PAGE coupled with autoradiography. Four soluble proteins, M/sub r/ 15, 15.5, 23 and 25 kd, were found to specific to cold-acclimated seedlings and were present throughout the acclimation period. Two soluble proteins, M/sub r/ 41, 60 kd, appeared transiently in plants acclimated for 3 days. One membrane protein, M/sub r/ 29 kd, appears on day-3 and peaks on day 14 of acclimation. It declines thereafter. Studies are in progress to determine whether these cold acclimation-specific proteins are required for cold acclimation.

  19. Protein

    MedlinePlus

    ... Search for: Harvard T.H. Chan School of Public Health Email People Departments Calendar Careers Give my.harvard ... Nutrition Source Harvard T.H. Chan School of Public Health > The Nutrition Source > What Should I Eat? > Protein ...

  20. Protein

    MedlinePlus

    ... Go lean with protein. • Choose lean meats and poultry. Lean beef cuts include round steaks (top loin, ... main dishes. • Use nuts to replace meat or poultry, not in addition to meat or poultry (i. ...

  1. A Surface Pattern on MALDI Steel Plate for One-Step In-Situ Self-Desalting and Enrichment of Peptides/Proteins

    NASA Astrophysics Data System (ADS)

    Wang, Sheng; Xiao, Chunsheng; Li, Ying; Ling, Ling; Chen, Xuesi; Guo, Xinhua

    2017-03-01

    We report a novel strategy to achieve simultaneous one-step in-situ self-desalting and enrichment (OISE) of peptides/proteins on a facilely fabricated patterned MALDI steel plate with a circular paraffin-steel-polystyrene structure. The OISE plate could efficiently segregate salts from both analytes and matrices while retaining both analyte and matrix concentrate, and facilitating them to form homogeneous co-crystals on the centrally located polystyrene pattern. With the OISE plate, high quality and reproducible spectra could be obtained for low abundance peptides even in the presence of high salt concentrations (200 mM NH4HCO3, 1 M NaCl, or 400 mM urea). Using this strategy, a significant sensitivity enhancement was gained over traditional MALDI plate. The practical utility of this method was further demonstrated by the successful profiling of BSA digests and human serum.

  2. rugose (rg), a Drosophila A kinase anchor protein, is required for retinal pattern formation and interacts genetically with multiple signaling pathways.

    PubMed Central

    Shamloula, Hoda K; Mbogho, Mkajuma P; Pimentel, Angel C; Chrzanowska-Lightowlers, Zosia M A; Hyatt, Vanneta; Okano, Hideyuki; Venkatesh, Tadmiri R

    2002-01-01

    In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways. PMID:12072466

  3. A Surface Pattern on MALDI Steel Plate for One-Step In-Situ Self-Desalting and Enrichment of Peptides/Proteins

    NASA Astrophysics Data System (ADS)

    Wang, Sheng; Xiao, Chunsheng; Li, Ying; Ling, Ling; Chen, Xuesi; Guo, Xinhua

    2017-01-01

    We report a novel strategy to achieve simultaneous one-step in-situ self-desalting and enrichment (OISE) of peptides/proteins on a facilely fabricated patterned MALDI steel plate with a circular paraffin-steel-polystyrene structure. The OISE plate could efficiently segregate salts from both analytes and matrices while retaining both analyte and matrix concentrate, and facilitating them to form homogeneous co-crystals on the centrally located polystyrene pattern. With the OISE plate, high quality and reproducible spectra could be obtained for low abundance peptides even in the presence of high salt concentrations (200 mM NH4HCO3, 1 M NaCl, or 400 mM urea). Using this strategy, a significant sensitivity enhancement was gained over traditional MALDI plate. The practical utility of this method was further demonstrated by the successful profiling of BSA digests and human serum.

  4. Chronic Maternal Low-Protein Diet in Mice Affects Anxiety, Night-Time Energy Expenditure and Sleep Patterns, but Not Circadian Rhythm in Male Offspring

    PubMed Central

    Mahadevan, Sangeetha K.; Fiorotto, Marta L.; Van den Veyver, Ignatia B.

    2017-01-01

    Offspring of murine dams chronically fed a protein-restricted diet have an increased risk for metabolic and neurobehavioral disorders. Previously we showed that adult offspring, developmentally exposed to a chronic maternal low-protein (MLP) diet, had lower body and hind-leg muscle weights and decreased liver enzyme serum levels. We conducted energy expenditure, neurobehavioral and circadian rhythm assays in male offspring to examine mechanisms for the body-weight phenotype and assess neurodevelopmental implications of MLP exposure. C57BL/6J dams were fed a protein restricted (8%protein, MLP) or a control protein (20% protein, C) diet from four weeks before mating until weaning of offspring. Male offspring were weaned to standard rodent diet (20% protein) and single-housed until 8–12 weeks of age. We examined body composition, food intake, energy expenditure, spontaneous rearing activity and sleep patterns and performed behavioral assays for anxiety (open field activity, elevated plus maze [EPM], light/dark exploration), depression (tail suspension and forced swim test), sociability (three-chamber), repetitive (marble burying), learning and memory (fear conditioning), and circadian behavior (wheel-running activity during light-dark and constant dark cycles). We also measured circadian gene expression in hypothalamus and liver at different Zeitgeber times (ZT). Male offspring from separate MLP exposed dams had significantly greater body fat (P = 0.03), less energy expenditure (P = 0.004), less rearing activity (P = 0.04) and a greater number of night-time rest/sleep bouts (P = 0.03) compared to control. MLP offspring displayed greater anxiety-like behavior in the EPM (P<0.01) but had no learning and memory deficit in fear-conditioning assay (P = 0.02). There was an effect of time on Per1, Per 2 and Clock circadian gene expression in the hypothalamus but not on circadian behavior. Thus, transplacental and early developmental exposure of dams to chronic MLP reduces

  5. Spatio-temporal patterning of arginyl-tRNA protein transferase (ATE) contributes to gametophytic development in a moss.

    PubMed

    Schuessele, Christian; Hoernstein, Sebastian N W; Mueller, Stefanie J; Rodriguez-Franco, Marta; Lorenz, Timo; Lang, Daniel; Igloi, Gabor L; Reski, Ralf

    2016-02-01

    The importance of the arginyl-tRNA protein transferase (ATE), the enzyme mediating post-translation arginylation of proteins in the N-end rule degradation (NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes. We characterize ATE:GUS reporter lines as well as ATE mutants in P. patens to study the impact and function of arginylation on moss development and physiology. ATE protein abundance is spatially and temporally regulated in P. patens by hormones and light and is highly abundant in meristematic cells. Further, the amount of ATE transcript is regulated during abscisic acid signalling and downstream of auxin signalling. Loss-of-function mutants exhibit defects at various levels, most severely in developing gametophores, in chloroplast starch accumulation and senescence. Thus, arginylation is necessary for moss gametophyte development, in contrast to the situation in flowering plants. Our analysis further substantiates the conservation of the N-end rule pathway components in land plants and highlights lineage-specific features. We introduce moss as a model system to characterize the role of the NERD pathway as an additional layer of complexity in eukaryotic development.

  6. Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3

    PubMed Central

    Smith, Jarrett; Calidas, Deepika; Schmidt, Helen; Lu, Tu; Rasoloson, Dominique; Seydoux, Geraldine

    2016-01-01

    RNA granules are non-membrane bound cellular compartments that contain RNA and RNA binding proteins. The molecular mechanisms that regulate the spatial distribution of RNA granules in cells are poorly understood. During polarization of the C. elegans zygote, germline RNA granules, called P granules, assemble preferentially in the posterior cytoplasm. We present evidence that P granule asymmetry depends on RNA-induced phase separation of the granule scaffold MEG-3. MEG-3 is an intrinsically disordered protein that binds and phase separates with RNA in vitro. In vivo, MEG-3 forms a posterior-rich concentration gradient that is anti-correlated with a gradient in the RNA-binding protein MEX-5. MEX-5 is necessary and sufficient to suppress MEG-3 granule formation in vivo, and suppresses RNA-induced MEG-3 phase separation in vitro. Our findings suggest that MEX-5 interferes with MEG-3’s access to RNA, thus locally suppressing MEG-3 phase separation to drive P granule asymmetry. Regulated access to RNA, combined with RNA-induced phase separation of key scaffolding proteins, may be a general mechanism for controlling the formation of RNA granules in space and time. DOI: http://dx.doi.org/10.7554/eLife.21337.001 PMID:27914198

  7. Expression patterns of Oct4, Cdx2, Tead4, and Yap1 proteins during blastocyst formation in embryos of the marsupial, Monodelphis domestica Wagner.

    PubMed

    Morrison, J T; Bantilan, N S; Wang, V N; Nellett, K M; Cruz, Y P

    2013-05-01

    The marsupial blastocyst forms in an entirely different manner from its eutherian counterpart, involving cell-zona rather than cell-cell adhesion during the 8- to-16-cell transition. While the eutherian blastocyst consists of a spherical trophoblast completely enveloping a pluripotent inner cell mass, or pluriblast, the marsupial blastocyst forms initially as a bowl-shaped monolayer of cells lining the zona pellucida at the embryonic pole (ep). This monolayer contains a small patch of centrally positioned pluriblast cells edged with trophoblast cells that later coalesce at the abembryonic pole. Using immunocytochemistry, we examined the localization of the proteins Oct4, Cdx2, Tead4, Sox2, and Yap1 in opossum embryos to determine if their temporal expression pattern differed from that in the mouse, given the important differences in cell behavior preceding blastocyst formation in these mammals. Our results indicate that these proteins are expressed in similar temporal patterns despite the topological differences between mouse and opossum cleavage-stage embryos and blastocysts. That the Hippo-pathway protein Yap1 localized specifically around the approximately 128-cell stage to opossum trophoblast nuclei but remained in the cytoplasm of pluriblast cells suggests that this transcriptional regulator participates in allocating cells to the trophoblast lineage, as it does in mouse. Interestingly, in both mouse and opossum embryos, expression of the pluripotency marker Oct4 persisted after Cdx2, which signals trophoblast specification, began to be expressed in trophoblast cells. This and the observation that Cdx2 is present in opossum embryos well before blastomere-zona adhesion even occurs suggests that the proteins studied may have other roles in early mammalian embryonic development.

  8. Inverse Association of General Joint Hypermobility With Hand and Knee Osteoarthritis and Serum Cartilage Oligomeric Matrix Protein Levels

    PubMed Central

    Chen, Hsiang-Cheng; Shah, Svati H.; Li, Yi-Ju; Stabler, Thomas V.; Jordan, Joanne M.; Kraus, Virginia Byers

    2013-01-01

    Objective Extensive joint hypermobility, lower serum cartilage oligomeric matrix protein (COMP), and early-onset osteoarthritis (OA) are phenotypes of inherited pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED). However, few studies have evaluated the association between articular hypermobility and primary OA. Therefore, we evaluated this association and tested the hypothesis that COMP level is associated with hypermobility in OA and non-OA individuals. Methods Two separate cohorts were available for analysis, the extended CARRIAGE family and a subset of the GOGO sib pair cohort. In the CARRIAGE family, we performed hand and knee examinations, hypermobility evaluations (Beighton criteria), and obtained sera for COMP and hyaluronan (HA). COMP and HA, extensive joint radiographic and hypermobility data were also available for the GOGO cohort. Results The prevalence of hypermobility was 13% in the CARRIAGE and (5%) in the GOGO cohort. In the CARRIAGE family, hypermobility was associated with a significantly lower prevalence of hand (especially proximal interphalangeal joint) and knee OA, and lower mean serum COMP in both the total cohort and non-hand OA subgroups. These results were further validated in the GOGO subsets without radiographic OA where hypermobility was also associated with a significantly lower mean serum COMP (p<0.01). Serum HA did not differ on the basis of hypermobility in either cohort. Conclusions We report an inverse relationship of hypermobility, hand and knee OA, and show that hypermobility is associated with lower serum COMP levels. Genetic variations of the COMP gene may account for some subgroups of benign joint hypermobility. PMID:19035482

  9. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins.

    PubMed

    Rosario, Karyna; Schenck, Ryan O; Harbeitner, Rachel C; Lawler, Stephanie N; Breitbart, Mya

    2015-01-01

    Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA) viruses that encode a conserved replication initiator protein (Rep) in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA) viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap) encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs), which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses.

  10. Notch and Delta mRNAs in early-stage and mid-stage Drosophila embryos exhibit complementary patterns of protein producing potentials

    PubMed Central

    Shepherd, Andrew; Wesley, Uma; Wesley, Cedric

    2010-01-01

    Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3′ processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early-stage and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3′ UTR and mRNA 3′ processing factors. PMID:20201103

  11. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins

    PubMed Central

    Rosario, Karyna; Schenck, Ryan O.; Harbeitner, Rachel C.; Lawler, Stephanie N.; Breitbart, Mya

    2015-01-01

    Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA) viruses that encode a conserved replication initiator protein (Rep) in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA) viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap) encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs), which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses. PMID:26217327

  12. Analysis of the expression pattern of the carrier protein transthyretin and its receptor megalin in the human scalp skin and hair follicles: hair cycle-associated changes.

    PubMed

    Adly, Mohamed A

    2010-12-01

    Transthyretin is a serum and cerebrospinal fluid protein synthesized early in development by the liver, choroid plexus and several other tissues. It is a carrier protein for the antioxidant vitamins, retinol, and thyroid hormones. Transthyretin helps internalize thyroxine and retinol-binding protein into cells by binding to megalin, which is a multi-ligand receptor expressed on the luminal surface of various epithelia. We investigated the expression of transthyretin and its receptor megalin in the human skin; however, their expression pattern in the hair follicle is still to be elucidated. This study addresses this issue and tests the hypothesis that "the expression of transthyretin and megalin undergoes hair follicle cycle-dependent changes." A total of 50 normal human scalp skin biopsies were examined (healthy females, 53-62 years) using immunofluorescence staining methods and real-time PCR. In each case, 50 hair follicles were analyzed (35, 10, and 5 follicles in anagen, catagen, and telogen, respectively). Transthyretin and megalin were prominently expressed in the human scalp skin and hair follicles, on both gene and protein levels. The concentrations of transthyretin and megalin were 0.12 and 0.03 Ul/ml, respectively, as indicated by PCR. The expression showed hair follicle cycle-associated changes i.e., strong expression during early and mature anagen, very weak expression during catagen and moderate expression during telogen. The expression values of these proteins in the anagen were statistically significantly higher than those of either catagen or telogen hair follicles (P ≤ 0.001). This study provides the first morphologic indication that transthyretin and megalin are variably expressed in the human scalp skin and hair follicles. It also reports variations in the expression of these proteins during hair follicle cycling. The clinical ramifications of these findings are open for further investigations.

  13. Improvement of reduced serum cartilage oligomeric matrix protein levels in systemic juvenile idiopathic arthritis patients treated with the anti-interleukin-6 receptor monoclonal antibody tocilizumab.

    PubMed

    Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

    2009-01-01

    In this study, we determined serum cartilage oligomeric matrix protein (COMP) levels in systemic juvenile idiopathic arthritis (sJIA) patients during both the active and the remission phases to investigate how the growth cartilage turnover changed under tocilizumab treatment. Specimens were collected from 201 healthy children under 16 years of age with no growth impairment, and paired sera were collected from 11 sJIA patients treated with tocilizumab. Disease activity was assessed from white blood cell count, erythrocyte sedimentation rate, C-reactive protein, and ferritin, and the COMP concentration was determined by sandwich enzyme-linked immunosorbent assay. Serum COMP concentrations were found independent of age, and the mean value in healthy children was 17.74+/-5.6 U/L. The mean serum COMP in sJIA patients during the active phase was 10.75+/-3.9 U/L, lower than that of healthy children. The mean serum COMP in the remission phase (14.89+/-3.9 U/L) was significantly higher than that in the active period (P<0.05). These results suggested that in sJIA patients, a reduced serum COMP concentration is a useful marker of active disease and growth impairment, and that the growth cartilage turnover suppressed during the active phase is improved in the remission phase under tocilizumab treatment.

  14. Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson's disease patients.

    PubMed

    Lippolis, Rosa; Siciliano, Rosa Anna; Pacelli, Consiglia; Ferretta, Anna; Mazzeo, Maria Fiorella; Scacco, Salvatore; Papa, Francesco; Gaballo, Antonio; Dell'Aquila, Claudia; De Mari, Michele; Papa, Sergio; Cocco, Tiziana

    2015-09-01

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.

  15. Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease

    PubMed Central

    Williams, Marshall V.; Cox, Brandon; Ariza, Maria Eugenia

    2016-01-01

    The human herpesviruses are ubiquitous viruses and have a prevalence of over 90% in the adult population. Following a primary infection they establish latency and can be reactivated over a person’s lifetime. While it is well accepted that human herpesviruses are implicated in numerous diseases ranging from dermatological and autoimmune disease to cancer, the role of lytic proteins in the pathophysiology of herpesvirus-associated diseases remains largely understudies. Only recently have we begun to appreciate the importance of lytic proteins produced during reactivation of the virus, in particular the deoxyuridine triphosphate nucleotidohydrolases (dUTPase), as key modulators of the host innate and adaptive immune responses. In this review, we provide evidence from animal and human studies of the Epstein–Barr virus as a prototype, supporting the notion that herpesviruses dUTPases are a family of proteins with unique immunoregulatory functions that can alter the inflammatory microenvironment and thus exacerbate the immune pathology of herpesvirus-related diseases including myalgic encephalomyelitis/chronic fatigue syndrome, autoimmune diseases, and cancer. PMID:28036046

  16. Herpesviruses dUTPases: A New Family of Pathogen-Associated Molecular Pattern (PAMP) Proteins with Implications for Human Disease.

    PubMed

    Williams, Marshall V; Cox, Brandon; Ariza, Maria Eugenia

    2016-12-28

    The human herpesviruses are ubiquitous viruses and have a prevalence of over 90% in the adult population. Following a primary infection they establish latency and can be reactivated over a person's lifetime. While it is well accepted that human herpesviruses are implicated in numerous diseases ranging from dermatological and autoimmune disease to cancer, the role of lytic proteins in the pathophysiology of herpesvirus-associated diseases remains largely understudies. Only recently have we begun to appreciate the importance of lytic proteins produced during reactivation of the virus, in particular the deoxyuridine triphosphate nucleotidohydrolases (dUTPase), as key modulators of the host innate and adaptive immune responses. In this review, we provide evidence from animal and human studies of the Epstein-Barr virus as a prototype, supporting the notion that herpesviruses dUTPases are a family of proteins with unique immunoregulatory functions that can alter the inflammatory microenvironment and thus exacerbate the immune pathology of herpesvirus-related diseases including myalgic encephalomyelitis/chronic fatigue syndrome, autoimmune diseases, and cancer.

  17. A peptidomic analysis of human milk digestion in the infant stomach reveals protein-specific degradation patterns.

    PubMed

    Dallas, David C; Guerrero, Andrés; Khaldi, Nora; Borghese, Robyn; Bhandari, Aashish; Underwood, Mark A; Lebrilla, Carlito B; German, J Bruce; Barile, Daniela

    2014-06-01

    In vitro digestion of isolated milk proteins results in milk peptides with a variety of actions. However, it remains unclear to what degree protein degradation occurs in vivo in the infant stomach and whether peptides previously annotated for bioactivity are released. This study combined nanospray LC separation with time-of-flight mass spectrometry, comprehensive structural libraries, and informatics to analyze milk from 3 human mothers and the gastric aspirates from their 4- to 12-d-old postpartum infants. Milk from the mothers contained almost 200 distinct peptides, demonstrating enzymatic degradation of milk proteins beginning either during lactation or between milk collection and feeding. In the gastric samples, 649 milk peptides were identified, demonstrating that digestion continues in the infant stomach. Most peptides in both the intact milk and gastric samples were derived from β-casein. The numbers of peptides from β-casein, lactoferrin, α-lactalbumin, lactadherin, κ-casein, serum albumin, bile salt-associated lipase, and xanthine dehydrogenase/oxidase were significantly higher in the gastric samples than in the milk samples (P < 0.05). A total of 603 peptides differed significantly in abundance between milk and gastric samples (P < 0.05). Most of the identified peptides have previously identified biologic activity. Gastric proteolysis occurs in the term infant in the first 2 wk of life, releasing biologically active milk peptides with immunomodulatory and antibacterial properties of clinical relevance to the proximal intestinal tract. Data are available via ProteomeXchange (identifier PXD000688).

  18. High-frequency retrotransposition of a marked I factor in Drosophila melanogaster correlates with a dynamic expression pattern of the ORF1 protein in the cytoplasm of oocytes.

    PubMed Central

    Seleme, M; Busseau, I; Malinsky, S; Bucheton, A; Teninges, D

    1999-01-01

    To study the expression of the I factor, a non-long-terminal-repeat retrotransposon responsible for I-R hybrid dysgenesis in Drosophila melanogaster, we have tagged the ORF1 protein (ORF1p) by inserting the HA epitope in its N-terminal region. In transgenic flies, this modification is compatible with a high rate of autonomous transposition and allows direct estimation of the transposition frequency. I factor transposes in the germline of females (SF) that are daughters from crosses between I strain males (which contain active copies of the I factor) and R strain females (which do not). We analyzed the expression pattern of ORF1p by indirect immunofluorescence. Its expression correlates with retrotransposition. During oogenesis ORF1p appears unexpectedly as a cytoplasmic product, which accumulates with a specific pattern into the oocyte. A comparison of the expression patterns under conditions that modify the transposing activity of the element clarifies some aspects of I-factor functioning in the transposition process. PMID:9927467

  19. The Alzheimer's Disease-Associated R47H Variant of TREM2 Has an Altered Glycosylation Pattern and Protein Stability

    PubMed Central

    Park, Ji-Seon; Ji, In Jung; Kim, Dong-Hou; An, Hyun Joo; Yoon, Seung-Yong

    2017-01-01

    The R47H coding variant of the triggering receptor expressed on myeloid cells-2 (TREM2) increases the risk of Alzheimer's disease (AD) similar to apolipoprotein E4. TREM2 R47H has recently been shown to have impaired binding to damage-associated lipid or apolipoprotein ligands. However, it is not known how this R47H variant affects the biochemical characteristics of TREM2 and alters the pathogenesis of AD. We previously reported that TREM2-R47H has a slightly different glycosylation pattern from wild-type. A more detailed characterization in our present study confirms that TREM2 R47H has an altered glycosylation pattern and reduced stability. TREM2 R47H shows different glycosylation profiles from analysis using monensin or kifunensine treatment which were confirmed by mass spectrometry. The solubility of TREM2 R47H and its cleaved products such as intracellular domain (ICD) is also decreased, increasing its proteasomal and lysosomal degradation. The different biochemical characteristics of TREM2 R47H, including glycosylation, solubility and processing, may offer insights into a future therapeutic strategy for AD. PMID:28149270

  20. Construction and Analysis of cDNA Libraries from the Antennae of Batocera horsfieldi and Expression Pattern of Putative Odorant Binding Proteins

    PubMed Central

    Li, Hui; Zhang, Aijun; Chen, Li-Zhen; Zhang, Guoan; Wang, Man-Qun

    2014-01-01

    A high-quality cDNA library was constructed from female and male antenna of the longhorned beetle, Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae), a serious pest of Populus (Salicales: Salicaceae). The titer was approximately 2.37 × 106 pfu/mL, and this complies with the test requirement. From the libraries, 692 clones were selected randomly, sequenced, and further analyzed, and the recombinational efficiency reached 93.85%. By alignment and cluster analysis, we identified four odorant binding proteins, two pheromone-binding proteins (have the characteristic six conserved cysteine residues), four Minus-C odorant binding proteins (lost two conserved cysteines), and three chemosensory proteins. In this study, we describe the identification and characterization of four new cDNAs that encode Minus-C odorant binding proteins (Minus-C OBPs) from B. horsfieldi antennal cDNA libraries. Our investigation focused on the expression pattern of the Minus-C OBP genes in various tissues in both sexes at different developmental stages, using reverse transcription PCR (RT-PCR) and real-time PCR (qPCR) strategies. Minus-C OBP1, 2, and 3 were expressed in all tested tissues, with the exception of the head (without antenna, labial palps, and maxillary palps). Minus-C OBP4 was expressed in the antenna, legs, and abdomen, but not in the labial palps, maxillary palps, or head. The qPCR results revealed Minus- C OBPs were expressed in the antenna throughout the adult life, and that the transcript levels of these genes depended on the sex, age, and mating status of adults. PMID:25373204

  1. Characterization of Mus musculus Papillomavirus 1 Infection In Situ Reveals an Unusual Pattern of Late Gene Expression and Capsid Protein Localization

    PubMed Central

    Handisurya, Alessandra; Day, Patricia M.; Thompson, Cynthia D.; Buck, Christopher B.; Pang, Yuk-Ying S.; Lowy, Douglas R.

    2013-01-01

    Full-length genomic DNA of the recently identified laboratory mouse papillomavirus 1 (MusPV1) was synthesized in vitro and was used to establish and characterize a mouse model of papillomavirus pathobiology. MusPV1 DNA, whether naked or encapsidated by MusPV1 or human papillomavirus 16 (HPV 16) capsids, efficiently induced the outgrowth of papillomas as early as 3 weeks after application to abraded skin on the muzzles and tails of athymic NCr nude mice. High concentrations of virions were extracted from homogenized papillomatous tissues and were serially passaged for >10 generations. Neutralization by L1 antisera confirmed that infectious transmission was capsid mediated. Unexpectedly, the skin of the murine back was much less susceptible to virion-induced papillomas than the muzzle or tail. Although reporter pseudovirions readily transduced the skin of the back, infection with native MusPV1 resulted in less viral genome amplification and gene expression on the back, including reduced expression of the L1 protein and very low expression of the L2 protein, results that imply skin region-specific control of postentry aspects of the viral life cycle. Unexpectedly, L1 protein on the back was predominantly cytoplasmic, while on the tail the abundant L1 was cytoplasmic in the lower epithelial layers and nuclear in the upper layers. Nuclear localization of L1 occurred only in cells that coexpressed the minor capsid protein, L2. The pattern of L1 protein staining in the infected epithelium suggests that L1 expression occurs earlier in the MusPV1 life cycle than in the life cycle of high-risk HPV and that virion assembly is regulated by a previously undescribed mechanism. PMID:24067981

  2. Microtubule-associated protein AtMPB2C plays a role in organization of cortical microtubules, stomata patterning, and tobamovirus infectivity.

    PubMed

    Ruggenthaler, Pia; Fichtenbauer, Daniela; Krasensky, Julia; Jonak, Claudia; Waigmann, Elisabeth

    2009-03-01

    AtMPB2C is the Arabidopsis (Arabidopsis thaliana) homolog of MPB2C, a microtubule-associated host factor of tobacco mosaic virus movement protein that was been previously identified in Nicotiana tabacum. To analyze the endogenous function of AtMPB2C and its role in viral infections, transgenic Arabidopsis plant lines stably overexpressing green fluorescent protein (GFP)-AtMPB2C were established. The GFP-AtMPB2C fusion protein was detectable in various cell types and organs and localized at microtubules in a punctuate pattern or in filaments. To determine whether overexpression impacted on the cortical microtubular cytoskeleton, GFP-AtMPB2C-overexpressing plants were compared to known microtubular marker lines. In rapidly elongated cell types such as vein cells and root cells, GFP-AtMPB2C overexpression caused highly unordered assemblies of cortical microtubules, a disturbed, snake-like microtubular shape, and star-like crossing points of microtubules. Phenotypically, GFP-AtMPB2C transgenic plants showed retarded growth but were viable and fertile. Seedlings of GFP-AtMPB2C transgenic plants were characterized by clockwise twisted leaves, clustered stomata, and enhanced drought tolerance. GFP-AtMPB2C-overexpressing plants showed increased resistance against oilseed rape mosaic virus, a close relative of tobacco mosaic virus, but not against cucumber mosaic virus when compared to Arabidopsis wild-type plants. These results suggest that AtMPB2C is involved in the alignment of cortical microtubules, the patterning of stomata, and restricting tobamoviral infections.

  3. Alterations in the fatty acid profile, antioxidant enzymes and protein pattern of Biomphalaria alexandrina snails exposed to the pesticides diazinon and profenfos.

    PubMed

    Bakry, Fayez A; El-Hommossany, Karem; Abd El-Atti, Mahmoud; Ismail, Somaya M

    2016-04-01

    The use of pesticides is widespread in agricultural activities. These pesticides may contaminate the irrigation and drainage systems during agriculture activities and pests' control and then negatively affect the biotic and a biotic component of the polluted water courses. The present study aimed to evaluate the effect of the pesticides diazinon and profenfos on some biological activities of Biomphalaria alexandrina snails such as fatty acid profile, some antioxidant enzymes (thioredoxin reductase (TrxR), sorbitol dehydrogenase (SDH), superoxide dismutase (SOD), catalase (CAT) as well as glutathione reductase (GR) and lipid peroxidation (LP)) and protein patterns in snails' tissues exposed for 4 weeks to LC10 of diazinon and profenfos. The results showed that the two pesticides caused considerable reduction in survival rates and egg production of treated snails. Identification of fatty acid composition in snail tissues treated with diazinon and profenfos pesticides was carried out using gas-liquid chromatography (GLC). The results declared alteration in fatty acid profile, fluctuation in percentage of long chain and short chain fatty acid contributions either saturated or unsaturated ones, and a decrease in total lipid content in tissues of snails treated with these pesticides. The data demonstrate that there was a significant inhibition in the activities of tissues SOD, CAT, glutathione reductase (GR), TrxR, and SDH in tissues of treated snails, while a significant elevation was detected in LP as compared to the normal control. On the other hand, the electrophoretic pattern of total protein showed differences in number and molecular weights of protein bands due to the treatment of snails. It was concluded that the residues of diazinon and profenfos pesticides in aquatic environments have toxic effects onB. alexandrina snails.

  4. Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

    PubMed

    Kennedy, Chelsey E; Krieger, Kari Beth; Sutovsky, Miriam; Xu, Wei; Vargovič, Peter; Didion, Bradley A; Ellersieck, Mark R; Hennessy, Madison E; Verstegen, John; Oko, Richard; Sutovsky, Peter

    2014-05-01

    Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility.

  5. Gene expression pattern and immunoreactive protein localization of LGR7 receptor in human endometrium throughout the menstrual cycle.

    PubMed

    Luna, José Juan; Riesewijk, Anne; Horcajadas, José A; Van Os Rd, Roselin de; Domínguez, Francisco; Mosselman, Sietse; Pellicer, Antonio; Simón, Carlos

    2004-02-01

    Relaxin (RLX) is a pregnancy-associated polypeptide hormone. In non-pregnant women, the peak of circulating relaxin coincides with the window of endometrial receptivity and both in vivo and in vitro experiments showed that it plays a role in the decidualization process. Recently, two receptors, LGR7 and LGR8, have been identified as high affinity receptors for relaxin. Here we describe LGR7 mRNA and protein expression in human endometrium using semi-quantitative and quantitative fluorescent PCR (Q-PCR) and immunohistochemical analyses. Three different experimental designs were used. First, endometrial biopsies from five different phases of the menstrual cycle were analysed. Secondly, we assessed the early luteal phase in more detail. Finally we analysed the expression at LH+2 (2 days after the natural LH surge, pre-receptive endometrium) versus LH+7 (receptive endometrium) within the same menstrual cycle from the same patient to avoid inter-cycle or inter-person variations in gene expression. Our results indicate that there is no consistent regulation of LGR7 mRNA expression, neither during the menstrual cycle nor during the early-mid-luteal phase. In general, we observed a large degree of variation in LGR7 mRNA expression levels between patients. LGR7 immunoreactive protein was identified in all stages of the menstrual cycle. LGR7 protein was localized in both the epithelial and the stromal compartments, except for the mid-luteal phase when the expression was restricted to the endometrial epithelium. We conclude that no consistent regulation of LGR7 mRNA expression can be detected in human endometrium during the menstrual cycle.

  6. Aging, Alzheimer's, and APOE genotype influence the expression and neuronal distribution patterns of microtubule motor protein dynactin-P50

    PubMed Central

    Aboud, Orwa; Parcon, Paul A.; DeWall, K. Mark; Liu, Ling; Mrak, Robert E.; Griffin, W. Sue T.

    2015-01-01

    Reports from neural cell cultures and experimental animal studies provide evidence of age- and disease-related changes in retrograde transport of spent or misfolded proteins destined for degradation or recycling. However, few studies address these issues in human brain from those who either age without dementia and overt neuropathology, or succumb to Alzheimer's; especially as such propensity may be influenced by APOE genotype. We studied the expression and distribution of the dynein subunit dynactin-P50, the β amyloid precursor protein (βAPP), and hyperphosphorylated tau (P-tau) in tissues and tissue sections of brains from non-demented, neuropathology-free patients and from Alzheimer patients, with either APOE ε3,3 or APOE ε4,4. We found that advanced age in patients without dementia or neuropathological change was associated with coordinated increases in dynactin-P50 and βAPP in neurons in pyramidal layers of the hippocampus. In contrast, in Alzheimer's, βAPP and dynactin were significantly reduced. Furthermore, the dynactin-P50 and βAPP that was present was located primarily in dystrophic neurites in Aβ plaques. Tissues from Alzheimer patients with APOE ε3,3 had less P-tau, more βAPP, dynactin-P50, and synaptophysin than did tissues from Alzheimer patients carrying APOE ε4,4. It is logical to conclude, then, that as neurons age successfully, there is coordination between retrograde delivery and maintenance and repair, as well as between retrograde delivery and degradation and/or recycling of spent proteins. The buildup of proteins slated for repair, synaptic viability, transport, and re-cycling in neuron soma and dystrophic neurites suggest a loss of this coordination in Alzheimer neurons. Inheritance of APOE ε3,3 rather than APOE ε4,4, is associated with neuronal resilience, suggestive of better repair capabilities, more synapses, more efficient transport, and less hyperphosphorylation of tau. We conclude that even in disease the ε3 allele is

  7. Differential sensing using proteins: exploiting the cross-reactivity of serum albumin to pattern individual terpenes and terpenes in perfume.

    PubMed

    Adams, Michelle M; Anslyn, Eric V

    2009-12-02

    There has been a growing interest in the use of differential sensing for analyte classification. In an effort to mimic the mammalian senses of taste and smell, which utilize protein-based receptors, we have introduced serum albumins as nonselective receptors for recognition of small hydrophobic molecules. Herein, we employ a sensing ensemble consisting of serum albumins, a hydrophobic fluorescent indicator (PRODAN), and a hydrophobic additive (deoxycholate) to detect terpenes. With the aid of linear discriminant analysis, we successfully applied our system to differentiate five terpenes. We then extended our terpene analysis and utilized our sensing ensemble for terpene discrimination within the complex mixtures found in perfume.

  8. Ontogenic expression pattern and genetic polymorphisms of the retinol-binding protein 4 (RBP4) gene in Erlang mountainous chickens.

    PubMed

    Yin, Hua-Dong; Gilbert, Elizabeth R; Chen, Shi-Yi; Li, Di-Yan; Zhang, Zhi-Chao; Wang, Yan; Liu, Yi-Ping; Zhu, Qing

    2013-09-10

    Retinol-binding protein 4 (RBP4) is the only circulatory transport protein for vitamin A. Based on the essential role of vitamin A in chicken reproduction, we measured RBP4 mRNA abundance in Erlang mountainous chickens. We also identified and analyzed the gene polymorphism and its effect on reproduction traits among 349 chickens. The expression of RBP4 mRNA showed specific developmental changes and striking differences among tissues. The mRNA abundance was greatest (P<0.05) in the liver, intermediate in the ovary, kidney, small intestine, oviduct and heart, and lowest in the hypothalamus and pituitary, as compared to all other tissues (P<0.05). We detected one single nucleotide polymorphism (g.19942455C>G) in intron 2 of the RBP4 gene. Three genotypes (CC, CG and GG) were identified, with a significant effect of genotype on the age at first egg (AFE), first egg weight (FEW), total eggs at 300 days (TE300), highest continuous laying days (HCLD) and average laying interval (ALI). The GG genotype, where chickens display earlier AFE, more TE300, longer HCLD and shorter ALI, would be genetically advantageous and its selection may improve reproduction traits. These results suggested that the RBP4 gene might play an important role in reproduction traits in chickens.

  9. Different temporal patterns in the expressions of bone morphogenetic proteins and noggin during astroglial scar formation after ischemic stroke.

    PubMed

    Shin, Jin A; Kang, Jihee Lee; Lee, Kyung-Eun; Park, Eun-Mi

    2012-05-01

    Bone morphogenetic proteins (BMPs) and their antagonists have roles in scar formation and regeneration after central nervous system injuries. However, temporal changes in their expression during astroglial scar formation in the ischemic brain are unknown. Here, we examined protein levels of BMP2, BMP7, and their antagonist noggin in the ischemic brain up to 4 weeks after experimental stroke in mice. BMP2 and BMP7 levels were increased from 1 to 4 weeks in the ischemic brain, and their expression was associated with astrogliosis. BMP7 expression was more intense and co-localized in reactive astrocytes in the ischemic subcortex at 1 week. Noggin expression began to increase after 2 weeks and was further increased at 4 weeks only in the ischemic subcortex, but the intensity was weak compared to the intensity of BMPs. Noggin was co-localized mainly in activated microglia. These findings show that expression of BMPs and noggin differed over time, in intensity and in types of cell, and suggest that BMPs and noggin have different roles in the processes of glial scar formation and neurorestoration in the ischemic brain.

  10. Molecular characterization, phylogenetic analysis and expression patterns of five protein arginine methyltransferase genes of channel catfish, Ictalurus punctatus (Rafinesque).

    PubMed

    Yeh, Hung-Yueh; Klesius, Phillip H

    2012-08-01

    Protein arginine methylation, catalyzed by protein arginine methyltransferases (PRMT), has recently emerged as an important modification in the regulation of gene expression. In this communication, we identified and characterized the channel catfish orthologs to human PRMT 1, 3, 4 and 5, and PRMT4 like. Each PRMT nucleic acid sequence has an open reading frame (ORF) and 3'-untranslated regions. Each ORF appears to encode 361, 587 and 458 amino acid residues for PRMT1, PRMT4 and variant, respectively. The partial ORF of PRMT3 and PRMT5 encode 292 and 563 amino acids, respectively. By comparison with the human counterparts, each channel catfish PRMT also has conserved domains. For expression profile, the channel catfish PRMT1 transcript was detected by RT-PCR in spleens, anterior kidneys, livers, intestines, skin and gills of fish examined. Except in liver, the PRMT3 transcript was detected in all catfish tissues examined. However, the PRMT4 cDNA was detected in livers from all three catfish and gills from two fish, but not other tissues. This information will enable us to further elucidate PRMT functions in channel catfish.

  11. DNA methylation patterns of protein-coding genes and long non-coding RNAs in males with schizophrenia.

    PubMed

    Liao, Qi; Wang, Yunliang; Cheng, Jia; Dai, Dongjun; Zhou, Xingyu; Zhang, Yuzheng; Li, Jinfeng; Yin, Honglei; Gao, Shugui; Duan, Shiwei

    2015-11-01

    Schizophrenia (SCZ) is one of the most complex mental illnesses affecting ~1% of the population worldwide. SCZ pathogenesis is considered to be a result of genetic as well as epigenetic alterations. Previous studies have aimed to identify the causative genes of SCZ. However, DNA methylation of long non-coding RNAs (lncRNAs) involved in SCZ has not been fully elucidated. In the present study, a comprehensive genome-wide analysis of DNA methylation was conducted using samples from two male patients with paranoid and undifferentiated SCZ, respectively. Methyl-CpG binding domain protein-enriched genome sequencing was used. In the two patients with paranoid and undifferentiated SCZ, 1,397 and 1,437 peaks were identified, respectively. Bioinformatic analysis demonstrated that peaks were enriched in protein-coding genes, which exhibited nervous system and brain functions. A number of these peaks in gene promoter regions may affect gene expression and, therefore, influence SCZ-associated pathways. Furthermore, 7 and 20 lncRNAs, respectively, in the Refseq database were hypermethylated. According to the lncRNA dataset in the NONCODE database, ~30% of intergenic peaks overlapped with novel lncRNA loci. The results of the present study demonstrated that aberrant hypermethylation of lncRNA genes may be an important epigenetic factor associated with SCZ. However, further studies using larger sample sizes are required.

  12. Mutual control of intracellular localisation of the patterning proteins AtMYC1, GL1 and TRY/CPC in Arabidopsis.

    PubMed

    Pesch, Martina; Schultheiß, Ilka; Digiuni, Simona; Uhrig, Joachim F; Hülskamp, Martin

    2013-08-01

    Trichome and root hair patterning is governed by a gene regulatory network involving TTG1 and several homologous MYB and bHLH proteins. The bHLH proteins GL3 and EGL3 are core components that serve as a regulatory platform for the activation of downstream genes. In this study we show that a homologue of GL3 and EGL3, AtMYC1, can regulate the intracellular localisation of GL1 and TRY. AtMYC1 protein is predominantly localised in the cytoplasm and can relocate GL1 from the nucleus into the cytoplasm. Conversely, AtMYC1 can be recruited into the nucleus by TRY and CPC, concomitant with a strong accumulation of TRY and CPC in the nucleus. When AtMYC1 is targeted to the nucleus or cytoplasm by nuclear localisation or export signals (NLS or NES), respectively, the intracellular localisation of GL1 and TRY also changes accordingly. The biological significance of this intracellular localisation is suggested by the finding that the efficiency of rescue of trichome number is significantly altered in NES and NLS fusions as compared with wild-type AtMYC1. Genetic analysis of mutants and overexpression lines supports the hypothesis that AtMYC1 represses the activity of TRY and CPC.

  13. Collagen XII and XIV, new partners of cartilage oligomeric matrix protein in the skin extracellular matrix suprastructure.

    PubMed

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-06-29

    The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.

  14. Pattern recognition of monocyte chemoattractant protein-1 (MCP-1) in whole blood samples using new platforms based on nanostructured materials

    NASA Astrophysics Data System (ADS)

    Stefan-van Staden, Raluca-Ioana; Gugoasa, Livia Alexandra; Biris, Alexandru Radu

    2015-09-01

    Four stochastic microsensors based on nanostructured materials (graphene, maltodextrin (MD), and diamond) integrated in miniaturized platforms were proposed. Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory cytokine whose main function is to regulate cell trafficking. It is correlated with the incidence of cardiovascular diseases and obesity, and was used as the model analyte in this study. The screening of whole blood samples for MCP-1 can be done for concentrations ranging from 10-12 to 10-8 g mL-1. The method was used for both qualitative and quantitative assessments of MCP-1 in whole blood samples. The lowest quantification limits for the assay of MCP-1 (1 pg mL-1) were reached when the microsensors based on protoporphyrin IX/Graphene-Au-3 and on MD/Graphene were employed in the platform design.

  15. Molecular characterization of two small heat shock protein genes in rice: their expression patterns, localizations, networks, and heterogeneous overexpressions.

    PubMed

    Ham, Deok-Jae; Moon, Jun-Chul; Hwang, Sun-Goo; Jang, Cheol Seong

    2013-09-28

    Heat stress is an example of a severe abiotic stress that plants can suffer in the field, causing a significant detrimental effect on their growth and productivity. Understanding the mechanism of plant response to heat stress is important for improving the productivity of crop plants under global warming. We used a microarray dataset that is deposited in the public database to evaluate plant responses to heat stress, and we selected the top 10 genes that are highly expressed under heat stress in rice. Two genes, OsSHSP1 (Os03g16030) and OsSHSP2 (Os01g04380), were selected for further study. These genes were highly induced in response to salt and drought but not in response to cold. In addition, OsSHSP1 and OsSHSP2 gene transcripts were induced under abscisic acid and salicylic acid but not under jasmonic acid and ethylene. Subcellular localization of proteins of 35S::OsSHSP1 were associated with the cytosol, whereas those of and 35S::OsSHSP2 were associated with the cytosol and nucleus. Heterogeneous overexpression of both genes exhibited higher germination rates than those of wild-type plants under the salt treatment, but not under heat or drought stress, supporting a hypothesis regarding functional specialization of members of small heat-shock protein family over evolutionary time. The network of both genes harboring nine sHSPs as well as at least 13 other chaperone genes might support the idea of a role for sHSPs in the chaperone network. Our findings might provide clues to shed light on the molecular functions of OsSHSP1 and OsSHSP2 in response to abiotic stresses, especially heat stress.

  16. Strong functional patterns in the evolution of eukaryotic genomes revealed by the reconstruction of ancestral protein domain repertoires

    PubMed Central

    2011-01-01

    Background Genome size and complexity, as measured by the number of genes or protein domains, is remarkably similar in most extant eukaryotes and generally exhibits no correlation with their morphological complexity. Underlying trends in the evolution of the functional content and capabilities of different eukaryotic genomes might be hidden by simultaneous gains and losses of genes. Results We reconstructed the domain repertoires of putative ancestral species at major divergence points, including the last eukaryotic common ancestor (LECA). We show that, surprisingly, during eukaryotic evolution domain losses in general outnumber domain gains. Only at the base of the animal and the vertebrate sub-trees do domain gains outnumber domain losses. The observed gain/loss balance has a distinct functional bias, most strikingly seen during animal evolution, where most of the gains represent domains involved in regulation and most of the losses represent domains with metabolic functions. This trend is so consistent that clustering of genomes according to their functional profiles results in an organization similar to the tree of life. Furthermore, our results indicate that metabolic functions lost during animal evolution are likely being replaced by the metabolic capabilities of symbiotic organisms such as gut microbes. Conclusions While protein domain gains and losses are common throughout eukaryote evolution, losses oftentimes outweigh gains and lead to significant differences in functional profiles. Results presented here provide additional arguments for a complex last eukaryotic common ancestor, but also show a general trend of losses in metabolic capabilities and gain in regulatory complexity during the rise of animals. PMID:21241503

  17. Pattern of expression of transforming growth factor-beta 4 mRNA and protein in the developing chicken embryo.

    PubMed

    Jakowlew, S B; Ciment, G; Tuan, R S; Sporn, M B; Roberts, A B

    1992-12-01

    Expression of TGF-beta 4 mRNA and protein was studied in the developing chicken embryo using specific cDNA probes and antibodies for chicken TGF-beta 4. Expression of TGF-beta 4 mRNA was detected by day 4 of incubation (Hamburger and Hamilton stage 22, E4) by RNA Northern blot analysis and increased with developmental age until day 12 of incubation (stage 38, E12) where it was detected in every embryonic tissue examined, with expression being highest in smooth muscle and lowest in the kidney. The steady-state level of expression of TGF-beta 4 mRNA remained relatively constant in most embryonic tissues through day 19 (stage 45, E19). In situ hybridization analysis detected TGF-beta 4 mRNA as early as the "definitive primitive streak" stage (stage 4); during neurulation (stage 10), TGF-beta 4 mRNA was detected in all three germ layers, including neuroectoderm. Following neurulation, TGF-beta 4 mRNA was detected in the neural tube, notochord, ectoderm, endoderm, sclerotome, and myotome, but not dermotome at stage 16. By day 6 of incubation (stage 29, E6), TGF-beta 4 mRNA was localized in several tissues including heart, lung, and gizzard. Immunohistochemical staining analysis also showed expression of TGF-beta 4 protein in all three germ layers as early as stage 4 in various cell types in qualitatively similar locations as TGF-beta 4 mRNA. These results suggest that TGF-beta 4 may play an important role in the development of many tissues in the chicken.

  18. Distribution pattern of versican, link protein and hyaluronic acid in the rat periodontal ligament during experimental tooth movement.

    PubMed

    Sato, R; Yamamoto, H; Kasai, K; Yamauchi, M

    2002-02-01

    The ability of the periodontal ligament (PDL) to rapidly remodel is the basis of orthodontic tooth movement. During the tooth movement, matrix proteoglycans (PGs) may play important roles in spatial, mechanical and biological aspects for the maintenance and repair of the PDL. The aim of this study was to characterize the distribution of a large hyaluronic acid (HA)-binding proteoglycan, versican, link protein (LP) and HA in the rat molar PDL during experimental tooth movement by histochemical and immunohistochemical methods. Experimental tooth movement was performed according to Waldo's method. Histologically, regressive changes, such as decrease of fibroblasts and collagen fibers and exudative change of edema were observed in the compressive side and progressive changes, such as proliferation of fibroblasts and collagen fibers, in the strain side one day after treatment. By 3 days after tooth movement, regressive or progressive changes were not observed in either side. Using monoclonal antibodies specific to versican core protein or LP, the positive immunoreactivity for both molecules was constantly observed throughout the PDL. After the experimental force was applied to the tooth, however, the immunostainings of versican and LP became significantly intense only in the compressive side but decreased in the strain side. The intensity in the compressive side was strongest one day after the force was applied and gradually diminished thereafter. HA of both sides did not change during experimental tooth movement. Since HA is present in the PDL, large amounts of versican and LP expressed in the compressive side may create large hydrated aggregates via their association with HA that dissipates the compressive force applied to this tissue.

  19. Cellular Redox Imbalance and Changes of Protein S-glutathionylation Patterns Are Associated with Senescence Induced by Oncogenic H-Ras

    PubMed Central

    Urbanelli, Lorena; Magini, Alessandro; Magherini, Francesca; Pugnaloni, Armanda; Piva, Francesco; Modesti, Alessandra; Emiliani, Carla; Principato, Giovanni

    2012-01-01

    H-Ras oncogene requires deregulation of additional oncogenes or inactivation of tumor suppressor proteins to increase cell proliferation rate and transform cells. In fact, the expression of the constitutively activated H-RasV12 induces cell growth arrest and premature senescence, which act like barriers in pre-neoplastic lesions. In our experimental model, human fibroblasts transfected with H-RasV12 show a dramatic modification of morphology. H-RasV12 expressing cells also show premature senescence followed by cell death, induced by autophagy and apoptosis. In this context, we provide evidence that in H-RasV12 expressing cells, the premature senescence is associated with cellular redox imbalance as well as with altered post-translation protein modification. In particular, redox imbalance is due to a strong reduction of total antioxidant capacity, and significant decrease of glutathione level. As the reversible addition of glutathione to cysteinyl residues of proteins is an important post-translational regulative modification, we investigated S-glutathionylation in cells expressing active H-Ras. In this contest we observed different S-glutathionylation patterns in control and H-RasV12 expressing cells. Particularly, the GAPDH enzyme showed S-glutathionylation increase and significant enzyme activity depletion in H-Ras V12 cells. In conclusion, we proposed that antioxidant defense reduction, glutathione depletion and subsequent modification of S-glutathionylation of target proteins contribute to arrest cell growth, leading to death of fibroblasts expressing constitutively active H-Ras oncogene, thus acting as oncogenic barriers that obstacle the progression of cell transformation. PMID:23284910

  20. Quantitative assessment of chromatin immunoprecipitation grade antibodies directed against histone modifications reveals patterns of co-occurring marks on histone protein molecules.

    PubMed

    Peach, Sally E; Rudomin, Emily L; Udeshi, Namrata D; Carr, Steven A; Jaffe, Jacob D

    2012-05-01

    The defining step in most chromatin immunoprecipitation (ChIP) assays is the use of an antibody to enrich for a particular protein or histone modification state associated with segments of chromatin. The specificity of the antibody is critical to the interpretation of the experiment, yet this property is rarely reported. Here, we present a quantitative method using mass spectrometry to characterize the specificity of key histone H3 modification-targeting antibodies that have previously been used to characterize the "histone code." We further extend the use of these antibody reagents to the observation of long range correlations among disparate histone modifications. Using purified human histones representing the mixture of chromatin states present in living cells, we were able to quantify the degree of target enrichment and the specificity of several commonly used, commercially available ChIP grade antibodies. We found significant differences in enrichment efficiency among various reagents directed against four frequently studied chromatin marks: H3K4me2, H3K4me3, H3K9me3, and H3K27me3. For some antibodies, we also detected significant off target enrichment of alternate modifications at the same site (i.e., enrichment of H3K4me2 by an antibody directed against H3K4me3). Through cluster analysis, we were able to recognize patterns of co-enrichment of marks at different sites on the same histone protein. Surprisingly, these co-enrichments corresponded well to "canonical" chromatin states that are exemplary of activated and repressed regions of chromatin. Altogether, our findings suggest that 1) the results of ChIP experiments need to be evaluated with caution given the potential for cross-reactivity of the commonly used histone modification recognizing antibodies, 2) multiple marks with consistent biological interpretation exist on the same histone protein molecule, and 3) some components of the histone code may be transduced on single proteins in living cells.

  1. Introns Structure Patterns of Variation in Nucleotide Composition in Arabidopsis thaliana and Rice Protein-Coding Genes

    PubMed Central

    Ressayre, Adrienne; Glémin, Sylvain; Montalent, Pierre; Serre-Giardi, Laurana; Dillmann, Christine; Joets, Johann

    2015-01-01

    Plant genomes present a continuous range of variation in nucleotide composition (G + C content). In coding regions, G + C-poor species tend to have unimodal distributions of G + C content among genes within genomes and slight 5′–3′ gradients along genes. In contrast, G + C-rich species display bimodal distributions of G + C content among genes and steep 5′–3′ decreasing gradients along genes. The causes of these peculiar patterns are still poorly understood. Within two species (Arabidopsis thaliana and rice), each representative of one side of the continuum, we studied the consequences of intron presence on coding region and intron G + C content at different scales. By properly taking intron structure into account, we showed that, in both species, intron presence is associated with step changes in nucleotide, codon, and amino acid composition. This suggests that introns have a barrier effect structuring G + C content along genes and that previous continuous characterizations of the 5′–3′ gradients were artifactual. In external gene regions (located upstream first or downstream last introns), species-specific factors, such as GC-biased gene conversion, are shaping G + C content whereas in internal gene regions (surrounded by introns), G + C content is likely constrained to remain within a range common to both species. PMID:26450849

  2. Introns Structure Patterns of Variation in Nucleotide Composition in Arabidopsis thaliana and Rice Protein-Coding Genes.

    PubMed

    Ressayre, Adrienne; Glémin, Sylvain; Montalent, Pierre; Serre-Giardi, Laurana; Dillmann, Christine; Joets, Johann

    2015-10-07

    Plant genomes present a continuous range of variation in nucleotide composition (G + C content). In coding regions, G + C-poor species tend to have unimodal distributions of G + C content among genes within genomes and slight 5'-3' gradients along genes. In contrast, G + C-rich species display bimodal distributions of G + C content among genes and steep 5'-3' decreasing gradients along genes. The causes of these peculiar patterns are still poorly understood. Within two species (Arabidopsis thaliana and rice), each representative of one side of the continuum, we studied the consequences of intron presence on coding region and intron G + C content at different scales. By properly taking intron structure into account, we showed that, in both species, intron presence is associated with step changes in nucleotide, codon, and amino acid composition. This suggests that introns have a barrier effect structuring G + C content along genes and that previous continuous characterizations of the 5'-3' gradients were artifactual. In external gene regions (located upstream first or downstream last introns), species-specific factors, such as GC-biased gene conversion, are shaping G + C content whereas in internal gene regions (surrounded by introns), G + C content is likely constrained to remain within a range common to both species.

  3. Painful locking of the wrist in a patient with pseudoachondroplasia confirmed by COMP mutation

    PubMed Central

    Ideta, Hirokazu; Uchiyama, Shigeharu; Hayashi, Masanori; Kosho, Tomoki; Nakamura, Yukio; Kato, Hiroyuki

    2017-01-01

    We report the case of a 40-year-old woman with pseudoachondroplasia (PSACH), with a heterozygous mutation (c.806A > G, p.Asp269Gly) located in the Type 3 repeats domain of the cartilage oligomeric matrix protein gene, who complained of the unusual symptom of painful locking of the wrist. Her condition was caused by a non-traumatic enlargement of the extensor carpi radialis longus (ECRL) and brevis (ECRB) tendons along with bulbous swelling of the synoviums around them. Surgical treatment resolved these unusual tendon-related symptoms. Repetitive mechanical loading of the wrist in daily activities, including distal intersection tenosynovitis between the extensor pollicis longus tendon and ECRL and ECRB tendons, may have contributed to changes in the structural integrity of the tendons. We should pay more attention to tendon-related symptoms in patients with PSACH. PMID:28044000

  4. Increased pentosidine, an advanced glycation end product, in serum and synovial fluid from patients with knee osteoarthritis and its relation with cartilage oligomeric matrix protein

    PubMed Central

    Senolt, L; Braun, M; Olejarova, M; Forejtova, S; Gatterova, J; Pavelka, K

    2005-01-01

    Background: Pentosidine, an advanced glycation end product, increasingly accumulates in articular cartilage with age, and contributes to the pathogenesis of osteoarthritis (OA). Increased pentosidine concentrations are associated with inflammatory disorders—for example, rheumatoid arthritis. Objective: To compare pentosidine serum concentrations in patients with knee OA and in healthy volunteers and to determine a relationship between pentosidine and cartilage oligomeric matrix protein (COMP)—a marker of articular cartilage destruction. Methods: Paired serum and synovial fluid samples were obtained by arthrocentesis from 38 patients with knee OA and from 38 healthy volunteers. Pentosidine concentration was measured by reverse phase high performance liquid chromatography with fluorescent detection and COMP was determined by sandwich ELISA. Results: Significantly increased serum pentosidine (p<0.01) and COMP (p<0.05) levels were detected in the patients with OA compared with the control group. Serum pentosidine correlated significantly with synovial fluid pentosidine (p<0.001). Pentosidine in synovial fluid (p<0.05) and in serum (p<0.05) correlated significantly with synovial fluid COMP. Pentosidine and COMP concentrations did not correlate significantly with the radiological stage of the disease. Conclusion: Increased pentosidine serum concentration in patients with OA and its correlation with the cartilage destruction marker COMP in synovial fluid suggests that pentosidine may be important in OA pathology and is a new potential OA marker. PMID:15897309

  5. The pattern of urinary stone disease in Leeds and in the United Kingdom in relation to animal protein intake during the period 1960-1980.

    PubMed

    Robertson, W G; Peacock, M

    1982-01-01

    Studies on the occurrence of urinary stone disease in Leeds between 1960 and 1980 show that there was an increase in the number of stones formed during the period 1960-1970, a fall between 1972 and a subsequent rise between 1977 and 1980. The fluctuations in stone incidence were accounted for almost entirely by changes in the number of 'pure' calcium oxalate stones and, to a lesser extent, the number of uric acid stones produced. The incidence patterns of these types of stone closely reflected changes in the consumption of animal protein in the population as a whole during the same period. There were no parallel changes either in the number of stones consisting of a mixture of calcium oxalate and calcium phosphate or in the number of infection stones.

  6. Effect of nutritional status and dietary patterns on human serum C-reactive protein and interleukin-6 concentrations.

    PubMed

    Smidowicz, Angelika; Regula, Julita

    2015-11-01

    The inflammatory process plays an important role in the pathogenesis of many chronic diseases, such as cardiovascular diseases, diabetes mellitus type 2, and metabolic syndrome. Serum C-reactive protein (CRP) and interleukin-6 (IL-6) are widely tested inflammatory markers involved in the development of these diseases. Several studies indicate a relation between nutritional status and the concentrations of human high-sensitivity CRP and IL-6. Similarly, the role of diet in reducing inflammation and thereby modulating the risk of non-communicable diseases is supported by numerous studies. This review focuses on the effects of the selected nutrition models in humans on the concentrations of CRP and IL-6. It seems that the Mediterranean diet model is most effective in inhibiting inflammation. The Dietary Approaches to Stop Hypertension model and the plant nutrition model also have proven to be beneficial. The data on low-fat and low-carbohydrate diets are inconclusive. Comprehensive studies are necessary, taking into account the cumulative effect of dietary and other factors on the inflammatory process.

  7. CpG site DNA methylation patterns reveal a novel regulatory element in the mouse prion protein gene

    PubMed Central

    DALAI, Wuyun; MATSUO, Eiko; TAKEYAMA, Natsumi; KAWANO, Junichi; SAEKI, Keiichi

    2016-01-01

    The cellular isoform of the prion protein (PrPC) plays critical roles in the development of prion disorders. Although PrP mRNA is ubiquitously present in a tissue-specific manner, the DNA methylation of PrP gene (Prnp) is still unknown. In this study, we demonstrated that the CpG island (CGI, positioned at −218 to +152 bp from the transcriptional start site) including the Prnp core promoter region was completely unmethylated in all tested tissues. On the other hand, CpG methylation in the CGI shore region (positioned at −599 to −238 bp) occurred in various tissue- and site-specific proportions. Interestingly, the correlation analysis between CpG methylation status and PrP mRNA levels showed that one CpG site methylation at −576 was negatively correlated with the PrP mRNA level (Pearson’s r = −0.374, P=0.035). Taken together, our results suggest that Prnp is a typical housekeeping gene and various methylation frequencies of the CGI shore region are likely to affect Prnp expression in a tissue-specific manner. PMID:27666463

  8. Effect of Nutritional Status and Dietary Patterns on Human Serum C-Reactive Protein and Interleukin-6 Concentrations12

    PubMed Central

    Smidowicz, Angelika; Regula, Julita

    2015-01-01

    The inflammatory process plays an important role in the pathogenesis of many chronic diseases, such as cardiovascular diseases, diabetes mellitus type 2, and metabolic syndrome. Serum C-reactive protein (CRP) and interleukin-6 (IL-6) are widely tested inflammatory markers involved in the development of these diseases. Several studies indicate a relation between nutritional status and the concentrations of human high-sensitivity CRP and IL-6. Similarly, the role of diet in reducing inflammation and thereby modulating the risk of non-communicable diseases is supported by numerous studies. This review focuses on the effects of the selected nutrition models in humans on the concentrations of CRP and IL-6. It seems that the Mediterranean diet model is most effective in inhibiting inflammation. The Dietary Approaches to Stop Hypertension model and the plant nutrition model also have proven to be beneficial. The data on low-fat and low-carbohydrate diets are inconclusive. Comprehensive studies are necessary, taking into account the cumulative effect of dietary and other factors on the inflammatory process. PMID:26567198

  9. Genome-Wide Comparison of Magnaporthe Species Reveals a Host-Specific Pattern of Secretory Proteins and Transposable Elements

    PubMed Central

    Gowda, Malali

    2016-01-01

    Blast disease caused by the Magnaporthe species is a major factor affecting the productivity of rice, wheat and millets. This study was aimed at generating genomic information for rice and non-rice Magnaporthe isolates to understand the extent of genetic variation. We have sequenced the whole genome of the Magnaporthe isolates, infecting rice (leaf and neck), finger millet (leaf and neck), foxtail millet (leaf) and buffel grass (leaf). Rice and finger millet isolates infecting both leaf and neck tissues were sequenced, since the damage and yield loss caused due to neck blast is much higher as compared to leaf blast. The genome-wide comparison was carried out to study the variability in gene content, candidate effectors, repeat element distribution, genes involved in carbohydrate metabolism and SNPs. The analysis of repeat element footprints revealed some genes such as naringenin, 2-oxoglutarate 3-dioxygenase being targeted by Pot2 and Occan, in isolates from different host species. Some repeat insertions were host-specific while other insertions were randomly shared between isolates. The distributions of repeat elements, secretory proteins, CAZymes and SNPs showed significant variation across host-specific lineages of Magnaporthe indicating an independent genome evolution orchestrated by multiple genomic factors. PMID:27658241

  10. Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition

    PubMed Central

    Chang, Chung-I; Ihara, Kentaro; Chelliah, Yogarany; Mengin-Lecreulx, Dominique; Wakatsuki, Soichi; Deisenhofer, Johann

    2005-01-01

    The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. We have determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-Å resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. We showed that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity. PMID:16006509

  11. Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition.

    PubMed

    Chang, Chung-I; Ihara, Kentaro; Chelliah, Yogarany; Mengin-Lecreulx, Dominique; Wakatsuki, Soichi; Deisenhofer, Johann

    2005-07-19

    The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. We have determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-A resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. We showed that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity.

  12. Distinct patterns of cleavage and translocation of cell cycle control proteins in CD95-induced and p53-induced apoptosis.

    PubMed Central

    Park, Weon Seo; Jung, Kyeong Cheon; Chung, Doo Hyun; Nam, Woo-Dong; Choi, Won Jin; Bae, Youngmee

    2003-01-01

    Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction (R) point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would therefore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression. PMID:12923319

  13. Comparative study of sugar fermentation and protein expression patterns of two Lactobacillus plantarum strains grown in three different media.

    PubMed

    Plumed-Ferrer, Carme; Koistinen, Kaisa M; Tolonen, Tiina L; Lehesranta, Satu J; Kärenlampi, Sirpa O; Mäkimattila, Elina; Joutsjoki, Vesa; Virtanen, Vesa; von Wright, Atte

    2008-09-01

    A comparative study of two strains of Lactobacillus plantarum (REB1 and MLBPL1) grown in commercial medium (MRS broth), cucumber juice, and liquid pig feed was performed to explore changes to the metabolic pathways of these bacteria, using a proteomics approach (two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry) combined with analyses of fermentable sugars and fermentation end products. The protein expression showed that even with an excess of glucose in all media, both strains could metabolize different carbohydrates simultaneously and that hexoses could also be used via a phosphoketolase pathway with preferential expression in liquid feed. Sugar analyses showed that the fermentation of sugars was homolactic for all media, with some heterolactic activity in liquid feed, as shown by the production of acetate. Cucumber juice (the medium with the highest glucose content) showed the lowest hexose consumption (10%), followed by liquid feed (33%) and MRS broth (50%). However, bacterial growth was significantly higher in cucumber juice and liquid feed than in MRS broth. This discrepancy was due to the growth benefit obtained from the utilization of the malate present in cucumber juice and liquid feed. Despite different growth conditions, the synthesis of essential cellular components and the stress response of the bacteria were unaffected. This study has improved our understanding of the mechanisms involved in the growth performance of an appropriate lactic acid bacterium strain to be used for food and feed fermentation, information that is of crucial importance to obtain a high-quality fermented product.

  14. Mutation of the Rice Narrow leaf1 Gene, Which Encodes a Novel Protein, Affects Vein Patterning and Polar Auxin Transport1[OA

    PubMed Central

    Qi, Jing; Qian, Qian; Bu, Qingyun; Li, Shuyu; Chen, Qian; Sun, Jiaqiang; Liang, Wenxing; Zhou, Yihua; Chu, Chengcai; Li, Xugang; Ren, Fugang; Palme, Klaus; Zhao, Bingran; Chen, Jinfeng; Chen, Mingsheng; Li, Chuanyou

    2008-01-01

    The size and shape of the plant leaf is an important agronomic trait. To understand the molecular mechanism governing plant leaf shape, we characterized a classic rice (Oryza sativa) dwarf mutant named narrow leaf1 (nal1), which exhibits a characteristic phenotype of narrow leaves. In accordance with reduced leaf blade width, leaves of nal1 contain a decreased number of longitudinal veins. Anatomical investigations revealed that the culms of nal1 also show a defective vascular system, in which the number and distribution pattern of vascular bundles are altered. Map-based cloning and genetic complementation analyses demonstrated that Nal1 encodes a plant-specific protein with unknown biochemical function. We provide evidence showing that Nal1 is richly expressed in vascular tissues and that mutation of this gene leads to significantly reduced polar auxin transport capacity. These results indicate that Nal1 affects polar auxin transport as well as the vascular patterns of rice plants and plays an important role in the control of lateral leaf growth. PMID:18562767

  15. The Barley Uniculme4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning1[OPEN

    PubMed Central

    Tavakol, Elahe; Okagaki, Ron; Verderio, Gabriele; Shariati J., Vahid; Hussien, Ahmed; Bilgic, Hatice; Scanlon, Mike J.; Todt, Natalie R.; Close, Timothy J.; Druka, Arnis; Waugh, Robbie; Steuernagel, Burkhard; Ariyadasa, Ruvini; Himmelbach, Axel; Stein, Nils; Muehlbauer, Gary J.

    2015-01-01

    Tillers are vegetative branches that develop from axillary buds located in the leaf axils at the base of many grasses. Genetic manipulation of tillering is a major objective in breeding for improved cereal yields and competition with weeds. Despite this, very little is known about the molecular genetic bases of tiller development in important Triticeae crops such as barley (Hordeum vulgare) and wheat (Triticum aestivum). Recessive mutations at the barley Uniculme4 (Cul4) locus cause reduced tillering, deregulation of the number of axillary buds in an axil, and alterations in leaf proximal-distal patterning. We isolated the Cul4 gene by positional cloning and showed that it encodes a BROAD-COMPLEX, TRAMTRACK, BRIC-À-BRAC-ankyrin protein closely related to Arabidopsis (Arabidopsis thaliana) BLADE-ON-PETIOLE1 (BOP1) and BOP2. Morphological, histological, and in situ RNA expression analyses indicate that Cul4 acts at axil and leaf boundary regions to control axillary bud differentiation as well as the development of the ligule, which separates the distal blade and proximal sheath of the leaf. As, to our knowledge, the first functionally characterized BOP gene in monocots, Cul4 suggests the partial conservation of BOP gene function between dicots and monocots, while phylogenetic analyses highlight distinct evolutionary patterns in the two lineages. PMID:25818702

  16. Matrilin-3 mutations that cause chondrodysplasias interfere with protein trafficking while a mutation associated with hand osteoarthritis does not

    PubMed Central

    Otten, C; Wagener, R; Paulsson, M; Zaucke, F

    2005-01-01

    Several mutations in the extracellular matrix protein matrilin-3 cause a heterogeneous disease spectrum affecting skeletal tissues. We introduced three disease causing point mutations leading to single amino acid exchanges (R116W, T298M, C299S) in matrilin-3 and expressed the corresponding proteins in primary articular chondrocytes to elucidate pathogenic mechanisms at the cellular level. Expression levels, processing, and the secretion pattern of a mutation linked to hand osteoarthritis (T298M) were similar to the wildtype protein, whereas the two other mutants were poorly expressed and hardly detectable in supernatants of transiently transfected cells. Using immunofluorescence staining, we demonstrated that mutants R116W and C299S are retained and accumulate within the endoplasmatic reticulum (ER). Their further trafficking to the Golgi compartment seems to be disturbed, whereas T298M is secreted normally. In cells transfected with the wildtype and T298M constructs, a matrilin-3 containing filamentous network was formed surrounding the cells, whereas in the case of R116W and C299S such structures were completely absent. These observations are similar to those for mutations in the cartilage oligomeric matrix protein (COMP) leading to multiple epiphyseal dysplasia and pseudoachondroplasia suggesting that retention and accumulation of cartilage proteins in the ER might be a general mechanism involved in the pathogenesis of chondrodysplasias. PMID:16199550

  17. The Cerberus/Dan-family protein Charon is a negative regulator of Nodal signaling during left-right patterning in zebrafish.

    PubMed

    Hashimoto, Hisashi; Rebagliati, Michael; Ahmad, Nadira; Muraoka, Osamu; Kurokawa, Tadahide; Hibi, Masahiko; Suzuki, Tohru

    2004-04-01

    We have isolated a novel gene, charon, that encodes a member of the Cerberus/Dan family of secreted factors. In zebrafish, Fugu and flounder, charon is expressed in regions embracing Kupffer's vesicle, which is considered to be the teleost fish equivalent to the region of the mouse definitive node that is required for left-right (L/R) patterning. Misexpression of Charon elicited phenotypes similar to those of mutant embryos defective in Nodal signaling or embryos overexpressing Antivin(Atv)/Lefty1, an inhibitor for Nodal and Activin. Charon also suppressed the dorsalizing activity of all three of the known zebrafish Nodal-related proteins (Cyclops, Squint and Southpaw), indicating that Charon can antagonize Nodal signaling. Because Southpaw functions in the L/R patterning of lateral plate mesoderm and the diencephalon, we asked whether Charon is involved in regulating L/R asymmetry. Inhibition of Charon's function by antisense morpholino oligonucleotides (MOs) led to a loss of L/R polarity, as evidenced by bilateral expression of the left side-specific genes in the lateral plate mesoderm (southpaw, cyclops, atv/lefty1, lefty2 and pitx2) and diencephalon (cyclops, atv/lefty1 and pitx2), and defects in early (heart jogging) and late (heart looping) asymmetric heart development, but did not disturb the notochord development or the atv/lefty1-mediated midline barrier function. MO-mediated inhibition of both Charon and Southpaw led to a reduction in or loss of the expression of the left side-specific genes, suggesting that Southpaw is epistatic to Charon in left-side formation. These data indicate that antagonistic interactions between Charon and Nodal (Southpaw), which take place in regions adjacent to Kupffer's vesicle, play an important role in L/R patterning in zebrafish.

  18. Mortality Patterns of Simulium vittatum Larvae (Diptera: Simuliidae) Following Exposure to Insecticidal Proteins Produced by Bacillus thuringiensis var. israelensis.

    PubMed

    Iburg, Joseph P; Gray, Elmer W; Noblet, Raymond

    2015-03-01

    Products containing insecticidal crystalline proteins (ICPs) produced by Bacillus thuringiensis var. israelensis (Bti ICPs) are used to suppress vector and nuisance populations of black flies. The efficacy of an application of these products is often determined by a posttreatment evaluation of larval mortality. Larvae are typically removed from the substrate at some point in time after application of the product and mortality is determined. The time necessary for the effects of Bti ICPs to cause morality in exposed larvae can vary, and there is little consensus on how long operators should wait before evaluating larval mortality. This study was conducted to provide more information to larvicide applicators when performing posttreatment evaluations. Simulium vittatum larvae were exposed to Bti ICPs under controlled conditions and the mortality was monitored over time. Larvae exposed to operational concentrations of ICPs exhibited maximum mortality, approximately 87%, after 4 h. Exposure of larvae to 1/3 of that concentration resulted in similar mortality; however, the maximum mortality was not reached until 8 h postexposure. Additional experiments revealed that maximum mortality and time to maximum mortality can also be affected by components in the larval medium. Larval mortality was compared between larvae exposed to Bti ICPs in moderately hard water, medium containing 50 parts per million (ppm) of kaolinite, and medium containing 50 ppm of cellulose. The clay material had no significant effect on larval mortality or time to achieve maximum mortality. When cellulose was present in the medium, the time to maximum mortality was increased 50% and overall mortality was reduced by more than 40%.

  19. Daily Expression Pattern of Protein-Encoding Genes and Small Noncoding RNAs in Synechocystis sp. Strain PCC 6803

    PubMed Central

    Beck, Christian; Hertel, Stefanie; Rediger, Anne; Lehmann, Robert; Wiegard, Anika; Kölsch, Adrian; Heilmann, Beate; Georg, Jens; Hess, Wolfgang R.

    2014-01-01

    Many organisms harbor circadian clocks with periods close to 24 h. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. These rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes. The oscillator of photosynthetic cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, linked to a complex regulatory network. Synechocystis sp. strain PCC 6803 possesses the standard cyanobacterial kaiABC gene cluster plus multiple kaiB and kaiC gene copies and antisense RNAs for almost every kai transcript. However, there is no clear evidence of circadian rhythms in Synechocystis sp. PCC 6803 under various experimental conditions. It is also still unknown if and to what extent the multiple kai gene copies and kai antisense RNAs affect circadian timing. Moreover, a large number of small noncoding RNAs whose accumulation dynamics over time have not yet been monitored are known for Synechocystis sp. PCC 6803. Here we performed a 48-h time series transcriptome analysis of Synechocystis sp. PCC 6803, taking into account periodic light-dark phases, continuous light, and continuous darkness. We found that expression of functionally related genes occurred in different phases of day and night. Moreover, we found day-peaking and night-peaking transcripts among the small RNAs; in particular, the amounts of kai antisense RNAs correlated or anticorrelated with those of their respective kai target mRNAs, pointing toward the regulatory relevance of these antisense RNAs. Surprisingly, we observed that the amounts of 16S and 23S rRNAs in this cyanobacterium fluctuated in light-dark periods, showing maximum accumulation in the dark phase. Importantly, the amounts of all transcripts, including small noncoding RNAs, did not show any rhythm under continuous light or darkness, indicating the absence

  20. Simple, Automated, High Resolution Mass Spectrometry Method to Determine the Disulfide Bond and Glycosylation Patterns of a Complex Protein

    PubMed Central

    Pike, Gennett M.; Madden, Benjamin J.; Melder, Deborah C.; Charlesworth, M. Cristine; Federspiel, Mark J.

    2011-01-01

    Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The avian sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A avian sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein. PMID:21454567

  1. Control of germline torso expression by the BTB/POZ domain protein pipsqueak is required for embryonic terminal patterning in Drosophila.

    PubMed

    Grillo, Marco; Furriols, Marc; Casanova, Jordi; Luschnig, Stefan

    2011-02-01

    Early embryogenesis in Drosophila melanogaster is controlled by maternal gene products, which are deposited in the egg during oogenesis. It is not well understood how maternal gene expression is controlled during germline development. pipsqueak (psq) is a complex locus that encodes several nuclear protein variants containing a PSQ DNA-binding domain and a BTB/POZ domain. Psq proteins are thought to regulate germline gene expression through epigenetic silencing. While psq was originally identified as a posterior-group gene, we show here a novel role of psq in embryonic terminal patterning. We characterized a new psq loss-of-function allele, psq(rum), which specifically affects signaling by the Torso (Tor) receptor tyrosine kinase (RTK). Using genetic epistasis, gene expression analyses, and rescue experiments, we demonstrate that the sole function impaired by the psq(rum) mutation in the terminal system is an essential requirement for controlling transcription of the tor gene in the germline. In contrast, the expression of several other maternal genes, including those encoding Tor pathway components, is not affected by the mutation. Rescue of the psq(rum) terminal phenotype does not require the BTB/POZ domain, suggesting that the PSQ DNA-binding domain can function independently of the BTB/POZ domain. Our finding that tor expression is subject to dedicated transcriptional regulation suggests that different maternal genes may be regulated by multiple distinct mechanisms, rather than by a general program controlling nurse-cell transcription.

  2. Development of a three-dimensional multiscale agent-based tumor model: simulating gene-protein interaction profiles, cell phenotypes and multicellular patterns in brain cancer.

    PubMed

    Zhang, Le; Athale, Chaitanya A; Deisboeck, Thomas S

    2007-01-07

    Experimental evidence suggests that epidermal growth factor receptor (EGFR)-mediated activation of the signaling protein phospholipase Cgamma plays a critical role in a cancer cell's phenotypic decision to either proliferate or to migrate at a given point in time. Here, we present a novel three-dimensional multiscale agent-based model to simulate this cellular decision process in the context of a virtual brain tumor. Each tumor cell is equipped with an EGFR gene-protein interaction network module that also connects to a simplified cell cycle description. The simulation results show that over time proliferative and migratory cell populations not only oscillate but also directly impact the spatio-temporal expansion patterns of the entire cancer system. The percentage change in the concentration of the sub-cellular interaction network's molecular components fluctuates, and, for the 'proliferation-to-migration' switch we find that the phenotype triggering molecular profile to some degree varies as the tumor system grows and the microenvironment changes. We discuss potential implications of these findings for experimental and clinical cancer research.

  3. Blood/Brain Biomarkers of Inflammation After Stroke and Their Association With Outcome: From C-Reactive Protein to Damage-Associated Molecular Patterns.

    PubMed

    Bustamante, Alejandro; Simats, Alba; Vilar-Bergua, Andrea; García-Berrocoso, Teresa; Montaner, Joan

    2016-10-01

    Stroke represents one of the most important causes of disability and death in developed countries. However, there is a lack of prognostic tools in clinical practice to monitor the neurological condition and predict the final outcome. Blood biomarkers have been proposed and studied in this indication; however, no biomarker is currently used in clinical practice. The stroke-related neuroinflammatory processes have been associated with a poor outcome in stroke, as well as with poststroke complications. In this review, we focus on the most studied blood biomarkers of this inflammatory processes, cytokines, and C-reactive protein, evaluating its association with outcome and complications in stroke through the literature, and performing a systematic review on the association of C-reactive protein and functional outcome after stroke. Globally, we identified uncertainty with regard to the association of the evaluated biomarkers with stroke outcome, with little added value on top of clinical predictors such as age or stroke severity, which makes its implementation unlikely in clinical practice for global outcome prediction. Regarding poststroke complications, despite being more practical scenarios in which to make medical decisions following a biomarker prediction, not many studies have been performed, although there are now some candidates for prediction of poststroke infections. Finally, as potential new candidates, we reviewed the pathophysiological actions of damage-associated molecular patterns as triggers of the neuroinflammatory cascade of stroke, and their possible use as biomarkers.

  4. Changes in the distribution pattern of Claudin tight junction proteins during the progression of mouse skin tumorigenesis

    PubMed Central

    Arabzadeh, Azadeh; Troy, Tammy-Claire; Turksen, Kursad

    2007-01-01

    Background Despite the fact that morphological and physiological observations suggest that the tight junction (TJ)-based permeability barrier is modified/disrupted in tumorigenesis, the role of members of the Claudin (Cldn) family of TJ proteins is not well-understood. Using a well-established two-stage chemical carcinogenesis model, we investigated the temporal and spatial changes in expression of those Cldns that we have previously demonstrated to be important in epidermal differentiation and the formation of the epidermal permeability barrier, i.e., Cldn1, Cldn6, Cldn11, Cldn12 and Cldn18. Methods The lower dorsal backskin of mice was treated topically with 7,12-dimethylbenz(a)anthracene (DMBA; 0.25 mg/ml in acetone) and following a 10-day incubation period, 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 25 μg/ml in acetone) was applied three times a week to the same area. Backskin samples were dissected 2, 4, 6, 8 and 12 weeks after the initiation of the experimental protocol and immunohistochemistry was performed on sections using antibodies against the following: Cldn1, Cldn6, Cldn11, Cldn12, Cldn18, Ki67 and CD3. Results Our data indicate that along with the changes in epidermal cell morphology and differentiation that occur during tumor formation, there is a dramatic change in Cldn distribution consistent with cell polarity and barrier selectivity changes. Specifically, in the early stages of DMBA/TPA treatment, the suprabasal-specific Cldns occupy an expanded zone of expression corresponding to an increased number of suprabasal epidermal cell layers. As tumorigenesis progressed, the number of suprabasal epidermal layers positive for Cldn6, Cldn11, Cldn12 and Cldn18 was reduced, especially in the lower strata of the expanded suprabasal zone. In addition, a variably reduced cell membrane association of those differentiation-specific Cldns was observed, especially within the infiltrating epidermal structures. In contrast, Cldn1 (which is normally expressed in

  5. The Hcp proteins fused with diverse extended-toxin domains represent a novel pattern of antibacterial effectors in type VI secretion systems.

    PubMed

    Ma, Jiale; Pan, Zihao; Huang, Jinhu; Sun, Min; Lu, Chengping; Yao, Huochun

    2017-01-06

    The type VI secretion system (T6SS) is a widespread molecular weapon deployed by many bacterial species to target eukaryotic host cells or rival bacteria. Using a dynamic injection mechanism, diverse effectors can be delivered by T6SS directly into recipient cells. Here, we report a new family of T6SS effectors encoded by extended Hcps carrying diverse toxin domains. Bioinformatic analyses revealed that these Hcps with C-terminal extension toxins, designated as Hcp-ET, exist widely in the Enterobacteriaceae. To verify our findings, Hcp-ET1 was tested for its antibacterial effect, and showed effective inhibition of target cell growth via the predicted HNH-DNase activity by T6SS-dependent delivery. Further studies showed that Hcp-ET2 mediated interbacterial antagonism via a Tle1 phospholipase (encoded by DUF2235 domain) activity. Notably, comprehensive analyses of protein homology and genomic neighborhoods revealed that Hcp-ET3-4 is fused with 2 toxin domains (Pyocin S3 and Colicin-DNase) C-terminally, and its encoding gene is followed 3 duplications of the cognate immunity genes. However, some bacteria encode a separated hcp-et3 and an orphan et4 (et4O1) genes caused by a termination-codon mutation in the fusion region between Pyocin S3 and Colicin-DNase encoding fragments. Our results demonstrated that both of these toxins had antibacterial effects. Further, all duplications of the cognate immunity protein contributed to neutralize the DNase toxicity of Pyocin S3 and Colicin, which has not been reported previously. In conclusion, we propose that Hcp-ET proteins are polymorphic T6SS effectors, and thus present a novel encoding pattern of T6SS effectors.

  6. Changes in the protein patterns in pea (Pisum sativum L.) roots under the influence of long- and short-term chilling stress and post-stress recovery.

    PubMed

    Badowiec, Anna; Swigonska, Sylwia; Weidner, Stanisław

    2013-10-01

    Amongst many factors restricting geographical distribution of plants and crop productivity, low temperature is one of the most important. To gain better understanding of the molecular response of germinating pea (Pisum sativum L.) to low temperature, we investigated the influence of long and short chilling stress as well as post-stress recovery on the alterations in the root proteomes. The impact of long stress was examined on the pea seeds germinating in the continuous chilling conditions of 10 °C for 8 days (LS). To examine the impact of short stress, pea seeds germinating for 72 h in the optimal temperature of 20 °C were subjected to 24-h chilling (SS). Additionally, both stress treatments were followed by 24 h of recovery in the optimal conditions (accordingly LSR and SR). Using the 2D gel electrophoresis and MALDI-TOF MS protein identification, it was revealed, that most of the proteins undergoing regulation under the applied conditions were implicated in metabolism, protection against stress, cell cycle regulation, cell structure maintenance and hormone synthesis, which altogether may influence root growth and development in the early stages of plant life. The obtained results have shown that most of detected alterations in the proteome patterns of pea roots are dependent on stress duration. However, there are some analogical response pathways which are triggered regardless of stress length. The functions of proteins which accumulation has been changed by chilling stress and post-stress recovery are discussed here in relation to their impact on pea roots development.

  7. Adaptive expansion of the maize maternally expressed gene (Meg) family involves changes in expression patterns and protein secondary structures of its members

    PubMed Central

    2014-01-01

    Background The Maternally expressed gene (Meg) family is a locally-duplicated gene family of maize which encodes cysteine-rich proteins (CRPs). The founding member of the family, Meg1, is required for normal development of the basal endosperm transfer cell layer (BETL) and is involved in the allocation of maternal nutrients to growing seeds. Despite the important roles of Meg1 in maize seed development, the evolutionary history of the Meg cluster and the activities of the duplicate genes are not understood. Results In maize, the Meg gene cluster resides in a 2.3 Mb-long genomic region that exhibits many features of non-centromeric heterochromatin. Using phylogenetic reconstruction and syntenic alignments, we identified the pedigree of the Meg family, in which 11 of its 13 members arose in maize after allotetraploidization ~4.8 mya. Phylogenetic and population-genetic analyses identified possible signatures suggesting recent positive selection in Meg homologs. Structural analyses of the Meg proteins indicated potentially adaptive changes in secondary structure from α-helix to β-strand during the expansion. Transcriptomic analysis of the maize endosperm indicated that 6 Meg genes are selectively activated in the BETL, and younger Meg genes are more active than older ones. In endosperms from B73 by Mo17 reciprocal crosses, most Meg genes did not display parent-specific expression patterns. Conclusions Recently-duplicated Meg genes have different protein secondary structures, and their expressions in the BETL dominate over those of older members. Together with the signs of positive selections in the young Meg genes, these results suggest that the expansion of the Meg family involves potentially adaptive transitions in which new members with novel functions prevailed over older members. PMID:25084677

  8. Upregulation of heat shock proteins and the promotion of damage-associated molecular pattern signals in a colorectal cancer model by modulated electrohyperthermia.

    PubMed

    Andocs, Gabor; Meggyeshazi, Nora; Balogh, Lajos; Spisak, Sandor; Maros, Mate Elod; Balla, Peter; Kiszner, Gergo; Teleki, Ivett; Kovago, Csaba; Krenacs, Tibor

    2015-01-01

    In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHT-provoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168- and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals. Significant cell death response upon mEHT treatment was accompanied by the early upregulation (4-h post-treatment) of heat shock protein (Hsp70 and Hsp90) mRNA levels. In situ, the treatment resulted in spatiotemporal occurrence of a DAMP protein signal sequence featured by the significant cytoplasmic to cell membrane translocation of calreticulin at 4 h, Hsp70 between 14 and 24 h and Hsp90 between 24- and 216-h post-treatment. The release of high-mobility group box1 protein (HMGB1) from tumor cell nuclei from 24-h post-treatment and its clearance from tumor cells by 48 h was also detected. Our results suggest that mEHT treatment can induce a DAMP-related signal sequence in colorectal cancer xenografts that may be relevant for promoting immunological cell death response, which need to be further tested in immune-competent animals.

  9. PPIM: A Protein-Protein Interaction Database for Maize1

    PubMed Central

    Wu, Aibo; Xu, Xin-Jian; Lu, Le; Liu, Jingdong; Cao, Yongwei; Chen, Luonan; Wu, Jun; Zhao, Xing-Ming

    2016-01-01

    Maize (Zea mays) is one of the most important crops worldwide. To understand the biological processes underlying various traits of the crop (e.g. yield and response to stress), a detailed protein-protein interaction (PPI) network is highly demanded. Unfortunately, there are very few such PPIs available in the literature. Therefore, in this work, we present the Protein-Protein Interaction Database for Maize (PPIM), which covers 2,762,560 interactions among 14,000 proteins. The PPIM contains not only accurately predicted PPIs but also those molecular interactions collected from the literature. The database is freely available at http://comp-sysbio.org/ppim with a user-friendly powerful interface. We believe that the PPIM resource can help biologists better understand the maize crop. PMID:26620522

  10. Patterns of Broken Patterns

    NASA Astrophysics Data System (ADS)

    Field, R. W.; Park, G. B.; Changala, P. B.; Baraban, J. H.; Stanton, J. F.; Merer, A. J.

    2013-06-01

    Spectroscopy - it is all about patterns. Some patterns look so indescribably complicated that, unlike pornography, you do not know one when you see one. It is tempting to say that, at high vibrational excitation, interactions among normal mode basis states are so strong and widespread that all patterns are obliterated. But this is not true. When normal mode frequencies are in near integer multiple ratios, polyads emerge. A polyad is a robust pattern often comprising many vibrational eigenstates. Each such pattern might span many hundreds of cm^{-1}, and it is inevitable that several unrelated polyad patterns overlap. When polyads overlap, it might seem impossible to disentangle them. However, the key to disentanglement is that polyads come in families in which successive generations are related by harmonic oscillator matrix element selection and scaling rules. Families of polyads are described by families of scaling-based effective Hamiltonian matrices, {H}^{{eff}}. No matter how complex and overlapped, the polyad {H}^{{eff}} serves as a magic decoder for picking out the polyad pattern. Sometimes the polyad patterns are systematically broken (a meta-pattern), owing to proximity to an isomerization barrier, as occurs in highly excited bending levels of the S_{1} state of HCCH, which encode the trans-cis minimum energy isomerization path. Quantum Chemists often dismiss {H}^{{eff}} models, precisely because they are models that do not express the full dimensionality of the complete Hamiltonian. But an {H}^{{eff}} explains rather than describes. Shunning {H}^{{eff}}s is like throwing out the baby with the bath water. Don't do it!

  11. A C1q Domain Containing Protein from Scallop Chlamys farreri Serving as Pattern Recognition Receptor with Heat-Aggregated IgG Binding Activity

    PubMed Central

    Wang, Leilei; Wang, Lingling; Zhang, Huan; Zhou, Zhi; Siva, Vinu S.; Song, Linshen