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Sample records for protein comp patterns

  1. Non-collagenous protein screening in the human chondrodysplasias: link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin.

    PubMed

    Stanescu, V; Do, T P; Chaminade, F; Maroteaux, P; Stanescu, R

    1994-05-15

    A gel-electrophoretic screening for link proteins, cartilage oligomeric matrix protein (COMP), and fibromodulin abnormalities was performed in fetuses, newborn infants, and children with various types of chondrodysplasia. Microdissected freeze-dried sections of upper tibial growth cartilage were extracted with 4M guanidinium chloride in the presence of proteolysis inhibitors. After dialysis against 8M urea, the extracts were submitted to stepwise ion-exchange chromatography to separate the large proteoglycans (aggrecans) from the other components. The latter were analyzed by gel electrophoresis, electrotransferred onto nitrocellulose membranes, and reacted with specific antibodies. Control samples from individuals with apparently normal growth were analyzed in the same runs. Two link protein bands with abnormal electrophoretic migration were found in a sporadic case of spondylometaphyseal dysplasia, Kozlowski type. Three link protein bands with the same migration as in the control samples were found in thanatophoric dysplasia, homozygous achondroplasia, achondrogenesis type II, hypochondrogenesis, Goldblatt syndrome, Desbuquois dysplasia, pseudoachondroplasia, and diastrophic dysplasia. In several pathologic cases with normal electrophoretic pattern of the link proteins, small link protein fragments appeared after reduction. The gel electrophoretic pattern of COMP was studied in thanatophoric dysplasia, diastrophic dysplasia, homozygous achondroplasia, fibrochondrogenesis, hypochondrogenesis, Goldblatt syndrome, and Kniest dysplasia. In all these cases the pattern was the same as in the control samples. The main band of fibromodulin had a normal migration rate in fibrochondrogenesis, Desbuquois dysplasia, Kniest dysplasia, and pseudoachondroplasia. It was delayed in diastrophic dysplasia. PMID:8030664

  2. 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

  3. Characterization of a pseudoachondroplasia-associated mutation (His587-->Arg) in the C-terminal, collagen-binding domain of cartilage oligomeric matrix protein (COMP).

    PubMed Central

    Spitznagel, Luitgard; Nitsche, D Patric; Paulsson, Mats; Maurer, Patrik; Zaucke, Frank

    2004-01-01

    We have introduced a pseudoachondroplasia-associated mutation (His(587)-->Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579-595 and it was assumed that the His(587)-->Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His(587)-->Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 degrees C. Our results suggest that the His(587)-->Arg mutation is not itself deleterious to the structure and collagen-binding of COMP. PMID:14580238

  4. 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

  5. Pattern Formation without Patterning Proteins in Cyanobacteria

    NASA Astrophysics Data System (ADS)

    Allard, Jun; Rutenberg, Andrew

    2006-03-01

    Filaments of cyanobacteria respond to nitrogen starvation by differentiating one cell in ten into a heterocyst, which is devoted to fixing atmospheric nitrogen. This is an example of self-organized pattern formation. We present a dynamical model explaining the initial selection of heterocysts in mutated cyanobacteria that are effectively without normal patterning proteins. Our simulations of this model produce distributions of heterocyst spacings that are consistent with experimental data, and lead to new qualitative predictions on the mechanisms of pattern formation in filamentous cyanobacteria. We discuss possible experimental tests of our results.

  6. Comp Curriculum Guide.

    ERIC Educational Resources Information Center

    Forsberg, Sara J.; And Others

    This curriculum guide describes and gives procedures for implementing the Comp Curriculum, which is composed of objectives categorized by age (birth to 5) and domains of developmental tasks (communication, self care, motor and problem solving), for handicapped and nonhandicapped children. (The objectives themselves are listed in a separate…

  7. Codon Bias Patterns of E. coli’s Interacting Proteins

    PubMed Central

    Dilucca, Maddalena; Cimini, Giulio; Semmoloni, Andrea; Deiana, Antonio; Giansanti, Andrea

    2015-01-01

    Synonymous codons, i.e., DNA nucleotide triplets coding for the same amino acid, are used differently across the variety of living organisms. The biological meaning of this phenomenon, known as codon usage bias, is still controversial. In order to shed light on this point, we propose a new codon bias index, CompAI, that is based on the competition between cognate and near-cognate tRNAs during translation, without being tuned to the usage bias of highly expressed genes. We perform a genome-wide evaluation of codon bias for E.coli, comparing CompAI with other widely used indices: tAI, CAI, and Nc. We show that CompAI and tAI capture similar information by being positively correlated with gene conservation, measured by the Evolutionary Retention Index (ERI), and essentiality, whereas, CAI and Nc appear to be less sensitive to evolutionary-functional parameters. Notably, the rate of variation of tAI and CompAI with ERI allows to obtain sets of genes that consistently belong to specific clusters of orthologous genes (COGs). We also investigate the correlation of codon bias at the genomic level with the network features of protein-protein interactions in E.coli. We find that the most densely connected communities of the network share a similar level of codon bias (as measured by CompAI and tAI). Conversely, a small difference in codon bias between two genes is, statistically, a prerequisite for the corresponding proteins to interact. Importantly, among all codon bias indices, CompAI turns out to have the most coherent distribution over the communities of the interactome, pointing to the significance of competition among cognate and near-cognate tRNAs for explaining codon usage adaptation. Notably, CompAI may potentially correlate with translation speed measurements, by accounting for the specific delay induced by wobble-pairing between codons and anticodons. PMID:26566157

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

    2007-01-01

    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. PMID:18058715

  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. Pseudoachondroplasia/COMP - translating from the bench to the bedside.

    PubMed

    Posey, Karen LaShea; Alcorn, Joseph L; Hecht, Jacqueline T

    2014-07-01

    Pseudoachondroplasia (PSACH) is a skeletal dysplasia characterized by disproportionate short stature, small hands and feet, abnormal joints and early onset osteoarthritis. PSACH is caused by mutations in thrombospondin-5 (TSP-5, also known as cartilage oligomeric matrix protein or COMP), a pentameric extracellular matrix protein primarily expressed in chondrocytes and musculoskeletal tissues. The thrombospondin gene family is composed of matricellular proteins that associate with the extracellular matrix (ECM) and regulate processes in the matrix. Mutations in COMP interfere with calcium-binding, protein conformation and export to the extracellular matrix, resulting in inappropriate intracellular COMP retention. This accumulation of misfolded protein is cytotoxic and triggers premature death of chondrocytes during linear bone growth, leading to shortened long bones. Both in vitro and in vivo models have been employed to study the molecular processes underlying development of the PSACH pathology. Here, we compare the strengths and weaknesses of current mouse models of PSACH and discuss how the resulting phenotypes may be translated to clinical therapies. PMID:24892720

  15. Two-Dimensional Protein Patterns in Heterodera glycines

    PubMed Central

    Ferris, V. R.; Ferris, J. M.; Murdock, L. L.

    1985-01-01

    Two-dimensional polyacrylamide gel electrophoretic protein patterns of H. glycines from southern Indiana (Posey County) and northern Indiana (Pulaski County) were largely similar, but many differences existed. The pattern of the Posey isolate was similar to patterns from isolates collected in other areas of the United States. Unique dense protein spots in the pattern of an isolate from Hokkaido, Japan, distinguished it from patterns of six U.S. isolates. PMID:19294120

  16. Protein surface patterning using nanoscale PEG hydrogels.

    PubMed

    Hong, Ye; Krsko, Peter; Libera, Matthew

    2004-12-01

    We have used focused electron-beam cross-linking to create nanosized hydrogels and thus present a new method with which to bring the attractive biocompatibility associated with macroscopic hydrogels into the submicron length-scale regime. Using amine-terminated poly(ethylene glycol) thin films on silicon substrates, we generate nanohydrogels with lateral dimensions of order 200 nm which can swell by a factor of at least five, depending on the radiative dose. With the focused electron beam, high-density arrays of such nanohydrogels can be flexibly patterned onto silicon surfaces. Significantly, the amine groups remain functional after e-beam exposure, and we show that they can be used to covalently bind proteins and other molecules. We use bovine serum albumin to amplify the number of amine groups, and we further demonstrate that different proteins can be covalently bound to different hydrogel pads on the same substrate to create multifunctional surfaces useful in emerging bio/proteomic and sensor technologies.

  17. Network based approaches reveal clustering in protein point patterns

    NASA Astrophysics Data System (ADS)

    Parker, Joshua; Barr, Valarie; Aldridge, Joshua; Samelson, Lawrence E.; Losert, Wolfgang

    2014-03-01

    Recent advances in super-resolution imaging have allowed for the sub-diffraction measurement of the spatial location of proteins on the surfaces of T-cells. The challenge is to connect these complex point patterns to the internal processes and interactions, both protein-protein and protein-membrane. We begin analyzing these patterns by forming a geometric network amongst the proteins and looking at network measures, such the degree distribution. This allows us to compare experimentally observed patterns to models. Specifically, we find that the experimental patterns differ from heterogeneous Poisson processes, highlighting an internal clustering structure. Further work will be to compare our results to simulated protein-protein interactions to determine clustering mechanisms.

  18. 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

  19. 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

  20. Selective memory generalization by spatial patterning of protein synthesis

    PubMed Central

    O’Donnell, Cian; Sejnowski, Terrence J.

    2014-01-01

    Summary Protein synthesis is crucial for both persistent synaptic plasticity and long-term memory. De novo protein expression can be restricted to specific neurons within a population, and to specific dendrites within a single neuron. Despite its ubiquity, the functional benefits of spatial protein regulation for learning are unknown. We used computational modeling to study this problem. We found that spatially patterned protein synthesis can enable selective consolidation of some memories but forgetting of others, even for simultaneous events that are represented by the same neural population. Key factors regulating selectivity include the functional clustering of synapses on dendrites, and the sparsity and overlap of neural activity patterns at the circuit level. Based on these findings we proposed a novel two-step model for selective memory generalization during REM and slow-wave sleep. The pattern-matching framework we propose may be broadly applicable to spatial protein signaling throughout cortex and hippocampus. PMID:24742462

  1. 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

  2. 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. PMID:26699430

  3. 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.

  4. Protein patterning: a comparison of direct spotting versus microcontact printing

    NASA Astrophysics Data System (ADS)

    Clancy, Kathryn F. A.; Nicolau, Dan V.

    2015-03-01

    Protein microarrays are used various research areas including drug discovery, diagnosis, and analysis of protein-ligand interactions. Their efficacy depends on a well-defined pattern of immobilized proteins that also have retained their bioactivity. Protein microarrays are classically fabricated using the robotic spotting drop method ("pin printing"), which can lead to spots with uneven protein concentration within the spotted area, leading to difficult to quantify readings. Among the alternative techniques, microcontact printing (μCP) with a poly(dimethylsiloxane) (PDMS) stamp appears to deliver more defined protein patterns on surfaces, while maintaining bioactivity for a wide range of proteins. Here we have quantitatively compared the distribution of fluorescently labeled proteins deposited using direct pipetting, pin printing and μCP printing with flat stamps onto various functionalized glass surfaces of different contact angles through fluorescent microscopy. The uniformity of the deposited protein spots across deposition techniques was also qualitatively analyzed. It was found that with the use of either the direct pipetting or pin printing techniques that protein concentration on surfaces varied largely across surfaces with different contact angles, whereas adsorption did not vary significantly when using the μCP printing Furthermore, when μCP printing was performed with flat relief structures the spot inhomogeneity was lower than when classical methods were used, and even less so when a pyramid relief structure was used. This suggests that μCP printing with pyramid relief structures could produce protein patterns on various surfaces and with increased spot uniformity to enable more reliable protein microarrays.

  5. Geometry sensing by self-organized protein patterns.

    PubMed

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

    2012-09-18

    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.

  6. Turn prediction in proteins using a pattern-matching approach.

    PubMed

    Cohen, F E; Abarbanel, R M; Kuntz, I D; Fletterick, R J

    1986-01-14

    We extend the use of amino acid sequence patterns [Cohen, F.E., Abarbanel, R. M., Kuntz, I. D., & Fletterick, R. J. (1983) Biochemistry 22, 4894-4904] to the identification of turns in globular proteins. The approach uses a conservative strategy, combined with a hierarchical search (strongest patterns first) and length-dependent masking, to achieve high accuracy (95%) on a test set of proteins of known structure. Applying the same procedure to homologous families gives a 90% success rate. Straightforward changes are suggested to improve the predictive power. The computer program, written in Lisp, provides a general pattern-recognition language well suited for a number of investigations of protein and nucleic acid sequences. PMID:3754149

  7. 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.

  8. 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

  9. Pattern recognition methods for protein functional site prediction.

    PubMed

    Yang, Zheng Rong; Wang, Lipo; Young, Natasha; Trudgian, Dave; Chou, Kuo-Chen

    2005-10-01

    Protein functional site prediction is closely related to drug design, hence to public health. In order to save the cost and the time spent on identifying the functional sites in sequenced proteins in biology laboratory, computer programs have been widely used for decades. Many of them are implemented using the state-of-the-art pattern recognition algorithms, including decision trees, neural networks and support vector machines. Although the success of this effort has been obvious, advanced and new algorithms are still under development for addressing some difficult issues. This review will go through the major stages in developing pattern recognition algorithms for protein functional site prediction and outline the future research directions in this important area. PMID:16248799

  10. 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.

  11. Humidified microcontact printing of proteins: universal patterning of proteins on both low and high energy surfaces.

    PubMed

    Ricoult, Sébastien G; Nezhad, Amir Sanati; Knapp-Mohammady, Michaela; Kennedy, Timothy E; Juncker, David

    2014-10-14

    Microcontact printing (μCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits μCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified μCP (HμCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HμCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HμCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HμCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted μCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles. PMID:25222734

  12. Humidified microcontact printing of proteins: universal patterning of proteins on both low and high energy surfaces.

    PubMed

    Ricoult, Sébastien G; Nezhad, Amir Sanati; Knapp-Mohammady, Michaela; Kennedy, Timothy E; Juncker, David

    2014-10-14

    Microcontact printing (μCP) of proteins is widely used for biosensors and cell biology but is constrained to printing proteins adsorbed to a low free energy, hydrophobic surface to a high free energy, hydrophilic surface. This strongly limits μCP as harsh chemical treatments are required to form a high energy surface. Here, we introduce humidified μCP (HμCP) of proteins which enables universal printing of protein on any smooth surface. We found that by flowing water in proximity to proteins adsorbed on a hydrophilized stamp, the water vapor diffusing through the stamp enables the printing of proteins on both low and high energy surfaces. Indeed, when proteins are printed using stamps with increasing spacing between water-filled microchannels, only proteins adjacent to the channels are transferred. The vapor transport through the stamp was modeled, and by comparing the humidity profiles with the protein patterns, 88% relative humidity in the stamp was identified as the threshold for HμCP. The molecular forces occurring between PDMS, peptides, and glass during printing were modeled ab initio to confirm the critical role water plays in the transfer. Using HμCP, we introduce straightforward protocols to pattern multiple proteins side-by-side down to nanometer resolution without the need for expensive mask aligners, but instead exploiting self-alignment effects derived from the stamp geometry. Finally, we introduce vascularized HμCP stamps with embedded microchannels that allow printing proteins as arbitrary, large areas patterns with nanometer resolution. This work introduces the general concept of water-assisted μCP and opens new possibilities for "solvent-assisted" printing of proteins and of other nanoparticles.

  13. 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.

  14. Functional module identification in protein interaction networks by interaction patterns

    PubMed Central

    Wang, Yijie; Qian, Xiaoning

    2014-01-01

    Motivation: Identifying functional modules in protein–protein interaction (PPI) networks may shed light on cellular functional organization and thereafter underlying cellular mechanisms. Many existing module identification algorithms aim to detect densely connected groups of proteins as potential modules. However, based on this simple topological criterion of ‘higher than expected connectivity’, those algorithms may miss biologically meaningful modules of functional significance, in which proteins have similar interaction patterns to other proteins in networks but may not be densely connected to each other. A few blockmodel module identification algorithms have been proposed to address the problem but the lack of global optimum guarantee and the prohibitive computational complexity have been the bottleneck of their applications in real-world large-scale PPI networks. Results: In this article, we propose a novel optimization formulation LCP2 (low two-hop conductance sets) using the concept of Markov random walk on graphs, which enables simultaneous identification of both dense and sparse modules based on protein interaction patterns in given networks through searching for LCP2 by random walk. A spectral approximate algorithm SLCP2 is derived to identify non-overlapping functional modules. Based on a bottom-up greedy strategy, we further extend LCP2 to a new algorithm (greedy algorithm for LCP2) GLCP2 to identify overlapping functional modules. We compare SLCP2 and GLCP2 with a range of state-of-the-art algorithms on synthetic networks and real-world PPI networks. The performance evaluation based on several criteria with respect to protein complex prediction, high level Gene Ontology term prediction and especially sparse module detection, has demonstrated that our algorithms based on searching for LCP2 outperform all other compared algorithms. Availability and implementation: All data and code are available at http://www.cse.usf.edu/∼xqian/fmi/slcp2hop

  15. Pattern Formation by Electrostatic Self-Organization of Membrane Proteins

    NASA Astrophysics Data System (ADS)

    Boedec, G.; Jaeger, M.; Homble, F.; Leonetti, M.

    2012-07-01

    The electric activity of biological cells and organs such as heart for example is at the origin of various phenomena of pattern formation. The electric membrane potential appears as the order parameter to characterize these spatiotemporal dynamics. A kind of patterns is characterized by a stationary spatial modulation of membrane potential along the cell, breaking a symmetry of the system. They are associated to transcellular currents. A mechanism proposed in literature is based on the coupling of the electric current produced by membrane proteins and their electrophoretic mobilities. Beyond its classical linear stability analysis, the numerical and theoretical analysis of this model offers a variety of spatiotemporal dynamics. Firstly, the background in the modelization of electric phenomena is recalled. Secondly, the analysis is focused on two nonlinear dynamics.

  16. Pbx Homeodomain Proteins: TALEnted regulators of Limb Patterning and Outgrowth

    PubMed Central

    Capellini, Terence D.; Zappavigna, Vincenzo; Selleri, Licia

    2011-01-01

    Limb development has long provided an excellent model for understanding the genetic principles driving embryogenesis. Studies utilizing chick and mouse have led to new insights into limb patterning and morphogenesis. Recent research has centered on the regulatory networks underlying limb development. Here, we discuss the hierarchical, overlapping, and iterative roles of Pbx family members in appendicular development that have emerged from genetic analyses in the mouse. Pbx genes are essential in determining limb bud positioning, early bud formation, limb axes establishment and coordination, and patterning and morphogenesis of most elements of the limb and girdle. Pbx proteins directly regulate critical effectors of limb and girdle development, including morphogen-encoding genes like Shh in limb posterior mesoderm, and transcription factor-encoding genes like Alx1 in pre-scapular domains. Interestingly, at least in limb buds, Pbx appear to act not only as Hox cofactors, but also in the upstream control of 5' HoxA/D gene expression. PMID:21416555

  17. 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

  18. 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.

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

    PubMed

    Cruz-Landim, Carminda da; Dallacqua, Rodrigo Pires

    2005-01-01

    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. PMID:16342031

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

    PubMed Central

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

    2016-01-01

    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. PMID:27052788

  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. Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

    PubMed Central

    Hou, Yanxia; Genua, Maria; Garçon, Laurie-Amandine; Buhot, Arnaud; Calemczuk, Roberto; Bonnaffé, David; Lortat-Jacob, Hugues; Livache, Thierry

    2014-01-01

    In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins. PMID:25286325

  3. Protein expression patterns of the yeast mating response.

    PubMed

    Yuan, Haiyu; Zhang, Rongfei; Shao, Bin; Wang, Xuan; Ouyang, Qi; Hao, Nan; Luo, Chunxiong

    2016-06-13

    Microfluidics, in combination with time-lapse microscopy, is a transformative technology that significantly enhances our ability to monitor and probe biological processes in living cells. However, high-throughput microfluidic devices mostly require sophisticated preparatory and setup work and are thus hard to adopt by non-experts. In this work, we designed an easy-to-use microfluidic chip, which enables tracking of 48 GFP-tagged yeast strains, with each strain under two different stimulus conditions, in a single experiment. We used this technology to investigate the dynamic pattern of protein expression during the yeast mating differentiation response. High doses of pheromone induce cell cycle arrest and the shmoo morphology, whereas low doses of pheromone lead to elongation and chemotrophic growth. By systematically analyzing the protein dynamics of 156 pheromone-regulated genes, we identified groups of genes that are preferentially induced in response to low-dose pheromone (elongation during growth) or high-dose pheromone (shmoo formation and cell cycle arrest). The protein dynamics of these genes may provide insights into the mechanisms underlying the differentiation switch induced by different doses of pheromone. PMID:27177258

  4. Donor Heart Treatment With COMP-Ang1 Limits Ischemia-Reperfusion Injury and Rejection of Cardiac Allografts.

    PubMed

    Syrjälä, S O; Nykänen, A I; Tuuminen, R; Raissadati, A; Keränen, M A I; Arnaudova, R; Krebs, R; Koh, G Y; Alitalo, K; Lemström, K B

    2015-08-01

    The major cause of death during the first year after heart transplantation is primary graft dysfunction due to preservation and ischemia-reperfusion injury (IRI). Angiopoietin-1 is a Tie2 receptor-binding paracrine growth factor with anti-inflammatory properties and indispensable roles in vascular development and stability. We used a stable variant of angiopoietin-1 (COMP-Ang1) to test whether ex vivo intracoronary treatment with a single dose of COMP-Ang1 in donor Dark Agouti rat heart subjected to 4-h cold ischemia would prevent microvascular dysfunction and inflammatory responses in the fully allogeneic recipient Wistar Furth rat. COMP-Ang1 reduced endothelial cell-cell junction disruption of the donor heart in transmission electron microscopy during 4-h cold ischemia, improved myocardial reflow, and reduced microvascular leakage and cardiomyocyte injury of transplanted allografts during IRI. Concurrently, the treatment reduced expression of danger signals, dendritic cell maturation markers, endothelial cell adhesion molecule VCAM-1 and RhoA/Rho-associated protein kinase activation and the influx of macrophages and neutrophils. Furthermore, COMP-Ang1 treatment provided sustained anti-inflammatory effects during acute rejection and prevented the development of cardiac fibrosis and allograft vasculopathy. These results suggest donor heart treatment with COMP-Ang1 having important clinical implications in the prevention of primary and subsequent long-term injury and dysfunction in cardiac allografts. PMID:25932532

  5. Protein patterning by microcontact printing using pyramidal PDMS stamps.

    PubMed

    Filipponi, Luisa; Livingston, Peter; Kašpar, Ondřej; Tokárová, Viola; Nicolau, Dan V

    2016-02-01

    Micro-contact printing, μCP, is a well-established soft-lithography technique for printing biomolecules. μCP uses stamps made of Poly(dimethylsiloxane), PDMS, made by replicating a microstructured silicon master fabricated by semiconductor manufacturing processes. One of the problems of the μCP is the difficult control of the printing process, which, because of the high compressibility of PDMS, is very sensitive to minute changes in the applied pressure. This over-sensitive response leads to frequent and/or uncontrollable collapse of the stamps with high aspect ratios, thus decreasing the printing accuracy and reproducibility. Here we present a straightforward methodology of designing and fabricating PDMS structures with an architecture which uses the collapse of the stamp to reduce, rather than enlarge the variability of the printing. The PDMS stamp, organized as an array of pyramidal micro-posts, whose ceiling collapses when pressed on a flat surface, replicates the structure of the silicon master fabricated by anisotropic wet etching. Upon application of pressure, depending on the size of, and the pitch between, the PDMS pyramids, an air gap is formed surrounding either the entire array, or individual posts. The printing technology, which also exhibits a remarkably low background noise for fluorescence detection, may find applications when the clear demarcation of the shapes of protein patterns and the distance between them are critical, such as microarrays and studies of cell patterning. PMID:26782964

  6. Model for evaluating patterned charge regulation contribution to electrostatic interactions between proteins

    NASA Astrophysics Data System (ADS)

    Hollenbeck, Dawn; Martini, K. Michael; Langner, Andreas; Ross, David; Harkin, Anthony; Nelson, Edward; Thurston, George

    2010-03-01

    We study the pattern-specific work of charging for two spherical model proteins in close proximity in ionic solution, using a grand-canonical partition function together with a coarse-grained, linear Debye-Huckel model to calculate the needed work of charging for each possible proton occupancy configuration. We seek to delineate a parameter-space phase diagram to characterize the circumstances under which patterned charge regulation, attractions due to heterogeneous protein charging patterns, and screened net protein charge could individually dominate the electrostatic portion of the interaction between model particles. Within the model, we place titratable residues in accordance with the tertiary protein structure, as is done in the case of a single protein within the Tanford-Kirkwood protein electrostatics model. We use Monte-Carlo simulation and analytical work to evaluate how the local statistics of the charging patterns on each protein respond to close proximity and relative orientation of neighboring proteins.

  7. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

    PubMed

    Hsiao, Tony W; Tresco, Patrick A; Hlady, Vladimir

    2015-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels.

  8. Astrocytes alignment and reactivity on collagen hydrogels patterned with ECM proteins.

    PubMed

    Hsiao, Tony W; Tresco, Patrick A; Hlady, Vladimir

    2015-01-01

    To modulate the surface properties of collagen and subsequent cell-surface interactions, a method was developed to transfer protein patterns from glass coverslips to collagen type I hydrogel surfaces. Two proteins and one proteoglycan found in central nervous system extracellular matrix as well as fibrinogen were patterned in stripes onto collagen hydrogel and astrocytes were cultured on these surfaces. The addition of the stripe protein patterns to hydrogels created astrocyte layers in which cells were aligned with underlying patterns and had reduced chondroitin sulfate expression compared to the cells grown on collagen alone. Protein patterns were covalently cross-linked to the collagen and stable over four days in culture with no visible cellular modifications. The present method can be adapted to transfer other types of protein patterns from glass coverslips to collagen hydrogels. PMID:25477179

  9. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    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 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.

  11. 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.

  12. 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.

  13. 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.

  14. Easy Fabrication of Thin Membranes with Through Holes. Application to Protein Patterning

    PubMed Central

    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 cm2. 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. PMID:22952944

  15. 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. PMID:22952944

  16. 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.

  17. 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

  18. Effect of hair dyes and bleach on the hair protein patterns as revealed by isoelectric focusing.

    PubMed

    Nagai, A; Komoriya, H; Bunai, Y; Yamada, S; Jiang, X Y; Ohya, I

    1991-06-01

    The effect of hair dyes, i.e., temporary, semi-permanent, or permanent hair dyes, or hair bleach on the isoelectric focusing (IEF) hair protein patterns was studied. A permanent hair dye (metallic, alkaline oxidative, or acidic oxidative) and hair bleach induced changes in the IEF hair protein patterns and in the intensity of hair protein bands. The changes in the IEF patterns, caused by the alkaline oxidative dye or the bleach, are considered to result from the combined effect of an alkaline agent and an oxidative agent in the alkaline oxidative dye and in the hair bleach.

  19. Effect of hair dyes and bleach on the hair protein patterns as revealed by isoelectric focusing.

    PubMed

    Nagai, A; Komoriya, H; Bunai, Y; Yamada, S; Jiang, X Y; Ohya, I

    1991-06-01

    The effect of hair dyes, i.e., temporary, semi-permanent, or permanent hair dyes, or hair bleach on the isoelectric focusing (IEF) hair protein patterns was studied. A permanent hair dye (metallic, alkaline oxidative, or acidic oxidative) and hair bleach induced changes in the IEF hair protein patterns and in the intensity of hair protein bands. The changes in the IEF patterns, caused by the alkaline oxidative dye or the bleach, are considered to result from the combined effect of an alkaline agent and an oxidative agent in the alkaline oxidative dye and in the hair bleach. PMID:1889397

  20. COMP-1 promotes competitive advantage of nematode sperm

    PubMed Central

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

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.05423.001 PMID:25789512

  1. PatternQuery: web application for fast detection of biomacromolecular structural patterns in the entire Protein Data Bank.

    PubMed

    Sehnal, David; Pravda, Lukáš; Svobodová Vařeková, Radka; Ionescu, Crina-Maria; Koča, Jaroslav

    2015-07-01

    Well defined biomacromolecular patterns such as binding sites, catalytic sites, specific protein or nucleic acid sequences, etc. precisely modulate many important biological phenomena. We introduce PatternQuery, a web-based application designed for detection and fast extraction of such patterns. The application uses a unique query language with Python-like syntax to define the patterns that will be extracted from datasets provided by the user, or from the entire Protein Data Bank (PDB). Moreover, the database-wide search can be restricted using a variety of criteria, such as PDB ID, resolution, and organism of origin, to provide only relevant data. The extraction generally takes a few seconds for several hundreds of entries, up to approximately one hour for the whole PDB. The detected patterns are made available for download to enable further processing, as well as presented in a clear tabular and graphical form directly in the browser. The unique design of the language and the provided service could pave the way towards novel PDB-wide analyses, which were either difficult or unfeasible in the past. The application is available free of charge at http://ncbr.muni.cz/PatternQuery. PMID:26013810

  2. PatternQuery: web application for fast detection of biomacromolecular structural patterns in the entire Protein Data Bank.

    PubMed

    Sehnal, David; Pravda, Lukáš; Svobodová Vařeková, Radka; Ionescu, Crina-Maria; Koča, Jaroslav

    2015-07-01

    Well defined biomacromolecular patterns such as binding sites, catalytic sites, specific protein or nucleic acid sequences, etc. precisely modulate many important biological phenomena. We introduce PatternQuery, a web-based application designed for detection and fast extraction of such patterns. The application uses a unique query language with Python-like syntax to define the patterns that will be extracted from datasets provided by the user, or from the entire Protein Data Bank (PDB). Moreover, the database-wide search can be restricted using a variety of criteria, such as PDB ID, resolution, and organism of origin, to provide only relevant data. The extraction generally takes a few seconds for several hundreds of entries, up to approximately one hour for the whole PDB. The detected patterns are made available for download to enable further processing, as well as presented in a clear tabular and graphical form directly in the browser. The unique design of the language and the provided service could pave the way towards novel PDB-wide analyses, which were either difficult or unfeasible in the past. The application is available free of charge at http://ncbr.muni.cz/PatternQuery.

  3. Differentiation of Campylobacter species by protein banding patterns in polyacrylamide slab gels.

    PubMed

    Ferguson, D A; Lambe, D W

    1984-09-01

    Soluble protein extracts of 37 catalase-positive strains of Campylobacter species were examined by polyacrylamide slab gel electrophoresis (PAGE). Electrophoretic banding patterns showed good correlation with biochemical tests and with available DNA homology data in distinguishing species of Campylobacter but did not differentiate subspecies or biotypes. PAGE patterns indicated that Campylobacter coli is a distinct species. Furthermore, the PAGE patterns indicated that C. jejuni and nalidixic acid-resistant thermophilic Campylobacter species (C. laridis) are each distinct species. The protein banding patterns of C. fetus subsp. venerealis and C. fetus subsp. fetus strains were distinctly different from those of the three thermophilic species. PMID:6490829

  4. Two-dimensional Protein Patterns in Labronema, Aporcelaimellus, and Eudorylaimus (Nematoda: Dorylaimida)

    PubMed Central

    Ferris, V. R.; Ferris, J. M.; Murdock, L. L.; Faghihi, J.

    1987-01-01

    Two-dimensional polyacrylamide gel electrophoretic patterns of proteins for two isolates of Labronema from Indiana were nearly identical to the pattern for L. vulvapapillatum from Europe. The pattern for a nominal isolate of L. pacificum from Florida was very different from the patterns of nominal L. pacificum isolates from Hawaii and Fiji (which had patterns very similar to each other). Patterns for four other isolates (in Eudorylaimus and Aporcelaimellus) were different from the Labronema patterns and from each other, although some constellations of protein spots were shared among all the isolates. The study demonstrates the utility of 2-D PAGE for clarifying taxonomic problems that cannot be resolved using classical morphological data alone. PMID:19290167

  5. Application of Gap-Constraints Given Sequential Frequent Pattern Mining for Protein Function Prediction

    PubMed Central

    Park, Hyeon Ah; Kim, Taewook; Li, Meijing; Shon, Ho Sun; Park, Jeong Seok; Ryu, Keun Ho

    2015-01-01

    Objectives Predicting protein function from the protein–protein interaction network is challenging due to its complexity and huge scale of protein interaction process along with inconsistent pattern. Previously proposed methods such as neighbor counting, network analysis, and graph pattern mining has predicted functions by calculating the rules and probability of patterns inside network. Although these methods have shown good prediction, difficulty still exists in searching several functions that are exceptional from simple rules and patterns as a result of not considering the inconsistent aspect of the interaction network. Methods In this article, we propose a novel approach using the sequential pattern mining method with gap-constraints. To overcome the inconsistency problem, we suggest frequent functional patterns to include every possible functional sequence—including patterns for which search is limited by the structure of connection or level of neighborhood layer. We also constructed a tree-graph with the most crucial interaction information of the target protein, and generated candidate sets to assign by sequential pattern mining allowing gaps. Results The parameters of pattern length, maximum gaps, and minimum support were given to find the best setting for the most accurate prediction. The highest accuracy rate was 0.972, which showed better results than the simple neighbor counting approach and link-based approach. Conclusion The results comparison with other approaches has confirmed that the proposed approach could reach more function candidates that previous methods could not obtain. PMID:25938021

  6. 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.

  7. Invariant patterns in crystal lattices: Implications for protein folding algorithms

    SciTech Connect

    HART,WILLIAM E.; ISTRAIL,SORIN

    2000-06-01

    Crystal lattices are infinite periodic graphs that occur naturally in a variety of geometries and which are of fundamental importance in polymer science. Discrete models of protein folding use crystal lattices to define the space of protein conformations. Because various crystal lattices provide discretizations of the same physical phenomenon, it is reasonable to expect that there will exist invariants across lattices related to fundamental properties of the protein folding process. This paper considers whether performance-guaranteed approximability is such an invariant for HP lattice models. The authors define a master approximation algorithm that has provable performance guarantees provided that a specific sublattice exists within a given lattice. They describe a broad class of crystal lattices that are approximable, which further suggests that approximability is a general property of HP lattice models.

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    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.

  10. 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

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

    PubMed

    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

  12. Different evolutionary patterns of classical swine fever virus envelope proteins.

    PubMed

    Li, Yan; Yang, Zexiao; Zhang, Mingwang

    2016-03-01

    Classical swine fever virus (CSFV) is the causative agent of classical swine fever, which is a highly contagious disease of the domestic pig as well as wild boar. The proteins E(rns), E1, and E2 are components of the viral envelope membrane. They are also implicated in virus attachment and entry, replication, and (or) anti-immune response. Here, we studied the genetic variations of these envelope proteins in the evolution of CSFV. The results reveal that the envelope proteins underwent different evolutionary fates. In E(rns) and E1, but not E2, a number of amino acid sites experienced functional divergence. Furthermore, the diversification in E(rns) and E1 was generally episodic because the divergence-related changes of E1 only occurred with the separation of 2 major groups of CSFV and that of E(rns) took place with the division of 1 major group. The major divergence-related sites of E(rns) are located on one of the substrate-binding regions of the RNase domain and C-terminal extension. These functional domains have been reported to block activation of the innate immune system and attachment and entry into host cells, respectively. Our results may shed some light on the divergent roles of the envelope proteins. PMID:26911308

  13. 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

  14. 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.

  15. 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.

  16. A Novel Technique for Micro-patterning Proteins and Cells on Polyacrylamide Gels

    PubMed Central

    Tang, Xin; Ali, M. Yakut; Saif, M. Taher A.

    2012-01-01

    Spatial patterning of proteins (extracellular matrix, ECM) for living cells on polyacrylamide (PA) hydrogels has been technically challenging due to the compliant nature of the hydrogels and their aqueous environment. Traditional micro-fabrication process is not applicable. Here we report a simple, novel and general method to pattern a variety of commonly used cell adhesion molecules, i.e. Fibronectin (FN), Laminin (LN) and Collagen I (CN), etc. on PA gels. The pattern is first printed on a hydrophilic glass using polydimethylsiloxane (PDMS) stamp and micro-contact printing (μCP). Pre-polymerization solution is applied on the patterned glass and is then sandwiched by a functionalized glass slide, which covalently binds to the gel. The hydrophilic glass slide is then peeled off from the gel when the protein patterns detach from the glass, but remain intact with the gel. The pattern is thus transferred to the gel. The mechanism of pattern transfer is studied in light of interfacial mechanics. It is found that hydrophilic glass offers strong enough adhesion with ECM proteins such that a pattern can be printed, but weak enough adhesion such that they can be completely peeled off by the polymerized gel. This balance is essential for successful pattern transfer. As a demonstration, lines of FN, LN and CN with widths varying from 5–400 μm are patterned on PA gels. Normal fibroblasts (MKF) are cultured on the gel surfaces. The cell attachment and proliferation are confined within these patterns. The method avoids the use of any toxic chemistry often used to pattern different proteins on gel surfaces. PMID:23002394

  17. 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.

  18. Integrated reactive ion etching to pattern cross-linked hydrophilic polymer structures for protein immobilization

    NASA Astrophysics Data System (ADS)

    Bhatnagar, Parijat; Strickland, Aaron D.; Kim, Il; Malliaras, George G.; Batt, Carl A.

    2007-04-01

    Patterning of cross-linked hydrophilic polymer features using reactive ion etching (RIE) capable of covalently immobilizing proteins has been achieved. Projection photolithography was used to pattern photoresist to create micromolds. Vapor phase molecular self-assembly of polymerizable monolayer in molds allowed covalent binding of hydrogel on surface during free-radical polymerization. Excess hydrogel blanket film was consumed with oxygen RIE resulting into hydrogel pattern of 1μm size aligned to prefabricated silicon oxide structures. Proteins were finally coupled through their primary amine groups selectively to acid functionalized hydrogel features through stable amide linkages using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and N-hydroxysulfosuccinimide.

  19. Creating two-dimensional patterned substrates for protein and cell confinement.

    PubMed

    Johnson, Dawn M; LaFranzo, Natalie A; Maurer, Joshua A

    2011-01-01

    Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.(1) While microcontact printing can be employed to directly print a large number of molecules, including proteins,(2) DNA,(3) and silanes,(4) the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.(5) This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern. The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.(6) These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior(7) to the creation of microelectronics.(8) While other types of monolayer chemistry have been used for cell culture studies, including work from

  20. Predicting the Binding Patterns of Hub Proteins: A Study Using Yeast Protein Interaction Networks

    PubMed Central

    Andorf, Carson M.; Honavar, Vasant; Sen, Taner Z.

    2013-01-01

    Background Protein-protein interactions are critical to elucidating the role played by individual proteins in important biological pathways. Of particular interest are hub proteins that can interact with large numbers of partners and often play essential roles in cellular control. Depending on the number of binding sites, protein hubs can be classified at a structural level as singlish-interface hubs (SIH) with one or two binding sites, or multiple-interface hubs (MIH) with three or more binding sites. In terms of kinetics, hub proteins can be classified as date hubs (i.e., interact with different partners at different times or locations) or party hubs (i.e., simultaneously interact with multiple partners). Methodology Our approach works in 3 phases: Phase I classifies if a protein is likely to bind with another protein. Phase II determines if a protein-binding (PB) protein is a hub. Phase III classifies PB proteins as singlish-interface versus multiple-interface hubs and date versus party hubs. At each stage, we use sequence-based predictors trained using several standard machine learning techniques. Conclusions Our method is able to predict whether a protein is a protein-binding protein with an accuracy of 94% and a correlation coefficient of 0.87; identify hubs from non-hubs with 100% accuracy for 30% of the data; distinguish date hubs/party hubs with 69% accuracy and area under ROC curve of 0.68; and SIH/MIH with 89% accuracy and area under ROC curve of 0.84. Because our method is based on sequence information alone, it can be used even in settings where reliable protein-protein interaction data or structures of protein-protein complexes are unavailable to obtain useful insights into the functional and evolutionary characteristics of proteins and their interactions. Availability We provide a web server for our three-phase approach: http://hybsvm.gdcb.iastate.edu. PMID:23431393

  1. Detonation wave curvature of cast Comp B and PBXN-111

    NASA Astrophysics Data System (ADS)

    Lemar, E. R.; Forbes, J. W.

    1994-07-01

    Detonation wave profiles for cast Comp B and PBXN-111 have been fitted accurately over the entire wave fronts using a series expansion of the natural logarithm of a Bessel function. The fit equation has been used to obtain the angle of the detonation front as a function of position and the radii of curvature used in Wood-Kirkwood zone length calculations. The results obtained from the fit equation agree with results obtained previously for PBXN-111. Since the fit equation gives a functional form for the detonation wave across the whole charge diameter, it can be used to test the results obtained from detonation theories and code calculations.

  2. Protein patterns of the Brassica rapa ovules and seeds under altered gravity.

    PubMed

    Sozinov, Igor; Kozub, Natalia; Popova, Antonina

    2004-07-01

    Electrophoretic investigation of protein patterns of Brassica rapa L. ovules and seeds from plants grown under clinorotation and in the laboratory control was carried out. Ovules at different stages (7 and 18 days after pollination) and mature seeds were analyzed. Polymorphism of seed storage proteins of B. rapa was taken into consideration in analysis of changes in ovule protein patterns under clinorotation. The appearance of a protein component in the region of about 43 kDa was detected in protein patterns of 7-day-old and 18-day-old ovules in the clinostat variants. Under altered gravity, in 18-day-old ovules, the appearance of a protein in the region of about 70 kDa was also revealed. The appearance of the protein component with the similar mobility (about 43 kDa) in ovules of different age from plants grown at clinorotation suggests that synthesis of this protein may be associated with the plant response to altered gravity. However, the investigation of the nature of this protein and its role requires further research to rule out its appearance because of genotypic differences between ovules of the control and experimental variants.

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

    PubMed

    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 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. PMID:27595425

  4. 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

  5. Adhesion of adipose-derived mesenchymal stem cells to glycosaminoglycan surfaces with different protein patterns.

    PubMed

    Soares da Costa, Diana; Márquez-Posadas, Maria del Carmen; Araujo, Ana R; Yang, Yuan; Merino, Santos; Groth, Thomas; Reis, Rui L; Pashkuleva, Iva

    2015-05-13

    Proteins and glycosaminoglycans (GAGs) are the main constituents of the extracellular matrix (ECM). They act in synergism and are equally critical for the development, growth, function, or survival of an organism. In this work, we developed surfaces that display these two classes of biomacromolecules, namely, GAGs and proteins, in a spatially controlled fashion. The generated surfaces can be used as a minimalistic but straightforward model aiding the elucidation of cell-ECM interactions. GAGs (hyaluronic acid and heparin) were covalently bound to amino functionalized surfaces, and albumin or fibronectin was patterned by microcontact printing on top of them. We demonstrate that adipose-derived stem cells (ASCs) can adhere either on the protein or on the GAG pattern as a function of the patterned molecules. ASCs found on the GAG pattern had different morphology and expressed different surface markers than the cells adhered on the protein pattern. ASCs morphology and spreading were also dependent on the size of the pattern. These results show that the developed supports can also be used for ASCs differentiation into different lineages.

  6. Regular Patterns for Proteome-Wide Distribution of Protein Abundance across Species

    PubMed Central

    Jiang, Ying; Ying, Wantao; Wu, Songfeng; Zhu, Yunping; Liu, Siqi; Yang, Pengyuan; Qian, Xiaohong; He, Fuchu

    2012-01-01

    A proteome of the bio-entity, including cell, tissue, organ, and organism, consists of proteins of diverse abundance. The principle that determines the abundance of different proteins in a proteome is of fundamental significance for an understanding of the building blocks of the bio-entity. Here, we report three regular patterns in the proteome-wide distribution of protein abundance across species such as human, mouse, fly, worm, yeast, and bacteria: in most cases, protein abundance is positively correlated with the protein's origination time or sequence conservation during evolution; it is negatively correlated with the protein's domain number and positively correlated with domain coverage in protein structure, and the correlations became stronger during the course of evolution; protein abundance can be further stratified by the function of the protein, whereby proteins that act on material conversion and transportation (mass category) are more abundant than those that act on information modulation (information category). Thus, protein abundance is intrinsically related to the protein's inherent characters of evolution, structure, and function. PMID:22427835

  7. Protein Adsorption on Patterned Hydroxyapatite Thin Films Fabricated by Pulsed Laser Deposition

    NASA Astrophysics Data System (ADS)

    Kusunoki, Masanobu; Kawasima, Masami; Nishikawa, Hiroaki; Morimoto, Koichi; Hayami, Takashi; Hontsu, Shigeki; Kawai, Tomoji

    2005-02-01

    Protein adsorption on hydroxyapatite (HAP) thin film was investigated before and after patterning. Hydroxyapatite thin film 100 nm thick was deposited by pulsed laser deposition. The film was patterned by photolithography and wet etching with HCl solution. Proteins (phospholyrase b, bovine serum albumin, and others) labeled with fluorescein isothiocyanate (FITC) were used as the reagent. After the HAP film was soaked in the reagent and washed with pure water, a conspicuous contrast in FITC was observed between the HAP pattern and the glass substrate (or photoresist). This behavior showed that the biocompatibility of the HAP thin film was not influenced by the patterning process. Our technique for HAP thin film is adaptable for applications involving biosensors as electronic devices and scaffolds for tissue culture.

  8. 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.

  9. QuasiMotiFinder: protein annotation by searching for evolutionarily conserved motif-like patterns.

    PubMed

    Gutman, Roee; Berezin, Carine; Wollman, Roy; Rosenberg, Yossi; Ben-Tal, Nir

    2005-07-01

    Sequence signature databases such as PROSITE, which include amino acid segments that are indicative of a protein's function, are useful for protein annotation. Lamentably, the annotation is not always accurate. A signature may be falsely detected in a protein that does not carry out the associated function (false positive prediction, FP) or may be overlooked in a protein that does carry out the function (false negative prediction, FN). A new approach has emerged in which a signature is replaced with a sequence profile, calculated based on multiple sequence alignment (MSA) of homologous proteins that share the same function. This approach, which is superior to the simple pattern search, essentially searches with the sequence of the query protein against an MSA library. We suggest here an alternative approach, implemented in the QuasiMotiFinder web server (http://quasimotifinder.tau.ac.il/), which is based on a search with an MSA of homologous query proteins against the original PROSITE signatures. The explicit use of the average evolutionary conservation of the signature in the query proteins significantly reduces the rate of FP prediction compared with the simple pattern search. QuasiMotiFinder also has a reduced rate of FN prediction compared with simple pattern searches, since the traditional search for precise signatures has been replaced by a permissive search for signature-like patterns that are physicochemically similar to known signatures. Overall, QuasiMotiFinder and the profile search are comparable to each other in terms of performance. They are also complementary to each other in that signatures that are falsely detected in (or overlooked by) one may be correctly detected by the other.

  10. Characterization of a nanoscale S-layer protein based template for biomolecular patterning.

    PubMed

    Wong, Wing Sze; Yung, Pun To

    2014-01-01

    Well organized template for biomolecular conjugation is the foundation for biosensing. Most of the current devices are fabricated using lithographic patterning processes and self-assembly monolayer (SAM) methods. However, the research toward developing a sub-10 nm patterned, self-regenerated template on various types of substrates is limited, mainly due to the limited functional groups of the building material. Bacterial surface layer proteins (S-layer proteins) can self-assemble into ordered lattice with regular pore sizes of 2-8 nm on different material supports and interfaces. The ordered structure can regenerate after extreme variations of solvent conditions. In this work, we developed a nanoscale biomolecular template based on S-layer proteins on gold surface for fabrication of sensing layer in biosensors. S-layer proteins were isolated from Bacillus cereus, Lysinibacillus sphaericus and Geobacillus stearothermophilus. Protein concentrations were measured by Bradford assay. The protein purities were verified by SDS-PAGE, showing molecular weights ranging from 97-135 kDa. The hydrophilicity of the substrate surface was measured after surface treatments of protein recrystallization. Atomic force microscopic (AFM) measurement was performed on substrate surface, indicating a successful immobilization of a monolayer of S-layer protein with 8-9 nm height on gold surface. The template can be applied on various material supports and acts as a self-regenerated sensing layer of biosensors in the future. PMID:25570568

  11. 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. PMID:24823688

  12. 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

  13. Laser trapping and patterning of protein microcrystals: Toward highly integrated protein microarrays

    NASA Astrophysics Data System (ADS)

    Hosokawa, Yoichiroh; Matsumura, Satoshi; Masuhara, Hiroshi; Ikeda, Keiko; Shimo-oka, Ai; Mori, Hajime

    2004-09-01

    Some insect virus infections occlude into a crystalline matrix consisting of a protein named polyhedrin. The shape of the matrix is a cubic polyhedron of the size of a few micrometers. Recently it was shown that these polyhedra could immobilize various functional proteins within them. Therefore, the polyhedron is interesting as an element in a protein chip. In this work, individual polyhedra were arrayed and bonded under a microscope by focused laser beams, with the aim of fabricating a highly integrated protein chip. The polyhedron was trapped and transferred to a suitable position on a polymer substrate by optical trapping with a 1064nmNd3+:YAG (YAG, yttrium aluminum garnet) laser. To bond the polyhedron on the substrate, the polymer surface was mechanically and chemically modified by multiphoton absorption of a 120fs, 800nm femtosecond Ti: sapphire laser, which results in strong adhesion of the polyhedron to the substrate. The arraying and bonding of polyhedra were successful, to a precision of about 1μm, with this procedure. The biological activity of polyhedra after these laser irradiations was confirmed by the fluorescence of green fluorescent protein occluded in the polyhedrin matrix.

  14. 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…

  15. Constitutive patterns of gene expression regulated by RNA-binding proteins

    PubMed Central

    2014-01-01

    Background RNA-binding proteins regulate a number of cellular processes, including synthesis, folding, translocation, assembly and clearance of RNAs. Recent studies have reported that an unexpectedly large number of proteins are able to interact with RNA, but the partners of many RNA-binding proteins are still uncharacterized. Results We combined prediction of ribonucleoprotein interactions, based on catRAPID calculations, with analysis of protein and RNA expression profiles from human tissues. We found strong interaction propensities for both positively and negatively correlated expression patterns. Our integration of in silico and ex vivo data unraveled two major types of protein–RNA interactions, with positively correlated patterns related to cell cycle control and negatively correlated patterns related to survival, growth and differentiation. To facilitate the investigation of protein–RNA interactions and expression networks, we developed the catRAPID express web server. Conclusions Our analysis sheds light on the role of RNA-binding proteins in regulating proliferation and differentiation processes, and we provide a data exploration tool to aid future experimental studies. PMID:24401680

  16. SplitPocket: identification of protein functional surfaces and characterization of their spatial patterns.

    PubMed

    Tseng, Yan Yuan; Dupree, Craig; Chen, Z Jeffrey; Li, Wen-Hsiung

    2009-07-01

    SplitPocket (http://pocket.uchicago.edu/) is a web server to identify functional surfaces of protein from structure coordinates. Using the Alpha Shape Theory, we previously developed an analytical approach to identify protein functional surfaces by the geometric concept of a split pocket, which is a pocket split by a binding ligand. Our geometric approach extracts site-specific spatial information from coordinates of structures. To reduce the search space, probe radii are designed according to the physicochemical textures of molecules. The method uses the weighted Delaunay triangulation and the discrete flow algorithm to obtain geometric measurements and spatial patterns for each predicted pocket. It can also measure the hydrophobicity on a surface patch. Furthermore, we quantify the evolutionary conservation of surface patches by an index derived from the entropy scores in HSSP (homology-derived secondary structure of proteins). We have used the method to examine approximately 1.16 million potential pockets and identified the split pockets in >26,000 structures in the Protein Data Bank. This integrated web server of functional surfaces provides a source of spatial patterns to serve as templates for predicting the functional surfaces of unbound structures involved in binding activities. These spatial patterns should also be useful for protein functional inference, structural evolution and drug design.

  17. Distinct expression patterns of ICK/MAK/MOK protein kinases in the intestine implicate functional diversity.

    PubMed

    Chen, Tufeng; Wu, Di; Moskaluk, Christopher A; Fu, Zheng

    2013-01-01

    ICK/MRK (intestinal cell kinase/MAK-related kinase), MAK (male germ cell-associated kinase), and MOK (MAPK/MAK/MRK-overlapping kinase) are closely related serine/threonine protein kinases in the protein kinome. The biological functions and regulatory mechanisms of the ICK/MAK/MOK family are still largely elusive. Despite significant similarities in their catalytic domains, they diverge markedly in the sequence and structural organization of their C-terminal non-catalytic domains, raising the question as to whether they have distinct, overlapping, or redundant biological functions. In order to gain insights into their biological activities and lay a fundamental groundwork for functional studies, we investigated the spatio-temporal distribution patterns and the expression dynamics of ICK/MAK/MOK protein kinases in the intestine. We found that ICK/MAK/MOK proteins display divergent expression patterns along the duodenum-to-colon axis and during postnatal murine development. Furthermore, they are differentially partitioned between intestinal epithelium and mesenchyme. A significant increase in the protein level of ICK, but not MAK, was induced in human primary colon cancer specimens. ICK protein level was up-regulated whereas MOK protein level was down-regulated in mouse intestinal adenomas as compared with their adjacent normal intestinal mucosa. These data suggest distinct roles for ICK/MAK/MOK protein kinases in the regulation of intestinal neoplasia. Taken together, our findings demonstrate that the expressions of ICK/MAK/MOK proteins in the intestinal tract can be differentially and dynamically regulated, implicating a significant functional diversity within this group of protein kinases.

  18. 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

  19. 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.

  20. 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.

  1. Diversity in transcripts and translational pattern of stress proteins in marine extremophiles.

    PubMed

    Ambily Nath, I V; Loka Bharathi, P A

    2011-03-01

    Extremophiles occur in a diverse range of habitats, from the frigid waters of Antarctic to the superheated plumes of hydrothermal vents. Their in-depth study could provide important insights into the biochemical, ecological and evolutionary aspects of marine microbes. The cellular machinery of such extreme-lovers could be highly flexible to cope with such harsh environments. Extreme conditions of temperature, pressure, salinity, pH, oxidative stress, radiation, etc., above the physiological tolerance level can disrupt the natural conformation of proteins in the cell. The induction of stress proteins (heat/cold shock proteins/salt stress proteins/pressure-induced proteins) plays a vital role in the acclimatization of extremophiles. The present review focuses on the in vitro studies conducted on the transcripts and translational pattern of stress proteins in extremophiles. Though some proteins are unique, a commonality in stress resistance mechanism has been observed, for example, the universal occurrence of HSP60, 70 and the expression of metabolic and DNA repair proteins. The review highlights that among all the stressful conditions, salt/osmotic stress evokes the expression of highest number of transcripts/proteins while psychrophilic condition the least.

  2. Association Between Dietary Pattern and Serum C-Reactive Protein in Japanese Men and Women

    PubMed Central

    Nanri, Hinako; Nakamura, Kazuyo; Hara, Megumi; Higaki, Yasuki; Imaizumi, Takeshi; Taguchi, Naoto; Sakamoto, Tatsuhiko; Horita, Mikako; Shinchi, Koichi; Tanaka, Keitaro

    2011-01-01

    Background Dietary pattern may influence the risks of cardiovascular disease, atherosclerosis, type 2 diabetes, and metabolic syndrome through its effects on inflammation. We evaluated the association between dietary pattern and serum high-sensitivity C-reactive protein (hs-CRP) in a Japanese population. Methods In this cross-sectional analysis, we used baseline data from 3905 men and 5640 women (age 40–69 years) who participated in a population-based cohort study between November 2005 and December 2007. Participants with possible inflammation-related diseases, current analgesic use, high hs-CRP levels (≥3000 ng/mL) or extreme dietary energy intake were excluded. We used 46 items from a validated short food frequency questionnaire and examined major dietary patterns by factor analysis. Results We identified 5 dietary patterns: healthy (high in vegetables and fruit), Western (high in meat and fried foods), seafood (high in shellfish, squid, fish, etc.), bread (high in bread and low in rice), and dessert (high in confections and fruit). After adjustment for age, alcohol use, smoking, physical activity, and body mass index, hs-CRP levels in men were inversely associated with the healthy, bread, and dessert patterns (P-trend: 0.01, 0.06, and <0.01, respectively) and positively associated with the seafood pattern (P-trend = 0.02). In women, hs-CRP levels were inversely associated with the healthy pattern (P-trend = 0.06) and positively associated with the Western pattern (P-trend = 0.06). Conclusions The healthy dietary pattern may be associated with suppressed inflammation in Japanese men and women, independently of body mass index and other factors. The sex-specific associations of hs-CRP with other dietary patterns (eg, the seafood pattern) require further study. PMID:21325731

  3. 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

  4. Proteomic profiling reveals a severely perturbed protein expression pattern in aged skeletal muscle.

    PubMed

    O'Connell, Kathleen; Gannon, Joan; Doran, Philip; Ohlendieck, Kay

    2007-08-01

    Extended longevity is often accompanied by frailty and increased susceptibility to a variety of crippling disorders. One of the most striking features of human aging is sarcopenia, which is defined as the age-related decline in skeletal muscle mass and strength. Although various metabolic and functional defects in aging muscle fibres have been described over the last decade, it is not known whether a pathophysiological hierarchy exists within degenerative pathways leading to muscle wasting. Hence, in order to identify novel biomarkers of age-dependent skeletal muscle degeneration, we have here applied mass spectrometry-based proteomics for studying global muscle protein expression patterns. As a model system of sarcopenia, we have employed crude extracts from senescent rat gastrocnemius muscle, as compared to young adult tissue preparations. Using the highly sensitive protein dye Deep Purple for the analysis of the 2-D separated muscle proteome and peptide mass fingerprinting for the identification of individual protein spots, a differential expression pattern was observed for contractile proteins, metabolic factors, regulatory components and heat shock elements. A drastic increase was shown for alpha B-crystallin, myosin light chain MLC-1, phosphoglycerate kinase, adenylate kinase, triosephosphate isomerase, albumin, aconitase and nucleoside-diphosphate kinase in aged fibres. In contrast, the expression of pyruvate kinase, aldolase, creatine kinase, transferrin, alpha-tropomyosin and myosin light chain MLC-3 was decreased in old skeletal muscle. Comparative 2-D immunoblotting of selected candidate proteins has confirmed the effect of aging on the skeletal muscle proteome. These findings demonstrate a severely perturbed protein expression pattern in aged skeletal muscle, which reflects the underlying molecular alterations causing a drastic decline of muscle strength in the senescent organism. In the long-term, the systematic deduction of abnormal protein expression

  5. Interaction between Cartilage Oligomeric Matrix Protein and Extracellular Matrix Protein 1 Mediates Endochondral Bone Growth

    PubMed Central

    Kong, Li; Tian, Qingyun; Guo, Fengjin; Mucignat, Maria T.; Perris, Roberto; Sercu, Sandy; Merregaert, Joseph; Di Cesare, Paul E.; Liu, Chuan-ju

    2010-01-01

    In an effort to define the biological functions of COMP, a functional genetic screen was performed. This led to the identification of extracellular matrix protein 1 (ECM1) as a novel COMP-associated partner. COMP directly binds to ECM1 both in vitro and in vivo. The EGF domain of COMP and the C-terminus of ECM1 mediate the interaction between them. COMP and ECM1 Colocalize in the Growth Plates in Vivo. ECM1 inhibits chondrocyte hypertrophy, matrix mineralization, and endochondral bone formation, and COMP overcomes the inhibition by ECM1. In addition, COMP-mediated neutralization of ECM1 inhibition depends on their interaction, since COMP largely fails to overcome the ECM1 inhibition in the presence of the EGF domain of COMP, which disturbs the association of COMP and ECM1. These findings provide the first evidence linking the association of COMP and ECM1 and the biological significance underlying the interaction between them in regulating endochondral bone growth. PMID:20138147

  6. Common and distinctive localization patterns of Crumbs polarity complex proteins in the mammalian eye

    PubMed Central

    Kim, Jin Young; Song, Ji Yun; Karnam, Santi; Park, Jun Young; Lee, Jamie JH; Kim, Seonhee; Cho, Seo-Hee

    2016-01-01

    Crumbs polarity complex proteins are essential for cellular and tissue polarity, and for adhesion of epithelial cells. In epithelial tissues deletion of any of three core proteins disrupts localization of the other proteins, indicating structural and functional interdependence among core components. Despite previous studies of function and co-localization that illustrated the properties that these proteins share, it is not known whether an individual component of the complex plays a distinct role in a unique cellular and developmental context. In order to investigate this question, we primarily used confocal imaging to determine the expression and subcellular localization of the core Crumbs polarity complex proteins during ocular development. Here we show that in developing ocular tissues core Crumbs polarity complex proteins, Crb, Pals1 and Patj, generally appear in an overlapping pattern with some exceptions. All three core complex proteins localize to the apical junction of the retinal and lens epithelia. Pals1 is also localized in the Golgi of the retinal cells and Patj localizes to the nuclei of the apically located subset of progenitor cells. These findings suggest that core Crumbs polarity complex proteins exert common and independent functions depending on cellular context. PMID:25636444

  7. Electrophoretic separation of purified myelin: a method to improve the protein pattern resolving.

    PubMed

    Ravera, Silvia; Bartolucci, Martina; Barbarito, Giulia; Calzia, Daniela; Panfoli, Isabella

    2013-01-01

    Myelin sheath is a lipid-rich membrane, consisting of 70% lipid and 30% proteins, that is involved in physiological and pathological processes. For this reason its protein composition has been often investigated, principally by two-dimensional electrophoresis; however, the consistent lipid content makes it difficult to obtain good proteins separation. To improve the resolution of myelin proteins in a denaturing monodimensional gel electrophoresis, we examined several mixtures for the denaturation of the sample, utilizing different detergents and reducing agents. The definition of the protein pattern was analyzed by both "Blue Silver" Coomassie staining and Western Blot analysis against myelin basic protein, one of the most represented myelin proteins. The best resolution is observed when the sample was incubated with a mixture containing 1.25% dithiothreitol, 4 M urea, and 1% dodecyl maltoside or 1 % 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, prior to addition of denaturing agents. In conclusion, this work describes a novel method to improve the separation of myelin proteins in a monodimensional gel electrophoresis. It may be also useful for investigating other lipid-rich samples. PMID:23464917

  8. Electrophoretic separation of purified myelin: a method to improve the protein pattern resolving.

    PubMed

    Ravera, Silvia; Bartolucci, Martina; Barbarito, Giulia; Calzia, Daniela; Panfoli, Isabella

    2013-01-01

    Myelin sheath is a lipid-rich membrane, consisting of 70% lipid and 30% proteins, that is involved in physiological and pathological processes. For this reason its protein composition has been often investigated, principally by two-dimensional electrophoresis; however, the consistent lipid content makes it difficult to obtain good proteins separation. To improve the resolution of myelin proteins in a denaturing monodimensional gel electrophoresis, we examined several mixtures for the denaturation of the sample, utilizing different detergents and reducing agents. The definition of the protein pattern was analyzed by both "Blue Silver" Coomassie staining and Western Blot analysis against myelin basic protein, one of the most represented myelin proteins. The best resolution is observed when the sample was incubated with a mixture containing 1.25% dithiothreitol, 4 M urea, and 1% dodecyl maltoside or 1 % 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, prior to addition of denaturing agents. In conclusion, this work describes a novel method to improve the separation of myelin proteins in a monodimensional gel electrophoresis. It may be also useful for investigating other lipid-rich samples.

  9. Proteins linked to autosomal dominant and autosomal recessive disorders harbor characteristic rare missense mutation distribution patterns.

    PubMed

    Turner, Tychele N; Douville, Christopher; Kim, Dewey; Stenson, Peter D; Cooper, David N; Chakravarti, Aravinda; Karchin, Rachel

    2015-11-01

    The role of rare missense variants in disease causation remains difficult to interpret. We explore whether the clustering pattern of rare missense variants (MAF < 0.01) in a protein is associated with mode of inheritance. Mutations in genes associated with autosomal dominant (AD) conditions are known to result in either loss or gain of function, whereas mutations in genes associated with autosomal recessive (AR) conditions invariably result in loss-of-function. Loss-of-function mutations tend to be distributed uniformly along protein sequence, whereas gain-of-function mutations tend to localize to key regions. It has not previously been ascertained whether these patterns hold in general for rare missense mutations. We consider the extent to which rare missense variants are located within annotated protein domains and whether they form clusters, using a new unbiased method called CLUstering by Mutation Position. These approaches quantified a significant difference in clustering between AD and AR diseases. Proteins linked to AD diseases exhibited more clustering of rare missense mutations than those linked to AR diseases (Wilcoxon P = 5.7 × 10(-4), permutation P = 8.4 × 10(-4)). Rare missense mutation in proteins linked to either AD or AR diseases was more clustered than controls (1000G) (Wilcoxon P = 2.8 × 10(-15) for AD and P = 4.5 × 10(-4) for AR, permutation P = 3.1 × 10(-12) for AD and P = 0.03 for AR). The differences in clustering patterns persisted even after removal of the most prominent genes. Testing for such non-random patterns may reveal novel aspects of disease etiology in large sample studies. PMID:26246501

  10. 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.

  11. Biomimetic Polymer Brushes Containing Tethered Acetylcholine Analogs for Protein and Hippocampal Neuronal Cell Patterning

    PubMed Central

    Zhou, Zhaoli; Yu, Panpan; Geller, Herbert M.; Ober, Christopher K.

    2013-01-01

    This paper describes a method to control neuronal cell adhesion and differentiation with both chemical and topographic cues by using a spatially defined polymer brush pattern. First, biomimetic methacrylate polymer brushes containing tethered neurotransmitter acetylcholine functionalities in the form of dimethylaminoethyl methacrylate (DMAEMA), or free hydroxyl-terminated poly(ethylene glycol) (PEG) units were prepared using the “grown from” method through surface-initiated atom transfer radical polymerization (SI-ATRP) reactions. The surface properties of the resulting brushes were thoroughly characterized with various techniques and hippocampal neuronal cell culture on the brush surfaces exhibit cell viability and differentiation comparable to, or even better than, those on commonly used poly-L-lysine coated glass coverslips. The polymer brushes were then patterned via UV photolithography techniques to provide specially designed surface features with different sizes (varying from 2 µm to 200 µm) and orientations (horizontal and vertical). Protein absorption experiments and hippocampal neuronal cell culture tests on the brush patterns showed that both protein and neurons can adhere to the patterns and therefore be guided by such patterns. These results also demonstrate that, because of their unique chemical composition and well-defined nature, the developed polymer brushes may find many potential applications in cell-material interactions studies and neural tissue engineering. PMID:23336729

  12. Comparative study of electrophoretic patterns of latex proteins from clones of Hevea brasiliensis.

    PubMed

    Arreguín, B; Lara, P; Rodríguez, R

    1988-07-01

    Latex serum proteins from Hevea brasiliensis were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins from whole serum and fractions isolated by gel chromatography on Ultrogel AcA 44 were analyzed. No qualitative clonal differences were found in the protein patterns of whole latex or in the fractions but laser densitometry revealed reliable quantitative differences in protein composition. Reproducible mobilities and molecular weights of selected bands were obtained both within single gels as well as in different gels, analyzing several lots of latex received at various times from a Hevea experimental field station. The clones compared were IAN 710, GV 31, GV 42; the first one had the highest rubber yields. PMID:3234370

  13. Protein interaction patterns in different cellular environments are revealed by in-cell NMR

    PubMed Central

    Barbieri, Letizia; Luchinat, Enrico; Banci, Lucia

    2015-01-01

    In-cell NMR allows obtaining atomic-level information on biological macromolecules in their physiological environment. Soluble proteins may interact with the cellular environment in different ways: either specifically, with their functional partners, or non-specifically, with other cellular components. Such behaviour often causes the disappearance of the NMR signals. Here we show that by introducing mutations on the human protein profilin 1, used here as a test case, the in-cell NMR signals can be recovered. In human cells both specific and non-specific interactions are present, while in bacterial cells only the effect of non-specific interactions is observed. By comparing the NMR signal recovery pattern in human and bacterial cells, the relative contribution of each type of interaction can be assessed. This strategy allows detecting solution in-cell NMR spectra of soluble proteins without altering their fold, thus extending the applicability of in-cell NMR to a wider range of proteins. PMID:26399546

  14. Sex dependent alterations in the protein characterization patterns of Haemonchus contortus.

    PubMed

    Jaiswal, Amit Kumar; Sudan, Vikrant; Pandey, Vijay; Singh, Amit; Gaur, Ruchi Singh; Kanojiya, Dharmendra; Nigam, Rajesh; Shanker, Daya

    2016-09-01

    The aim of the study was to highlight the sex dependent differences in the electrophoretic protein patterns of male and female Haemonchus contortus worms SDS based polyacrylamide gels of both male and female worms were run side by side for comparison. A total of 33 and 35 polypeptides were detected in polyacrylamide gels stained with coomassie brilliant blue R-250, respectively. Besides many of the fundamental homologies in protein profile, some of the polypeptides specific to either sex were also observed. Most of the characteristic polypeptides were of low molecular weight. These polypeptides needs deeper unrevealing regarding the nature of protein, through well planned zymographic studies, so as to ascertain the true nature and/or type of protein involved in those bands. This will help us in better understanding of parasite immunology and sex influenced differences amongst the worm and the possible variations in their pathogenesis contributed thereof, if any. PMID:27605828

  15. Developmental pattern of rat intestinal brush-border enzymic proteins along the villus--crypt axis.

    PubMed

    Simon, P M; Kedinger, M; Raul, F; Grenier, J F; Haffen, K

    1979-02-15

    At various postnatal stages, intestinal epithelial cells were isolated sequentially from villus tip to crypt base by successive EDTA treatments. According to the localization of marker enzymic activities, isolated cells were pooled into three cell compartments: villus (V), lower villus and upper crypt (VC) and crypt (C). Purified brush-border-membrane proteins were separated by 7.5%-polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Enzymic activities could be assigned to some protein bands: maltase/glucoamylase (protein band 3), sucrase-isomaltase (protein bands 3 and 6), lactase (protein band 5) and alkaline phosphatase (region of protein bands 8 and 9). The findings suggest the following. (1) Sucrase-isomaltase activities appeared in compartment C at 17 days with a simultaneous increase of the pre-existing protein band 3 and appearance of a well-defined protein band in position 6; the enzymic complex remained still present in the crypt cells until adulthood. From the day 21 onwards, sucrase-isomaltase was detected in compartments VC and V. (2) Lactase was only present in the three cell compartments until day 21; at this developmental stage its activity completely disappeared from compartment C, in spite of the persistence of a weak protein band. (3) Alkaline phosphatase activity could be detected as a single peak corresponding to protein band 9 in all three cell compartments until day 21; thereafter it was replaced by two peaks of activity showing a less precise correlation with the well-defined protein bands 8 and 9. In the crypt cells of the adult rat, however, the preweaning situation, which was regularly observed, is an unexpected phenomenon. (4) Maltase and glucoamylase did not display any marked qualitative or quantitative modifications either along the villus-crypt axis or during the period of postnatal development studied. Evidence is given from the present data that each brush-border enzyme investigated has a specific

  16. Protein assembly onto patterned microfabricated devices through enzymatic activation of fusion pro-tag.

    PubMed

    Lewandowski, Angela T; Yi, Hyunmin; Luo, Xiaolong; Payne, Gregory F; Ghodssi, Reza; Rubloff, Gary W; Bentley, William E

    2008-02-15

    We report a versatile approach for covalent surface-assembly of proteins onto selected electrode patterns of pre-fabricated devices. Our approach is based on electro-assembly of the aminopolysaccharide chitosan scaffold as a stable thin film onto patterned conductive surfaces of the device, which is followed by covalent assembly of the target protein onto the scaffold surface upon enzymatic activation of the protein's "pro-tag." For our demonstration, the model target protein is green fluorescent protein (GFP) genetically fused with a pentatyrosine pro-tag at its C-terminus, which assembles onto both two-dimensional chips and within fully packaged microfluidic devices in situ and under flow. Our surface-assembly approach enables spatial selectivity and orientational control under mild experimental conditions. We believe that our integrated approach harnessing genetic manipulation, in situ enzymatic activation, and electro-assembly makes it advantageous for a wide variety of bioMEMS and biosensing applications that require facile "biofunctionalization" of microfabricated devices. PMID:17625789

  17. Biocompatible patterning of proteins on wettability gradient surface by thermo-transfer printing.

    PubMed

    Kim, Sungho; Ryu, Yong-Sang; Suh, Jeng-Hun; Keum, Chang-Min; Sohn, Youngjoo; Lee, Sin-Doo

    2014-08-01

    We develop a simple and biocompatible method of patterning proteins on a wettability gradient surface by thermo-transfer printing. The wettability gradient is produced on a poly(dimethylsiloxane) (PDMS)-modified glass substrate through the temperature gradient during thermo-transfer printing. The water contact angle on the PDMS-modified surface is found to gradually increase along the direction of the temperature gradient from a low to a high temperature region. Based on the wettability gradient, the gradual change in the adsorption and immobilization of proteins (cholera toxin B subunit) is achieved in a microfluidic cell with the PDMS-modified surface. PMID:25936059

  18. 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

  19. Analysis of the expression patterns, subcellular localisations and interaction partners of Drosophila proteins using a pigP protein trap library

    PubMed Central

    Lowe, Nick; Rees, Johanna S.; Roote, John; Ryder, Ed; Armean, Irina M.; Johnson, Glynnis; Drummond, Emma; Spriggs, Helen; Drummond, Jenny; Magbanua, Jose P.; Naylor, Huw; Sanson, Bénédicte; Bastock, Rebecca; Huelsmann, Sven; Trovisco, Vitor; Landgraf, Matthias; Knowles-Barley, Seymour; Armstrong, J. Douglas; White-Cooper, Helen; Hansen, Celia; Phillips, Roger G.; Lilley, Kathryn S.; Russell, Steven; St Johnston, Daniel

    2014-01-01

    Although we now have a wealth of information on the transcription patterns of all the genes in the Drosophila genome, much less is known about the properties of the encoded proteins. To provide information on the expression patterns and subcellular localisations of many proteins in parallel, we have performed a large-scale protein trap screen using a hybrid piggyBac vector carrying an artificial exon encoding yellow fluorescent protein (YFP) and protein affinity tags. From screening 41 million embryos, we recovered 616 verified independent YFP-positive lines representing protein traps in 374 genes, two-thirds of which had not been tagged in previous P element protein trap screens. Over 20 different research groups then characterized the expression patterns of the tagged proteins in a variety of tissues and at several developmental stages. In parallel, we purified many of the tagged proteins from embryos using the affinity tags and identified co-purifying proteins by mass spectrometry. The fly stocks are publicly available through the Kyoto Drosophila Genetics Resource Center. All our data are available via an open access database (Flannotator), which provides comprehensive information on the expression patterns, subcellular localisations and in vivo interaction partners of the trapped proteins. Our resource substantially increases the number of available protein traps in Drosophila and identifies new markers for cellular organelles and structures. PMID:25294943

  20. Unexpected Deposition Patterns of Recombinant Proteins in Post-Endoplasmic Reticulum Compartments of Wheat Endosperm1

    PubMed Central

    Arcalis, Elsa; Marcel, Sylvain; Altmann, Friedrich; Kolarich, Daniel; Drakakaki, Georgia; Fischer, Rainer; Christou, Paul; Stoger, Eva

    2004-01-01

    Protein transport within cereal endosperm cells is complicated by the abundance of endoplasmic reticulum (ER)-derived and vacuolar protein bodies. For wheat storage proteins, two major transport routes run from the ER to the vacuole, one bypassing and one passing through the Golgi. Proteins traveling along each route converge at the vacuole and form aggregates. To determine the impact of this trafficking system on the fate of recombinant proteins expressed in wheat endosperm, we used confocal and electron microscopy to investigate the fate of three recombinant proteins containing different targeting information. KDEL-tagged recombinant human serum albumin, which is retrieved to the ER lumen in leaf cells, was deposited in prolamin aggregates within the vacuole of endosperm cells, most likely following the bulk of endogenous glutenins. Recombinant fungal phytase, a glycoprotein designed for secretion, was delivered to the same compartment, with no trace of the molecule in the apoplast. Glycan analysis revealed that this protein had passed through the Golgi. The localization of human serum albumin and phytase was compared to that of recombinant legumin, which contains structural targeting information directing it to the vacuole. Uniquely, legumin accumulated in the globulin inclusion bodies at the periphery of the prolamin bodies, suggesting a different mode of transport and/or aggregation. Our results demonstrate that recombinant proteins are deposited in an unexpected pattern within wheat endosperm cells, probably because of the unique storage properties of this tissue. Our data also confirm that recombinant proteins are invaluable tools for the analysis of protein trafficking in cereals. PMID:15489278

  1. Effect of altered eating pattern on serum fructosamine: total protein ratio and plasma glucose level.

    PubMed

    Ch'ng, S L; Cheah, S H; Husain, R; Duncan, M T

    1989-05-01

    The effect of alteration of eating pattern during Ramadan on body mass index (BMI), serum fructosamine: total protein ratio (F/TP), and glucose level in 18 healthy male Asiatic Moslems were studied. The results showed a significant decrease (p less than 0.025) in F/TP at the second week of Ramadan in 11 subjects who experienced continuous decrease in BMI throughout Ramadan. The remaining 7 subjects showed no significant changes in BMI and F/TP. No evidence of hypoglycaemia was observed in the subjects during the study. Serum fructosamine: total protein ratio in subjects with altered eating pattern preferably should be interpreted along with the change in body mass index.

  2. Phylogenetic Analyses in Dorylaimida Using Data from 2-D Protein Patterns

    PubMed Central

    Ferris, V. R.; Ferris, J. M.

    1988-01-01

    Data from two-dimensional protein patterns for nine dorylaimid isolates were analyzed using PAUP, a computer program for inferring phylogenies under the principle of maximum parsimony. With a variety of available options, including branch swapping and rooting, essentially the same tree was obtained. When isolates of the genus Labronema were analyzed alone, all trees obtained had the same topology, although tree length varied considerably, depending on whether a hypothetical ancestral taxon was included. PMID:19290191

  3. Conversion of amino-acid sequence in proteins to classical music: search for auditory patterns

    PubMed Central

    2007-01-01

    We have converted genome-encoded protein sequences into musical notes to reveal auditory patterns without compromising musicality. We derived a reduced range of 13 base notes by pairing similar amino acids and distinguishing them using variations of three-note chords and codon distribution to dictate rhythm. The conversion will help make genomic coding sequences more approachable for the general public, young children, and vision-impaired scientists. PMID:17477882

  4. Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals

    NASA Astrophysics Data System (ADS)

    Kirian, R. A.; White, T. A.; Holton, J. M.; Chapman, H. N.; Fromme, P.; Barty, A.; Lomb, L.; Aquila, A.

    2011-03-01

    A complete set of structure factors has been extracted from hundreds of thousands of femtosecond X-ray diffraction patterns from randomly oriented Photosystem I membrane protein nanocrystals, using the Monte Carlo method of intensity integration. The data, collected at the Linac Coherent Light Source, are compared with conventional single-crystal data collected at a synchrotron source, and the quality of each data set was found to be similar.

  5. Field observations of the persistence of Comp B explosives residues in a salt marsh impact area.

    PubMed

    Walsh, Marianne E; Taylor, Susan; Hewitt, Alan D; Walsh, Michael R; Ramsey, Charles A; Collins, Charles M

    2010-01-01

    Field observations of weathering Comp B (RDX/TNT 60/40) residue were made on a live-fire training range over four years. The Comp B residue was formed by low-order detonations of 120-mm mortar projectiles. Physical changes were the disaggregation of initially solid chunks into masses of smaller diameter pieces and formation of red phototransformation products that washed off with rain or tidal flooding. Disaggregation increased the surface area of the residue, thereby increasing the potential for dissolution. The bulk of the mass of Comp B was in the craters, but solid chunks were scattered asymmetrically up to 30m away. PMID:19883934

  6. Zipper encodes a putative integral membrane protein required for normal axon patterning during Drosophila neurogenesis.

    PubMed Central

    Zhao, D B; Côté, S; Jähnig, F; Haller, J; Jäckle, H

    1988-01-01

    During the development of the central nervous system, Drosophila embryo axons become organized in a stereo-typed fasciculation pattern. We have found that the zipper (zip) gene, initially identified on the basis of a defective larval cuticle in zip mutant embryos, is possibly involved in the establishment or maintenance of the axon pattern during the late stages of neurogenesis. The zip wild-type gene is expressed in the developing nervous system. It codes for a putative integral membrane protein. Both the molecular features of zipper and its biological effect in the nervous system of mutants suggest that zipper is an essential component for cell surface interactions involved in axon patterning, and that the cuticle phenotype of zip mutants is dependent on the primary defects observed in the nervous system. Images PMID:3402433

  7. 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

  8. Protein addressing on patterned microchip by coupling chitosan electrodeposition and 'electro-click' chemistry.

    PubMed

    Shi, Xiao-Wen; Qiu, Ling; Nie, Zhen; Xiao, Ling; Payne, Gregory F; Du, Yumin

    2013-12-01

    Many applications in proteomics and lab-on-chip analysis require methods that guide proteins to assemble at surfaces with high spatial and temporal control. Electrical inputs are particularly convenient to control, and there has been considerable effort to discover simple and generic mechanisms that allow electrical inputs to trigger protein assembly on-demand. Here, we report the electroaddressing of a protein to a patterned surface by coupling two generic electroaddressing mechanisms. First, we electrodeposit the stimuli-responsive film-forming aminopolysaccharide chitosan to form a hydrogel matrix at the electrode surface. After deposition, the matrix is chemically functionalized with alkyne groups. Second, we ''electro-click' an azide-tagged protein to the functionalized matrix using electrical signals to trigger conjugation by Huisgen 1,3-dipolar cycloadditions. Specifically, a cathodic potential is applied to the matrix-coated electrode to reduce Cu(II) to Cu(I) which is required for the click reaction. Using fluorescently-labeled bovine serum albumin as our model, we demonstrate that protein conjugation can be controlled spatially and temporally. We anticipate that the coupling of polysaccharide electrodeposition and electro-click chemistry will provide a simple and generic approach to electroaddress proteins within compatible hydrogel matrices.

  9. Protein and alkaloid patterns of the floral nectar in some solanaceous species.

    PubMed

    Kerchner, András; Darók, Judit; Bacskay, Ivett; Felinger, Attila; Jakab, Gábor; Farkas, Ágnes

    2015-09-01

    The family Solanaceae includes several melliferous plants, which tend to produce copious amounts of nectar. Floral nectar is a chemically complex aqueous solution, dominated by sugars, but minor components such as amino acids, proteins, flavonoids and alkaloids are present as well. This study aimed at analysing the protein and alkaloid profile of the nectar in seven solanaceous species. Proteins were examined with SDS-PAGE and alkaloids were analyzed with HPLC. The investigation of protein profile revealed significant differences in nectar-protein patterns not only between different plant genera, but also between the three Nicotiana species investigated. SDS-PAGE suggested the presence of several Nectarin proteins with antimicrobial activity in Nicotiana species. The nectar of all tobacco species contained the alkaloid nicotine, N. tabacum having the highest nicotine content. The nectar of Brugmansia suaveolens, Datura stramonium, Hyoscyamus niger and Lycium barbarum contained scopolamine, the highest content of which was measured in B. suaveolens. The alkaloid concentrations in the nectars of most solanaceous species investigated can cause deterrence in honeybees, and the nectar of N. rustica and N. tabacum can be considered toxic for honeybees. PMID:26344026

  10. Protein patterning by UV-induced photodegradation of poly(oligo(ethylene glycol) methacrylate) brushes.

    PubMed

    Alang Ahmad, Shahrul; Hucknall, Angus; Chilkoti, Ashutosh; Leggett, Graham J

    2010-06-15

    The UV photodegradation of protein-resistant poly(oligo(ethylene glycol) methacrylate) (POEGMA) bottle-brush films, grown on silicon oxide by surface-initiated atom radical transfer polymerization, was studied using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Exposure to light with a wavelength of 244 nm caused a loss of polyether units from the brush structure and the creation of aldehyde groups that could be derivatized with amines. An increase was measured in the coefficient of friction of the photodegraded polymer brush compared to the native brush, attributed to the creation of a heterogeneous surface film, leading to increased energy dissipation through film deformation and the creation of new polar functional groups at the surface. Exposure of the films through a photomask yielded sharp, well-defined patterns. Analysis of topographical images showed that physical removal of material occurred during exposure, at a rate of 1.35 nm J(-1) cm(2). Using fluorescence microscopy, the adsorption of labeled proteins onto the exposed surfaces was studied. It was found that protein strongly adsorbed to exposed areas, while the masked regions retained their protein resistance. Exposure of the film to UV light from a scanning near-field optical microscope yielded submicrometer-scale patterns. These data indicate that a simple, rapid, one-step photoconversion of the poly(OEGMA) brush occurs that transforms it from a highly protein-resistant material to one that adsorbs protein and can covalently bind amine-containing molecules and that this photoconversion can be spatially addressed with high spatial resolution.

  11. 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.

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

    DOE PAGESBeta

    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

  13. 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.

  14. Characterization of the pattern of ribosomal protein L19 production during the lifecycle of Leishmania spp.

    PubMed

    de Almeida-Bizzo, Janayna Hammes; Alves, Lysangela Ronalte; Castro, Felipe F; Garcia, Juliana Bório Ferreira; Goldenberg, Samuel; Cruz, Angela Kaysel

    2014-12-01

    Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of the L19 gene during development has been observed for all Leishmania species studied to date (L. major, L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19 overexpression affects negatively translation elongation or termination. The lack of correlation between L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during development in the various species investigated.

  15. Characterization of the pattern of ribosomal protein L19 production during the lifecycle of Leishmania spp.

    PubMed

    de Almeida-Bizzo, Janayna Hammes; Alves, Lysangela Ronalte; Castro, Felipe F; Garcia, Juliana Bório Ferreira; Goldenberg, Samuel; Cruz, Angela Kaysel

    2014-12-01

    Leishmania is a genus of protozoan parasites causing a wide clinical spectrum of diseases in humans. Analysis of a region of chromosome 6 from Leishmania major (Iribar et al.) showed that the transcript of a putative L19 ribosomal protein (RPL19) was most abundant at the amastigote stage. We therefore decided to characterize L19 protein abundance throughout the lifecycle of Leishmania. Differential expression of the L19 gene during development has been observed for all Leishmania species studied to date (L. major, L. braziliensis, L. donovani, and L. amazonensis). Immunoblotting with polyclonal antibodies against L. major RPL19 revealed that changes to L19 protein abundance follow a similar pattern in various species. The amount of L19 protein was higher in exponentially growing promastigotes than in stationary phase promastigotes. The L19 protein was barely detectable in amastigotes, despite the abundance of L19 transcripts observed in L. major at this stage. Immunofluorescence assays showed a granular, dispersed distribution of RPL19 throughout the cytoplasm. Subcellular fractionation confirmed the presence of the protein in the ribosomal fraction, but not in the cytosol of L. major. We generated a L. major transfectant bearing a plasmid-borne L19 gene. Overproduction of the L19 transcript and protein resulted in impaired growth of the transfectants in association with high polysome peaks. We also showed by metabolic labeling that L19 overexpressing clones display low rates of translation. These data suggest that L19 overexpression affects negatively translation elongation or termination. The lack of correlation between L19 transcript and protein abundances suggest that the translation of L19 is differentially controlled during development in the various species investigated. PMID:25290356

  16. Site-selective adsorption of protein induced by a metal pattern on a poly(ethylene terephthalate) surface.

    PubMed

    Cao, Jiali; Wu, Zhongkui; Li, Shaoying; Tang, Hongxiao; Mei, Qilin

    2013-11-01

    A novel technique for inducing site-selective adsorption of protein through constructing metal patterns on flexible poly(ethylene terephthalate) surfaces is presented. The substrates were first modified by vacuum ultraviolet (VUV) irradiation through a photomask to introduce regions with different functional groups. Then the designed metal patterns were constructed on the surfaces of VUV-treated substrates. The surface rearrangement was effectively prevented by constructing silver patterns on poly(ethylene terephthalate) surfaces, thus significantly improving the stability and selectivity of protein adsorption on the surfaces. Moreover, the protein-repulsive layer further reinforced the effect. Finally, protein patterns were successfully obtained. As confirmed by fluorescence microscope, field emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and static water contact angle measurement, the protein patterns possess both excellent selectivity and high fidelity. Feature size of the protein patterns surrounded by a protein-repulsive layer was exactly the same as that of the photomask. And the grain sizes of silver particles were approximately 50 nm. This work could potentially be used in various fields such as biomedicine, bioelectronic components, and tissue repair and replacement, where selective adsorption of protein is desired.

  17. Mapping functional group free energy patterns at protein occluded sites: nuclear receptors and G-protein coupled receptors.

    PubMed

    Lakkaraju, Sirish Kaushik; Yu, Wenbo; Raman, E Prabhu; Hershfeld, Alena V; Fang, Lei; Deshpande, Deepak A; MacKerell, Alexander D

    2015-03-23

    Occluded ligand-binding pockets (LBP) such as those found in nuclear receptors (NR) and G-protein coupled receptors (GPCR) represent a significant opportunity and challenge for computer-aided drug design. To determine free energies maps of functional groups of these LBPs, a Grand-Canonical Monte Carlo/Molecular Dynamics (GCMC/MD) strategy is combined with the Site Identification by Ligand Competitive Saturation (SILCS) methodology. SILCS-GCMC/MD is shown to map functional group affinity patterns that recapitulate locations of functional groups across diverse classes of ligands in the LBPs of the androgen (AR) and peroxisome proliferator-activated-γ (PPARγ) NRs and the metabotropic glutamate (mGluR) and β2-adreneric (β2AR) GPCRs. Inclusion of protein flexibility identifies regions of the binding pockets not accessible in crystal conformations and allows for better quantitative estimates of relative ligand binding affinities in all the proteins tested. Differences in functional group requirements of the active and inactive states of the β2AR LBP were used in virtual screening to identify high efficacy agonists targeting β2AR in Airway Smooth Muscle (ASM) cells. Seven of the 15 selected ligands were found to effect ASM relaxation representing a 46% hit rate. Hence, the method will be of use for the rational design of ligands in the context of chemical biology and the development of therapeutic agents.

  18. nWayComp: a genome-wide sequence comparison tool for multiple strains/species of phylogenetically related microorganisms.

    PubMed

    Yao, Jiqiang; Lin, Hong; Doddapaneni, Harshavardhan; Civerolo, Edwin L

    2007-01-01

    The increasing number of whole genomic sequences of microorganisms has led to the complexity of genome-wide annotation and gene sequence comparison among multiple microorganisms. To address this problem, we have developed nWayComp software that compares DNA and protein sequences of phylogenetically-related microorganisms. This package integrates a series of bioinformatics tools such as BLAST, ClustalW, ALIGN, PHYLIP and PRIMER3 for sequence comparison. It searches for homologous sequences among multiple organisms and identifies genes that are unique to a particular organism. The homologous gene sets are then ranked in the descending order of the sequence similarity. For each set of homologous sequences, a table of sequence identity among homologous genes along with sequence variations such as SNPs and INDELS is developed, and a phylogenetic tree is constructed. In addition, a common set of primers that can amplify all the homologous sequences are generated. The nWayComp package provides users with a quick and convenient tool to compare genomic sequences among multiple organisms at the whole-genome level. PMID:17688445

  19. 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

  20. 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.

  1. An LRR-only protein representing a new type of pattern recognition receptor in Chlamys farreri.

    PubMed

    Wang, Mengqiang; Wang, Lingling; Guo, Ying; Yi, Qilin; Song, Linsheng

    2016-01-01

    Accumulating evidence has demonstrated that leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein-protein interactions and were involved in the immune response. In the present study, an LRR-only protein (designed as CfLRRop-1) was cloned from Zhikong scallop Chlamys farreri. The complete cDNA sequence of CfLRRop-1 contained an open reading frame (ORF) of 1377 bp, which encoded a protein of 458 amino acids. An LRRNT motif, an LRR_7 motif and seven LRR motifs were found in the deduced amino acid sequence of CfLRRop-1. And these seven LRR motifs contained a conserved signature sequence LxxLxLxxNxL. The mRNA transcripts of CfLRRop-1 were constitutively expressed in all the tested tissues, including haemocytes, muscle, mantle, gill, hepatopancreas and gonad, with the highest expression level in hepatopancreas. After the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), the mRNA transcripts of CfLRRop-1 in haemocytes all increased firstly within the first 6 h and secondly during 12-24 h post stimulation. The mRNA expression level of CfLRRop-1 was continuously up-regulated, after the expression of CfTLR (previously identified Toll-like receptor in C. farreri) was suppressed via RNA interference (RNAi). The recombinant CfLRRop-1 protein could directly bind LPS, PGN, GLU and poly I:C, and induce the release of TNF-α in mixed primary cultured scallop haemocytes. These results collectively indicated that CfLRRop-1 would function as a powerful pattern recognition receptor (PRR) and play a pivotal role in the immune response of scallops.

  2. Overexpression of an F-box protein gene disrupts cotyledon vein patterning in Arabidopsis.

    PubMed

    Cui, Xianghuan; Xu, Xiaofeng; He, Yangyang; Du, Xiling; Zhu, Jian

    2016-05-01

    Plant vascular patterning is complex. However, the detailed molecular mechanism of vascular patterning is still unknown. In this study, FBXL, an Arabidopsis F-box motif gene, was isolated by using 3' rapid amplification of cDNA ends (RACE) technique. The gene contained a coding sequence of 1407 nucleotides coding 468 amino acid residues. Amino acid sequence analysis revealed that the gene encoded a protein harboring an F-box motif at the N terminus, an LRRs motif in the middle, and an FBD motif at the C terminus. FBXL promoter-β-glucuronidase (GUS) and 35S promoter-FBXL vectors were constructed and transformed into Arabidopsis thaliana to understand the function of the FBXL gene. GUS expression analysis indicated that FBXL was specifically expressed in the vascular tissues of the root, stem, leaf, and inflorescence. FBXL overexpression in Arabidopsis displayed an abnormal venation pattern in cotyledons. Furthermore, FBXL expression was not induced by exogenous auxin and its transcript accumulation did not overlap with the distribution of endogenous auxin. These results suggested that FBXL may be involved in cotyledon vein patterning via auxin-independent pathway. PMID:26901782

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

    PubMed Central

    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. PMID:25480915

  4. Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography.

    PubMed

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

    2016-01-26

    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 nanoscale, 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 nanoscale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and antitumor 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.

  5. 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.

  6. Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos.

    PubMed

    Borah, Supriya; Barrodia, Praveen; Swain, Rajeeb K

    2016-01-01

    The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut. PMID:26934290

  7. 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

  8. Light at night alters daily patterns of cortisol and clock proteins in female Siberian hamsters.

    PubMed

    Bedrosian, T A; Galan, A; Vaughn, C A; Weil, Z M; Nelson, R J

    2013-06-01

    Humans and other organisms have adapted to a 24-h solar cycle in response to life on Earth. The rotation of the planet on its axis and its revolution around the sun cause predictable daily and seasonal patterns in day length. To successfully anticipate and adapt to these patterns in the environment, a variety of biological processes oscillate with a daily rhythm of approximately 24 h in length. These rhythms arise from hierarchally-coupled cellular clocks generated by positive and negative transcription factors of core circadian clock gene expression. From these endogenous cellular clocks, overt rhythms in activity and patterns in hormone secretion and other homeostatic processes emerge. These circadian rhythms in physiology and behaviour can be organised by a variety of cues, although they are most potently entrained by light. In recent history, there has been a major change from naturally-occurring light cycles set by the sun, to artificial and sometimes erratic light cycles determined by the use of electric lighting. Virtually every individual living in an industrialised country experiences light at night (LAN) but, despite its prevalence, the biological effects of such unnatural lighting have not been fully considered. Using female Siberian hamsters (Phodopus sungorus), we investigated the effects of chronic nightly exposure to dim light on daily rhythms in locomotor activity, serum cortisol concentrations and brain expression of circadian clock proteins (i.e. PER1, PER2, BMAL1). Although locomotor activity remained entrained to the light cycle, the diurnal fluctuation of cortisol concentrations was blunted and the expression patterns of clock proteins in the suprachiasmatic nucleus and hippocampus were altered. These results demonstrate that chronic exposure to dim LAN can dramatically affect fundamental cellular function and emergent physiology.

  9. 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).

  10. 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.

  11. 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.

  12. 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'.

  13. 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.

  14. Protein immobilization on Ni(II) ion patterns prepared by microcontact printing and dip-pen nanolithography.

    PubMed

    Wu, Chien-Ching; Reinhoudt, David N; Otto, Cees; Velders, Aldrik H; Subramaniam, Vinod

    2010-02-23

    An indirect method of protein patterning by using Ni(II) ion templates for immobilization via a specific metal-protein interaction is described. A nitrilotriacetic acid (NTA)-terminated self-assembled monolayer (SAM) allows oriented binding of histidine-tagged proteins via complexation with late first-row transition metal ions, such as Ni(II). Patterns of nickel(II) ions were prepared on NTA SAM-functionalized glass slides by microcontact printing (microCP) and dip-pen nanolithography (DPN) to obtain micrometer and submicrometer scale patterns. Consecutive dipping of the slides in 6His-protein solutions resulted in the formation of protein patterns, as was subsequently proven by AFM and confocal fluorescence microscopy. This indirect method prevents denaturation of fragile biomolecules caused by direct printing or writing of proteins. Moreover, it yields well-defined patterned monolayers of proteins and, in principle, is indifferent for biomolecules with a high molecular weight. This approach also enabled us to characterize the transfer of Ni(II) ions on fundamental parameters of DPN, such as writing speeds and tip-surface contact times, while writing with the smallest possible ink "molecules" (i.e., metal ions). PMID:20104881

  15. Predicting protein fold pattern with functional domain and sequential evolution information.

    PubMed

    Shen, Hong-Bin; Chou, Kuo-Chen

    2009-02-01

    The fold pattern of a protein is one level deeper than its structural classification, and hence is more challenging and complicated for prediction. Many efforts have been made in this regard, but so far all the reported success rates are still under 70%, indicating that it is extremely difficult to enhance the success rate even by 1% or 2%. To address this problem, here a novel approach is proposed that is featured by combining the functional domain information and the sequential evolution information through a fusion ensemble classifier. The predictor thus developed is called PFP-FunDSeqE. Tests were performed for identifying proteins among their 27 fold patterns. Compared with the existing predictors tested by a same stringent benchmark dataset, the new predictor can, for the first time, achieve over 70% success rate. The PFP-FunDSeqE predictor is freely available to the public as a web server at http://www.csbio.sjtu.edu.cn/bioinf/PFP-FunDSeqE/.

  16. Expression patterns of five polymorphic membrane proteins during the Chlamydia abortus developmental cycle

    PubMed Central

    Wheelhouse, Nick; Sait, Michelle; Wilson, Kim; Aitchison, Kevin; McLean, Kevin; Smith, David G.E.; Longbottom, David

    2012-01-01

    It has been suggested that polymorphic membrane proteins (Pmps) belonging to the Type V autotransporter protein family play an important role in the pathogenesis of Chlamydia abortus (C. abortus; formerly Chlamydophila abortus) infection. In a previous study we demonstrated the expression of all the pmps at the transcriptional level. The purpose of this study was to measure the number of Pmp positive inclusions throughout the C. abortus developmental cycle to investigate heterogeneity in expression patterns. McCoy cells were infected with C. abortus and analysed for Pmp expression over a 72 h period by fluorescent immunocytochemistry. Pmp18D could be detected at all analysed time points, and could only be accurately quantified from 36 hpi while Pmp10G positive inclusions could be visualised from 36 hpi. Expression of Pmps 13G, 16G and 17G could only be visualised later in the cycle and within less than half of visualised inclusions. These results indicate that while expression of specific Pmps is constitutive (Pmp18D), the pattern of expression of other Pmps is more variable. This suggests that different members of the Pmp family may play different roles within the developmental cycle of the organism, with some (Pmps10G and 18D) having roles throughout the cycle, while the heterogeneity of expression of others may aid in antigenic variation. PMID:22776512

  17. 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. PMID:27417229

  18. Adaptation of Salmonella enterica Hadar under static magnetic field: effects on outer membrane protein pattern

    PubMed Central

    2012-01-01

    Background Salmonella enterica serovar Hadar (S. Hadar) is a highly prevalent foodborne pathogen and therefore a major cause of human gastroenteritis worldwide. Outer membrane proteins whose production is often regulated by environmental conditions also play important roles in the adaptability of bacterial pathogens to various environments. Results The present study investigated the adaptation of S. Hadar under the effect of acute static magnetic field exposure (200 mT, 9 h) and the impact on the outer membrane protein pattern. Via two-dimensional electrophoresis (2-DE) and LC-MS/MS spectrometry, we compared the proteome of enriched-outer membrane fraction before and after exposure to a magnetic field. A total of 11 proteins, displaying more than a two-fold change, were differentially expressed in exposed cells, among which 7 were up-regulated and 4 down-regulated. These proteins were involved in the integrity of cell envelope (TolB, Pal), in the response to oxidative stress (OmpW, dihydrolipoamide dehydrogenase, UspF), in the oxidative stress status (bacterioferritin), in virulence (OmpX, Yfgl) or in motility (FlgE and UspF). Complementary experiments associated the down-regulation of FlgE and UspF with an alteration of swarming, a flagella-driven motility, under SMF. Furthermore, the antibiotic disc diffusion method confirmed a decrease of gentamicin susceptibility in exposed cells. This decrease could be partly associated with the up-regulation of TolC, outer membrane component of an efflux pump. OmpA, a multifunctional protein, was up-regulated. Conclusions SMF (200 mT) seems to maintain the cell envelope integrity and to submit the exposed cells to an oxidative stress. Some alterations suggest an increase of the ability of exposed cells to form biofilms. PMID:22304719

  19. Pattern of mineralization after regenerative periodontal therapy with enamel matrix proteins.

    PubMed

    Bosshardt, Dieter D; Sculean, Anton; Donos, Nikolaos; Lang, Niklaus P

    2006-05-01

    A derivative (EMD) of enamel matrix proteins (EMPs) is used for periodontal regeneration because EMPs are believed to induce the formation of acellular extrinsic fiber cementum (AEFC). Other reports, however, indicate that EMPs have osteogenic potential. The aim of this study was to characterize the nature of the tissue that forms on the root surface following application of EMD. Ten human teeth affected by periodontitis and scheduled for extraction were treated with EMD. Four to six weeks later, they were extracted and processed for analysis by light microscopy and transmission electron microscopy. Immunocytochemistry with antibodies against bone sialoprotein (BSP) and osteopontin (OPN) was performed to determine the mineralization pattern. The newly formed tissues on the root were thick and contained embedded cells. Small mineralization foci were regularly seen, and large organic matrix patches were occasionally seen, but a distinct mineralization front was lacking. While labeling for BSP was always associated with small mineralization foci and large matrix patches, OPN labeling was seen inconsistently. It is concluded that tissues resembling either cellular intrinsic fiber cementum or a type of bone were observed. The mineralization pattern mostly resembled that found in bone, except for a few areas that exhibited a hitherto undescribed mineralization pattern. PMID:16674690

  20. XFINGER: a tool for searching and visualising protein fingerprints and patterns.

    PubMed

    Perkins, D N; Attwood, T K

    1996-04-01

    A tool for searching pattern and fingerprint databases is described. Fingerprints are groups of motifs excised from conserved regions of sequence alignments and used for iterative database scanning. The constituent motifs are thus encoded as small alignments in which sequence information is maximised with each database pass; they therefore differ from regular-expression patterns, in which alignments are reduced to single consensus sequences. Different database formats have evolved to store these disparate types of information, namely the PROSITE dictionary of patterns and the PRINTS fingerprint database, but programs have not been available with the flexibility to search them both. We have developed a facility to do this: the system allows query sequences to be scanned against either PROSITE, the full PRINTS database, or against individual fingerprints. The results of fingerprint searches are displayed simultaneously in both text and graphical windows to render them more tangible to the user. Where structural coordinates are available, identified motifs may be visualised in a 3D context. The program runs on Silicon Graphics machines using GL graphics libraries and on machines with X servers supporting the PEX extension: its use is illustrated here by depicting the location of low-density lipoprotein-binding (LDL) motifs and leucine-rich repeats in a mosaic G-protein-coupled receptor (GPCR).

  1. 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.

  2. The comP locus of Neisseria gonorrhoeae encodes a type IV prepilin that is dispensable for pilus biogenesis but essential for natural transformation.

    PubMed

    Wolfgang, M; van Putten, J P; Hayes, S F; Koomey, M

    1999-03-01

    The expression of type IV pili (Tfp) by Neisseria gonorrhoeae has been shown to be essential for natural genetic transformation at the level of sequence-specific uptake of DNA. All previously characterized mutants defective in this step of transformation either lack Tfp or are altered in the expression of Tfp-associated properties, such as twitching motility, autoagglutination and the ability to bind to human epithelial cells. To examine the basis for this relationship, we identified potential genes encoding polypeptides sharing structural similarities to PilE, the Tfp subunit, within the N. gonorrhoeae genome sequence database. We found that disruption of one such gene, designated comP (for competence-associated prepilin), leads to a severe defect in the capacity to take up DNA in a sequence-specific manner, but does not alter Tfp biogenesis or expression of the Tfp-associated properties of auto-agglutination, twitching motility and human epithelial cell adherence. Indirect evidence based on immunodetection suggests that ComP is expressed at very low levels relative to that of PilE. The process of DNA uptake in gonococci, therefore, is now known to require the expression of at least three distinct components: Tfp, the recently identified PilT protein and ComP.

  3. Expression pattern analysis of odorant-binding proteins in the pea aphid Acyrthosiphon pisum.

    PubMed

    De Biasio, Filomena; Riviello, Lea; Bruno, Daniele; Grimaldi, Annalisa; Congiu, Terenzio; Sun, Yu Feng; Falabella, Patrizia

    2015-04-01

    Odorant-binding proteins (OBPs) are soluble proteins mediating chemoreception in insects. In previous research, we investigated the molecular mechanisms adopted by aphids to detect the alarm pheromone (E)-β-farnesene and we found that the recognition of this and structurally related molecules is mediated by OBP3 and OBP7. Here, we show the differential expression patterns of 5 selected OBPs (OBP1, OBP3, OBP6, OBP7, OBP8) obtained performing quantitative RT-PCR and immunolocalization experiments in different body parts of adults and in the 5 developmental instars, including winged and unwinged morphs, of the pea aphid Acyrthosiphon pisum. The results provide an overall picture that allows us to speculate on the relationship between the differential expression of OBPs and their putative function. The expression of OBP3, OBP6, and OBP7 in the antennal sensilla suggests a chemosensory function for these proteins, whereas the constant expression level of OBP8 in all instars could suggest a conserved role. Moreover, OBP1 and OBP3 are also expressed in nonsensory organs. A light and scanning electron microscopy study of sensilla on different body parts of aphid, in particular antennae, legs, mouthparts, and cornicles-cauda, completes this research providing a guide to facilitate the mapping of OBP expression profiles.

  4. Zebrafish Thsd7a is a neural protein required for angiogenic patterning during development.

    PubMed

    Wang, Chieh-Huei; Chen, I-Hui; Kuo, Meng-Wei; Su, Pei-Tsu; Lai, Zih-Yin; Wang, Chian-Huei; Huang, Wei-Chang; Hoffman, Jana; Kuo, Calvin J; You, May-Su; Chuang, Yung-Jen

    2011-06-01

    Angiogenesis is a highly organized process under the control of guidance cues that direct endothelial cell (EC) migration. Recently, many molecules that were initially described as regulators of neural guidance were subsequently shown to also direct EC migration. Here, we report a novel protein, thrombospondin type I domain containing 7A (Thsd7a), that is a neural molecule required for directed EC migration during embryonic angiogenesis in zebrafish. Thsd7a is a vertebrate conserved protein. Zebrafish thsd7a transcript was detected along the ventral edge of the neural tube in the developing zebrafish embryos, correlating with the growth path of angiogenic intersegmental vessels (ISVs). Morpholino-knockdown of Thsd7a caused a lateral deviation of angiogenic ECs below the thsd7a-expressing sites, resulting in aberrant ISV patterning. Collectively, our study shows that zebrafish Thsd7a is a neural protein required for ISV angiogenesis, and suggests an important role of Thsd7a in the neurovascular interaction during zebrafish development.

  5. Altered temporal patterns of anxiety in aged and amyloid precursor protein (APP) transgenic mice

    PubMed Central

    Bedrosian, Tracy A.; Herring, Kamillya L.; Weil, Zachary M.; Nelson, Randy J.

    2011-01-01

    Both normal aging and dementia are associated with dysregulation of the biological clock, which contributes to disrupted circadian organization of physiology and behavior. Diminished circadian organization in conjunction with the loss of cholinergic input to the cortex likely contributes to impaired cognition and behavior. One especially notable and relatively common circadian disturbance among the aged is “sundowning syndrome,” which is characterized by exacerbated anxiety, agitation, locomotor activity, and delirium during the hours before bedtime. Sundowning has been reported in both dementia patients and cognitively intact elderly individuals living in institutions; however, little is known about temporal patterns in anxiety and agitation, and the neurobiological basis of these rhythms remains unspecified. In the present study, we explored the diurnal pattern of anxiety-like behavior in aged and amyloid precursor protein (APP) transgenic mice. We then attempted to treat the observed behavioral disturbances in the aged mice using chronic nightly melatonin treatment. Finally, we tested the hypothesis that time-of-day differences in acetylcholinesterase and choline acetyltransferase expression and general neuronal activation (i.e., c-Fos expression) coincide with the behavioral symptoms. Our results show a temporal pattern of anxiety-like behavior that emerges in elderly mice. This behavioral pattern coincides with elevated locomotor activity relative to adult mice near the end of the dark phase, and with time-dependent changes in basal forebrain acetylcholinesterase expression. Transgenic APP mice show a similar behavioral phenomenon that is not observed among age-matched wild-type mice. These results may have useful applications to the study and treatment of age- and dementia-related circadian behavioral disturbances, namely, sundowning syndrome. PMID:21709248

  6. [Identification and expression patterns of anterior silk gland specific cuticle protein Bm11721 in the silkworm (Bombyx mori)].

    PubMed

    Xie, Kang; Wang, Xin; Chen, Huifang; Li, Yi; Song, Qianru; Zhao, Ping

    2016-01-01

    The silk gland of silkworm is the organ of silk protein synthesis and secretion. According to the morphological and functional differences, silk gland can be divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). ASG is the place for silk proteins conformation changes although it cannot synthetize silk proteins. ASG has narrow luminal structures and rigid wall which consists of chitin and cuticle proteins so that it can provide the shearing force which plays an important role in the silk protein conformation changes. The objective of this study is to identify the new chitin binding proteins in ASG of silkworm (Bombyx mori), and to analyze their expression patterns in different tissues. We identified a cuticle protein with chitin binding domain Bml1721 (GenBank Accession No. NM-001173285.1) by chitin affinity chromatography column. We also expressed the recombinant protein as inclusion body using the prokaryotic expression system, and then successfully purified the recombinant protein by nickel affinity chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bm11721 was specifically expressed in ASG. Furthermore, the expression level of Bm 11721 protein was unchanged during the 5th instar. Immunofluorescence analysis revealed that Bm1 1721 was located in the ASG inner membrane. It is proposed that Bm11721 is a component of inner membrane and probably provides the shearing force for conformational changes.

  7. [Identification and expression patterns of anterior silk gland specific cuticle protein Bm11721 in the silkworm (Bombyx mori)].

    PubMed

    Xie, Kang; Wang, Xin; Chen, Huifang; Li, Yi; Song, Qianru; Zhao, Ping

    2016-01-01

    The silk gland of silkworm is the organ of silk protein synthesis and secretion. According to the morphological and functional differences, silk gland can be divided into anterior silk gland (ASG), middle silk gland (MSG) and posterior silk gland (PSG). ASG is the place for silk proteins conformation changes although it cannot synthetize silk proteins. ASG has narrow luminal structures and rigid wall which consists of chitin and cuticle proteins so that it can provide the shearing force which plays an important role in the silk protein conformation changes. The objective of this study is to identify the new chitin binding proteins in ASG of silkworm (Bombyx mori), and to analyze their expression patterns in different tissues. We identified a cuticle protein with chitin binding domain Bml1721 (GenBank Accession No. NM-001173285.1) by chitin affinity chromatography column. We also expressed the recombinant protein as inclusion body using the prokaryotic expression system, and then successfully purified the recombinant protein by nickel affinity chromatography column to generate the polyclonal antibodies. The expression patterns analysis in various tissues showed that both in transcriptional and protein levels Bm11721 was specifically expressed in ASG. Furthermore, the expression level of Bm 11721 protein was unchanged during the 5th instar. Immunofluorescence analysis revealed that Bm1 1721 was located in the ASG inner membrane. It is proposed that Bm11721 is a component of inner membrane and probably provides the shearing force for conformational changes. PMID:27363199

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

    PubMed Central

    Johnson, Gregory R.; Li, Jieyue; Shariff, Aabid; Rohde, Gustavo K.; Murphy, Robert F.

    2015-01-01

    Characterizing the spatial distribution of proteins directly from microscopy images is a difficult problem with numerous applications in cell biology (e.g. identifying motor-related proteins) and clinical research (e.g. identification of cancer biomarkers). Here we describe the design of a system that provides automated analysis of punctate protein patterns in microscope images, including quantification of their relationships to microtubules. We constructed the system using confocal immunofluorescence microscopy images from the Human Protein Atlas project for 11 punctate proteins in three cultured cell lines. These proteins have previously been characterized as being primarily located in punctate structures, but their images had all been annotated by visual examination as being simply “vesicular”. We were able to show that these patterns could be distinguished from each other with high accuracy, and we were able to assign to one of these subclasses hundreds of proteins whose subcellular localization had not previously been well defined. In addition to providing these novel annotations, we built a generative approach to modeling of punctate distributions that captures the essential characteristics of the distinct patterns. Such models are expected to be valuable for representing and summarizing each pattern and for constructing systems biology simulations of cell behaviors. PMID:26624011

  9. The application of counter immunoelectrophoresis (CIE) in ocular protein studies. Part I: time dependent deposition patterns of immunoregulatory proteins on anionic hydrogel contact lenses.

    PubMed

    Mann, Aisling M; Jones, Lyndon W; Tighe, Brian J

    2002-06-01

    This paper focuses on the effects of wear regime on the deposition pattern of important immunoregulatory proteins on FDA Group IV etafilcon-A lenses. Specifically, the aim was to assess the extent to which the daily disposable wear modality produces a different deposition of proteins from the conventional daily wear regime which is coupled with cleaning and disinfection. Counter immunoelectrophoresis (CIE) was employed to detect individual proteins in lens extracts from individual patients and focused on the analysis of five proteins, IgA, IgG, lactoferrin, albumin and kininogen. Deposition was monitored as a function of time; significantly lower deposition was detected on the daily disposable lenses.

  10. 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. PMID:26249371

  11. 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.

  12. Polaris, a protein involved in left-right axis patterning, localizes to basal bodies and cilia.

    PubMed

    Taulman, P D; Haycraft, C J; Balkovetz, D F; Yoder, B K

    2001-03-01

    Mutations in Tg737 cause a wide spectrum of phenotypes, including random left-right axis specification, polycystic kidney disease, liver and pancreatic defects, hydrocephalus, and skeletal patterning abnormalities. To further assess the biological function of Tg737 and its role in the mutant pathology, we identified the cell population expressing Tg737 and determined the subcellular localization of its protein product called Polaris. Tg737 expression is associated with cells possessing either motile or immotile cilia and sperm. Similarly, Polaris concentrated just below the apical membrane in the region of the basal bodies and within the cilia or flagellar axoneme. The data suggest that Polaris functions in a ciliogenic pathway or in cilia maintenance, a role supported by the loss of cilia on the ependymal cell layer in ventricles of Tg737(orpk) brains and by the lack of node cilia in Tg737(Delta2-3betaGal) mutants. PMID:11251073

  13. ParA-mediated plasmid partition driven by protein pattern self-organization

    PubMed Central

    Hwang, Ling Chin; Vecchiarelli, Anthony G; Han, Yong-Woon; Mizuuchi, Michiyo; Harada, Yoshie; Funnell, Barbara E; Mizuuchi, Kiyoshi

    2013-01-01

    DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown. We reconstituted and visualized ParA-mediated plasmid partition inside a DNA-carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB-stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion-ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid. PMID:23443047

  14. Serum pattern profiling for analyzing different types of stress by protein chip technology: a preliminary study.

    PubMed

    Liu, Hui; Hou, Diandong; Wu, Da; Yin, Hong; Wu, Xiaoyi

    2010-01-01

    ProteinChip is a widely accepted tool for exploring serum pattern profile to evaluate the risk of somatic diseases from different stressors. In this study, by using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-ToF), the serum proteome from mice under restraint and thermal stresses were profiled in detail and compared with the control group. Around 150 characteristic peaks were detected in all three groups, with m/z ranging from 1500 to 50,000, with most peaks being within the 2000 m/z to 20,000 m/z range. Compared with the control group, three significant protein peaks with m/z values of 2780, 3303 and 3450 appeared specifically in the restrained stress group and four other peaks with m/z values of 7500, 7811, 29,950 and 38,565 in the thermal stress group. Unexpectedly, no universal positive stress peaks were detected. These preliminary results clearly suggested that there might not be a common mechanism shared by various psychophysiological disorders under different stressors. By fast serum proteomics profiling, SELDI-ToF may be a convenient tool for evaluating the risk of stress-induced illness.

  15. 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.

  16. 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

  17. 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.

  18. 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. PMID:26968261

  19. 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.

  20. 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…

  1. 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...

  2. 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…

  3. 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

  4. 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

  5. Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals

    PubMed Central

    Kirian, Richard A.; White, Thomas A.; Holton, James M.; Chapman, Henry N.; Fromme, Petra; Barty, Anton; Lomb, Lukas; Aquila, Andrew; Maia, Filipe R. N. C.; Martin, Andrew V.; Fromme, Raimund; Wang, Xiaoyu; Hunter, Mark S.; Schmidt, Kevin E.; Spence, John C. H.

    2011-01-01

    A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single-shot X-ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle-size distribution. The data were collected at the Linac Coherent Light Source (the first hard-X-ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single ‘still’ diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal-size effects. These results were compared with single-crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation-damage reduction and for time-resolved diffraction studies at room temperature are discussed. PMID:21325716

  6. Expression pattern of the CsPK3 auxin-responsive protein kinase gene.

    PubMed

    Chono, M; Suzuki, Y; Nemoto, K; Yamane, H; Murofushi, N; Yamaguchi, I

    2001-03-01

    We have previously cloned a cDNA of a putative serine/threonine protein kinase gene named CsPK3 from cucumber, the mRNA level of which was up-regulated by auxin and down-regulated by light irradiation. To examine the CsPK3 gene expression in detail, we cloned a genomic DNA of CsPK3 gene and made transgenic tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plants containing the fused CsPK3 promoter-beta-glucuronidase gene. The beta-glucuronidase expression was detected in the shoot apex, vascular tissues, and the outermost layer of cortex. The histological distribution of CsPK3 mRNA in cucumber seedlings was supported by in situ hybridization, where the positive signals were observed in similar tissues as those observed by beta-glucuronidase staining. The responsiveness of the CsPK3 gene to auxin and light was also confirmed for beta-glucuronidase activity. The pattern of beta-glucuronidase staining changed during the development of the tobacco seedlings. The results of our experiment showed that CsPK3 was expressed in a wide variety of tissues and cells in which the developmental and growth controls by auxin are suggested.

  7. Alterations in the protein pattern of subcellular fractions isolated from Paramecium cells suppressed in phagocytosis.

    PubMed

    Surmacz, L; Wiejak, J; Wyroba, E

    2001-01-01

    SDS-PAGE and quantitative densitometric analysis revealed alterations in the protein pattern of subcellular fractions (100,000 x g) isolated from Paramecium aurelia (299s axenic) cells suppressed in phagocytosis as compared with the control. Two different agents were used to block phagocytosis: the beta-adrenergic antagonist-1-propranolol (200 microM) and inhibitor of calmodulin-dependent processes--trifluoperazine (20 microM). More than 40 polypeptides were identified in the cytosolic (soluble) fractions S1 and S2. A considerable decrease in band intensity was found for three polypeptides: by 60% for 87 kDa band, 52% for 75 kDa and 37% for 42 kDa in comparison to the control, when S2 fractions from propranolol-treated cells of equal load were quantified. TFP treatment evoked only a small decrease in the intensity of the same bands: 9%, 10% and 6%, respectively. The 42 kDa band was identified by Western blot analysis and chemiluminiscent detection to be actin. This result suggests that actin may be a primary target of pharmacological agents used in this study to inhibit Paramecium phagocytic activity.

  8. Endomembrane trafficking protein SEC24A regulates cell size patterning in Arabidopsis.

    PubMed

    Qu, Xian; Chatty, Prerana Rao; Roeder, Adrienne H K

    2014-12-01

    Size is a critical property of a cell, but how it is determined is still not well understood. The sepal epidermis of Arabidopsis (Arabidopsis thaliana) contains cells with a diversity of sizes ranging from giant cells to small cells. Giant cells have undergone endoreduplication, a specialized cell cycle in which cells replicate their DNA but fail to divide, becoming polyploid and enlarged. Through forward genetics, we have identified a new mutant with ectopic giant cells covering the sepal epidermis. Surprisingly, the mutated gene, SEC24A, encodes a coat protein complex II vesicle coat subunit involved in endoplasmic reticulum-to-Golgi trafficking in the early secretory pathway. We show that the ectopic giant cells of sec24a-2 are highly endoreduplicated and that their formation requires the activity of giant cell pathway genes LOSS OF GIANT CELLS FROM ORGANS, DEFECTIVE KERNEL1, and Arabidopsis CRINKLY4. In contrast to other trafficking mutants, cytokinesis appears to occur normally in sec24a-2. Our study reveals an unexpected yet specific role of SEC24A in endoreduplication and cell size patterning in the Arabidopsis sepal.

  9. Bone morphogenetic proteins, eye patterning, and retinocollicular map formation in the mouse

    PubMed Central

    Plas, Daniel T.; Dhande, Onkar; Lopez, Joshua E.; Murali, Deepa; Thaller, Christina; Henkemeyer, Mark; Furuta, Yasuhide; Overbeek, Paul; Crair, Michael C.

    2009-01-01

    Patterning events during early eye formation determine retinal cell fate and can dictate the behavior of retinal ganglion cell (RGC) axons as they navigate toward central brain targets. The temporally and spatially regulated expression of bone morphogenetic proteins (BMPs) and their receptors in the retina are thought to play a key role in this process, initiating gene expression cascades that distinguish different regions of the retina, particularly along the dorsoventral axis. Here, we examine the role of BMP and a potential downstream effector, EphB, in retinotopic map formation in the lateral geniculate nucleus (LGN) and superior colliculus (SC). RGC axon behaviors during retinotopic map formation in wild type mice are compared with those in several strains of mice with engineered defects of BMP and EphB signaling. Normal RGC axon sorting produces axon order in the optic tract that reflects the dorsoventral position of the parent RGCs in the eye. A dramatic consequence of disrupting BMP signaling is a missorting of RGC axons as they exit the optic chiasm. This sorting is not dependent on EphB. When BMP signaling in the developing eye is genetically modified, RGC order in the optic tract and targeting in the LGN and SC are correspondingly disrupted. These experiments show that BMP signaling regulates dorsoventral RGC cell fate, RGC axon behavior in the ascending optic tract and retinotopic map formation in the LGN and SC through mechanisms that are in part distinct from EphB signaling in the LGN and SC. PMID:18614674

  10. Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation.

    PubMed

    Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

    2007-01-01

    Erythroblast macrophage protein (Emp) mediates the attachment of erythroid cells to macrophages and is required for normal differentiation of both cell lineages. In erythroid cells, Emp is believed to be involved in nuclear extrusion, however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data show that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture shows that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data show that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation and suggest that Emp may be involved in multiple cellular functions.

  11. Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals.

    PubMed

    Kirian, Richard A; White, Thomas A; Holton, James M; Chapman, Henry N; Fromme, Petra; Barty, Anton; Lomb, Lukas; Aquila, Andrew; Maia, Filipe R N C; Martin, Andrew V; Fromme, Raimund; Wang, Xiaoyu; Hunter, Mark S; Schmidt, Kevin E; Spence, John C H

    2011-03-01

    A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single-shot X-ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle-size distribution. The data were collected at the Linac Coherent Light Source (the first hard-X-ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single 'still' diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal-size effects. These results were compared with single-crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation-damage reduction and for time-resolved diffraction studies at room temperature are discussed.

  12. Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals.

    PubMed

    Kirian, Richard A; White, Thomas A; Holton, James M; Chapman, Henry N; Fromme, Petra; Barty, Anton; Lomb, Lukas; Aquila, Andrew; Maia, Filipe R N C; Martin, Andrew V; Fromme, Raimund; Wang, Xiaoyu; Hunter, Mark S; Schmidt, Kevin E; Spence, John C H

    2011-03-01

    A complete set of structure factors has been extracted from hundreds of thousands of femtosecond single-shot X-ray microdiffraction patterns taken from randomly oriented nanocrystals. The method of Monte Carlo integration over crystallite size and orientation was applied to experimental data from Photosystem I nanocrystals. This arrives at structure factors from many partial reflections without prior knowledge of the particle-size distribution. The data were collected at the Linac Coherent Light Source (the first hard-X-ray laser user facility), to which was fitted a hydrated protein nanocrystal injector jet, according to the method of serial crystallography. The data are single 'still' diffraction snapshots, each from a different nanocrystal with sizes ranging between 100 nm and 2 µm, so the angular width of Bragg peaks was dominated by crystal-size effects. These results were compared with single-crystal data recorded from large crystals of Photosystem I at the Advanced Light Source and the quality of the data was found to be similar. The implications for improving the efficiency of data collection by allowing the use of very small crystals, for radiation-damage reduction and for time-resolved diffraction studies at room temperature are discussed. PMID:21325716

  13. 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. PMID:23477911

  14. WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning.

    PubMed

    Lee, M M; Schiefelbein, J

    1999-11-24

    The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

  15. pvSOAR: detecting similar surface patterns of pocket and void surfaces of amino acid residues on proteins.

    PubMed

    Binkowski, T Andrew; Freeman, Patrick; Liang, Jie

    2004-07-01

    Detecting similar protein surfaces provides an important route for discovering unrecognized or novel functional relationship between proteins. The web server pvSOAR (pocket and void Surfaces Of Amino acid Residues) provides an online resource to identify similar protein surface regions. pvSOAR can take a structure either uploaded by a user or obtained from the Protein Data Bank, and identifies similar surface patterns based on geometrically defined pockets and voids. It provides several search modes to compare protein surfaces by similarity in local sequence, local shape and local orientation. Statistically significant search results are reported for visualization and interactive exploration. pvSOAR can be used to predict biological functions of proteins with known three-dimensional structures but unknown biological roles. It can also be used to study functional relationship between proteins and for exploration of the evolutionary origins of structural elements important for protein function. We present an example using pvSOAR to explore the biological roles of a protein whose structure was solved by the structural genomics project. The pvSOAR web server is available at http://pvsoar.bioengr.uic.edu/.

  16. ADAMTS-12 Associates with and Degrades Cartilage Oligomeric Matrix Protein*

    PubMed Central

    Liu, Chuan-ju; Kong, Wei; Xu, Ke; Luan, Yi; Ilalov, Kiril; Sehgal, Bantoo; Yu, Shuang; Howell, Ronald D.; Cesare, Paul E. Di

    2006-01-01

    Loss of articular cartilage because of extracellular matrix breakdown is the hallmark of arthritis. Degradative fragments of cartilage oligomeric matrix protein (COMP), a prominent noncollagenous matrix component in articular cartilage, have been observed in the cartilage, synovial fluid, and serum of arthritis patients. The molecular mechanism of COMP degradation and the enzyme(s) responsible for it, however, remain largely unknown. ADAMTS-12 (a disintegrin and metalloprotease with thrombospondin motifs) was shown to associate with COMP both in vitro and in vivo. ADAMTS-12 selectively binds to only the epidermal growth factor-like repeat domain of COMP of the four functional domains tested. The four C-terminal TSP-1-like repeats of ADAMTS-12 are shown to be necessary and sufficient for its interaction with COMP. Recombinant ADAMTS-12 is capable of digesting COMP in vitro. The COMP-degrading activity of ADAMTS-12 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the level of ADAMTS-12 in the cartilage and synovium of patients with both osteoarthritis and rheumatoid arthritis is significantly higher than in normal cartilage and synovium. Together, these findings indicate that ADAMTS-12 is a new COMP-interacting and -degrading enzyme and thus may play an important role in the COMP degradation in the initiation and progression of arthritis. PMID:16611630

  17. WXG100 Protein Superfamily Consists of Three Subfamilies and Exhibits an α-Helical C-Terminal Conserved Residue Pattern

    PubMed Central

    Poulsen, Christian; Panjikar, Santosh; Holton, Simon J.; Wilmanns, Matthias; Song, Young-Hwa

    2014-01-01

    Members of the WXG100 protein superfamily form homo- or heterodimeric complexes. The most studied proteins among them are the secreted T-cell antigens CFP-10 (10 kDa culture filtrate protein, EsxB) and ESAT-6 (6 kDa early secreted antigen target, EsxA) from Mycobacterium tuberculosis. They are encoded on an operon within a gene cluster, named as ESX-1, that encodes for the Type VII secretion system (T7SS). WXG100 proteins are secreted in a full-length form and it is known that they adopt a four-helix bundle structure. In the current work we discuss the evolutionary relationship between the homo- and heterodimeric WXG100 proteins, the basis of the oligomeric state and the key structural features of the conserved sequence pattern of WXG100 proteins. We performed an iterative bioinformatics analysis of the WXG100 protein superfamily and correlated this with the atomic structures of the representative WXG100 proteins. We find, firstly, that the WXG100 protein superfamily consists of three subfamilies: CFP-10-, ESAT-6- and sagEsxA-like proteins (EsxA proteins similar to that of Streptococcus agalactiae). Secondly, that the heterodimeric complexes probably evolved from a homodimeric precursor. Thirdly, that the genes of hetero-dimeric WXG100 proteins are always encoded in bi-cistronic operons and finally, by combining the sequence alignments with the X-ray data we identify a conserved C-terminal sequence pattern. The side chains of these conserved residues decorate the same side of the C-terminal α-helix and therefore form a distinct surface. Our results lead to a putatively extended T7SS secretion signal which combines two reported T7SS recognition characteristics: Firstly that the T7SS secretion signal is localized at the C-terminus of T7SS substrates and secondly that the conserved residues YxxxD/E are essential for T7SS activity. Furthermore, we propose that the specific α-helical surface formed by the conserved sequence pattern including YxxxD/E motif is a key

  18. 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

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

    PubMed

    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.

  20. Dietary patterns and risk of elevated C-reactive protein concentrations 12 years later.

    PubMed

    Julia, Chantal; Meunier, Nathalie; Touvier, Mathilde; Ahluwalia, Namanjeet; Sapin, Vincent; Papet, Isabelle; Cano, Noël; Hercberg, Serge; Galan, Pilar; Kesse-Guyot, Emmanuelle

    2013-08-01

    Inflammation mediates several chronic diseases. Micronutrients can act on inflammation, either through modulating cytokine production or by scavenging by-products of activated white cells. Identifying dietary patterns (DP) reflecting these mechanisms and relating them to inflammation is of interest. The objective of the study was to identify DP specifically associated with intakes of nutrients potentially involved in inflammatory processes in a middle-aged population and investigate long-term associations between these DP and C-reactive protein (CRP) status assessed several years later. Subjects included in the Supplementation in Vitamins and Mineral Antioxidants 2 cohort study, having available data on dietary assessment carried out in 1994-5 and CRP measurement in 2007-9, were included in the analysis. DP were extracted with reduced rank regression (RRR), using antioxidant micronutrients and PUFA as response variables. Associations between CRP measurements >3 mg/l and extracted DP were then examined with logistic regression models providing OR and 95% CI. A total of 2031 subjects (53·2% women, mean follow-up duration: 12·5 years) were included in the analyses. Of the four extracted DP, a DP with high loading values of vegetables and vegetable oils, leading to high intakes of antioxidant micronutrients and essential fatty acids, was significantly and negatively associated with risk of elevated CRP (OR 0·88; 95% CI 0·78, 0·98). Conversely, a DP reflecting a high n-6:n-3 fatty acid intake ratio was positively and significantly associated with elevated CRP (adjusted OR 1·15; 95% CI 1·00, 1·32). DP extracted with RRR provide support for further exploration of relationships between dietary behaviour and inflammation.

  1. Expression Patterns and Function of Chromatin Protein HMGB2 during Mesenchymal Stem Cell Differentiation*

    PubMed Central

    Taniguchi, Noboru; Caramés, Beatriz; Hsu, Emily; Cherqui, Stephanie; Kawakami, Yasuhiko; Lotz, Martin

    2011-01-01

    The superficial zone (SZ) of articular cartilage is critical in maintaining tissue function and homeostasis and represents the site of the earliest changes in osteoarthritis (OA). The expression of chromatin protein HMGB2 is restricted to the SZ, which contains cells expressing mesenchymal stem cell (MSC) markers. Age-related loss of HMGB2 and gene deletion are associated with reduced SZ cellularity and early onset OA. This study addressed HMGB2 expression patterns in MSC and its role during differentiation. HMGB2 was detected at higher levels in human MSC as compared with human articular chondrocytes, and its expression declined during chondrogenic differentiation of MSC. Lentiviral HMGB2 transduction of MSC suppressed chondrogenesis as reflected by an inhibition of Col2a1 and Col10a1 expression. Conversely, in bone marrow MSC from Hmgb2−/− mice, Col10a1 was more strongly expressed than in wild-type MSC. This is consistent with in vivo results from mouse growth plates showing that Hmgb2 is expressed in proliferating and prehypertrophic zones but not in hypertrophic cartilage where Col10a1 is strongly expressed. Osteogenesis was also accelerated in Hmgb2−/− MSC. The expression of Runx2, which plays a major role in late stage chondrocyte differentiation, was enhanced in Hmgb2−/− MSC, and HMGB2 negatively regulated the stimulatory effect of Wnt/β-catenin signaling on the Runx2 proximal promoter. These results demonstrate that HMGB2 expression is inversely correlated with the differentiation status of MSC and that HMGB2 suppresses chondrogenic differentiation. The age-related loss of HMGB2 in articular cartilage may represent a mechanism responsible for the decline in adult cartilage stem cell populations. PMID:21890638

  2. 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.

  3. 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.

  4. Tissue specificity of the hormonal response in sex accessory tissues is associated with nuclear matrix protein patterns.

    PubMed

    Getzenberg, R H; Coffey, D S

    1990-09-01

    The DNA of interphase nuclei have very specific three-dimensional organizations that are different in different cell types, and it is possible that this varying DNA organization is responsible for the tissue specificity of gene expression. The nuclear matrix organizes the three-dimensional structure of the DNA and is believed to be involved in the control of gene expression. This study compares the nuclear structural proteins between two sex accessory tissues in the same animal responding to the same androgen stimulation by the differential expression of major tissue-specific secretory proteins. We demonstrate here that the nuclear matrix is tissue specific in the rat ventral prostate and seminal vesicle, and undergoes characteristic alterations in its protein composition upon androgen withdrawal. Three types of nuclear matrix proteins were observed: 1) nuclear matrix proteins that are different and tissue specific in the rat ventral prostate and seminal vesicle, 2) a set of nuclear matrix proteins that either appear or disappear upon androgen withdrawal, and 3) a set of proteins that are common to both the ventral prostate and seminal vesicle and do not change with the hormonal state of the animal. Since the nuclear matrix is known to bind androgen receptors in a tissue- and steroid-specific manner, we propose that the tissue specificity of the nuclear matrix arranges the DNA in a unique conformation, which may be involved in the specific interaction of transcription factors with DNA sequences, resulting in tissue-specific patterns of secretory protein expression.

  5. Conserved and divergent expression patterns of the proteolipid protein gene family in the amphibian central nervous system.

    PubMed

    Yoshida, M; Shan, W S; Colman, D R

    1999-07-01

    The recent discovery of a proteolipid protein gene family has revealed that its members are in fact widely distributed and are not exclusively associated with myelination. To date, three different gene products, DMalpha/DM-20/PLP, DMbeta/M6a, and DMgamma/M6b, have been isolated from certain primitive fish species, mouse, and human central nervous system (CNS). We cloned Xenopus laevis orthologues of DMbeta/M6a and DMgamma/M6b and investigated the expression patterns of these gene transcripts as well as that of PLP in developing Xenopus CNS. As is the case in shark and mouse, the mRNA encoding the major myelin integral protein, PLP, is first detected at stage 42/43 in tadpoles and is exclusively found in morphologically recognizable oligodendrocytes throughout the brain, while DMbeta mRNA is solely expressed in young presumptive neurons in the gray matter. There exist two distinct DMgamma mRNAs and, in contrast to these evolutionarily conserved expression patterns, DMgamma mRNAs distribute uniquely within the ventricular zone in young tadpoles (stage 25) through maturity. Furthermore, both DMbeta and DMgamma are expressed in the developing retina, and their distributions are different from one other. In Xenopus CNS, therefore, the expression patterns of three proteolipid proteins, PLP, DMbeta, and DMgamma, are distinct from each other, implying very different roles for their protein products within the cell populations in which they are expressed. PMID:10397631

  6. 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.

  7. ProSMoS server: a pattern-based search using interaction matrix representation of protein structures.

    PubMed

    Shi, Shuoyong; Chitturi, Bhadrachalam; Grishin, Nick V

    2009-07-01

    Assessing structural similarity and defining common regions through comparison of protein spatial structures is an important task in functional and evolutionary studies of proteins. There are many servers that compare structures and define sub-structures in common between proteins through superposition and closeness of either coordinates or contacts. However, a natural way to analyze a structure for experts working on structure classification is to look for specific three-dimensional (3D) motifs and patterns instead of finding common features in two proteins. Such motifs can be described by the architecture and topology of major secondary structural elements (SSEs) without consideration of subtle differences in 3D coordinates. Despite the importance of motif-based structure searches, currently there is a shortage of servers to perform this task. Widely known TOPS does not fully address this problem, as it finds only topological match but does not take into account other important spatial properties, such as interactions and chirality. Here, we implemented our approach to protein structure pattern search (ProSMoS) as a web-server. ProSMoS converts 3D structure into an interaction matrix representation including the SSE types, handednesses of connections between SSEs, coordinates of SSE starts and ends, types of interactions between SSEs and beta-sheet definitions. For a user-defined structure pattern, ProSMoS lists all structures from a database that contain this pattern. ProSMoS server will be of interest to structural biologists who would like to analyze very general and distant structural similarities. The ProSMoS web server is available at: http://prodata.swmed.edu/ProSMoS/. PMID:19420061

  8. ProSMoS server: a pattern-based search using interaction matrix representation of protein structures

    PubMed Central

    Shi, Shuoyong; Chitturi, Bhadrachalam; Grishin, Nick V.

    2009-01-01

    Assessing structural similarity and defining common regions through comparison of protein spatial structures is an important task in functional and evolutionary studies of proteins. There are many servers that compare structures and define sub-structures in common between proteins through superposition and closeness of either coordinates or contacts. However, a natural way to analyze a structure for experts working on structure classification is to look for specific three-dimensional (3D) motifs and patterns instead of finding common features in two proteins. Such motifs can be described by the architecture and topology of major secondary structural elements (SSEs) without consideration of subtle differences in 3D coordinates. Despite the importance of motif-based structure searches, currently there is a shortage of servers to perform this task. Widely known TOPS does not fully address this problem, as it finds only topological match but does not take into account other important spatial properties, such as interactions and chirality. Here, we implemented our approach to protein structure pattern search (ProSMoS) as a web-server. ProSMoS converts 3D structure into an interaction matrix representation including the SSE types, handednesses of connections between SSEs, coordinates of SSE starts and ends, types of interactions between SSEs and β-sheet definitions. For a user-defined structure pattern, ProSMoS lists all structures from a database that contain this pattern. ProSMoS server will be of interest to structural biologists who would like to analyze very general and distant structural similarities. The ProSMoS web server is available at: http://prodata.swmed.edu/ProSMoS/. PMID:19420061

  9. New algorithmic approaches to protein spot detection and pattern matching in two-dimensional electrophoresis gel databases.

    PubMed

    Pleissner, K P; Hoffmann, F; Kriegel, K; Wenk, C; Wegner, S; Sahlström, A; Oswald, H; Alt, H; Fleck, E

    1999-01-01

    Protein spot identification in two-dimensional electrophoresis gels can be supported by the comparison of gel images accessible in different World Wide Web two-dimensional electrophoresis (2-DE) gel protein databases. The comparison may be performed either by visual cross-matching between gel images or by automatic recognition of similar protein spot patterns. A prerequisite for the automatic point pattern matching approach is the detection of protein spots yielding the x(s),y(s) coordinates and integrated spot intensities i(s). For this purpose an algorithm is developed based on a combination of hierarchical watershed transformation and feature extraction methods. This approach reduces the strong over-segmentation of spot regions normally produced by watershed transformation. Measures for the ellipticity and curvature are determined as features of spot regions. The resulting spot lists containing x(s),y(s),i(s)-triplets are calculated for a source as well as for a target gel image accessible in 2-DE gel protein databases. After spot detection a matching procedure is applied. Both the matching of a local pattern vs. a full 2-DE gel image and the global matching between full images are discussed. Preset slope and length tolerances of pattern edges serve as matching criteria. The local matching algorithm relies on a data structure derived from the incremental Delaunay triangulation of a point set and a two-step hashing technique. For the incremental construction of triangles the spot intensities are considered in decreasing order. The algorithm needs neither landmarks nor an a priori image alignment. A graphical user interface for spot detection and gel matching is written in the Java programming language for the Internet. The software package called CAROL (http://gelmatching.inf.fu-berlin.de) is realized in a client-server architecture.

  10. 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.

  11. Simulated rainfall-driven dissolution of TNT, Tritonal, Comp B and Octol particles.

    PubMed

    Taylor, Susan; Lever, James H; Fadden, Jennifer; Perron, Nancy; Packer, Bonnie

    2009-05-01

    Live-fire military training can deposit millimeter-sized particles of high explosives (HE) on surface soils when rounds do not explode as intended. Rainfall-driven dissolution of the particles then begins a process whereby aqueous HE solutions can enter the soil and groundwater as contaminants. We dripped water onto individual particles of TNT, Tritonal, Comp B and Octol to simulate how surface-deposited HE particles might dissolve under the action of rainfall and to use the data to verify a model that predicts HE dissolution as a function of particle size, particle composition and rainfall rate. Particle masses ranged from 1.1 to 17 mg and drip rates corresponded to nominal rainfall rates of 6 and 12 mmh(-1). For the TNT and Tritonal particles, TNT solubility governed dissolution time scales, whereas the lower-solubility of RDX controlled the dissolution time of both RDX and TNT in Comp B. The large, low-solubility crystals of HMX slowed but did not control the dissolution of TNT in Octol. Predictions from a drop-impingement dissolution model agree well with dissolved-mass timeseries for TNT, Tritonal and Comp B, providing some confidence that the model will also work well when applied to the rainfall-driven, outdoor dissolution of these HE particles.

  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. Comp Curriculum.

    ERIC Educational Resources Information Center

    Forsberg, Sara J.; And Others

    This document provides lists of behavioral objectives intended to be used in planning learning experiences for children from birth to age 5. The objectives are arranged by age level under four general areas or domains: communication, self-care, motor and problem solving. Since basic objectives are included, the lists are also appropriate for…

  14. Structure and Glycosylation Patterns of Surface Proteins from Woodchuck Hepatitis Virus

    PubMed Central

    Tolle, Tanja K.; Glebe, Dieter; Linder, Monica; Linder, Dietmar; Schmitt, Sigrid; Geyer, Rudolf; Gerlich, Wolfram H.

    1998-01-01

    Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans. PMID:9811735

  15. Functional and expression pattern analysis of chemosensory proteins expressed in antennae and pheromonal gland of Mamestra brassicae.

    PubMed

    Jacquin-Joly, E; Vogt, R G; François, M C; Nagnan-Le Meillour, P

    2001-09-01

    Sequences coding for chemosensory proteins (CSP) CSPMbraA and CSPMbraB, soluble proteins of low mol. wt, have been amplified using polymerase chain reaction on antennal and pheromonal gland complementary DNAs. On the basis of their sequences, these proteins could be classed in the 'OS-D like' protein family whose first member was described in Drosophila, and that includes proteins characterized in chemosensory organs of many insect phylla, including our recent identification in Mamestra brassicae proboscis. Binding assays have shown that these proteins bind the pheromonal component (Z)-11-hexadecenyl-1-acetate (Z11-16:Ac) as well as (Z)-11-octadecenyl-1-acetate (Z11-18:Ac), an other putative component of the M. brassicae pheromonal blend. Furthermore, binding with fatty acids, but not with progesterone that is a structurally unrelated compound, leads to the hypothesis that the odorant-binding capability of the MbraCSPs may be restricted to fatty acids and/or to 16-18 carbon backbone skeletons. Thus, these proteins do not show the same highly binding specificity as the pheromone-binding proteins do. The CSP-related proteins appear homologous based on sequence identity, conserved cysteine residues and general patterns of expression. However, phylogenetic analyses suggest the presence of multiple classes of CSP within a given species and possible diversification of CSPs within different orders. This diversity perhaps contributes to the many CSP functions proposed in the literature. In M. brassicae, we localized the CSPMbraA expression to the sensilla trichodea, devoted to pheromone reception, suggesting a role in the chemosensory pathway. However, we also localized such proteins in the pheromonal gland, devoid of any chemosensory structure. This suggests that the M. brassicae CSP could be involved in transport of hydrophobic molecules through different aqueous media, such as the sensillar lymph, as well as the pheromonal gland cytosol.

  16. HER-3 targeting alters the dimerization pattern of ErbB protein family members in breast carcinomas

    PubMed Central

    Karamouzis, Michalis V.; Dalagiorgou, Georgia; Georgopoulou, Urania; Nonni, Afroditi; Kontos, Michalis; Papavassiliou, Athanasios G.

    2016-01-01

    Breast carcinogenesis is a multi-step process in which membrane receptor tyrosine kinases are crucial participants. Lots of research has been done on epidermal growth factor receptor (EGFR) and HER-2 with important clinical results. However, breast cancer patients present intrinsic or acquired resistance to available HER-2-directed therapies, mainly due to HER-3. Using new techniques, such as proximity ligation assay, herein we evaluate the dimerization pattern of HER-3 and the importance of context-dependent dimer formation between HER-3 and other HER protein family members. Additionally, we show that the efficacy of novel HER-3 targeting agents can be better predicted in certain breast cancer patient sub-groups based on the dimerization pattern of HER protein family members. Moreover, this model was also evaluated and reproduced in human paraffin-embedded breast cancer tissues. PMID:26716646

  17. 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.

  18. SEMModComp: An R Package for Calculating Likelihood Ratio Tests for Mean and Covariance Structure Models

    ERIC Educational Resources Information Center

    Levy, Roy

    2010-01-01

    SEMModComp, a software package for conducting likelihood ratio tests for mean and covariance structure modeling is described. The package is written in R and freely available for download or on request.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. Changes in Expression Pattern of Selected Endometrial Proteins following Mesenchymal Stem Cells Infusion in Mares with Endometrosis

    PubMed Central

    Mambelli, Lisley I.; Mattos, Rodrigo C.; Winter, Gustavo H. Z.; Madeiro, Dener S.; Morais, Bruna P.; Malschitzky, Eduardo; Miglino, Maria Angélica; Kerkis, Alexandre; Kerkis, Irina

    2014-01-01

    Mesenchymal stem cells (MSCs) due to their self-renewal potential and differentiation capacity are useful for tissue regeneration. Immunomodulatory and trophic properties of MSCs were demonstrated suggesting their use as medicinal signaling cells able to positively change local environment in injured tissue. Equine endometrosis is a progressive degenerative disease responsible for glandular alterations and endometrial fibrosis which causes infertility in mares. More precisely, this disease is characterized by phenotypic changes in the expression pattern of selected endometrial proteins. Currently, no effective treatment is available for endometrosis. Herein, we aimed at the evaluation of expression pattern of these proteins after allogeneic equine adipose tissue-derived multipotent mesenchymal stem cells (eAT-MSCs) infusion as well as at testing the capacity of these cells to promote endometrial tissue remodeling in mares with endometrosis. eAT-MSC (2×107/animal) were transplanted into mares’ uterus and control animals received only placebo. Uterine biopsies were collected before (day 0) and after (days 7, 21 and 60) cells transplantation. Conventional histopathology as well as expression analysis of such proteins as laminin, vimentin, Ki-67-antigen, α-smooth muscle actin (α-SMA) and cytokeratin 18 (CK18) have been performed before and after eAT-MSCs transplantation. We demonstrated that eAT-MSCs induced early (at day 7) remodeling of endometrial tissue microenvironment through changes observed in intra cellular and intra glandular localization of aforementioned proteins. We demonstrated that eAT-MSCs were able to positively modulate the expression pattern of studied secretory proteins as well as, to promote the induction of glandular epithelial cells proliferation suggesting local benefits to committed endometrial tissue environment after eAT-MSCs transplantation. PMID:24901368

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

    PubMed Central

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

    2016-01-01

    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. PMID:25697908

  5. 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.

  6. 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. PMID:25384250

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  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. Ovocalyxin-36 Is a Pattern Recognition Protein in Chicken Eggshell Membranes

    PubMed Central

    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). PMID:24391897

  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. PMID:26685787

  14. 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.

  15. A SELDI mass spectrometry study of experimental autoimmune encephalomyelitis: sample preparation, reproducibility, and differential protein expression patterns

    PubMed Central

    2013-01-01

    isoform 8 (MBP8) (14.2 kDa) levels were lower in EAE samples with advanced disease relative to controls, while an MBP fragment (12. 4kDa), likely due to calpain digestion, was increased in EAE relative to controls. The appearance of MBP in mitochondrially enriched fractions is due to tissue freezing and storage, as MBP was not found associated with mitochondria obtained from fresh tissue. Conclusions SELDI mass spectrometry can be employed to explore the proteome of a complex tissue (brain) and obtain protein profiles of differentially expressed proteins from protein fractions. Appropriate homogenization protocols and protein fractionation using anion exchange beads can be employed to reduce sample complexity without introducing significant additional variation into the SELDI mass spectra beyond that inherent in the SELDI- MS method itself. SELDI-MS coupled with principal component analysis and hierarchical cluster analysis provides protein patterns that can clearly distinguish the disease state from controls. However, identification of individual differentially expressed proteins requires a separate purification of the proteins of interest by polyacrylamide electrophoresis prior to trypsin digestion and peptide mass fingerprint analysis, and unambiguous identification of differentially expressed proteins can be difficult if protein bands consist of several proteins with similar molecular weights. PMID:23635033

  16. Strategic compliance planning using the NO{sub x}COMP computer model

    SciTech Connect

    Baublis, D.; Frazier, W.; Killen, D.; Copolo, T.; Flynn, D.; Nelson, J.

    1997-09-01

    Strategic planning for NO{sub x} compliance becomes more important as the NO{sub x} emission limits are reduced further for Phase 2 of the Clean Air Act Amendments of 1990 (CAAA). Utilities in sensitive areas such as the Ozone Transport Region (OTR), or others which may be affected by the activities of the Ozone Transport Assessment Group (OTAG) may face still lower limits than the current Title 4, Phase 2 regulations. Effective compliance strategy development must take into account baseline NO{sub x} emissions, actual or future regulatory limits, emissions averaging and bubbling schemes, unit capacity factors and economic parameters, as well as the potential of various NO{sub x} control techniques under consideration. Stone and Webster has developed and used the NO{sub x}COMP technical/economic computer model to aid utilities in determining the least-cost, least-risk strategy for compliance with the NO{sub x} provisions of the CAAA. Designed specifically to be used throughout the implementation of a NO{sub x} compliance program, NO{sub x}COMP can be updated with post-modification data. By inputting actual cost and emissions performance information, the impact to the utility compliance program can be determined and the strategy adjusted accordingly. Better, or worse than expected, performance can be evaluated based on overall compliance cost. NO{sub x}COMP uses input directly from the major load forecasting programs such as ENPRO and PROMOD, and when combined with the actual NO{sub x} vs. load profile for each boiler, develops projections of baseline annual NO{sub x} emissions. Least-cost compliance plans are determined by evaluating all applicable technologies to each unit, allowing marginal costs to be generated. For each scenario, the optimal NO{sub x} technology for each unit and associated costs are developed.

  17. 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.

  18. Cartilage Oligomeric Matrix Protein in Idiopathic Pulmonary Fibrosis

    PubMed Central

    Pandit, Kusum; Ben-Yehudah, Ahmi; Chu, Yanxia; Richards, Thomas; Sciurba, Joshua; Myerburg, Michael; Zhang, Yingze; Parwani, Anil V.; Gibson, Kevin F.; Kaminski, Naftali

    2013-01-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive and life threatening disease with median survival of 2.5–3 years. The IPF lung is characterized by abnormal lung remodeling, epithelial cell hyperplasia, myofibroblast foci formation, and extracellular matrix deposition. Analysis of gene expression microarray data revealed that cartilage oligomeric matrix protein (COMP), a non-collagenous extracellular matrix protein is among the most significantly up-regulated genes (Fold change 13, p-value <0.05) in IPF lungs. This finding was confirmed at the mRNA level by nCounter® expression analysis in additional 115 IPF lungs and 154 control lungs as well as at the protein level by western blot analysis. Immunohistochemical analysis revealed that COMP was expressed in dense fibrotic regions of IPF lungs and co-localized with vimentin and around pSMAD3 expressing cells. Stimulation of normal human lung fibroblasts with TGF-β1 induced an increase in COMP mRNA and protein expression. Silencing COMP in normal human lung fibroblasts significantly inhibited cell proliferation and negatively impacted the effects of TGF-β1 on COL1A1 and PAI1. COMP protein concentration measured by ELISA assay was significantly increased in serum of IPF patients compared to controls. Analysis of serum COMP concentrations in 23 patients who had prospective blood draws revealed that COMP levels increased in a time dependent fashion and correlated with declines in force vital capacity (FVC). Taken together, our results should encourage more research into the potential use of COMP as a biomarker for disease activity and TGF-β1 activity in patients with IPF. Hence, studies that explore modalities that affect COMP expression, alleviate extracellular matrix rigidity and lung restriction in IPF and interfere with the amplification of TGF-β1 signaling should be persuaded. PMID:24376648

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

    PubMed

    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

  1. Spatial patterns of gluten protein and polymer distribution in wheat grain.

    PubMed

    He, Jibin; Penson, Simon; Powers, Stephen J; Hawes, Chris; Shewry, Peter R; Tosi, Paola

    2013-07-01

    The starchy endosperm is the major storage tissue in the mature wheat grain and exhibits quantitative and qualitative gradients in composition, with the outermost cell layers being rich in protein, mainly gliadins, and the inner cells being low in protein but enriched in high-molecular-weight (HMW) subunits of glutenin. We have used sequential pearling to produce flour fractions enriched in particular cell layers to determine the protein gradients in four different cultivars grown at two nitrogen levels. The results show that the steepness of the protein gradient is determined by both genetic and nutritional factors, with three high-protein breadmaking cultivars being more responsive to the N treatment than a low-protein cultivar suitable for livestock feed. Nitrogen also affected the relative abundances of the three main classes of wheat prolamins: the sulfur-poor ω-gliadins showed the greatest response to nitrogen and increased evenly across the grain; the HMW subunits also increased in response to nitrogen but proportionally more in the outer layers of the starchy endosperm than near the core, while the sulfur-rich prolamins showed the opposite trend.

  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. 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

  4. Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: genes, expression patterns and protein interactions of two potential moonlighting proteins.

    PubMed

    Lorenzatto, Karina Rodrigues; Monteiro, Karina Mariante; Paredes, Rodolfo; Paludo, Gabriela Prado; da Fonsêca, Marbella Maria; Galanti, Norbel; Zaha, Arnaldo; Ferreira, Henrique Bunselmeyer

    2012-09-10

    Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.

  5. Molecular cloning and characterisation of a pattern recognition protein, lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) from Chinese shrimp Fenneropenaeus chinensis.

    PubMed

    Liu, Fengsong; Li, Fuhua; Dong, Bo; Wang, Xiaomei; Xiang, Jianhai

    2009-03-01

    A pattern recognition protein (PRP), lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte of Chinese shrimp Fenneropenaeus chinensis by the techniques of homology cloning and RACE. Analysis of nucleotide sequence revealed that the full-length cDNA of 1,275 bp has an open reading frame of 1,098 bp encoding a protein of 366 amino acids including a 17 amino acid signal peptide. Sequence comparison of the deduced amino acid sequence of F. chinensis LGBP showed a high identity of 94%, 90%, 87%, 72% and 63% with Penaeus monodon BGBP, Litopenaeus stylirostris LGBP, Marsupenaeu japonicus BGBP, Homarus gammarus BGBP and Pacifastacus leniusculus LGBP, respectively. The calculated molecular mass of the mature protein is 39,857 Da with a deduced pI of 4.39. Two putative integrin binding motifs, RGD (Arg-Gly-Asp) and a potential recognition motif for beta-1,3-linkage of polysaccharides were observed in LGBP sequence. RT-PCR analysis showed that LGBP gene expresses in haemocyte and hepatopancreas only, but not in other tissues. Capillary electrophoresis RT-PCR method was used to quantify the variation of mRNA transcription level during artificial infection with heat-killed Vibrio anguillarum and Staphylococcus aureusin. A significant enhancement of LGBP transcription was appeared at 6 h post-injection in response to bacterial infection. These results have provided useful information to understand the function of LGBP in shrimp. PMID:18163220

  6. Spatial Patterns of DNA Replication, Protein Synthesis, and Oxygen Concentration within Bacterial Biofilms Reveal Diverse Physiological States▿

    PubMed Central

    Rani, Suriani Abdul; Pitts, Betsey; Beyenal, Haluk; Veluchamy, Raaja Angathevar; Lewandowski, Zbigniew; Davison, William M.; Buckingham-Meyer, Kelli; Stewart, Philip S.

    2007-01-01

    It has long been suspected that microbial biofilms harbor cells in a variety of activity states, but there have been few direct experimental visualizations of this physiological heterogeneity. Spatial patterns of DNA replication and protein synthetic activity were imaged and quantified in staphylococcal biofilms using immunofluorescent detection of pulse-labeled DNA and also an inducible green fluorescent protein (GFP) construct. Stratified patterns of DNA synthetic and protein synthetic activity were observed in all three biofilm systems to which the techniques were applied. In a colony biofilm system, the dimensions of the zone of anabolism at the air interface ranged from 16 to 38 μm and corresponded with the depth of oxygen penetration measured with a microelectrode. A second zone of activity was observed along the nutrient interface of the biofilm. Much of the biofilm was anabolically inactive. Since dead cells constituted only 10% of the biofilm population, most of the inactive cells in the biofilm were still viable. Collectively, these results suggest that staphylococcal biofilms contain cells in at least four distinct states: growing aerobically, growing fermentatively, dead, and dormant. The variety of activity states represented in a biofilm may contribute to the special ecology and tolerance to antimicrobial agents of biofilms. PMID:17337582

  7. Formation of highly stable self-assembled alkyl phosphonic acid monolayers for the functionalization of titanium surfaces and protein patterning.

    PubMed

    Han, Xuemingyue; Sun, Xiangyu; He, Tao; Sun, Shuqing

    2015-01-01

    A protocol for the preparation of improved phosphonate monolayers on a titanium (Ti) substrate is presented. Zirconium ions were used to enhance the bonding between the phosphonate headgroup and the pretreated Ti surface. Contact angle and X-ray photoelectron spectroscopy were used to characterize self-assembled monolayers (SAMs) of alkylphosphonic acid that formed spontaneously on Zr-mediated Ti (Zr/Ti) surfaces. The surfaces that were treated with an aqueous solution of zirconium oxychloride showed significantly enhanced stability in buffer compared with those formed directly on the native oxidized Ti. A bifunctional molecule, 10-mercaptodecanyl phosphonic acid (MDPA), was also used to form SAMs on Zr/Ti surfaces using an identical method, which enabled us to regulate the surface functionality through the terminal functional group. Protein patterning on the surface was carried out using UV irradiation through a mask to selectively degrade regions of the MDPA molecules. The surface was then backfilled with a protein-resistant molecule in the exposed regions followed by selective immobilization of proteins to the unexposed areas using a heterobifunctional linker molecule. The presented strategy significantly improved the stability of the phosphonate SAMs on oxidized Ti surfaces, which provided an ideal approach foundation for biomolecular immobilization and patterning onto the Ti surfaces. Thus, this method provided a versatile platform to activate the surfaces of biomedical Ti implants.

  8. Cytokeratin and protein expression patterns in squamous cell carcinoma of the oral cavity provide evidence for two distinct pathogenetic pathways

    PubMed Central

    FROHWITTER, GESCHE; BUERGER, HORST; VAN DIEST, PAUL J.; KORSCHING, EBERHARD; KLEINHEINZ, JOHANNES; FILLIES, THOMAS

    2016-01-01

    Squamous cell carcinoma (SCC) of the oral cavity is a morphological heterogeneous disease. Various cytokeratin (CK) expression patterns with different prognostic values have been described, but little is known concerning the underlying biological cell mechanisms. Therefore, the present study investigated 193 cases of oral SCCs using immunohistochemistry for α/β/γ-catenin, glucose transporter 1, caspase-3, X-linked inhibitor of apoptosis protein, hypoxia inducible factor-1α, carbonic anhydrase 9, heat shock protein (hsp) 70, mast/stem cell growth factor receptor, p21, p27, p16, p53, B-cell lymphoma 6, epidermal growth factor receptor, cyclin D1 and CK1, 5/6, 8/18, 10, 14 and 19. Expression patterns were analyzed with biomathematical permutation analysis. The present results revealed a significant association between the expression of low-molecular weight CK8/18 and 19 and a high-tumor grade, β and γ-catenin expression, deregulated cell cycle proteins and a predominant localization of the tumor on the floor of the mouth. By contrast, expression of high-molecular weight CK1, 5/6, 10 and 14 was significantly associated with the expression of p21 and hsp70. In conclusion, the current study presents evidence for the existence of two parallel pathogenetic pathways in oral SCCs, characterized by the expression of low- and high-molecular weight CKs. Additional studies are required to demonstrate the extent that these results may be used to improve therapeutic regimens. PMID:27347109

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

    PubMed

    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

    2015-10-16

    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 Trp(67)-Glu(71)-Asp(80) inactivation triad within the channel structure and its bearing on the selectivity filter.

  11. Unraveling Patterns of Site-to-Site Synonymous Rates Variation and Associated Gene Properties of Protein Domains and Families

    PubMed Central

    Dimitrieva, Slavica; Anisimova, Maria

    2014-01-01

    In protein-coding genes, synonymous mutations are often thought not to affect fitness and therefore are not subject to natural selection. Yet increasingly, cases of non-neutral evolution at certain synonymous sites were reported over the last decade. To evaluate the extent and the nature of site-specific selection on synonymous codons, we computed the site-to-site synonymous rate variation (SRV) and identified gene properties that make SRV more likely in a large database of protein-coding gene families and protein domains. To our knowledge, this is the first study that explores the determinants and patterns of the SRV in real data. We show that the SRV is widespread in the evolution of protein-coding sequences, putting in doubt the validity of the synonymous rate as a standard neutral proxy. While protein domains rarely undergo adaptive evolution, the SRV appears to play important role in optimizing the domain function at the level of DNA. In contrast, protein families are more likely to evolve by positive selection, but are less likely to exhibit SRV. Stronger SRV was detected in genes with stronger codon bias and tRNA reusage, those coding for proteins with larger number of interactions or forming larger number of structures, located in intracellular components and those involved in typically conserved complex processes and functions. Genes with extreme SRV show higher expression levels in nearly all tissues. This indicates that codon bias in a gene, which often correlates with gene expression, may often be a site-specific phenomenon regulating the speed of translation along the sequence, consistent with the co-translational folding hypothesis. Strikingly, genes with SRV were strongly overrepresented for metabolic pathways and those associated with several genetic diseases, particularly cancers and diabetes. PMID:24896293

  12. 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

  13. 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

    2016-01-01

    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

  14. Comparison of protein expression pattern between the Plasmodium falciparum chloroquine-resistant RKL9 and chloroquine-sensitive MRC2 strains

    PubMed Central

    Antony, Hiasindh Ashmi; Pathak, Vrushali; Ghosh, Kanjaksha; Parija, Subhash Chandra

    2016-01-01

    Objective: The objective of this study was to compare the protein expression patterns of Plasmodium falciparum extracellular and intracellular proteins separated by two-dimensional electrophoresis (2-DE) from the chloroquine-sensitive (CQS) MRC2 strain and chloroquine-resistant (CQR) RKL9 strain. Materials and Methods: Both the extracellular protein (ECP) and intracellular protein (ICP) were extracted and solubilized. The proteins were separated by 2-DE, first based on their charges using isoelectric focusing and then their sizes by electrophoresis. The separated protein spots were detected by silver staining, and further, the protein spot density was analyzed by an image analysis software. Results: 2-DE separated the proteins extracted from the CQS and CQR strains based on their differentially expressed protein patterns. Extracellular Protein Analysis: A total of 109 and 77 protein spots were detected by image analysis of ECP extracted from MRC2 and RKL9 strains, respectively. There was a marked reduction in protein expression pattern in the CQR strain when compared with the CQS strain. Interestingly, 50 and 18 protein spots were uniquely expressed in MRC2 and RKL9 strains, respectively. When MRC2 strain-expressed proteins were taken as the control, 12 upregulated and 14 downregulated protein spots were observed in the RKL9 strain-extracted proteins. Intracellular Protein Analysis: ICP extracted from MRC2 and RKL9 strains showed 187 and 199 protein spots by an image analysis software, and a small enhancement of protein expression was measured when comparing the CQR strain with CQS strain. There were 67 and 79 unique protein spots detected in MRC2 and RKL9 strains, respectively. A total of 120 protein spots were similar when MRC2 proteins were taken as the control; among these protein spots, 40 upregulated and 22 downregulated protein spots were detected in RKL9 strain-expressed protein. Conclusions: Both these unique and matched protein spots might be molecularly

  15. Cell segmentation in time-lapse fluorescence microscopy with temporally varying sub-cellular fusion protein patterns.

    PubMed

    Bunyak, Filiz; Palaniappan, Kannappan; Chagin, Vadim; Cardoso, M

    2009-01-01

    Fluorescently tagged proteins such as GFP-PCNA produce rich dynamically varying textural patterns of foci distributed in the nucleus. This enables the behavioral study of sub-cellular structures during different phases of the cell cycle. The varying punctuate patterns of fluorescence, drastic changes in SNR, shape and position during mitosis and abundance of touching cells, however, require more sophisticated algorithms for reliable automatic cell segmentation and lineage analysis. Since the cell nuclei are non-uniform in appearance, a distribution-based modeling of foreground classes is essential. The recently proposed graph partitioning active contours (GPAC) algorithm supports region descriptors and flexible distance metrics. We extend GPAC for fluorescence-based cell segmentation using regional density functions and dramatically improve its efficiency for segmentation from O(N(4)) to O(N(2)), for an image with N(2) pixels, making it practical and scalable for high throughput microscopy imaging studies.

  16. 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

  17. A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins.

    PubMed

    Fu, Jianping; Schoch, Reto B; Stevens, Anna L; Tannenbaum, Steven R; Han, Jongyoon

    2007-02-01

    Microfabricated regular sieving structures hold great promise as an alternative to gels to improve the speed and resolution of biomolecule separation. In contrast to disordered porous gel networks, these regular structures also provide well defined environments ideal for the study of molecular dynamics in confining spaces. However, the use of regular sieving structures has, to date, been limited to the separation of long DNA molecules, however separation of smaller, physiologically relevant macromolecules, such as proteins, still remains a challenge. Here we report a microfabricated anisotropic sieving structure consisting of a two-dimensional periodic nanofluidic filter array. The designed structural anisotropy causes different-sized or -charged biomolecules to follow distinct trajectories, leading to efficient separation. Continuous-flow size-based separation of DNA and proteins, as well as electrostatic separation of proteins, was achieved, demonstrating the potential use of this device as a generic molecular sieving structure for an integrated biomolecule sample preparation and analysis system. PMID:18654231

  18. A patterned anisotropic nanofluidic sieving structure for continuous-flow separation of DNA and proteins

    NASA Astrophysics Data System (ADS)

    Fu, Jianping; Schoch, Reto B.; Stevens, Anna L.; Tannenbaum, Steven R.; Han, Jongyoon

    2007-02-01

    Microfabricated regular sieving structures hold great promise as an alternative to gels to improve the speed and resolution of biomolecule separation. In contrast to disordered porous gel networks, these regular structures also provide well defined environments ideal for the study of molecular dynamics in confining spaces. However, the use of regular sieving structures has, to date, been limited to the separation of long DNA molecules, however separation of smaller, physiologically relevant macromolecules, such as proteins, still remains a challenge. Here we report a microfabricated anisotropic sieving structure consisting of a two-dimensional periodic nanofluidic filter array. The designed structural anisotropy causes different-sized or -charged biomolecules to follow distinct trajectories, leading to efficient separation. Continuous-flow size-based separation of DNA and proteins, as well as electrostatic separation of proteins, was achieved, demonstrating the potential use of this device as a generic molecular sieving structure for an integrated biomolecule sample preparation and analysis system.

  19. Effects of growth patterns and dietary protein levels during rearing of broiler breeders on fertility, hatchability, embryonic mortality, and offspring performance.

    PubMed

    van Emous, R A; Kwakkel, R P; van Krimpen, M M; van den Brand, H; Hendriks, W H

    2015-04-01

    The objective of this study was to determine the effects of different growth patterns and dietary crude protein levels during rearing in broiler breeder females on fertility, hatchability, embryonic mortality, and offspring performance. A 2×3 factorial arrangement of treatments was used, with 2 growth patterns to reach a target body weight at 20 wk of age of 2,200 g (standard=standard growth pattern) or 2,400 g (high=high growth pattern), and 3 dietary protein levels (high=crude protein, high), (medium=crude protein, medium), and low=crude protein, low). Fresh egg composition and organ development in hatchlings were determined. Offspring of the different groups were reared until an age of 34 d and feed intake, body weight gain, mortality, and carcass composition were determined. In 29-wk-old high growth pattern breeders compared to standard growth pattern breeders, fertility and hatchability of set eggs were increased; embryonic mortality between d 1 and 9 was decreased whereas hatchability of fertile eggs was not affected. Breeders fed the medium crude protein diet showed a decreased hatchability of fertile eggs caused by an increased embryonic mortality between d 18 and 21 compared to breeders fed the high crude protein and low crude protein diets. Offspring of 29-wk-old high growth pattern breeders tended (P=0.059) to have a higher body weight at d 34 than offspring of standard growth pattern breeders, which was achieved by a tendency to a higher body weight gain (P=0.057). Offspring of breeders fed the medium and low crude protein diet showed a higher feed intake between d 18 and 27 and during the total growth period, as compared to offspring of high crude protein breeders. Male broilers of low crude protein breeders had higher breast meat yield than male broilers of high crude protein breeders, while breast meat yield of female broilers was not affected by dietary protein levels. This experiment showed that a higher growth pattern during the rearing period

  20. Temporal expression patterns of insulin-like growth factor binding protein-4 in the embryonic and postnatal rat brain

    PubMed Central

    2013-01-01

    Background IGFBP-4 has been considered as a factor involving in development of the central nervous system (CNS), but its role needs to be further clarified. In present study, the localization of IGFBP-4 expression in the embryonic forebrain, midbrain and hindbrain was determined using immunohistochemistry, and the levels of IGFBP-4 protein and mRNA were semi-quantified using RT-PCR and Western blot in the embryonic (forebrain, midbrain and hindbrain) and postnatal brain (cerebral cortex, cerebellum and midbrain). Results A clear immunoreactivity of IGFBP-4 covered almost the entire embryonic brain (forebrain, midbrain, hindbrain) from E10.5 to E18.5, except for the area near the ventricle from E14.5. The change of IGFBP-4 mRNA level was regularly from E10.5 to E18.5: its expression peaked at E13.5 and E14.5, followed by gradual decreasing from E15.5. The expression of IGFBP-4 protein was similar to that of mRNA in embryonic stage. After birth, the pattern of IGFBP-4 expression was shown to be rather divergent in different brain areas. In the cerebral cortex, the IGFBP-4 mRNA increased gradually after birth (P0), while the protein showed little changes from P0 to P28, but decreased significantly at P70. In the cerebellum, the IGFBP-4 mRNA decreased gradually from P0, reached the lowest level at P21, and then increased again. However, its protein level gradually increased from P0 to P70. In the midbrain, the IGFBP-4 mRNA first decreased and reached its lowest level at P28 before it increased, while the protein remained constant from P0 to P70. At P7, P14, P21, P28 and P70, the levels of IGFBP-4 mRNA in the cerebral cortex were significantly higher than that in the cerebellum or in the midbrain. Differently, the protein levels in the cerebellum were significantly higher than that either in the cerebral cortex or in the midbrain at P14, P21, P28 and P70. Conclusions The temporal expression pattern of IGFBP-4 in the embryonic brain from E10.5 to E18.5 was consistent

  1. Characterization of group A streptococci (Streptococcus pyogenes): correlation of M-protein and emm-gene type with T-protein agglutination pattern and serum opacity factor.

    PubMed

    Johnson, Dwight R; Kaplan, Edward L; VanGheem, Amy; Facklam, Richard R; Beall, Bernard

    2006-02-01

    Strain characterization of group A streptococci (GAS) has traditionally been based on serological identification of M protein. Additional tests to determine T-protein serotype and production of streptococcal serum opacity factor (SOF) provide important information both to aid in and to supplement M-protein serotyping. Advances in DNA-sequencing technology in the late twentieth century resulted in the development of a method for determining the M type of GAS from the sequence of the gene encoding M protein, the emm gene. Although emm-sequence typing has largely replaced M typing in many laboratories, information provided by T typing and SOF determination continues to provide valuable supplementary information for strain characterization. A comprehensive summary of the correlation of T pattern and SOF production with M type was last published in 1993, several years before emm typing became widely available. Since then, the ease of M-type identification afforded by emm typing has resulted in an increase in the number of confirmed M/emm types of more than 50 %. However, comprehensive information about T-protein serotype and the correlation of SOF production with these new M/emm types is not widely available. This report presents a comprehensive summary of this information, not only for newly described types, but also updated information for previously described types. This information was extracted from combined records from streptococcal reference laboratories at the University of Minnesota and at the Centers for Disease Control and Prevention in Atlanta. Data from more than 40,000 strains (representing uncomplicated GAS infections, systemic invasive infections and strains associated with non-suppurative sequelae, collected from the US and diverse locations worldwide) were analysed.

  2. A chemo-resistant protein expression pattern of glioblastoma cells (A172) to perillyl alcohol

    PubMed Central

    Fischer, Juliana de Saldanha da Gama; Carvalho, Paulo Costa; Fonseca, Clovis Orlando da; Liao, Lujian; Degrave, Wim M; Carvalho, Maria da Gloria da Costa; Yates, John R; Domont, Gilberto B

    2010-01-01

    Glioblastoma multiform (GBM) is by far the most malignant glioma. We have introduced a new treatment for GBMs that comprises the inhalation of a naturally occurring terpene with chemotherapeutic properties known as perillyl alcohol (POH). Clinical trial results on recurrent GBM patients showed that POH extends the average life by more than eight months, temporarily slows tumor growth, and in some cases even decreases tumor size. After approximately seven months the tumor continues to grow and leads to a dismal prognosis. To investigate how these tumors become resistant to POH we generated an A172 human glioblastoma cell culture tolerant to 0.06 mM of POH (A172r). We used Multidimensional Protein Identification Technology (MudPIT) to compare the protein expression profile of A172r cells to the established glioblastoma A172 cell line. Our results include a list of identified proteins unique to either the resistant or the non-resistant cell line. These proteins are related to cellular growth, negative apoptosis regulation, Ras pathway, and other key cellular functions that could be connected to the underlying mechanisms of resistance. PMID:20806975

  3. A close relative of the adrenoleukodystrophy (ALD) gene codes for a peroxisomal protein with a specific expression pattern.

    PubMed Central

    Lombard-Platet, G; Savary, S; Sarde, C O; Mandel, J L; Chimini, G

    1996-01-01

    Adrenoleukodystrophy (ALD), a severe demyelinating disease, is caused by mutations in a gene coding for a peroxisomal membrane protein (ALDP), which belongs to the superfamily of ATP binding cassette (ABC) transporters and has the structure of a half transporter. ALDP showed 38% sequence identity with another peroxisomal membrane protein, PMP70, up to now its closest homologue. We describe here the cloning and characterization of a mouse ALD-related gene (ALDR), which codes for a protein with 66% identity with ALDP and shares the same half transporter structure. The ALDR protein was overexpressed in COS cells and was found to be associated with the peroxisomes. The ALD and ALDR genes show overlapping but clearly distinct expression patterns in mouse and may thus play similar but nonequivalent roles. The ALDR gene, which appears highly conserved in man, is a candidate for being a modifier gene that could account for some of the extreme phenotypic variability of ALD. The ALDR gene is also a candidate for being implicated in one of the complementation groups of Zellweger syndrome, a genetically heterogeneous disorder of peroxisome biogenesis, rare cases of which were found to be associated with mutations in the PMP70 (PXMP1) gene. Images Fig. 1 Fig. 2 Fig. 3 PMID:8577752

  4. Evolution of expression patterns of two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila.

    PubMed

    Yasukawa, Jyunichiro; Tomioka, Sachiko; Aigaki, Toshiro; Matsuo, Takashi

    2010-11-01

    Odorant-binding proteins (OBPs) function in the perception of chemical signals together with odorant and taste receptors. Genes encoding OBPs form a large family in insect genomes. In Drosophila, the evolution of OBP gene repertoire has been well studied by comparisons of the whole genome sequences from 12 closely related species. In contrast, their expression patterns are known only in Drosophila melanogaster. Two OBP genes, Obp57d and Obp57e, arose by gene duplication at the early stage of D. melanogaster species group evolution, followed by the divergence of open reading frame (ORF) sequences from each other. While most species in the melanogaster group maintain both Obp57d and Obp57e, some species have lost either gene, suggesting that the birth-and-death process is a dominating pattern of evolution at the Obp57d/e locus. However, it has not been explored whether the expression patterns of these two OBP genes are diverged or conserved among species. Here, we examined the expression patterns of Obp57d and Obp57e in the selected species from the melanogaster group using a combination of reporter analysis, RNA in situ hybridization, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis. As previously reported for D. melanogaster, expression in the chemosensilla on the legs was observed in all the species examined. Unlike in D. melnanogaster, however, additional expression in the chemosensilla on the mouthparts was observed in some species including Drosophila pseudoobscura, which maintains an ancestral OBP gene at the Obp57d/e locus. This result shows that, as well as their ORF sequences, the expression patterns of Obp57d and Obp57e have diverged substantially between closely related Drosophila species. PMID:20637846

  5. 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

  6. Nano-patterned SERS substrate: application for protein analysis vs. temperature.

    PubMed

    Das, Gobind; Mecarini, Federico; Gentile, Francesco; De Angelis, Francesco; Mohan Kumar, Hg; Candeloro, Patrizio; Liberale, Carlo; Cuda, Giovanni; Di Fabrizio, Enzo

    2009-02-15

    We have illustrated the fabrication of nano-structures as a surface enhanced Raman scattering (SERS) substrate using electro-plating and electron-beam lithography techniques to obtain an array of gold nanograin-aggregate structures of diameter ranging between 80 and 100 nm with interstitial gap of 10-30 nm. The nanostructure based SERS substrate permits us to have better control and reproducibility on generation of plasmon polaritons. The calculation shows the possible detection of myoglobin concentration down to attomole. This SERS substrate is used to investigate the structural changes of different proteins; lysozyme, ribonuclease-B, bovin serum albumin and myoglobin in the temperature range between -65 and 90 degrees C. The in-depth analysis even for small conformational changes is performed using 2D Raman correlation analysis and difference Raman analysis in order to gain straightforward understanding of proteins undergoing thermodynamical perturbation.

  7. Protein expression patterns in two Spiraea species in response to cold treatment.

    PubMed

    Liu, H-M; Fang, L; Che, Y-S; Wu, F-Z; Yang, C-P

    2014-07-01

    We analyzed the different cold-resistance species Spiraea trichocarpa Nakai and Spiraea bumalda 'Goldmound' for low-temperature protein expression, protein types identification, and investigated the cold resistance mechanisms under different levels of low temperature by two-dimensional gel electrophoresis (2-DE) and mass spectrometry. An average of 668 and 559 protein spots were detected by 2-DE of S. bumalda 'Goldmound' and S. trichocarpa Nakai, respectively, under different low-temperature treatments. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy identified 48 proteins, with varying expression, related to metabolism, amino acid synthesis, transportation, stress responses and oxidation-reduction reactions. The results showed that the photosynthesis of S. bumalda 'Goldmound' had been affected, enzymes (RuBisCO large and small subunits) involved in the Calvin cycle were up- and down-regulated, and ATP synthase in photophosphorylation was down-regulated. Cytosolic malate dehydrogenase expression weakened in the TCA cycle, while amino acid synthesis strengthened. The activity of four antioxidant enzymes (superoxide dismutase [Cu-Zn], L-ascorbate peroxidase, glutathione peroxidase and peroxidase) was reduced under varying low temperatures. Enzymes (ribulose-bisphosphate carboxylase and RuBisCO small chain precursor) involved in the photosynthesis of S. trichocarpa Nakai showed obvious up- and down-regulation under low temperatures. Cold treatment influenced the photosynthesis of S. trichocarpa Nakai and S. bumalda 'Goldmound', but the results showed significant differences between the two species, which were supposed to the fact that low temperature modified the metabolic mechanisms and led to the weaker cold resistance in S. bumalda 'Goldmound' than in S. trichocarpa Nakai.

  8. 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.

  9. Localization of ecdysone receptor protein during colour pattern formation in wings of the butterfly Precis coenia (Lepidoptera: Nymphalidae) and co-expression with Distal-less protein.

    PubMed

    Koch, P Bernhardt; Merk, Rosi; Reinhardt, Ralf; Weber, Petra

    2003-01-01

    Butterfly wing colour patterns are determined during late larval and early pupal development, a part of metamorphosis controlled by ecdysteroid hormones via their nuclear hormone receptors. We have sequenced a fragment of the common regions of the ecdysone receptor (EcR) from the butterflies Precis coenia and Bicyclus anynanaand found high identities (83.5% to 100%) to EcR from a moth, Manduca sexta. In P. coenia, we sequenced a putative EcR-B1 isoform with 80.4% identity with the A/B-region of the M. sexta-EcR-B1. Consequently, we used antibodies generated against MsEcR-B1 to localise EcR protein during wing development of P. coenia. Nuclear staining of EcR was observed in different cell types during the course of colour pattern formation. Major observations are as follows: EcR is expressed in cell nuclei corresponding to wing lacunae and prospective veins. EcR is expressed early in pupal wing development in "focal" cells which are thought to release determining signals in a process leading to eyespot formation. Scale forming cells differentiate first and show EcR signal in the eyespot foci and most of the wing sheet, but not in areas corresponding to prospective eyespots. In the eyespots, 20-24 h after pupation, EcR expression seems to play a role in formation of scale rows preceding a later expression (28 h) in scale-forming cells. The results demonstrate that EcR is locally expressed in correlation to all major events of wing development and colour pattern formation. In particular, EcR expression patterns in prospective eyespots show that these are special pattern elements which are specified in concert with other factors of colour pattern formation such as the transcription factor Distal-less. In eyespot foci, Distal-less is expressed simultaneously with EcR, but clearly precedes EcR expression in eyespot scale-forming cells. This indicates a potential interaction between "short-range" signalling systems and "long-range" hormonal systems. PMID:12536321

  10. Localization of ecdysone receptor protein during colour pattern formation in wings of the butterfly Precis coenia (Lepidoptera: Nymphalidae) and co-expression with Distal-less protein.

    PubMed

    Koch, P Bernhardt; Merk, Rosi; Reinhardt, Ralf; Weber, Petra

    2003-01-01

    Butterfly wing colour patterns are determined during late larval and early pupal development, a part of metamorphosis controlled by ecdysteroid hormones via their nuclear hormone receptors. We have sequenced a fragment of the common regions of the ecdysone receptor (EcR) from the butterflies Precis coenia and Bicyclus anynanaand found high identities (83.5% to 100%) to EcR from a moth, Manduca sexta. In P. coenia, we sequenced a putative EcR-B1 isoform with 80.4% identity with the A/B-region of the M. sexta-EcR-B1. Consequently, we used antibodies generated against MsEcR-B1 to localise EcR protein during wing development of P. coenia. Nuclear staining of EcR was observed in different cell types during the course of colour pattern formation. Major observations are as follows: EcR is expressed in cell nuclei corresponding to wing lacunae and prospective veins. EcR is expressed early in pupal wing development in "focal" cells which are thought to release determining signals in a process leading to eyespot formation. Scale forming cells differentiate first and show EcR signal in the eyespot foci and most of the wing sheet, but not in areas corresponding to prospective eyespots. In the eyespots, 20-24 h after pupation, EcR expression seems to play a role in formation of scale rows preceding a later expression (28 h) in scale-forming cells. The results demonstrate that EcR is locally expressed in correlation to all major events of wing development and colour pattern formation. In particular, EcR expression patterns in prospective eyespots show that these are special pattern elements which are specified in concert with other factors of colour pattern formation such as the transcription factor Distal-less. In eyespot foci, Distal-less is expressed simultaneously with EcR, but clearly precedes EcR expression in eyespot scale-forming cells. This indicates a potential interaction between "short-range" signalling systems and "long-range" hormonal systems.

  11. Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity.

    PubMed

    Fan, Chunxin; Zhang, Shicui; Li, Lei; Chao, Yeqing

    2008-07-01

    Fibrinogen-related proteins (FREPs) containing fibrinogen-like (FBG) domain have been shown to be involved in immune responses in both invertebrates and vertebrates, but the underlying mechanisms remain ill-defined. In this study we isolated a cDNA encoding amphioxus (Branchiostoma belcheri) FREP homolog, BbFREP. BbFREP encoded a protein of 286 amino acids, which included a C-terminal FBG domain and clustered together with human fibrinogen beta and gamma chains. Quantitative real time PCR revealed that the expression of BbFREP was significantly up-regulated following challenge with lipopolysaccharides (LPS) or lipoteichoic acid (LTA). The recombinant BbFREP expressed in Pichia pastoris was able to specifically recognize the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LPS, peptidoglycan (PGN) and LTA, and displayed strong bacteriolytic activities against both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus. BbFREP was also able to bind to both E. coli and S. aureus. In situ hybridization indicated that BbFREP was mainly expressed in the hepatic caecum and hind-gut, agreeing basically with the primary expression of vertebrate FREP genes in the liver. All these suggest that BbFREP can function as a pattern recognition receptor with a bacteriolytic activity via interaction with LPS, LTA and PGN. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver, acting as a major tissue in acute phase response. PMID:18533266

  12. Profenofos induced modulation in physiological indices, genomic template stability and protein banding patterns of Anabaena sp. PCC 7120.

    PubMed

    Tiwari, Balkrishna; Chakraborty, Sindhunath; Singh, Savita; Mishra, Arun K

    2016-11-01

    To understand the mechanism underlying organophosphate pesticide toxicity, cyanobacterium Anabaena PCC 7120 was subjected to varied concentrations (0, 5, 10, 20 and 30 mg L(-1)) of profenofos and the effects were investigated in terms of changes in cellular physiology, genomic template stability and protein expression pattern. The supplementation of profenofos reduced the growth, total pigment content and photosynthetic efficiency of the test organism in a dose dependent manner with maximum toxic effect at 30 mg L(-1). The high fluorescence intensity of 2', 7' -dichlorofluorescin diacetate and increased production of malondialdehyde confirmed the prevalence of acute oxidative stress condition inside the cells of the cyanobacterium. Rapid amplified polymorphic DNA (RAPD) fingerprinting and SDS-PAGE analyses showed a significant alteration in the banding patterns of DNA and proteins respectively. A marked increase in superoxide dismutase, catalase, peroxidase activity and a concomitant reduction in glutathione content indicated their possible role in supporting the growth of Anabaena 7120 up to 20 mg L(-1). These findings suggest that the uncontrolled use of profenofos in the agricultural fields may not only lead to the destruction of the cyanobacterial population, but it would also disturb the nutrient dynamics and energy flow. PMID:27428931

  13. Distinct patterns of expression but similar biochemical properties of protein L-isoaspartyl methyltransferase in higher plants.

    PubMed

    Thapar, N; Kim, A K; Clarke, S

    2001-02-01

    Protein L-isoaspartyl methyltransferase is a widely distributed repair enzyme that initiates the conversion of abnormal L-isoaspartyl residues to their normal L-aspartyl forms. Here we show that this activity is expressed in developing corn (Zea mays) and carrot (Daucus carota var. Danvers Half Long) plants in patterns distinct from those previously seen in winter wheat (Triticum aestivum cv Augusta) and thale cress (Arabidopsis thaliana), whereas the pattern of expression observed in rice (Oryza sativa) is similar to that of winter wheat. Although high levels of activity are found in the seeds of all of these plants, relatively high levels of activity in vegetative tissues are only found in corn and carrot. The activity in leaves was found to decrease with aging, an unexpected finding given the postulated role of this enzyme in repairing age-damaged proteins. In contrast with the situation in wheat and Arabidopsis, we found that osmotic or salt stress could increase the methyltransferase activity in newly germinated seeds (but not in seeds or seedlings), whereas abscisic acid had no effect. We found that the corn, rice, and carrot enzymes have comparable affinity for methyl-accepting substrates and similar optimal temperatures for activity of 45 degrees C to 55 degrees C as the wheat and Arabidopsis enzymes. These experiments suggest that this enzyme may have specific roles in different plant tissues despite a common catalytic function.

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

    DOE PAGESBeta

    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

  15. Cadmium accumulation and protein binding patterns in tissues of the rainbow trout, Salmo gairdneri.

    PubMed Central

    Kay, J; Thomas, D G; Brown, M W; Cryer, A; Shurben, D; Solbe, J F; Garvey, J S

    1986-01-01

    Rainbow trout were exposed to defined levels of cadmium in their aquarium water for differing periods at a variety of near-lethal concentrations that ensured the survival of the majority of the fish. The gills, liver and kidney together accounted for 99% of the accumulated load of body cadmium in the fish under these conditions. Although the proportion of total cadmium present in the liver remained relatively constant throughout, the distribution of the remainder between gill and kidney altered with the time of exposure. The cadmium in all three organs was bound by two low molecular weight proteins distinct in character from metallothionein. The isoforms of metallothionein were also present but were found to bind only zinc and copper. By contrast, when trout were injected with cadmium intraperitoneally, most of the metal accumulated in the liver where it was sequestered by the two isoforms of metallothionein. Pre-exposure of the trout to either a low concentration of cadmium (for several months) or to an elevated concentration of zinc (for 5 days) allowed the animals to survive a subsequent exposure to a high, otherwise lethal concentration of cadmium. The proteins responsible for sequestration of the two metals were identified, but two different mechanisms seemed to be involved in the protection of the animals. The significance of these observations in terms of the induction of proteins and the prevention of the toxic effects of cadmium is considered. PMID:3709433

  16. spn-F encodes a novel protein that affects oocyte patterning and bristle morphology in Drosophila.

    PubMed

    Abdu, Uri; Bar, Dikla; Schüpbach, Trudi

    2006-04-01

    The anteroposterior and dorsoventral axes of the Drosophila embryo are established during oogenesis through the activities of Gurken (Grk), a Tgfalpha-like protein, and the Epidermal growth factor receptor (Egfr). spn-F mutant females produce ventralized eggs similar to the phenotype produced by mutations in the grk-Egfr pathway. We found that the ventralization of the eggshell in spn-F mutants is due to defects in the localization and translation of grk mRNA during mid-oogenesis. Analysis of the microtubule network revealed defects in the organization of the microtubules around the oocyte nucleus. In addition, spn-F mutants have defective bristles. We cloned spn-F and found that it encodes a novel coiled-coil protein that localizes to the minus end of microtubules in the oocyte, and this localization requires the microtubule network and a Dynein heavy chain gene. We also show that Spn-F interacts directly with the Dynein light chain Ddlc-1. Our results show that we have identified a novel protein that affects oocyte axis determination and the organization of microtubules during Drosophila oogenesis. PMID:16540510

  17. GELANAL, a personal computer-program to compare protein patterns on two-dimensional polyacrylamide gels.

    PubMed

    Klerk, H; Jespers, A

    1990-05-01

    Comparing and analyzing a series of two-dimensional gels by hand is troublesome and subjective. So far a number of systems for automatic analysis have been developed on mainly mainframe computers, using complex algorithms. This paper presents an inexpensive system, based on a simple Pascal program, to compare individual spots on two-dimensional gels using an IBM or compatible personal computer in a qualitative way. The accuracy of the method is demonstrated by comparing two patterns of the same extract from different runs.

  18. Tissue microarray analysis reveals a tight correlation between protein expression pattern and progression of esophageal squamous cell carcinoma

    PubMed Central

    Xue, Li-yan; Hu, Nan; Song, Yong-mei; Zou, Shuang-mei; Shou, Jian-zhong; Qian, Lu-xia; Ren, Li-qun; Lin, Dong-mei; Tong, Tong; He, Zu-gen; Zhan, Qi-min; Taylor, Philip R; Lu, Ning

    2006-01-01

    Background The development of esophageal squamous cell carcinoma (ESCC) progresses a multistage process, collectively known as precursor lesions, also called dysplasia (DYS) and carcinoma in situ (CIS), subsequent invasive lesions and final metastasis. In this study, we are interested in investigating the expression of a variety of functional classes of proteins in ESCC and its precursor lesions and characterizing the correlation of these proteins with ESCC malignant progression. Methods Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC were analyzed using immunohistochemistry on tissue microarray containing 205 ESCC and 173 adjacent precursor lesions as well as corresponding normal mucosa. To confirm the immunohistochemical results, three proteins, fascin, CK14 and laminin-5γ2, which were overexpressed in ESCC on tissue microarray, were detected in 12 ESCC cell lines by Western blot assay. Results In ESCC and its precursor lesions, FADD, CDC25B, fascin, CK14, laminin-5γ2 and SPARC were overexpressed, while Fas, caspase 8, CK4 and annexin I were underexpressed. The abnormalities of these proteins could be classified into different groups in relation to the stages of ESCC development. They were "early" corresponding to mild and moderate DYS with overexpression of fascin, FADD and CDC25B and underexpression of Fas, caspase 8, CK4 and annexin I, "intermediate" to severe DYS and CIS with overexpression of FADD and CK14, and "late" to invasive lesions (ESCC) and to advanced pTNM stage ESCC lesions with overexpression of CK14, laminin-5γ2 and SPARC. Conclusion Analyzing the protein expression patterns of Fas, FADD, caspase 8, CDC25B, fascin, CK14, CK4, annexin I, laminin-5γ2 and SPARC would be valuable to develop rational strategies for early detection of lesions at risk in advance as well as for prevention and treatment of ESCC. PMID:17187659

  19. Defined topologically-complex protein matrices to manipulate cell shape via three-dimensional fiber-like patterns

    PubMed Central

    Moraes, Christopher; Kim, Byoung Choul; Zhu, Xiaoyue; Mills, Kristen L.; Dixon, Angela R.; Thouless; Takayama, Shuichi

    2014-01-01

    Culturing cells in three-dimensional (3D) environments has been shown to significantly influence cell function, and may provide a more physiologically relevant environment within which to study the behavior of specific cell types. 3D tissues typically present a topologically complex fibrous adhesive environment, which is technically challenging to replicate in a controlled manner. Micropatterning technologies have provided significant insights into cell-biomaterial interactions, and can be used to create fiber-like adhesive structures, but are typically limited to flat culture systems; the methods are difficult to apply to topologically-complex surfaces. In this work, we utilize crack formation in multilayered microfabricated materials under applied strain to rapidly generate well-controlled and topologically complex ‘fiber-like’ adhesive protein patterns, capable of supporting cell culture and controlling cell shape on three-dimensional patterns. We first demonstrate that the features of the generated adhesive environments such as width, spacing and topology can be controlled, and that these factors influence cell morphology. The patterning technique is then applied to examine the influence of fiber structure on the nuclear morphology and actin cytoskeletal structure of cells cultured in a nanofibrous biomaterial matrix. PMID:24632936

  20. 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

    2014-01-01

    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.

  1. 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.

  2. Automated extraction of precise protein expression patterns in lymphoma by text mining abstracts of immunohistochemical studies

    PubMed Central

    Chang, Jia-Fu; Popescu, Mihail; Arthur, Gerald L.

    2013-01-01

    Background: In general, surgical pathology reviews report protein expression by tumors in a semi-quantitative manner, that is, -, -/+, +/-, +. At the same time, the experimental pathology literature provides multiple examples of precise expression levels determined by immunohistochemical (IHC) tissue examination of populations of tumors. Natural language processing (NLP) techniques enable the automated extraction of such information through text mining. We propose establishing a database linking quantitative protein expression levels with specific tumor classifications through NLP. Materials and Methods: Our method takes advantage of typical forms of representing experimental findings in terms of percentages of protein expression manifest by the tumor population under study. Characteristically, percentages are represented straightforwardly with the % symbol or as the number of positive findings of the total population. Such text is readily recognized using regular expressions and templates permitting extraction of sentences containing these forms for further analysis using grammatical structures and rule-based algorithms. Results: Our pilot study is limited to the extraction of such information related to lymphomas. We achieved a satisfactory level of retrieval as reflected in scores of 69.91% precision and 57.25% recall with an F-score of 62.95%. In addition, we demonstrate the utility of a web-based curation tool for confirming and correcting our findings. Conclusions: The experimental pathology literature represents a rich source of pathobiological information, which has been relatively underutilized. There has been a combinatorial explosion of knowledge within the pathology domain as represented by increasing numbers of immunophenotypes and disease subclassifications. NLP techniques support practical text mining techniques for extracting this knowledge and organizing it in forms appropriate for pathology decision support systems. PMID:23967385

  3. Expression patterns of CREB binding protein (CREBBP) and its methylated species during zebrafish development.

    PubMed

    Batut, Julie; Duboé, Carine; Vandel, Laurence

    2015-01-01

    Proper embryonic development requires a fine-tuned control of gene expression, which is achieved in part through the activity of transcription coactivators or corepressors. The nuclear coactivator cAMP-response element-binding protein (CREB) binding protein (CREBBP or CBP) interacts with numerous transcription factors and thereby plays a key role in various signaling pathways. Interestingly, in cell-based studies CREBBP activity is modulated by post-translational modifications such as methylation on arginine residues which is catalyzed by coactivator-associated arginine methyltransferase 1 (CARM1). However, whether and where CREBBP, and in particular its methylated forms, are expressed during development in vertebrates has not been addressed so far. Here, we analyzed the expression of the two crebbp genes (crebbpa & crebbpb) during zebrafish development using both RT-qPCR and in situ hybridization. We found that while crebbpa expression is higher in posterior, caudal nascent somites during somitogenesis, crebbpb accumulates in anterior, rostral, and more mature somites. In addition, crebbpa mRNA is enriched in the central myotome at 24 hpf indicating that its expression is spatially and temporally controlled. We next characterized the expression of CREBBP protein from blastula to gastrula stages by immunohistochemistry. We found that while CREBBP is clearly cytoplasmic in the early blastula, it becomes both cytoplasmic and nuclear at 30% epiboly before turning mainly nuclear during gastrulation. Of interest, CREBBP methylated species appear to be mainly nuclear from 30% epiboly to 6-somite stage. This suggests that methylation may regulate CREBBP import to the nucleus during zebrafish development and could therefore participate in the control of early developmental processes.

  4. 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

  5. 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

  6. 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

  7. A novel C1qDC protein acting as pattern recognition receptor in scallop Argopecten irradians.

    PubMed

    Wang, Leilei; Wang, Lingling; Kong, Pengfei; Yang, Jialong; Zhang, Huan; Wang, Mengqiang; Zhou, Zhi; Qiu, Limei; Song, Linsheng

    2012-08-01

    The C1q domain containing (C1qDC) proteins refer to a family of proteins containing the versatile charge pattern recognition globular C1q domain in the C-terminus, which could bind various ligands including PAMPs and trigger a serial of immune response. In this study, a novel C1qDC protein was identified from Argopecten irradians (designated as AiC1qDC-2). Its full-length cDNA was of 1062 bp with an open reading frame of 720 bp encoding a polypeptide of 240 amino acids containing a typical gC1q domain. This gC1q domain possessed the typical 10-stranded β-sandwich fold with a jelly-roll topology common to all C1q family members, and shared high homology with most of the other identified gC1q domains. The mRNA transcripts of AiC1qDC-2 were mainly detected in hepatopancreas, and also marginally detectable in mantle, gonad, adductor, gill and hemocytes. Its relative expression level in hemocytes was significantly up-regulated after challenges of fungi Pichia pastoris GS115 (P < 0.05), Gram-positive bacteria Micrococcus luteus (P < 0.05) and Gram-negative bacteria Vibrio anguillarum (P < 0.05). The recombinant protein of AiC1qDC-2 (rAiC1qDC-2) could bind various PAMPs, including LPS, PGN, polyI:C, mannan, β-1,3-glucan as well as Yeast-glucan, and displayed agglutinating activity to fungi P. pastoris GS115, Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli TOP10F' as well as V. anguillarum. All these results indicated that AiC1qDC-2 could function as a pattern recognition receptor to recognize various PAMPs on different pathogens in the innate immune responses of scallop, and provided new clues to understand the role of invertebrate C1qDC proteins in the ancient complement system.

  8. Preparation and photolithography of self-assembled monolayers of 10-mercaptodecanylphosphonic acid on glass mediated by zirconium for protein patterning.

    PubMed

    Han, Xuemingyue; Sun, Shuqing; He, Tao

    2013-08-01

    Self-assembled monolayers (SAMs) formed by adsorption of octadecylphosphonic acid (ODPA) on zirconium mediated glass substrates were prepared. In this sandwich structure, Zr(4+) was used as a bi-linker to bind phosphonic acid head group in ODPA to glass substrates. The contact angle of the as-prepared SAMs was measured to be around 104°. X-ray photoelectron spectroscopy (XPS) characterization indicated the modification of Zr(4+) on glass substrates was critical for the formation of reasonably dense, well-ordered SAMs similar in quality to those typically formed on other metal oxide surfaces. Bifunctional molecule, 10-mercaptodecanylphosphonic acid (MDPA), bearing thiol terminal groups for various chemical reactions, was synthesized and formed SAMs on glass using the same approach, which allowed us to control the surface chemistry and functionality through photooxidation of the thiol terminal group. Photopatterning of proteins was performed first by exposing the SAMs to UV light through a mask, followed by protein immobilization to the masked regions through a heterobifunctional linker, while the exposed areas prohibit nonspecific protein absorption. The present strategy, which combined the SAMs assembly and photolithography, offered a facile approach for the fabrication of biomolecule patterning and could be applied to construction of biochips and other applications.

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

    PubMed Central

    Loose, Martin; Mitchison, Timothy J.

    2014-01-01

    Bacterial cytokinesis is commonly initiated by the Z-ring, a cytoskeletal structure assembling 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 a negative regulation on 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. PMID:24316672

  10. Preparation and photolithography of self-assembled monolayers of 10-mercaptodecanylphosphonic acid on glass mediated by zirconium for protein patterning.

    PubMed

    Han, Xuemingyue; Sun, Shuqing; He, Tao

    2013-08-01

    Self-assembled monolayers (SAMs) formed by adsorption of octadecylphosphonic acid (ODPA) on zirconium mediated glass substrates were prepared. In this sandwich structure, Zr(4+) was used as a bi-linker to bind phosphonic acid head group in ODPA to glass substrates. The contact angle of the as-prepared SAMs was measured to be around 104°. X-ray photoelectron spectroscopy (XPS) characterization indicated the modification of Zr(4+) on glass substrates was critical for the formation of reasonably dense, well-ordered SAMs similar in quality to those typically formed on other metal oxide surfaces. Bifunctional molecule, 10-mercaptodecanylphosphonic acid (MDPA), bearing thiol terminal groups for various chemical reactions, was synthesized and formed SAMs on glass using the same approach, which allowed us to control the surface chemistry and functionality through photooxidation of the thiol terminal group. Photopatterning of proteins was performed first by exposing the SAMs to UV light through a mask, followed by protein immobilization to the masked regions through a heterobifunctional linker, while the exposed areas prohibit nonspecific protein absorption. The present strategy, which combined the SAMs assembly and photolithography, offered a facile approach for the fabrication of biomolecule patterning and could be applied to construction of biochips and other applications. PMID:23524079

  11. 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. PMID:26267515

  12. 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

  13. 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.

  14. Dynamic gene and protein expression patterns of the autism-associated Met receptor tyrosine kinase in the developing mouse forebrain

    PubMed Central

    Judson, Matthew C.; Bergman, Mica Y.; Campbell, Daniel B.; Eagleson, Kathie L.; Levitt, Pat

    2009-01-01

    The establishment of appropriate neural circuitry depends upon the coordination of multiple developmental events across space and time. These events include proliferation, migration, differentiation, and survival - all of which can be mediated by hepatocyte growth factor (HGF) signaling through the Met receptor tyrosine kinase. We previously found a functional promoter variant of the MET gene to be associated with autism spectrum disorder, suggesting that forebrain circuits governing social and emotional function may be especially vulnerable to developmental disruptions in HGF/Met signaling. However, little is known about the spatiotemporal distribution of Met expression in the forebrain during the development of such circuits. To advance our understanding of the neurodevelopmental influences of Met activation, we employed complementary Western blotting, in situ hybridization and immunohistochemistry to comprehensively map Met transcript and protein expression throughout perinatal and postnatal development of the mouse forebrain. Our studies reveal complex and dynamic spatiotemporal patterns of expression during this period. Spatially, Met transcript is localized primarily to specific populations of projection neurons within the neocortex and in structures of the limbic system, including the amygdala, hippocampus and septum. Met protein appears to be principally located in axon tracts. Temporally, peak expression of transcript and protein occurs during the second postnatal week. This period is characterized by extensive neurite outgrowth and synaptogenesis, supporting a role for the receptor in these processes. Collectively, these data suggest that Met signaling may be necessary for the appropriate wiring of forebrain circuits with particular relevance to social and emotional dimensions of behavior. PMID:19226509

  15. 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.

  16. Electrophoretic and spectroscopic characterization of the protein patterns formed in different surfactant solutions.

    PubMed

    Blanco, Elena; Ruso, Juan M; Prieto, Gerardo; Sarmiento, Félix

    2008-01-01

    The complexations between catalase and the sodium perfluorooctanoate/sodium octanoate and sodium perfluorooctanoate/sodium dodecanoate systems have been studied by a combination of electrophoresis and spectroscopy measurements. The numbers of adsorption sites on the protein were determined from the observed increases of the zeta-potential as a function of surfactant concentration in the regions where the adsorption was a consequence of the hydrophobic effect. The Gibbs energies of adsorption of the surfactants onto the protein were evaluated and the results show that for all systems, Gibbs energies are negative and larger, in absolute values, at low values of surfactant concentration where binding to the high energy sites takes place, and become less negative as more surfactant molecules bind, suggesting a saturation process. The role of hydrophobic interactions in these systems has been demonstrated to be the predominant. Spectroscopy measurements suggest conformational changes on catalase depending on the surfactant mixture as well as the mixed ratio. No isosbestic point or shifts have been found showing that catalase has spectrophotometrically one kind of binding site for these surfactant mixtures.

  17. Immunohistochemical expression pattern of MMR protein can specifically identify patients with colorectal cancer microsatellite instability.

    PubMed

    Amira, Arfaoui Toumi; Mouna, Trabelsi; Ahlem, Blel; Raoudha, Aloui; Majid, Ben Hmida; Amel, Hamza; Rachida, Zermani; Nadia, Kourdaa

    2014-07-01

    The microsatellite instability (MSI) pathway is found in most cases of hereditary nonpolyposis colorectal cancer (HNPCC) and in 12 % of sporadic colorectal cancer (CRC). It involves inactivation of deoxyribonucleic acid mismatch repair (MMR) genes MLH1, MSH2, PMS2, and MSH6. MMR germline mutation detections are an important supplement to HNPCC clinical diagnosis. It enables at-risk and mutation-positive relatives to be informed about their cancer risks and to benefit from intensive surveillance programs that have been proven to reduce the incidence of CRC. In this study, we analyzed for the first time in Tunisia the potential value of immunohistochemical assessment of MMR protein to identify microsatellite instability in CRC. We evaluate by immunohistochemistry MMR protein expression loss in tumoral tissue compared to positive expression in normal mucosa. Immunohistochemistry revealed loss of expression for MLH1, MSH2, MSH6, and PMS2 in 15, 21, 13, and 15 % of cases, respectively. Here, we report a more elevated frequency of MSI compared to data of the literature. In fact, by immunohistochemistry, 70 % of cases were shown to be MSS phenotype, whereas 30 % of cases, in our set, were instable. Moreover, according to molecular investigation, 71 % of cases were instable (MSI-H) and remaining cases were stable (29 %). Thus, we found a perfect association between MMR immunohistochemical analyses and MSI molecular investigation. Immunohistochemical analysis of MMR gene product expression may allow one to specifically identify MSI phenotype of patients with colorectal carcinomas.

  18. 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.

  19. 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.

  20. Developing consensus on the CompHP professional standards for health promotion in Europe.

    PubMed

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

    2012-12-01

    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 criteria, following the approach of the European Qualifications Framework. Three phases of consultation included an electronic consultation survey, focus groups and workshops, and an online consultation. The standards were revised at each stage following comments received. Responses from across Europe and beyond indicate high levels of agreement with the standards and support for their implementation in education and employment settings to accredit health promotion practitioners and raise the profile of health promotion in Europe.

  1. Temporal and spatial expression patterns of bone morphogenetic protein 3 in developing zebrafish.

    PubMed

    Ito-Amano, Midori; Nakamura, Yukio; Morisaki, Mika; He, Xinjun; Hayashi, Masanori; Watanapokasin, Ramida; Kato, Hiroyuki

    2014-01-01

    Bone morphogenetic proteins (BMPs) are important elements in bone biology. We herein report the expression profiles of zebrafish bmp3 (zbmp3) as demonstrated by real-time PCR and in situ hybridization. The expression of zbmp3 was highly detectable by real-time PCR from 1 day post-fertilization (1 dpf) to 2 weeks post-fertilization (2 wpf) and peaked at 1 wpf. For in situ hybridization experiments, zbmp3 was expressed in the otic vesicle at 1 dpf, 2 dpf, 3 dpf, and 5 dpf. It was also expressed in the pharyngeal arches, including the opercle, branchiostegal ray, and pectoral fins, at 2 dpf. Our results suggest that zbmp3 may play an important role in the skeletal biology of developing zebrafish.

  2. Differential Growth Patterns Among Healthy Infants Fed Protein Hydrolysate or Cow-Milk Formulas

    PubMed Central

    Ventura, Alison K.; Beauchamp, Gary K.

    2011-01-01

    OBJECTIVE: Infant formulas differ considerably in composition and sensory profiles. In this randomized study, we examined whether healthy infants fed an extensively protein hydrolysate formula (PHF) would differ in feeding behavior and growth from those fed cow-milk formula (CMF). PATIENTS AND METHODS: Infants were randomly assigned to be fed CMF or PHF between 0.5 and 7.5 months of age. Each month for 7 months, infants were weighed and measured and then videotaped while being fed their assigned formula. Anthropometric z scores were calculated by using World Health Organization growth standards. Multilevel linear growth and piecewise mixed-effects models compared trajectories for growth measures and formula acceptance. RESULTS: When compared with infants fed CMF, infants fed PHF had significantly lower weight-for-length z scores across ages 2.5 to 7.5 months. There were no differences in length-for-age z scores, which indicate that group differences resulted from gains in weight, not length. Infants fed PHF also had significantly slower weight gain velocity compared with infants fed CMF. During the monthly assessments, PHF-fed infants consumed less formula to satiation than did CMF-fed infants across the study period. Maternal ratings of infants' acceptance of the formula did not differ at any age. CONCLUSIONS: z-score trajectories indicate that CMF-fed infants' weight gain was accelerated, whereas PHF-fed infants' weight gain was normative. Whether such differences in growth are because of differences in the protein content or amino acid profile of the formulas and, in turn, metabolism is unknown. Research on the long-term consequences of these early growth differences is needed. PMID:21187303

  3. 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

  4. Nep1-like proteins from three kingdoms of life act as a microbe-associated molecular pattern in Arabidopsis.

    PubMed

    Oome, Stan; Raaymakers, Tom M; Cabral, Adriana; Samwel, Simon; Böhm, Hannah; Albert, Isabell; Nürnberger, Thorsten; Van den Ackerveken, Guido

    2014-11-25

    Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by a wide range of plant-associated microorganisms. They are best known for their cytotoxicity in dicot plants that leads to the induction of rapid tissue necrosis and plant immune responses. The biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis encodes 10 different noncytotoxic NLPs (HaNLPs) that do not cause necrosis. We discovered that these noncytotoxic NLPs, however, act as potent activators of the plant immune system in Arabidopsis thaliana. Ectopic expression of HaNLP3 in Arabidopsis triggered resistance to H. arabidopsidis, activated the expression of a large set of defense-related genes, and caused a reduction of plant growth that is typically associated with strongly enhanced immunity. N- and C-terminal deletions of HaNLP3, as well as amino acid substitutions, pinpointed to a small central region of the protein that is required to trigger immunity, indicating the protein acts as a microbe-associated molecular pattern (MAMP). This was confirmed in experiments with a synthetic peptide of 24 aa, derived from the central part of HaNLP3 and corresponding to a conserved region in type 1 NLPs that induces ethylene production, a well-known MAMP response. Strikingly, corresponding 24-aa peptides of fungal and bacterial type 1 NLPs were also able to trigger immunity in Arabidopsis. The widespread phylogenetic distribution of type 1 NLPs makes this protein family (to our knowledge) the first proteinaceous MAMP identified in three different kingdoms of life.

  5. 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.

  6. Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes

    PubMed Central

    1975-01-01

    The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented. PMID:166079

  7. [Morphological characterization of annatto fruits 9bixa orellana L.) and its correspondence with protein and isoenzym patterns].

    PubMed

    Medina, A M; Michelangeli, C; Ramis, C; Díaz, A

    2001-01-01

    A group of 32 annatto genotypes collected in five Venezuelan regions (Oriente, Centro, Llanos, Andes and Amazonas) and in Brazil were used for morphological studies. The fruit variables with the greatest discriminatory power in the formation of groups were capsule size, spinosity and seed size. On the other hand, an association group among the variables spinosity, spine length, dehiscence and apex shape were formed, also a proportional association between capsule and seed size, and between dehiscent capsule and brown coloured seeds. Additionally, in order to discriminate morphological variables behaviour in respond to electrophoretic variables, a group of protein and isozyme bands associated with fruit characteristics were established. Therefore, a classification system of this species was possible using morphological studies of the capsules, even though a determined association relating morphological and molecular patterns was not found.

  8. Novel odorant-binding proteins and their expression patterns in grasshopper, Oedaleus asiaticus.

    PubMed

    Zhang, Shuo; Pang, Baoping; Zhang, Long

    2015-05-01

    Insects use olfaction to detect exogenous odors and adapt to environments. In their olfaction systems, odorant-binding proteins (OBPs) are believed to be a key component. The unique OBP system of each species reflects the evolution of chemosensation of insects with habits. Here, we for the first time identified 15 OBPs, OasiOBP1-15, of a grasshopper, Oedaleus asiaticus, that lives in the grasslands of Northern China and is closely related to the locust, Locusta migratoria. OasiOBP9 and OasiOBP10 are specifically expressed in the antennae. Other OBPs are expressed in the antennae as well as other chemosensory organs, such as the mouthparts and wings. Significantly more OasiOBP7 was detected in male than female antennae, but there are 9 OBPs that were more expressed in female than male antennae by quantitative real-time PCR. Phylogenetic analysis indicated that most of the O. asiaticus OBPs are similar to those of L. migratoria, but some are substantially different. This indicates that the OBPs originally evolved in a common ancestor, but their unique chemosensory systems are adapted to different ecosystems. PMID:25778868

  9. Competitive Immunoassays for Simultaneous Detection of Metabolites and Proteins Using Micromosaic Patterning

    PubMed Central

    Murphy, Brian M.; He, Xinya; Dandy, David; Henry, Charles S.

    2010-01-01

    New high-throughput immunoassay methods for rapid point-of-care diagnostic applications represent an unmet need and current focus of numerous innovative methods. We report a new micromosaic competitive immunoassay developed for the analysis of the thyroid hormone thyroxine (T4), inflammation biomarker C-reactive protein (CRP), and the oxidative damage marker 3-nitrotyrosine (BSA–3NT) on a silicon nitride substrate. To demonstrate the versatility of the method, both direct and indirect format competitive immunoassays were developed and could be applied simultaneously for single samples. Signals from standard solutions were fit to a logistic equation, allowing simultaneous detection of T4 (7.7–257.2 nM), CRP (0.3–4.2 µg/mL), and BSA–3NT (0.03–22.3 µg/mL). Total assay time including sample introduction, washing, and fluorescence measurement was less than 45 min. Dissociation constants for affinity pairs in the system have been estimated using regression. This proof-of-concept experiment shows that both small and macromolecular biomarkers can be quantified from a single sample using the method and suggests that groups of clinically related analytes may be analyzed by competitive micromosaic immunoassay techniques. PMID:18092765

  10. 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. PMID:15666213

  11. Multiple haplotype-resolved genomes reveal population patterns of gene and protein diplotypes

    PubMed Central

    Hoehe, Margret R.; Church, George M.; Lehrach, Hans; Kroslak, Thomas; Palczewski, Stefanie; Nowick, Katja; Schulz, Sabrina; Suk, Eun-Kyung; Huebsch, Thomas

    2014-01-01

    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. PMID:25424553

  12. Cadmium accumulation and protein binding patterns in tissues of the rainbow trout, Salmo gairdneri

    SciTech Connect

    Kay, J.; Thomas, D.G.; Brown, M.W.; Cryer, A.; Shurben, D.; Solbe, J.F.deL.G.; Garvey, J.S.

    1986-03-01

    Rainbow trout were exposed to defined levels of cadmium in their aquarium water for differing periods at a variety of near-lethal concentrations that ensured the survival of the majority of the fish. The gills, liver and kidney together accounted for 99% of the accumulated load of body cadmium in the fish under these conditions. Although the proportion of total cadmium present in the liver remained relatively constant throughout, the distribution of the remainder between gill and kidney altered with the time of exposure. The cadmium in all three organs was bound by two low molecular weight proteins distinct in character from metallothionein. By contrast, when trout were injected with cadmium intraperitoneally, most of the metal accumulated in the liver where it was sequestered by the two isoforms of metallothionein. Pre-exposure of the trout to either a low concentration of cadmium (for several months) or to an elevated concentration of zinc (for 5 days) allowed the animals to survive a subsequent exposure to a high, otherwise lethal concentration of cadmium.

  13. 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

  14. 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.

  15. 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.

  16. Biochemical markers and protein pattern analysis for canine coagulase-positive staphylococci and their distribution on dog skin.

    PubMed

    Chanchaithong, Pattrarat; Prapasarakul, Nuvee

    2011-08-01

    Coagulase-positive staphylococci (CoPS) including S. pseudintermedius, S. schleiferi subsp. coagulans and S. aureus are etiological agents of dermatitis in companion animals and can be zoonotic pathogens. To date no consensual biochemical marker for routine microbiological identification of these species has been identified. The aim of this study was to evaluate biochemical markers and compare the results with the approved molecular method, multiplex-PCR (M-PCR), and confirm their species-specific phenotypic characteristic by using SDS-PAGE. The distribution and frequency of CoPS species were also determined. Three hundred and thirty-seven canine CoPS isolates were obtained from the nasal mucosa, perineum and groins of 66 healthy dogs and were identified by the M-PCR as S. aureus (n=5), S. pseudintermedius (n=263) and S. schleiferi subsp. coagulans (n=69). Selected biochemical tests including the Voges-Proskauer test, mannitol broth fermentation, the assimilation of maltose, galactose, trahalose and lactose using broth medium, were successfully used to distinguish the three species of canine CoPS from other CoPS species. Additionally, species-specific protein patterns were also found to be useful for phenotypic differentiation, with good agreement with the results of M-PCR and the use of biochemical markers. S. aureus occured infrequently on dog skin while co-colonization with S. pseudintermedius and S. schleiferi subsp. coagulans was observed. We propose the use of consensual biochemical markers of canine CoPS with the presence of the unique protein patterns as an alternative tool for conventional laboratory use.

  17. Expression pattern of heat shock protein 90 gene of humphead snapper Lutjanus sanguineus during pathogenic Vibrio harveyi stress.

    PubMed

    Zhang, X Z; Dai, L P; Wu, Z H; Jian, J C; Lu, Y S

    2011-07-01

    The full-length cDNA of heat shock protein 90 (HSP90) of humphead snapper Lutjanus sanguineus, designated as rsHSP90, was cloned by rapid amplification of complementary (c)DNA ends (RACE) techniques with the primers designed from the known expressed sequence tag (EST) sequence identified from the subtracted cDNA library of the head kidney of L. sanguineus. Sequence analysis showed that the full-length cDNA of rsHSP90 was 2745 bp, containing a 5' terminal untranslated region (UTR) of 99 bp, a 3' terminal UTR of 471 bp and an open reading frame (ORF) of 2175 bp encoding a polypeptide of 725 amino acids. On the basis of the deduced amino acid sequence, the theoretical molecular mass of rsHSP90 was calculated to be 83·18 kDa with an isoelectric point of 4·79. Moreover, five classical HSP90 family signatures were found in the amino acids sequence of rsHSP90 by PredictProtein. Basic local-alignment search-tool (BLAST) analysis revealed that the amino acids sequence of rsHSP90 had the highest similarity of 97% when compared with other HSP90s. Fluorescent real-time quantitative reverse-transcription (RT)-PCR was used to examine the expression pattern of rsHSP90 in eight kinds of tissues and organs of L. sanguineus challenged with Vibrio harveyi. There was a clear time-dependent expression pattern of rsHSP90 in head kidney, spleen and thymus after bacterial challenge and the expression of messenger (m)RNA reached the maximum level at the time points of 9, 15 and 24 h, respectively. The up-regulated mRNA expression of rsHSP90 in L. sanguineus after bacterial challenge indicated that rsHSP90 was inducible and might be involved in immune response.

  18. Effects of Dicto-Comp and Dictation on the Writing Skill of Female Adult Iranian EFL Learners

    ERIC Educational Resources Information Center

    Adel, Rahil; Hashemian, Mahmood

    2015-01-01

    This study was an attempt to clarify and remind L2 learners/teachers of 2 kinds of writing: dicto-comp and dictation. We explored the effect of controlled writing on the accuracy of the writing of adult Iranian EFL learners. Prior to the study, the homogeneity of 30 adult EFL learners was checked through an OPT test. Thirty participants were…

  19. 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…

  20. Using Construct Validity To Evaluate Assessment Instruments: A Comparison of the ACT-COMP Exam and the ETS Academic Profile.

    ERIC Educational Resources Information Center

    Pike, Gary; Banta, Trudy W.

    The purpose of this paper is (1) to discuss a set of standards that can be used to evaluate potential assessment instruments; and (2) to use these standards to evaluate the American College Testing Program's College Outcomes Measures Program (ACT-COMP) and the Educational Testing Service (ETS) Academic Profile. Using the work of S. Messick (1975,…

  1. The pattern of protein synthesis induced by heat-shock of the moderately halophilic bacterium Chromobacterium marismortui: protective effect of high salt concentration against the thermal shock.

    PubMed

    Katinakis, P

    1989-01-01

    The protein synthetic response to heat shock of the moderately halophilic bacterium Chromobacterium marismortui was examined. Upon exposure to elevated temperature there is an increased synthesis of a specific subset of proteins (heat shock proteins-hsps) in the molecular weight region of 15 to 90 kD, while normal protein synthesis is severely repressed. The synthesis of hsps reaches a maximum 5 min after heat shock at 42 degrees C. Cells recovered their normal protein synthesis patterns rapidly upon returning to their normal growth temperature following heat shock. When cells grown in 2.5M NaCl were challenged with heat shock at 42 degrees C, the synthesis of some normal proteins was permitted. Furthermore, growth in high salt concentration resulted in an extension of the upper temperature limits at which C. marismortui could synthesize hsps. Adaptation of C. marismortui to decreasing salinity stimulated the synthesis of new proteins distinct from the hsps.

  2. 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.

  3. Myotube Formation on Micro-patterned Glass: Intracellular Organization and Protein Distribution in C2C12 Skeletal Muscle Cells

    PubMed Central

    Yamamoto, Daniel L.; Csikasz, Robert I.; Li, Yu; Sharma, Gunjana; Hjort, Klas; Karlsson, Roger; Bengtsson, Tore

    2008-01-01

    Proliferation and fusion of myoblasts are needed for the generation and repair of multinucleated skeletal muscle fibers in vivo. Studies of myocyte differentiation, cell fusion, and muscle repair are limited by an appropriate in vitro muscle cell culture system. We developed a novel cell culture technique [two-dimensional muscle syncytia (2DMS) technique] that results in formation of myotubes, organized in parallel much like the arrangement in muscle tissue. This technique is based on UV lithography–produced micro-patterned glass on which conventionally cultured C2C12 myoblasts proliferate, align, and fuse to neatly arranged contractile myotubes in parallel arrays. Combining this technique with fluorescent microscopy, we observed alignment of actin filament bundles and a perinuclear distribution of glucose transporter 4 after myotube formation. Newly formed myotubes contained adjacently located MyoD-positive and MyoD-negative nuclei, suggesting fusion of MyoD-positive and MyoD-negative cells. In comparison, the closely related myogenic factor Myf5 did not exhibit this pattern of distribution. Furthermore, cytoplasmic patches of MyoD colocalized with bundles of filamentous actin near myotube nuclei. At later stages of differentiation, all nuclei in the myotubes were MyoD negative. The 2DMS system is thus a useful tool for studies on muscle alignment, differentiation, fusion, and subcellular protein localization. (J Histochem Cytochem 56:881–892, 2008) PMID:18574252

  4. Identification of SHRUBBY, a SHORT-ROOT and SCARECROW interacting protein that controls root growth and radial patterning.

    PubMed

    Koizumi, Koji; Gallagher, Kimberly L

    2013-03-01

    The timing and extent of cell division is particularly important for the growth and development of multicellular organisms. Roots of the model organism Arabidopsis thaliana have been widely studied as a paradigm for organ development in plants. In the Arabidopsis root, the plant-specific GRAS family transcription factors SHORT-ROOT (SHR) and SCARECROW (SCR) are key regulators of root growth and of the asymmetric cell divisions that separate the ground tissue into two separate layers: the endodermis and cortex. To elucidate the role of SHR in root development, we identified 17 SHR-interacting proteins. Among those isolated was At5g24740, which we named SHRUBBY (SHBY). SHBY is a vacuolar sorting protein with similarity to the gene responsible for Cohen syndrome in humans. Hypomorphic alleles of shby caused poor root growth, decreased meristematic activity and defects in radial patterning that are characterized by an increase in the number of cell divisions in the ground tissue that lead to extra cells in the cortex and endodermis, as well as additional cell layers. Analysis of genetic and molecular markers indicates that SHBY acts in a pathway that partially overlaps with SHR, SCR, PLETHORA1 and PLETHORA2 (PLT1 and PLT2). The shby-1 root phenotype was partially phenocopied by treatment of wild-type roots with the proteosome inhibitor MG132 or the gibberellic acid (GA) synthesis inhibitor paclobutrazol (PAC). Our results indicate that SHBY controls root growth downstream of GA in part through the regulation of SHR and SCR.

  5. Application of power spectra patterns in Fourier transform square wave voltammetry to evaluate electrode kinetics of surface-confined proteins.

    PubMed

    Fleming, Barry D; Barlow, Nicola L; Zhang, Jie; Bond, Alan M; Armstrong, Fraser A

    2006-05-01

    This paper describes an application of Fourier transform (FT) voltammetry that provides a quantitative evaluation of the electron-transfer kinetics of protein molecules attached to electrode surfaces. The potential waveform applied in these experiments consists of a large-amplitude square wave of frequency f superimposed onto the traditional triangular voltage used in dc cyclic voltammetry. The resultant current-time response, when Fourier transformed into the frequency domain, provides patterns of data at the even harmonic frequencies that arise from nonlinearity in the Faradaic response. These even harmonic contributions are ideally suited for kinetic evaluation of electron-transfer processes because they are highly selective to quasi-reversible behavior (insensitive to reversible or irreversible processes) and almost devoid of background charging current. Inverse FT methods can then be used to provide the wave shapes of the dc as well as the ac voltammetric components and other characteristics employed to detect the level of nonideality present relative to theoretical models based upon noninteracting surface-confined molecules. The new form of data evaluation has been applied to the electron-transfer properties of a typical biological electron carrier, the blue copper protein azurin, immobilized on polycrystalline gold electrodes modified with self-assembled monolayers of different length alkanethiols. Details of the electrode kinetics (rates of electron transfer, dispersion, and charge-transfer coefficients) as a function of alkanethiol, apparent surface coverage, and capacitance are all deduced from the square wave (FT-inverse FT) protocol, and the implications of these findings are considered.

  6. Fatal familial insomnia with an unusual prion protein deposition pattern: an autopsy report with an experimental transmission study.

    PubMed

    Sasaki, K; Doh-ura, K; Wakisaka, Y; Tomoda, H; Iwaki, T

    2005-02-01

    We recently performed a post-mortem examination on a Japanese patient who had a prion protein gene mutation responsible for fatal familial insomnia (FFI). The patient initially developed cerebellar ataxia, but finally demonstrated insomnia, hyperkinetic delirium, autonomic signs and myoclonus in the late stage of the illness. Histological examination revealed marked neuronal loss in the thalamus and inferior olivary nucleus; however, prion protein (PrP) deposition was not proved in these lesions by immunohistochemistry. Instead, PrP deposition and spongiform change were both conspicuous within the cerebral cortex, whereas particular PrP deposition was also observed within the cerebellar cortex. The abnormal protease-resistant PrP (PrP(res)) molecules in the cerebral cortex of this case revealed PrP(res) type 2 pattern and were compatible with those of FFI cases, but the transmission study demonstrated that a pathogen in this case was different from that in a case with classical FFI. By inoculation with homogenate made from the cerebral cortex, the disease was transmitted to mice, and neuropathological features that were distinguishable from those previously reported were noted. These findings indicate the possibility that a discrete pathogen was involved in the disease in this case. We suggest that not only the genotype of the PrP gene and some other as yet unknown genetic factors, but also the variation in pathogen strains might be responsible for the varying clinical and pathological features of this disease.

  7. 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

  8. DEX1, a Novel Plant Protein, Is Required for Exine Pattern Formation during Pollen Development in Arabidopsis1

    PubMed Central

    Paxson-Sowders, Dawn M.; Dodrill, Craig H.; Owen, Heather A.; Makaroff, Christopher A.

    2001-01-01

    To identify factors that are required for proper pollen wall formation, we have characterized the T-DNA-tagged, dex1 mutation of Arabidopsis, which results in defective pollen wall pattern formation. This study reports the isolation and molecular characterization of DEX1 and morphological and ultrastructural analyses of dex1 plants. DEX1 encodes a novel plant protein that is predicted to be membrane associated and contains several potential calcium-binding domains. Pollen wall development in dex1 plants parallels that of wild-type plants until the early tetrad stage. In dex1 plants, primexine deposition is delayed and significantly reduced. The normal rippling of the plasma membrane and production of spacers observed in wild-type plants is also absent in the mutant. Sporopollenin is produced and randomly deposited on the plasma membrane in dex1 plants. However, it does not appear to be anchored to the microspore and forms large aggregates on the developing microspore and the locule walls. Based on the structure of DEX1 and the phenotype of dex1 plants, several potential roles for the protein are proposed. PMID:11743117

  9. 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}.

  10. Alterations in penicillin binding protein gene of Streptococcus pneumoniae and their correlation with susceptibility patterns.

    PubMed

    Ohsaki, Yoshinobu; Tachibana, Mineji; Nakanishi, Kyoko; Nakao, Shoko; Saito, Kumiko; Toyoshima, Eri; Sato, Maki; Takahashi, Toru; Osanai, Shinobu; Itoh, Yoshihisa; Kikuchi, Kenjiro

    2003-08-01

    Penicillin binding protein (pbp) gene alterations of 328 clinical isolates of Streptococcus pneumoniae were examined for a correlation with their antibiotic-resistance. The frequency of penicillin G (PEN-G) resistance was determined to clarify susceptibility to several antibiotics, namely PEN-G, ampicillin, sulbactam/ampicillin, cefozopram, panipenem (PAPM), clarithromycin (CLR), azithromycin (AZM) and levofloxacin (LVX). Oligonucleotide primers for three pbp genes (pbp1a, pbp2x and pbp2b) were used to detect mutations in pbp. Of the strains, 25.9% were classified as Pen-Gs, 68.0% as Pen-Gir and 6.1% as Pen-Gr. The polymerase chain reaction product for wild-type pbp1a was found in 185 isolates, that for wild-type pbp2x was found in 66 isolates and that for wild-type pbp2b was found in 213 isolates. None of these three genes was detectable in 100 isolates while all of them were detected in 64 isolates (1aw/2xw/2bw). Of those 64 isolates with 1aw/2xw/2bw, the minimum inhibitory concentration (MIC) of PEN-G was < or =0.06 mg/l for 54 isolates and 0.12 mg/l for 10 isolates. Of the 272 strains for which the MIC of PAPM was < or =0.03 mg/l, there were 85 Pen-Gs, 184 Pen-Gir and three Pen-Gr isolates. Three strains for which the MIC of LVX was > or =4.0 mg/l included one Pen-Gs and two Pen-Gir isolates. The MICs of CLR correlated significantly with those of AZM. The MIC of CLR was > or =1 mg/l for 216 isolates, and the MIC of AZM was > or =1 mg/l for 244 of them. These data suggested that PAPM may be effective against S. pneumoniae infection, although acquisition of resistance should be considered. LVX also seemed to be effective against S. pneumoniae.

  11. Genome organization, phylogenies, expression patterns, and three-dimensional protein models of two acetylcholinesterase genes from the red flour beetle.

    PubMed

    Lu, Yanhui; Pang, Yuan-Ping; Park, Yoonseong; Gao, Xiwu; Yao, Jianxiu; Zhang, Xin; Zhu, Kun Yan

    2012-01-01

    Since the report of a paralogous acetylcholinesterase (AChE, EC3.1.1.7) gene in the greenbug (Schizaphis graminum) in 2002, two different AChE genes (Ace1 and Ace2) have been identified in each of at least 27 insect species. However, the gene models of Ace1 and Ace2, and their molecular properties have not yet been comprehensively analyzed in any insect species. In this study, we sequenced the full-length cDNAs, computationally predicted the corresponding three-dimensional protein models, and profiled developmental stage and tissue-specific expression patterns of two Ace genes from the red flour beetle (Tribolium castaneum; TcAce1 and TcAce2), a globally distributed major pest of stored grain products and an emerging model organism. TcAce1 and TcAce2 encode 648 and 604 amino acid residues, respectively, and have conserved motifs including a choline-binding site, a catalytic triad, and an acyl pocket. Phylogenetic analysis show that both TcAce genes are grouped into two insect Ace clusters and TcAce1 is completely diverged from TcAce2, suggesting that these two genes evolve from their corresponding Ace gene lineages in insect species. In addition, TcAce1 is located on chromosome 5, whereas TcAce2 is located on chromosome 2. Reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR analyses indicate that both genes are virtually transcribed in all the developmental stages and predominately expressed in the insect brain. Our computational analyses suggest that the TcAce1 protein is a robust acetylcholine (ACh) hydrolase and has susceptibility to sulfhydryl agents whereas the TcAce2 protein is not a catalytically efficient ACh hydrolase.

  12. Correlations Between Amino Acids at Different Sites in Local Sequences of Protein Fragments with Given Structural Patterns

    NASA Astrophysics Data System (ADS)

    Lu, Wen; Liu, Hai-yan

    2007-02-01

    Ample evidence suggests that the local structures of peptide fragments in native proteins are to some extent encoded by their local sequences. Detecting such local correlations is important but it is still an open question what would be the most appropriate method. This is partly because conventional sequence analyses treat amino acid preferences at each site of a protein sequence independently, while it is often the inter-site interactions that bring about local sequence-structure correlations. Here a new scheme is introduced to capture the correlation between amino acid preferences at different sites for different local structure types. A library of nine-residue fragments is constructed, and the fragments are divided into clusters based on their local structures. For each local structure cluster or type, chi-square tests are used to identify correlated preferences of amino acid combinations at pairs of sites. A score function is constructed including both the single site amino acid preferences and the dual-site amino acid combination preferences, which can be used to identify whether a sequence fragment would have a strong tendency to form a particular local structure in native proteins. The results show that, given a local structure pattern, dual-site amino acid combinations contain different information from single site amino acid preferences. Representative examples show that many of the statistically identified correlations agree with previously-proposed heuristic rules about local sequence-structure correlations, or are consistent with physical-chemical interactions required to stabilize particular local structures. Results also show that such dual-site correlations in the score function significantly improves the Z-score matching a sequence fragment to its native local structure relative to non-native local structures, and certain local structure types are highly predictable from the local sequence alone if inter-site correlations are considered.

  13. 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.

  14. The RGSGR amino acid motif of the intercellular signalling protein, HetN, is required for patterning of heterocysts in Anabaena sp. strain PCC 7120.

    PubMed

    Higa, Kelly C; Rajagopalan, Ramya; Risser, Douglas D; Rivers, Orion S; Tom, Sasa K; Videau, Patrick; Callahan, Sean M

    2012-02-01

    Nitrogen-fixing heterocysts are arranged in a periodic pattern on filaments of the cyanobacterium Anabaena sp. strain PCC 7120 under conditions of limiting combined nitrogen. Patterning requires two inhibitors of heterocyst differentiation, PatS and HetN, which work at different stages of differentiation by laterally suppressing levels of an activator of differentiation, HetR, in cells adjacent to source cells. Here we show that the RGSGR sequence in the 287-amino-acid HetN protein, which is shared by PatS, is critical for patterning. Conservative substitutions in any of the five amino acids lowered the extent to which HetN inhibited differentiation when overproduced and altered the pattern of heterocysts in filaments with an otherwise wild-type genetic background. Conversely, substitution of amino acids comprising the putative catalytic triad of this predicted reductase had no effect on inhibition or patterning. Deletion of putative domains of HetN suggested that the RGSGR motif is the primary component of HetN required for both its inhibitory and patterning activity, and that localization to the cell envelope is not required for patterning of heterocysts. The intercellular signalling proteins PatS and HetN use the same amino acid motif to regulate different stages of heterocyst patterning.

  15. Results from the Intergovernmental Panel on Climatic Change Photochemical Model Intercomparison (PhotoComp)

    NASA Astrophysics Data System (ADS)

    Olson, Jennifer; Prather, Michael; Berntsen, Terje; Carmichael, Gregory; Chatfield, Robert; Connell, Peter; Derwent, Richard; Horowitz, Larry; Jin, Shengxin; Kanakidou, Maria; Kasibhatla, Prasad; Kotamarthi, Rao; Kuhn, Michael; Law, Kathy; Penner, Joyce; Perliski, Lori; Sillman, Sanford; Stordal, Frode; Thompson, Anne; Wild, Oliver

    1997-03-01

    Results from the Intergovernmental Panel on Climatic Change (IPCC) tropospheric photochemical model intercomparison (PhotoComp) are presented with a brief discussion of the factors that may contribute to differences in the modeled behaviors of HOx cycling and the accompanying O3 tendencies. PhotoComp was a tightly controlled model experiment in which the IPCC 1994 assessment sought to determine the consistency among models that are used to predict changes in tropospheric ozone, an important greenhouse gas. Calculated tropospheric photodissociation rates displayed significant differences, with a root-mean-square (rms) error of the reported model results ranging from about ±6-9% of the mean (for O3 and NO2) to up to ±15% (H2O2 and CH2O). Models using multistream methods in radiative transfer calculations showed distinctly higher rates for photodissociation of NO2 and CH2O compared to models using two-stream methods, and this difference accounted for up to one third of the rms error for these two rates. In general, some small but systematic differences between models were noted for the predicted chemical tendencies in cases that did not include reactions of nomnethane hydrocarbons (NMHC). These differences in modeled O3 tendencies in some cases could be identified, for example, as being due to differences in photodissociation rates, but in others they could not and must be ascribed to unidentified errors. O3 tendencies showed rms errors of about ±10% in the moist, surface level cases with NOx concentrations equal to a few tens of parts per trillion by volume. Most of these model to model differences can be traced to differences in the destruction of O3 due to reaction with HO2. Differences in HO2, in turn, are likely due to (1) inconsistent reaction rates used by the models for the conversion of HO2 to H2O2 and (2) differences in the model-calculated photolysis of H2O2 and CH2O. In the middle tropospheric "polluted" scenario with NOx concentrations larger than a

  16. Expression pattern of the apoptosis-stimulating protein of p53 family in p53+ human breast cancer cell lines

    PubMed Central

    2013-01-01

    Background The apoptosis-stimulating protein of p53 (ASPP) family comprises three members, namely, ASPP1, ASPP2, and iASPP. They regulate the promotive effect of p53 on apoptosis. Breast cancer (BC) remains as one of the leading causes of cancer or cancer-related mortality among women. However, the relationship between the ASPP family members and p53, as well as the dissemination and expression pattern of ASPP family members in p53+ BC, has not been elucidated. Our objectives are to detect the expression of ASPP family members in p53+ BC cell lines and determine its significance in tumor cell apoptosis. Methods The mRNA expression of ASPP family members in five p53+ BC cell lines was detected through RT-PCR and assayed using Quality-one software. The p53 protein expression was detected by immunohistochemistry. Afterward, the apoptosis indices of the five BC cell lines were detected by flow cytometry. Results The iASPP mRNA was expressed in Bcap-37, MCF-7, and HBL-100. Compared with the human peripheral blood mononuclear cells, significant differences were found in the ASPP1 mRNA in Bcap-37, MDA-MB-231, MCF-7, and HBL-100 (p < 0.05), except that in ZR-75-30 (p > 0.05). The ASPP2 mRNA was expressed in MDA-MB-231, Bcap-37, and MCF-7, but not in HBL-100 and ZR-75-30. The p53 protein was expressed in five breast cancer cell lines. ZR-75-30 and MDA-MB-231 apoptosis indices were higher than those of other breast cancer cell line and peripheral blood mononuclear cells (p < 0.01). Conclusions The mRNA expression of ASPP family members varied in the five p53+ BC cell lines. The results also verified that the family members have an important function in apoptosis, which was promoted by p53 protein. ZR-75-30 BC showed high apoptosis index, without expression of any ASPP family members, indicating that the pathway of apoptosis in this cell line may be related to other cell transduction pathway. MDA-MB-231, Bcap37, and MCF-7 cell lines all expressed ASPP1/2. However, the

  17. Two-dimensional gel electrophoresis pattern (pH 6-11) and identification of water-soluble barley seed and malt proteins by mass spectrometry.

    PubMed

    Bak-Jensen, Kristian Sass; Laugesen, Sabrina; Roepstorff, Peter; Svensson, Birte

    2004-03-01

    A protocol was established for two-dimensional gel electrophoresis (2-DE) of barley seed and malt proteins in the pH range of 6-11. Proteins extracted from flour in a low-salt buffer were focused after cup-loading onto IPG strips. Successful separation in the second dimension was achieved using gradient gels in a horizontal SDS-PAGE system. Silver staining of gels visualized around 380 (seed) and 500 (malt) spots. Thirty-seven different proteins from seeds were identified in 60 spots, among these 46 were visualized also in the malt 2-D pattern. Proteins were identified by peptide mass fingerprinting and by tandem MS sequencing after in-gel digestion by trypsin. In addition, the N-terminal sequence of 10 different proteins from 11 spots was determined after electroblotting to a polyvinylidene difluoride (PVDF) membrane. Five identified proteins (in 9 spots) are involved in glycolysis, 12 in defence against pathogens (21 spots), 4 in storage, folding, and synthesis of proteins, and in nitrogen metabolism (5 spots), 6 in carbohydrate metabolism (11 spots), and 4 in stress and detoxification (9 spots). Six proteins (7 spots) were not grouped in these categories, and 3 were not ascribed a function. The presented 2-D patterns and identifications will be used to describe proteome differences between cultivars and changes during malting. PMID:14997495

  18. 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.

  19. 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." PMID:25677525

  20. 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."

  1. 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

  2. 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

  3. Colonization pattern of the biocontrol strain Pseudomonas chlororaphis MA 342 on barley seeds visualized by using green fluorescent protein.

    PubMed

    Tombolini, R; van der Gaag, D J; Gerhardson, B; Jansson, J K

    1999-08-01

    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.

  4. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein

    PubMed Central

    Liu, Chuan-ju; Kong, Wei; Ilalov, Kiril; Yu, Shuang; Xu, Ke; Prazak, Lisa; Fajardo, Marc; Sehgal, Bantoo; Di Cesare, Paul E.

    2006-01-01

    Degradative fragments of cartilage oligomeric matrix protein (COMP) have been observed in arthritic patients. The physiological enzyme(s) that degrade COMP, however, remain unknown. We performed a yeast two-hybrid screen (Y2H) to search for proteins that associate with COMP to identify an interaction partner that might degrade it. One screen using the epidermal growth factor (EGF) domain of COMP as bait led to the discovery of ADAMTS-7. Rat ADAMTS-7 is composed of 1595 amino acids, and this protein exhibits higher expression in the musculoskeletal tissues. COMP binds directly to ADAMTS-7 in vitro and in native articular cartilage. ADAMTS-7 selectively interacts with the EGF repeat domain but not with the other three functional domains of COMP, whereas the four C-terminal TSP motifs of ADAMTS-7 are required and sufficient for association with COMP. The recombinant catalytic domain and intact ADAMTS-7 are capable of digesting COMP in vitro. The enzymatic activity of ADAMTS-7 requires the presence of Zn2+ and appropriate pH (7.5-9.5), and the concentration of ADAMTS-7 in cartilage and synovium of patients with rheumatoid arthritis is significantly increased as compared to normal cartilage and synovium. ADAMTS-7 is the first metalloproteinase found to bind directly to and degrade COMP.—Liu, C., Kong, W., Ilalov, K., Yu, S., Xu, K., Prazak, L., Fajardo, M., Sehgal, B., Di Cesare, P. E. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J. 20, E129 -E140 (2006) PMID:16585064

  5. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities.

  6. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

    PubMed Central

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  7. Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins.

    PubMed

    Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

    2016-01-01

    The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200

  8. Effect of different amino acid patterns on semen quality of boars fed with low-protein diets.

    PubMed

    Ren, Bo; Cheng, Xu; Wu, De; Xu, Sheng-Yu; Che, Lian-Qiang; Fang, Zheng-Feng; Lv, Gang; Dong, Hong-Jun; Lin, Yan

    2015-10-01

    The objective of the present study was to determine the effect of different amino acid patterns on the semen quality of boars fed with low-protein diets. Twenty-four boars were randomly divided into 4 groups (HP, LP1, LP2, and LP3). HP boars received 17% crude protein diet with a lysine:threonine:tryptophan:arginine (Lys:Thr:Trp:Arg) ratio of 100:50:20:104. Other boars received 13% CP and similar Lys levels (0.84%) with Lys:Thr:Trp:Arg ratios of 100:50:20:71, 100:76:38:71, and 100:76:38:120 for LP1, LP2, and LP3, respectively. These results showed sperm motility in the LP3 group was higher than in HP group during the 13-22 week period. The total sperm number, acrosome integrity ratio, and the effective total sperm number in LP3 and LP2 was higher than in other groups, and the abnormality ratio was lower than in other groups during the 13-18 week period. During 19-22 week period, in LP1 and LP3 groups, total sperm number and effective total sperm number were higher than in other groups, abnormality ratio was lower, and acrosome integrity ratio was higher than in the HP group. Nitric oxide synthase activity of seminal plasma and nitric oxide concentration of spermatozoa were significantly higher in the LP3 group than in other groups. Furthermore, mRNA expression of androgen receptor in testes was up-regulated in LP3. In conclusion, we suggest that the optimum ratio of Lys:Thr:Trp:Arg in a 13% CP diet for boars is 100:76:38:120, which results in similar or better reproductive performances than a 17% CP diet.

  9. Human salivary gland acinar cells spontaneously form three-dimensional structures and change the protein expression patterns.

    PubMed

    Chan, Yen-Hui; Huang, Tsung-Wei; Young, Tai-Horng; Lou, Pei-Jen

    2011-11-01

    Applying tissue engineering principles to design an auto-secretory device is a potential solution for patients suffering loss of salivary gland function. However, the largest challenge in implementing this solution is the primary culture of human salivary gland cells, because the cells are highly differentiated and difficult to expand in vitro. This situation leads to the lack of reports on the in vitro cell biology and physiology of human salivary gland cells. This study used a low-calcium culture system to selectively cultivate human parotid gland acinar (PGAC) cells from tissues with high purity in cell composition. This condition enables PGAC cells to continuously proliferate and retain the phenotypes of epithelial acinar cells to express secreting products (α-amylase) and function-related proteins (aquaporin-3, aquaporin-5, and ZO-1). Notably, when the cells reached confluence, three-dimensional (3D) cell aggregates were observed in crowded regions. These self-formed cell spheres were termed post-confluence structures (PCSs). Unexpectedly, despite being cultured in the same media, cells in PCSs exhibited higher expression levels and different expression patterns of function-related proteins compared to the two-dimensional (2D) cells. Translocation of aquoporin-3 from cytosolic to alongside the cell boundaries, and of ZO-1 molecules to the boundary of the PCSs were also observed. These observations suggest that when PGAC cells cultured on the 2D substrate would form PCSs without the help of 3D scaffolds and retain certain differentiation and polarity. This phenomenon implies that it is possible to introduce 2D substrates instead of 3D scaffolds into artificial salivary gland tissue engineering.

  10. 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

  11. Granulin-Epithelin Precursor (GEP) Binds Directly to ADAMTS-7 and ADAMTS-12 and Inhibits Their Degradation of Cartilage Oligomeric Matrix Protein

    PubMed Central

    Guo, Fengjin; Lai, Yongjie; Tian, Qingyun; Lin, Edward A.; Kong, Li; Liu, Chuan-ju

    2010-01-01

    Objective ADAMTS-7 and ADAMTS-12, two members of the ADAMTS (adisintegrin and metalloprotease with thrombospondin motifs) family, associate with and degrade COMP by binding to the EGF domain of COMP. Granulin-epithelin precursor (GEP) is a secreted growth factor that mediates tissue regeneration, tumorigenesis, and inflammation, and may play an important role in the pathogenesis of arthritis through mechanisms yet unknown. GEP also binds to the EGF domain of COMP. This study is to determine 1) whether there exists a protein interaction network between GEP, ADAMTS-7/-12 and COMP; 2) whether GEP interferes with the interactions between ADAMTS-7/-12 metalloproteinases and COMP substrate, including the cleavage of COMP; 3) whether GEP affects TNFα-mediated induction of ADAMTS-7/-12 expression and COMP degradation; and 4) whether GEP levels are altered during the progression of arthritis. Methods Yeast two-hybrid, in vitro GST pull down and co-immunoprecipitation assays were used to 1) examine the interactions between GEP, ADAMTS-7/-12 and COMP, and 2) map the binding sites required for the interactions between GEP and ADAMTS-7/-12; Immunoflouresence cell staining was performed to visualize the sub-cellular localization of GEP and ADAMTS-7/-12; an in vitro digestion assay was employed to determine whether GEP inhibits ADAMTS-7/-12-mediated digestion of COMP; The role of GEP in inhibiting TNFα-induced ADAMTS-7/-12 expression and COMP degradation in cartilage explants was also analyzed. Results GEP binds directly to ADAMTS-7 and ADAMTS-12 in vitro and in chondrocytes, and the four C-terminal TSP motifs of ADAMTS-7/-12 and each granulin unit of GEP mediate their interactions. Additionally, GEP co-localizes with ADAMTS-7 and ADAMTS-12 on the cell surface of chondrocytes. More importantly, GEP inhibits COMP degradation by ADAMTS-7/-12 in a dose-dependent manner through the following two mechanisms: a) competitive inhibition through direct protein-protein interactions with

  12. [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. PMID:26137796

  13. Splitting placodes: effects of bone morphogenetic protein and Activin on the patterning and identity of mouse incisors.

    PubMed

    Munne, Pauliina M; Felszeghy, Szabolcs; Jussila, Maria; Suomalainen, Marika; Thesleff, Irma; Jernvall, Jukka

    2010-01-01

    The single large rodent incisor in each jaw quadrant is evolutionarily derived from a mammalian ancestor with many small incisors. The embryonic placode giving rise to the mouse incisor is considerably larger than the molar placode, and the question remains whether this large incisor placode is a developmental requisite to make a thick incisor. Here we used in vitro culture system to experiment with the molecular mechanism regulating tooth placode development and how mice have thick incisors. We found that large placodes are prone to disintegration and formation of two to three small incisor placodes. The balance between one large or multiple small placodes was altered through the regulation of bone morphogenetic protein (BMP) and Activin signaling. Exogenous Noggin, which inhibits BMP signaling, or exogenous Activin cause the development of two to three incisors. These incisors were more slender than normal incisors. Additionally, two inhibitor molecules, Sostdc1 and Follistatin, which regulate the effects of BMPs and Activin and have opposite expression patterns, are likely to be involved in the incisor placode regulation in vivo. Furthermore, inhibition of BMPs by recombinant Noggin has been previously suggested to cause a change in the tooth identity from the incisor to the molar. This evidence has been used to support a homeobox code in determining tooth identity. Our work provides an alternative interpretation, where the inhibition of BMP signaling can lead to splitting of the large incisor placode and the formation of partly separate incisors, thereby acquiring molar-like morphology without a change in tooth identity.

  14. Hydrolysis and Sulfation Pattern Effects on Release of Bioactive Bone Morphogenetic Protein-2 from Heparin-Based Microparticles

    PubMed Central

    Tellier, Liane E.; Miller, Tobias; McDevitt, Todd C.; Temenoff, Johnna S.

    2015-01-01

    Glycosaminoglycans (GAGs) such as heparin are promising materials for growth factor delivery due to their ability to efficiently bind positively charged growth factors including bone morphogenetic protein-2 (BMP-2) through their negatively charged sulfate groups. Therefore, the goal of this study was to examine BMP-2 release from heparin-based microparticles (MPs) after first, incorporating a hydrolytically degradable crosslinker and varying heparin content within MPs to alter MP degradation and second, altering the sulfation pattern of heparin within MPs to vary BMP-2 binding and release. Using varied MP formulations, it was found that the time course of MP degradation for 1 wt% heparin MPs was ~4 days slower than 10 wt% heparin MPs, indicating that MP degradation was dependent on heparin content. After incubating 100 ng BMP-2 with 0.1 mg MPs, most MP formulations loaded BMP-2 with ~50% efficiency and significantly more BMP-2 release (60% of loaded BMP-2) was observed from more sulfated heparin MPs (MPs with ~100% and 80% of native sulfation). Similarly, BMP-2 bioactivity in more sulfated heparin MP groups was at least four-fold higher than soluble BMP-2 and less sulfated heparin MP groups, as determined by an established C2C12 cell alkaline phosphatase (ALP) assay. Ultimately, the two most sulfated 10 wt% heparin MP formulations were able to efficiently load and release BMP-2 while enhancing BMP-2 bioactivity, making them promising candidates for future growth factor delivery applications.

  15. [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.

  16. Use of amino acid profiles of preterm and term human milks in evaluating scoring patterns for routine protein quality assessment of infant formulas.

    PubMed

    Sarwar, G; Darling, P; Ujiie, M; Botting, H G; Pencharz, P B

    1996-01-01

    Reports on the amino acid composition of human milk vary considerably with respect to concentrations of sulfur amino acids. Often, analyses forego tryptophan determination. A complete analysis of protein and amino acid concentrations was performed on human milk samples (5-10 days postpartum) collected from mothers of preterm (gestations of 25-32 weeks) and term (gestations of > 36 weeks) infants. Careful attention was given to quantitate amino acids such as cysteine and tryptophan, which are vulnerable to acidic hydrolysis conditions. Differences in concentrations of total amino acids (expressed on protein basis) between preterm and term milks were small, despite the higher true protein content of preterm milk versus term milk (19.20 versus 12.60 g/L). The methionine + cyst(e)ine contents of term and preterm milks (3.72-3.84 g/100 g protein) were comparable with those reported in 1991 by the Food and Agricultural Organization/World Health Organization (FAO/WHO) for mature human milk (4.20 g/100 g protein) but higher than those reported in 1991 by the European Commission (2.9 g/100 g protein). The amino acid pattern of human milk obtained in this study confirms that the 1991 FAO/WHO amino acid scoring pattern for predicting protein quality of infant formulas is representative of the amino acid quality of both preterm and term human milks.

  17. Expression patterns of cyclin D1 and related proteins regulating G1-S phase transition in uveal melanoma and retinoblastoma

    PubMed Central

    Coupland, S; Bechrakis, N; Schuler, A; Anagnostopoulos, I; Hummel, M; Bornfeld, N; Stein, H

    1998-01-01

    , differ also in their immunohistochemical pattern for markers of the G1-S phase transition of the cell cycle. The results of the present study support the concept of (a) an autoregulatory loop between pRB and cyclin D1 in tumours with a functional pRB and the disruption of this loop in the presence of pRB mutation, as well as (b) a checkpoint mechanism in late G1, whose regulation via loss of p16 or pRB, or overexpression of cyclin D1 constitutes a common pathway to malignancy. Further, the results raise the possibility of cyclin D1 overexpression having a role in the pathogenesis of uveal melanoma.

 Keywords: cyclin D1; retinoblastoma protein; antigens; antibodies; bipolar cells; uveal melanoma; retinoblastoma PMID:9828785

  18. Determination of the N-glycosylation patterns of seed proteins: applications to determine the authenticity and substantial equivalence of genetically modified (GM) crops.

    PubMed

    Marsh, Justin T; Tryfona, Theodora; Powers, Stephen J; Stephens, Elaine; Dupree, Paul; Shewry, Peter R; Lovegrove, Alison

    2011-08-24

    Methods have been developed to determine the N-glycosylation pattern of proteins at the single-seed level in two different biological systems. These were the well-characterized and widely consumed storage protein phaseolin from several species of Phaseolus (bean) and the α-amylase inhibitor from the same Phaseolus species expressed transgenically in pea. The N-glycosylation pattern of the α-amylase inhibitor expressed transgenically in pea was different from that of the inhibitor present in common bean (P. vulgaris), the species of origin of the gene. However, multivariate analysis showed that the differences in N-glycan patterns between the α-amylase inhibitors from common bean and pea were less than those between the inhibitors from common bean and two related bean species, lima bean (Phaseolus lunatus) and tepary bean (Phaseolus acutifolius).

  19. Biphasic Temporal and Spatial Induction Patterns of Defense-Related mRNAs and Proteins in Fungus-Infected Parsley Leaves.

    PubMed Central

    Reinold, S.; Hahlbrock, K.

    1996-01-01

    Previous experiments using in situ RNA hybridization have shown that the mRNAs encoding phenylalanine ammonia-lyase, 4-coumarate:coenzyme A ligase, and pathogenesis-related protein 1 accumulated transiently around fungal infection sites in parsley (Petroselinum crispum) leaf buds. These studies have now been extended by (a) analyzing different stages of the infection process and (b) monitoring the timing of appearance and the spatial distribution of the proteins as well as the corresponding mRNAs. An early and short period of mRNA induction throughout a large portion of the infected leaf was followed by a longer period, during which the mRNA levels remained high in a more localized area around the site of fungal penetration with sharp borders toward the surrounding tissue. This biphasic pattern of mRNA accumulation was followed after some delay by the same pattern of protein accumulation. PMID:12226380

  20. 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. PMID:27101757

  1. A Novel Gli3 Enhancer Controls the Gli3 Spatiotemporal Expression Pattern through a TALE Homeodomain Protein Binding Site ▿‡

    PubMed Central

    Coy, Sarah; Caamaño, Jorge H.; Carvajal, Jaime; Cleary, Michael L.; Borycki, Anne-Gaëlle

    2011-01-01

    The zinc finger transcription factor Gli3 is an essential mediator of hedgehog signaling. Gli3 has a dynamic expression pattern during embryonic development. In the neural tube, Gli3 transcripts are patterned along the anteroposterior and dorsoventral axes such that the initial broad expression in the posterior neural tube becomes dorsally restricted as neurogenesis takes place. Little is known about the molecular mechanisms that regulate this dynamic expression. Here, we report on a phylogenetic analysis of the Gli3 locus that uncovered a novel regulatory element, HCNE1. HCNE1 contains a compound Pbx/Meis binding site that binds Pbx and Meis/Prep proteins in vitro and in vivo. We show that HCNE1 recapitulates Gli3 expression in the developing neural tube and that mutations in the Pbx/Meis binding site affect the spatiotemporal control of HCNE1 transcriptional activity. Ectopic expression or loss of function of Pbx and Meis/Prep proteins in the chick and mouse embryo results in aberrant expression of endogenous Gli3 transcripts. We propose a novel role for TALE proteins in establishing the correct spatiotemporal expression pattern of Gli3 in the vertebrate spinal cord, thus implicating TALE transcription factors in early embryonic patterning events controlled by Sonic hedgehog signaling. PMID:21262763

  2. 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.

  3. Synthesis and secretion of plasma proteins by embryonic chick hepatocytes: changing patterns during the first three days of culture

    PubMed Central

    1978-01-01

    A simple model system is described for studying synthesis of plasma proteins. The system is based on chick embryo hepatocytes in primary monolayer culture which synthesize a broad spectrum of plasma proteins and secrete them into the culture medium. The secreted proteins are stable and consist almost exclusively of plasma proteins. The cultured cells are nonproliferating hepatic parenchymal cells whose cell mass remains constant in culture. By a modification of Laurell's rocket immunoelectrophoresis, the secreted plasma proteins can be detected in nanogram amounts in 3 microliter of unconcentrated culture medium. Kinetics of secretion are obtained by sequential assay of proteins accumulating in the medium. In this system it is demonstrated that: (a) intracellular plasma protein levels are equivalent to less than 5% of the daily secretion; (b) synthesis and secretion are continuous; and (c) the overall half-time for plasma protein movement along the secretory pathway is less than 10 min. From these results, it follows that the rate at which the plasma proteins are secreted gives a valid estimate of their rate of synthesis. This feature of the culture and the sensitivity of the assay allow routine measurements of plasma protein synthesis without disruption of the cells and without the use of radioisotopes. It is shown, furthermore, that the overall rate of plasma protein synthesis in cultured hepatocytes is constant over a 3- day period and is similar to that of the intact liver. 3,000,000 cells, containing 1 mg cell protein, synthesize 0.2 mg of plasma proteins daily, amounting to one-fifth of hepatocellular protein synthesis. Under the conditions used, albumin synthesis steadily decreases with culture time whereas the synthesis of many other plasma proteins increases. The observed phenotypic changes and reorganization of plasma protein synthesis illustrate how the system may be exploited for studying the regulatory processes governing plasma protein synthesis. PMID

  4. Analysis of two-dimensional electrophoretic patterns of proteins obtained from the sera of normal and tumor-bearing nude rats.

    PubMed

    Yoshida, M; Itoh, M; Imai, T; Tanimoto, Y; Sakurabayashi, I; Furuya, S

    1991-01-01

    To classify two-dimensional electrophoresis (2-DE) patterns of normal and abnormal samples, a new parameter, named shift value, is introduced. The shift value is defined by the product of three indices; differences in density, differences in area, and the Euclid distance between peaks of matched glycoprotein spots in the 2-DE patterns. Shift values obtained from the differences between the 2-DE patterns of tumor-bearing and normal sera (control) were always found to be larger than those obtained from two control patterns. The shift value obtained from glycoprotein spots could be more effectively used to distinguish normal and abnormal patterns than that obtained from simple proteins. In our earlier attempts to compare serum proteins of normal and tumor patients, we could not discriminate differences caused by genetic background. In order to circumvent this difficulty, we employed an inbred strain of nude rats into which various types of human cancer had been implanted. Reliability of the analysis is enhanced by using a polyacrylamide gel backed with a silanized glass support and an automatic 2-DE apparatus.

  5. 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.

  6. 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.

  7. Gene expression patterns in response to pathogen challenge and interaction with hemolin suggest that the Yippee protein of Antheraea pernyi is involved in the innate immune response.

    PubMed

    Sun, Yu; Dai, Lishang; Sun, Yuxuan; Wang, Lei; Qian, Cen; Wei, Guoqing; Zhu, Bao-Jian; Liu, Chao-Liang

    2016-07-01

    Yippee was first identified as a protein that physically interacts with the Hemolin protein of Hyalophora cecropia. In this study, we identified a gene with a 366bp open reading frame (ORF) that encodes a 121 amino acid protein containing a conserved Yippee domain. We named this gene Ap-Yippee (Yippee gene from Antheraea pernyi), and investigated the role of the protein in the host immune response. A recombinant Ap-Yippee protein was expressed in Escherichia coli cells, and polyclonal antibodies were produced against the recombinant protein. Real-time PCR and a Western blot analysis revealed that Ap-Yippee is expressed in the hemocytes, Malpighian tubules, midgut, silk gland, epidermis, and fat bodies of A. pernyi, with the highest expression level observed in Malpighian tubules. The fifth instar larvae of A. pernyi were challenged by injecting them with nucleopolyhedrovirus (AP-NPV), the Gram-negative bacterium E. coli, the Gram-positive bacterium Micrococcus luteus, or the entomopathogenic fungus, Beauveria bassiana. These challenges with diverse pathogens resulted in differential expression patterns of the protein. A knockdown of the Ap-Yippee gene by small interfering RNA (siRNA) transfection had a significant influence on the expression of the hemolin in the pupae which was confirmed by qRT-PCR and Western blot. Furthermore, a possible protein-protein interaction between Ap-Yippee and Hemolin was explored by Far-Western blotting. Therefore, our data suggest that the Ap-Yippee protein is involved in a pathway that regulates the immune response of insects. PMID:27261060

  8. 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.

  9. Changes in the acinar activity patterns of phosphoenolpyruvate carboxykinase in livers of male and female rats upon feeding a high protein and a high fat diet.

    PubMed

    Wimmer, M; Luttringer, C; Colombi, M

    1990-01-01

    Phosphoenolpyruvate carboxykinase (PEPCK) activity was investigated in relation to its localization within the liver acinus of male and female rats after feeding either a high protein diet (77%) or a high fat diet (52%). Both diets led to sex-dependent changes in activity and acinar distribution patterns. In male rats high protein diet provoked a shift in the acinar activity pattern towards the perivenous parts of the acinus without increase in average activity. Yet in the livers of females the activity was increased in all parts of the acinus, but to a greater extent in the perivenous parts of the acinus. Feeding a high fat diet increased PEPCK activity in males and to an even greater extent in females. In both sexes the increase was greater in the perivenous zone when compared to the changes within the periportal zone. The results are discussed in relation to changes in the concentrations of glucoregulatory hormones.

  10. 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. PMID:27004646

  11. Effect of caffeine on the expression pattern of water-soluble proteins in rice (Oryza sativa) seedlings.

    PubMed

    Deng, Wei-Wei; Sasamoto, Hamako; Ashihara, Hiroshi

    2015-05-01

    It has been suggested that caffeine acts as an allelochemical which influences the germination and growth of plants. The effect of caffeine on the expression profiles of proteins was investigated in shoot-root axes of rice (Oryza sativa) seedlings. Two-dimensional difference gel electrophoresis combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight/Time of Flight Mass Spectrometry was employed for the separation and identification of proteins. The results indicated that amounts of 51 protein spots were reduced and 14 were increased by treatment with 1 mM caffeine. Twelve rice seedling proteins were identified. Down-regulated proteins were β-tubulin, sucrose synthase, glyceraldehyde-3-phosphate dehydrogenase, reversibly glycosylated polypeptide/α-1,4-glucan protein synthase and cytoplasmic malate dehydrogenase. In contrast, up-regulated proteins were alanyl-aminopeptidase, acetyl-CoA carboxylase, adenine phosphoribosyltransferase, NAD-malate dehydrogenase, ornithine carbamoyltransferase, glucose-6-phosphate isomerase and nuclear RNA binding protein. Possible alternation of metabolism caused by caffeine is discussed with the protein expression data.

  12. 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)

  13. Protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteins are the major structural and functional components of all cells in the body. They are macromolecules that comprise 1 or more chains of amino acids that vary in their sequence and length and are folded into specific 3-dimensional structures. The sizes and conformations of proteins, therefor...

  14. Detection of restricted predominant epitopes of Theiler's murine encephalomyelitis virus capsid proteins expressed in the lambda gt11 system: differential patterns of antibody reactivity among different mouse strains.

    PubMed

    Crane, M A; Jue, C; Mitchell, M; Lipton, H; Kim, B S

    1990-05-01

    Intracerebral injection of mice with Theiler's murine encephalomyelitis virus results in chronic demyelination in susceptible strains, and serves as a model system for the study of multiple sclerosis. The role of individual epitopes in the disease process remains to be elucidated. Random fragments of DNA from the viral capsid protein genome covering the coding regions from VP1, VP2, and VP3 have been expressed in the lambda gt11 vector system. Fusion proteins from the clones were expressed and probed with antibodies from both resistant and susceptible strains of mice. Each strain displays a distinctive pattern with certain fusion proteins recognized by all of the strains and others recognized uniquely by either the susceptible or the resistant strains.

  15. 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.

  16. Stimulation of whole body protein synthesis by insulin in neonates is dependent on the pattern of amino acids available

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insulin stimulates muscle protein synthesis in neonatal pigs. To determine insulin's effects on whole body protein turnover, (13)C-leucine was infused for 4 hr during hyperinsulinemic (0, 30, 100, 1000 ng/(kg(0.66)/min))-euglycemic-euaminoacidemic clamps in fasted 7-d-old pigs (n=5/dose). Trophami...

  17. 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

  18. The Effect of Water, Sugars, and Proteins on the Pattern of Ice Nucleation and Propagation in Acclimated and Nonacclimated Canola Leaves1

    PubMed Central

    Gusta, L.V.; Wisniewski, M.; Nesbitt, N.T.; Gusta, M.L.

    2004-01-01

    Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2°C h−1) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone. PMID:15247390

  19. The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves.

    PubMed

    Gusta, L V; Wisniewski, M; Nesbitt, N T; Gusta, M L

    2004-07-01

    Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2 degrees C h(-1)) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone.

  20. Basic nuclear protein pattern and chromatin condensation in the male germ cells of a tropical abalone, Haliotis asinina.

    PubMed

    Suphamungmee, Worawit; Apisawetakan, Somjai; Weerachatyanukul, Wattana; Wanichanon, Chaitip; Sretarugsa, Prapee; Poomtong, Tanes; Sobhon, Prasert

    2005-02-01

    The basic nuclear proteins (BNPs) in spermatozoa of a tropical abalone, Haliotis asinina, were composed of a majority of protamine-like (PL) protein and a small amount of histones H1 and H4. Abalone H1 and PL proteins exhibited strong immunological cross reactivities among themselves as well as with chick H5 and calf thymus H1. Thus, all these proteins may belong to the same family. Immunolocalization by indirect immunofluorescence and immunoelectron microscopy indicated that H1 and H4 were present in all steps of the male germ cells, however, with decreasing amount in late stage cells, particularly spermatids and spermatozoa. On the other hand, PL was present in middle step cells (secondary spermatocytes) with increasing amount in spermatids and spermatozoa when the chromatin became tightly packed. Thus, PL may be involved in the condensation of chromatin in the spermatozoa of this species.

  1. The effect of albumin fusion patterns on the production and bioactivity of the somatostatin-14 fusion protein in Pichia pastoris.

    PubMed

    Ding, Yuedi; Fan, Jun; Li, Wenxin; Yang, Runlin; Peng, Ying; Deng, Lili; Wu, Yu; Fu, Qiang

    2013-08-01

    Somatostatin is a natural inhibitor of growth hormone, and its analogues are clinically used for the therapy of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndrome. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins, and Pichia pastoris was used as an expression system. Three fusion proteins, (somatostatin (SS)14)2-human serum albumin (HSA), (SS14)3-HSA, and HSA-(SS14)3, were constructed with different fusion copies of somatostatin-14 and fusion orientations. The expression level of (SS14)3-HSA and HSA-(SS14)3 was much lower than (SS14)2-HSA due to the additional fusion of the somatostatin-14 molecule. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard of somatostatin-14, all three fusion proteins were able to inhibit growth hormone secretion in the blood, with (SS14)2-HSA being the most effective one. On the whole, (SS14)2-HSA was the most effective protein in both production level and bioactivity, and increasing the number of small protein copies fused to HSA may not be a suitable method to improve the protein bioactivity. PMID:23712794

  2. 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. PMID:24259685

  3. 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.

  4. 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

  5. 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.

  6. Discovery of EST-SSRs in lung cancer: tagged ESTs with SSRs lead to differential amino acid and protein expression patterns in cancerous tissues.

    PubMed

    Bakhtiarizadeh, Mohammad Reza; Ebrahimi, Mansour; Ebrahimie, Esmaeil

    2011-01-01

    Tandem repeats are found in both coding and non-coding sequences of higher organisms. These sequences can be used in cancer genetics and diagnosis to unravel the genetic basis of tumor formation and progression. In this study, a possible relationship between SSR distributions and lung cancer was studied by comparative analysis of EST-SSRs in normal and lung cancerous tissues. While the EST-SSR distribution was similar between tumorous tissues, this distribution was different between normal and tumorous tissues. Trinucleotides tandem repeats were highly different; the number of trinucleotides in ESTs of lung cancer was 3 times higher than normal tissue. Significant negative correlation between normal and cancerous tissue showed that cancerous tissue generates different types of trinucleotides. GGC and CGC were the more frequent expressed trinucleotides in cancerous tissue, but these SSRs were not expressed in normal tissue. Similar to the EST level, the expression pattern of EST-SSRs-derived amino acids was significantly different between normal and cancerous tissues. Arg, Pro, Ser, Gly, and Lys were the most abundant amino acids in cancerous tissues, and Leu, Cys, Phe, and His were significantly more abundant in normal tissues than in cancerous tissues. Next, the putative functions of triplet SSR-containing genes were analyzed. In cancerous tissue, EST-SSRs produce different types of proteins. Chromodomain helicase DNA binding proteins were one of the major protein products of EST-SSRs in the cancerous library, while these proteins were not produced from EST-SSRs in normal tissue. For the first time, the findings of this study confirmed that EST-SSRs in normal lung tissues are different than in unhealthy tissues, and tagged ESTs with SSRs cause remarkable differences in amino acid and protein expression patterns in cancerous tissue. We suggest that EST-SSRs and EST-SSRs differentially expressed in cancerous tissue may be suitable candidate markers for lung cancer

  7. Relating material surface heterogeneity to protein adsorption: the effect of annealing of micro-contact-printed OTS patterns

    PubMed Central

    HODGKINSON, GERALD; HLADY, VLADIMIR

    2009-01-01

    We have investigated the influence of micrometer- and sub-micrometer-scale surface heterogeneities in patterned octadecyltrichlorosilane (OTS) films on human serum albumin (HSA) adsorption and its spatial distribution. 5-μm-wide OTS patterns were created on glass substrates by micro-contact printing and in some instances subsequent annealing was used to alter OTS molecule distribution within the patterns. Scanning force microscopy (SFM), advancing water contact angles and water vapor condensation figures were used to characterize the OTS films and to assess the nature of the heterogeneities within the various surface areas. High-resolution fluorescence microscopy was used to record images of fluorescently labeled albumin on OTS patterned films and fluorescence intensity was quantified and converted into the adsorbed amount. Adsorbed albumin was also characterized through SFM measurements. Combined SFM topography and lateral force microscopy (LFM) imaging revealed that micro-contact printing of OTS onto glass both replicated the stamp pattern and created small islands within the non-stamped regions between the patterns. The OTS coverage within stamped regions was not fully continuous but improved with subsequent annealing. Annealing also resulted in OTS island growth within the non-stamped regions and decreased average wettability on both the stamped and non-stamped areas. The extent of albumin adsorption was not proportional to OTS coverage, but correlated with the sub-μm distribution of OTS chains. We inferred that the surface distribution of ligands such as OTS on a sub-μm length scale determines the nature of albumin adsorption and its kinetics. PMID:19693285

  8. 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

  9. 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.

  10. Proteins

    NASA Astrophysics Data System (ADS)

    Regnier, Fred E.; Gooding, Karen M.

    Because of the complexity of cellular material and body fluids, it is seldom possible to analyze a natural product directly. Qualitative and quantitative analyses must often be preceded by some purification step that separates the molecular species being examined from interfering materials. In the case of proteins, column liquid chromatography has been used extensively for these fractionations. With the advent of gel permeation, cation exchange, anion exchange, hydrophobic, and affinity chromatography, it became possible to resolve proteins through their fundamental properties of size, charge, hydrophobicity, and biological affinity. The chromatographic separations used in the early isolation and characterization of many proteins later became analytical tools in their routine analysis. Unfortunately, these inherently simple and versatile column chromatographic techniques introduced in the 50s and 60s have a severe limitation in routine analysis-separation time. It is common to encounter 1-24 h separation times with the classical gel-type supports.

  11. 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

  12. Deposition pattern and subcellular distribution of disease-associated prion protein in cerebellar organotypic slice cultures infected with scrapie

    PubMed Central

    Wolf, Hanna; Hossinger, André; Fehlinger, Andrea; Büttner, Sven; Sim, Valerie; McKenzie, Debbie; Vorberg, Ina M.

    2015-01-01

    Organotypic cerebellar slices represent a suitable model for characterizing and manipulating prion replication in complex cell environments. Organotypic slices recapitulate prion pathology and are amenable to drug testing in the absence of a blood-brain-barrier. So far, the cellular and subcellular distribution of disease-specific prion protein in organotypic slices is unclear. Here we report the simultaneous detection of disease-specific prion protein and central nervous system markers in wild-type mouse cerebellar slices infected with mouse-adapted prion strain 22L. The disease-specific prion protein distribution profile in slices closely resembles that in vivo, demonstrating granular spot like deposition predominately in the molecular and Purkinje cell layers. Double immunostaining identified abnormal prion protein in the neuropil and associated with neurons, astrocytes and microglia, but absence in Purkinje cells. The established protocol for the simultaneous immunohistochemical detection of disease-specific prion protein and cellular markers enables detailed analysis of prion replication and drug efficacy in an ex vivo model of the central nervous system. PMID:26581229

  13. 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. PMID:27125786

  14. Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns

    PubMed Central

    1990-01-01

    A major cytoskeletal polypeptide (Mr approximately 46,000; protein IT) of human intestinal epithelium was characterized by biochemical and immunological methods. The polypeptide, which was identified as a specific and genuine mRNA product by translation in vitro, reacted, in immunoblotting after SDS-PAGE, only with one of numerous cytokeratin (CK) antisera tested but with none of many monoclonal CK antibodies. In vitro, it formed heterotypic complexes with the type II CK 8, as shown by blot binding assays and gel electrophoresis in 4 M urea, and these complexes assembled into intermediate filaments (IFs) under appropriate conditions. A chymotrypsin-resistant Mr approximately 38,000 core fragment of protein IT could be obtained from cytoskeletal IFs, indicating its inclusion in a coiled coil. Antibodies raised against protein IT decorated typical CK fibril arrays in normal and transformed intestinal cells. Four proteolytic peptide fragments obtained from purified polypeptide IT exhibited significant amino acid sequence homology with corresponding regions of coils I and II of the rod domain of several other type I CKs. Immunocytochemically, the protein was specifically detected as a prominent component of intestinal and gastric foveolar epithelium, urothelial umbrella cells, and Merkel cells of epidermis. Sparse positive epithelial cells were noted in the thymus, bronchus, gall bladder, and prostate gland. The expression of protein IT was generally maintained in primary and metastatic colorectal carcinomas as well as in cell cultures derived therefrom. A corresponding protein was also found in several other mammalian species. We conclude that polypeptide IT is an integral IF component which is related, though somewhat distantly, to type I CKs, and, therefore, we propose to add it to the human CK catalogue as CK 20. PMID:1696264

  15. The Adenovirus L4-33K Protein Regulates both Late Gene Expression Patterns and Viral DNA Packaging

    PubMed Central

    Wu, Kai; Guimet, Diana

    2013-01-01

    The adenovirus (Ad) L4-33K protein has been linked to disparate functions during infection. L4-33K is a virus-encoded alternative RNA splicing factor which activates splicing of viral late gene transcripts that contain weak 3′ splice sites. Additionally, L4-33K has been indicated to play a role in adenovirus assembly. We generated and characterized an Ad5 L4-33K mutant virus to further explore its function(s) during infection. Infectivity, viral genome replication, and most viral gene expression of the L4-33K mutant virus are comparable to those of the wild-type virus, except for a prominent decrease in the levels of the late proteins IIIa and pVI. The L4-33K mutant virus produces only empty capsids, indicating a defect in viral DNA packaging. We demonstrate that L4-33K does not preferentially bind to viral packaging sequences in vivo, and mutation of L4-33K does not interfere with the binding of the known viral packaging proteins IVa2, L4-22K, L1-52/55K, and IIIa to the packaging sequences in vivo. Collectively, these results demonstrate that the phenotype of an Ad5 L4-33K mutant virus is complex. The L4-33K protein regulates the accumulation of selective Ad late gene mRNAs and is involved in the proper transition of gene expression during the late phase of infection. The L4-33K protein also plays a role in adenovirus morphogenesis by promoting the packaging of the viral genome into the empty capsid. These results demonstrate the multifunctional nature of the L4-33K protein and its involvement in several different and critical aspects of viral infection. PMID:23552425

  16. Automation in clinical microbiology: a new approach to identifying micro-organisms by automated pattern matching of proteins labelled with 35S-methionine.

    PubMed Central

    Tabaqchali, S; Silman, R; Holland, D

    1987-01-01

    A new rapid automated method for the identification and classification of microorganisms is described. It is based on the incorporation of 35S-methionine into cellular proteins and subsequent separation of the radiolabelled proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein patterns produced were species specific and reproducible, permitting discrimination between the species. A large number of Gram negative and Gram positive aerobic and anaerobic organisms were successfully tested. Furthermore, there were sufficient differences within species between the protein profiles to permit subdivision of the species. New typing schemes for Clostridium difficile, coagulase negative staphylococci, and Staphylococcus aureus, including the methicillin resistant strains, could thus be introduced; this has provided the basis for useful epidemiological studies. To standardise and automate the procedure an automated electrophoresis system and a two dimensional scanner were developed to scan the dried gels directly. The scanner is operated by a computer which also stores and analyses the scan data. Specific histograms are produced for each bacterial species. Pattern recognition software is used to construct databases and to compare data obtained from different gels: in this way duplicate "unknowns" can be identified. Specific small areas showing differences between various histograms can also be isolated and expanded to maximise the differences, thus providing differentiation between closely related bacterial species and the identification of differences within the species to provide new typing schemes. This system should be widely applied in clinical microbiology laboratories in the near future. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 PMID:3312300

  17. 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.

  18. Arabidopsis WD REPEAT DOMAIN55 Interacts with DNA DAMAGED BINDING PROTEIN1 and Is Required for Apical Patterning in the Embryo[C][W

    PubMed Central

    Bjerkan, Katrine N.; Jung-Roméo, Sabrina; Jürgens, Gerd; Genschik, Pascal; Grini, Paul E.

    2012-01-01

    CUL4-RING ubiquitin E3 ligases (CRL4s) were recently shown to exert their specificity through the binding of various substrate receptors, which bind the CUL4 interactor DNA DAMAGED BINDING PROTEIN1 (DDB1) through a WDxR motif. In a segregation-based mutagenesis screen, we identified a WDxR motif–containing protein (WDR55) required for male and female gametogenesis and seed development. We demonstrate that WDR55 physically interacts with Arabidopsis thaliana DDB1A in planta, suggesting that WDR55 may be a novel substrate recruiter of CRL4 complexes. Examination of mutants revealed a failure in the fusion of the polar cells in embryo sac development, in addition to embryo and endosperm developmental arrest at various stages ranging from the zygote stage to the globular stage. wdr55-2 embryos suggest a defect in the transition to bilateral symmetry in the apical embryo domain, further supported by aberrant apical embryo localization of DORNROESCHEN, a direct target of the auxin response factor protein MONOPTEROS. Moreover, the auxin response pattern, as determined using the synthetic auxin-responsive reporter ProDR5:GREEN FLUORESCENT PROTEIN, was shifted in the basal embryo and suspensor but does not support a strong direct link to auxin response. Interestingly, the observed embryo and endosperm phenotype is reminiscent of CUL4 or DDB1A/B loss of function and thus may support a regulatory role of a putative CRL4WDR55 E3 ligase complex. PMID:22447688

  19. 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

  20. The Drosophila CPEB Protein Orb2 Has a Novel Expression Pattern and Is Important for Asymmetric Cell Division and Nervous System Function

    PubMed Central

    Hafer, Nathaniel; Xu, Shuwa; Bhat, Krishna Moorthi; Schedl, Paul

    2011-01-01

    Cytoplasmic polyadenylation element binding (CPEB) proteins bind mRNAs to regulate their localization and translation. While the first CPEBs discovered were germline specific, subsequent studies indicate that CPEBs also function in many somatic tissues including the nervous system. Drosophila has two CPEB family members. One of these, orb, plays a key role in the establishment of polarity axes in the developing egg and early embryo, but has no known somatic functions or expression outside of the germline. Here we characterize the other Drosophila CPEB, orb2. Unlike orb, orb2 mRNA and protein are found throughout development in many different somatic tissues. While orb2 mRNA and protein of maternal origin are distributed uniformly in early embryos, this pattern changes as development proceeds and by midembryogenesis the highest levels are found in the CNS and PNS. In the embryonic CNS, Orb2 appears to be concentrated in cell bodies and mostly absent from the longitudinal and commissural axon tracts. In contrast, in the adult brain, the protein is seen in axonal and dendritic terminals. Lethal effects are observed for both RNAi knockdowns and orb2 mutant alleles while surviving adults display locomotion and behavioral defects. We also show that orb2 funtions in asymmetric division of stem cells and precursor cells during the development of the embryonic nervous system and mesoderm. PMID:21900268

  1. A Global View of Velocity Fluctuations in the Corona below 1.3 R ⊙ with CoMP

    NASA Astrophysics Data System (ADS)

    Morton, R. J.; Tomczyk, S.; Pinto, R. F.

    2016-09-01

    The Coronal Multi-channel Polarimeter (CoMP) has previously demonstrated the presence of Doppler velocity fluctuations in the solar corona. The observed fluctuations are thought to be transverse waves, i.e., highly incompressible motions whose restoring force is dominated by the magnetic tension, some of which demonstrate clear periodicity. We aim to exploit CoMP’s ability to provide high cadence observations of the off-limb corona to investigate the properties of velocity fluctuations in a range of coronal features, providing insight into how (whether) the properties of the waves are influenced by the varying magnetic topology in active regions, quiet Sun and open field regions. An analysis of Doppler velocity time-series of the solar corona from the 10747 Å Iron xiii line is performed, determining the velocity power spectrum and using it as a tool to probe wave behavior. Further, the average phase speed and density for each region are estimated and used to compute the spectra for energy density and energy flux. In addition, we assess the noise levels associated with the CoMP data, deriving analytic formulae for the uncertainty on Doppler velocity measurements and providing a comparison by estimating the noise from the data. It is found that the entire corona is replete with transverse wave behavior. The corresponding power spectra indicate that the observed velocity fluctuations are predominately generated by stochastic processes, with the spectral slope of the power varying between the different magnetic regions. Most strikingly, all power spectra reveal the presence of enhanced power occurring at ∼3 mHz, potentially implying that the excitation of coronal transverse waves by p-modes is a global phenomenon.

  2. Hydrolytic Enzyme Activities and Protein Pattern of Avocado Fruit Ripened in Air and in Low Oxygen, with and without Ethylene.

    PubMed

    Kanellis, A K; Solomos, T; Mattoo, A K

    1989-05-01

    The effect of 2.5% O(2) atmosphere with and without ethylene on the activities of hydrolytic enzymes associated with cell walls, and total protein profile during ripening of avocado fruits (Persea americana Mill., cv Hass) were investigated. The low 2.5% O(2) atmosphere prevented the rise in the activities of cellulase, polygalacturonase, and acid phosphatase in avocado fruits whose ripening was initiated with ethylene. Addition of 100 microliters per liter ethylene to low O(2) atmosphere did not alter these suppressive effects of 2.5% O(2). Furthermore, 2.5% O(2) atmosphere delayed the development of a number of polypeptides that appear during ripening of avocado fruits while at the same time new polypeptides accumulated. The composition of the extraction buffer and its pH greatly affected the recovery of cellulase activity and its total immunoreactive protein.

  3. 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.

  4. A Peptidomic Analysis of Human Milk Digestion in the Infant Stomach Reveals Protein-Specific Degradation Patterns123

    PubMed Central

    Dallas, David C.; Guerrero, Andrés; Khaldi, Nora; Borghese, Robyn; Bhandari, Aashish; Underwood, Mark A.; Lebrilla, Carlito B.; German, J. Bruce; Barile, Daniela

    2014-01-01

    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). PMID:24699806

  5. Construction and analysis of cDNA libraries from the antennae of Batocera horsfieldi and expression pattern of putative odorant binding proteins.

    PubMed

    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 realtime 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 MinusC 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

  6. 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

  7. Mitochondrial protein-derived cryptides: Are endogenous N-formylated peptides including mitocryptide-2 components of mitochondrial damage-associated molecular patterns?

    PubMed

    Marutani, Takayuki; Hattori, Tatsuya; Tsutsumi, Koki; Koike, Yusuke; Harada, Akihiko; Noguchi, Kosuke; Kiso, Yoshiaki; Mukai, Hidehito

    2016-11-01

    Recently, much attention has been paid to "nonclassical" bioactive peptides, which are fragmented peptides simultaneously produced during maturation and degradation of various functional proteins. We identified many fragmented peptides derived from various mitochondrial proteins including mitocryptide-1 and mitocryptide-2 that efficiently activate neutrophils. These endogenous, functionally active, fragmented peptides are referred to as "cryptides." Among them, mitocryptide-2 is an N-formylated cryptide cleaved from mitochondrial cytochrome b that is encoded in mitochondrial DNA (mtDNA). It is known that 13 proteins encoded in mtDNA are translated in mitochondria as N-formylated forms, suggesting the existence of endogenous N-formylated peptides other than mitocryptide-2. Here, we investigated the effects of N-formylated peptides presumably cleaved from mtDNA-encoded proteins other than cytochrome b on the functions of neutrophilic cells to elucidate possible regulation by endogenous N-formylated cryptides. Four N-formylated cryptides derived from cytochrome c oxidase subunit I and NADH dehydrogenase subunits 4, 5, and 6 among 12 peptides from mtDNA-encoded proteins efficiently induced not only migration but also β-hexosaminidase release, which is an indicator of neutrophilic phagocytosis, in HL-60 cells differentiated into neutrophilic cells. These activities were comparable to or higher than those induced by mitocryptide-2. Although endogenous N-formylated peptides that are contained in mitochondrial damage-associated molecular patterns (DAMPs) have yet to be molecularly identified, they have been implicated in innate immunity. Thus, N-formylated cryptides including mitocryptide-2 are first-line candidates for the contents of mitochondrial DAMPs to promote innate immune responses. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 580-587, 2016.

  8. Review: Soluble innate immune pattern-recognition proteins for clearing dying cells and cellular components: implications on exacerbating or resolving inflammation.

    PubMed

    Litvack, Michael L; Palaniyar, Nades

    2010-06-01

    Soluble innate immune pattern-recognition proteins (sPRPs) identify non-self or altered-self molecular patterns. Dying cells often display altered-self arrays of molecules on their surfaces. Hence, sPRPs are ideal for recognizing these cells and their components. Dying cell surfaces often contain, or allow the access to different lipids, intracellular glycoproteins and nucleic acids such as DNA at different stages of cell death. These are considered as 'eat me' signals that replace the native 'don't eat me' signals such as CD31, CD47 present on the live cells. A programmed cell death process such as apoptosis also generates cell surface blebs that contain intracellular components. These blebs are easily released for effective clearance or signalling. During late stages of cell death, soluble components are also released that act as 'find me' signal (e.g. LysoPC, nucleotides). The sPRPs such as collectins, ficolins, pentraxins, sCD14, MFG-E8, natural IgM and C1q can effectively identify some of these specific molecular patterns. The biological end-point is different depending on sPRP, tissue, stage of apoptosis and the type of cell death. The sPRPs that reside in the immune-privileged surfaces such as lungs often act as opsonins and enhance a silent clearance of dying cells and cellular material by macrophages and other phagocytic cells. Although the recognition of these materials by complement-activating proteins could amplify the opsonic signal, this pathway may aggravate inflammation. Clear understanding of the involvement of specific sPRPs in cell death and subsequent clearance of dying cell and their components is essential for devising appropriate treatment strategies for diseases involving infection, inflammation and auto-antibody generation.

  9. 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.

  10. 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.

  11. Bi-Ligand Surfaces with Oriented and Patterned Protein for Real-Time Tracking of Cell Migration

    PubMed Central

    Vernekar, Varadraj N.; Wallace, Charles S.; Wu, Mina; Chao, Joshua T.; O’Connor, Shannon K.; Raleigh, Aimee; Liu, Xiaji; Haugh, Jason M.; Reichert, William M.

    2014-01-01

    A bioactive platform for the quantitative observation of cell migration is presented by 1) presenting migration factors in a well-defined manner on 2-D substrates, and 2) enabling continuous cell tracking. Well-defined substrate presentation is achieved by correctly orienting immobilized proteins (chemokines and cell adhesion molecules), such that the active site is accessible to cell surface receptors. A thiol-terminated self-assembled monolayer on a silica slide was used as a base substrate for subsequent chemistry. The thiol-terminated surface was converted to an immobilized metal ion surface using a maleimido-nitrilotriacetic acid (NTA) cross-linker that bound Histidine-tagged recombinant proteins on the surface with uniform distribution and specific orientation. This platform was used to study the influence of surface-immobilized chemokine SDF-1α and cell adhesion molecule ICAM-1 on murine splenic B lymphocyte migration. While soluble SDF-1α induced trans-migration in a Boyden Chamber type chemotaxis assay, immobilized SDF-1α alone did not elicit significant surface-migration on our test-platform surface. Surface-immobilized cell adhesion protein, ICAM-1, in conjunction with activation enabled migration of this cell type on our surface. Controlled exposure to UV light was used to produce stable linear gradients of His-tagged recombinant SDF-1α co-immobilized with ICAM-1 following our surface chemistry approach. XPS and antibody staining showed defined gradients of outwardly oriented SDF-1α active sites. This test platform can be especially valuable for investigators interested in studying the influence of surface-immobilized factors on cell behavior and may also be used as a cell migration enabling platform for testing the effects of various diffusible agents. PMID:25262410

  12. 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

    2009-12-01

    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.

  13. The COMP-Ident Assessment Format. A Descriptive Manual and Comprehensive Process of Linking Screening, Assessment, and Programming for the Handicapped Preschooler.

    ERIC Educational Resources Information Center

    Bagnato, Stephen J.; And Others

    The COMP-Ident Assessment Format for developing assessment and intervention procedures to follow up initial screening of preschool handicapped children in rural areas is the second stage in the HICOMP Project. Linkage between assessment and programming includes describing the child's current levels of functioning in all areas; specifying long- and…

  14. P-glycoprotein and CYP1A protein expression patterns in Nile tilapia (Oreochromis niloticus) tissues after waterborne exposure to benzo(a)pyrene (BaP).

    PubMed

    Costa, Joana; Reis-Henriques, Maria Armanda; Wilson, Jonathan M; Ferreira, Marta

    2013-09-01

    The protein levels and tissue distribution patterns of P-glycoprotein (Pgp) and cytochrome P450 (CYP1A) were investigated in Nile tilapia (Oreochromis niloticus) after waterborne exposure to different benzo(a)pyrene (BaP) concentrations, using immunochemical approaches. The Pgp mammalian monoclonal antibody (mAb) C219 cross reacted with a ∼170kDa protein, almost exclusively localized to the bile canaliculi, while probing with the Pgp mammalian mAb C494, resulted in a positive reaction in liver, gills and intestine of Nile tilapia and covered a wider set of cell types. Levels of Pgp expression were not altered after in vivo exposure to BaP. CYP1A, detected with the mAb C10-7, reacted positively in liver, gills and intestine and followed a BaP dose-dependent fold induction. Taken together, these results indicate that CYP1A is involved in BaP metabolism in liver, gills and intestine, however, further studies are needed to elucidate the possible interaction of the efflux protein Pgp with BaP and/or its metabolites.

  15. Gene Expression Patterns Define Key Transcriptional Events InCell-Cycle Regulation By cAMP And Protein Kinase A

    SciTech Connect

    Zambon, Alexander C.; Zhang, Lingzhi; Minovitsky, Simon; Kanter, Joan R.; Prabhakar, Shyam; Salomonis, Nathan; Vranizan, Karen; Dubchak Inna,; Conklin, Bruce R.; Insel, Paul A.

    2005-06-01

    Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPTcAMP), a PKA-selective cAMP analog, alters the expression of approx equal to 4,500 of approx. equal to 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPTcAMP. Changes in mRNA and protein expression of several cell cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.

  16. Expression patterns of prion protein gene in differential genotypes sheep: quantification using molecular beacon real-time RT-PCR.

    PubMed

    Wang, Chuan; Wu, Run; Li, Fa-Di; Liu, Lei; Zhang, Xiao-Li; Zhao, Chun-Lin; Diao, Xiao-Long; Guan, Hong-Wei

    2011-06-01

    Determination of the transcription level of cellular prion protein (PrP(C)) is essential for understanding its role in organisms and revealing mechanism of susceptibility and resistance to scrapie. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail in digestive tract which is important during scrapie spread through oral route. Herein, we report on measurement of sheep PrP mRNA using absolute quantitative real-time RT-PCR. Total RNA was isolated from five different regions of the central nervous system (CNS), four regions of lymphoid system, eleven regions of digestive tract, and two reproductive organ tissues of eight sheep of two different genotypes (ARR/ARQ and ARH/ARQ) and PrP mRNA was quantified by real-time RT-PCR using molecular beacon. The results showed that highest levels of PrP mRNA were expressed in thalamus and cerebrum (P < 0.01) of CNS examined, followed by cerebellum, spinal cord, and brain stem. In peripheral organs examined, lymph tissue showed moderate level of PrP expression similar to that in digestive tract and reproduction organs. PrP expression levels in the same tissue of different genotype sheep had significant variation. Our study provided the first detail, tissue-specific and genotype-specific data of PrP mRNA expression in sheep for further studies of pathogenesis of prion diseases.

  17. 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.

  18. 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.

  19. Comparison of expression patterns of fibroblast growth factor 8, bone morphogenetic protein 4 and sonic hedgehog in jaw development of the house shrew, Suncus murinus.

    PubMed

    Ogi, Hidenao; Tabata, Makoto J; Yamanaka, Atsushi; Yasui, Kinya; Uemura, Masanori

    2002-01-01

    To elucidate the mechanism underlying jaw development in mammals, we used a new laboratory animal, Suncus murinus (house shrew, an insectivore) as the subject for the investigation, because Suncus has all types of teeth (incisor, canine, premolar and molar) in its upper and lower jaws and is thought to be a good model animal having a general mammalian tooth pattern. At the start, by use of degenerate primers we cloned Suncus homologues of fibroblast growth factor 8 (sFgf8), bone morphogenetic protein 4 (sBmp4) and sonic hedgehog (sShh) genes from cDNA library derived from whole Suncus embryos at day 12 (E12). Thereafter, we examined the expression patterns of these genes in the jaw development of Suncus E11-16 embryos (for mouse E9.5-12 embryos). sFgf8 and sBmp4 were expressed in E11 but not in E15 and onward during orofacial development. sShh was expressed from E11 onward, and its expression was increased in the orofacial area. The expression pattern of sFgf8 in the maxillary and mandibular arches of E14 coincided with the area of the presumptive tooth arch. However, sShh and sBmp4 were expressed only in the outer area (= buccal/labial side) of presumptive tooth arch. Thus, these 3 genes showed specific expression pattern in jaw development of Suncus, and their distributions did not overlap each other except in a few regions. These findings suggest that sFgf8, sBmp4 and sShh have a specific function respectively during jaw development in Suncus murinus.

  20. Pattern recognition of monocyte chemoattractant protein-1 (MCP-1) in whole blood samples using new platforms based on nanostructured materials.

    PubMed

    Stefan-van Staden, Raluca-Ioana; Gugoasa, Livia Alexandra; Biris, Alexandru Radu

    2015-09-28

    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. PMID:26183340

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. Evaluation of protein content, lysine and sulfur-containing amino acids content and electrophoretic patterns of soluble proteins for gamma-irradiated semolina before and after milling of durum wheat

    NASA Astrophysics Data System (ADS)

    Azzeh, F. S.; Amr, A. S.

    2009-11-01

    Influenced of gamma irradiation (0, 0.25, 1, 2.5, 5 and 10 kGy) on total nitrogen, lysine and sulfur-containing amino acids content and electrophoretic patterns of soluble proteins of semolina was studied. The effect of irradiation before and after milling on previous parameters was also investigated. Protein content of semolina was not affected with gamma irradiation before and after milling. Up to 10 kGy dose, cystine and methionine were not significantly changed, although they increased slightly with increasing irradiation dose. Lysine content decreased significantly ( P≤0.05) at irradiation dose higher than 5 kGy. At 10 kGy dose, lysine decreased 5% and 14% for irradiated semolina and that obtained from irradiated wheat grains, respectively. The bands number and intensity of soluble proteins decreased with increasing irradiation dose higher than 5 kGy, as shown on SDS-PAGE electrophoresis. Irradiated semolina and semolina obtained from irradiated wheat grains at 10 kGy showed 13 and 15 bands, respectively. Unirradiated sample showed 19 bands.

  6. 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

  7. 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

  8. 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.

  9. 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.

  10. A C1q domain containing protein from Crassostrea gigas serves as pattern recognition receptor and opsonin with high binding affinity to LPS.

    PubMed

    Jiang, Shuai; Li, Hui; Zhang, Daoxiang; Zhang, Huan; Wang, Lingling; Sun, Jinsheng; Song, Linsheng

    2015-08-01

    C1q proteins serve as pattern recognition receptors and involve in the pathogen recognition and complement pathway activation. In the present study, a novel C1q domain containing protein from Crassostrea gigas (designated CgC1qDC-1) was isolated by liposaccharide-Sepharose 6B affinity chromatography. The coding sequence of CgC1qDC-1 gene was determined by performing a homologous search of eight tryptic peptides identified by MALDI-TOF/TOF-MS against the genome of C. gigas. The coding sequence of CgC1qDC-1 was of 387 bp encoding a polypeptide of 128 amino acids containing a typical globular C1q domain. The globular C1q domain possessed eight β strands with a jelly-roll topology structure, which was similar to the structure of human gC1q domain. The mRNA transcripts of CgC1qDC-1 were dominantly expressed in mantle and hemocytes, while low expressed in hepatopancreas, gonad, gill and muscle. The expression level of CgC1qDC-1 increased drastically at 6 h after Vibrio splendidus stimulation, and then gradually fell to the normal level at about 24 h. ELISA assay quantified that CgC1qDC-1 bound to LPS with high binding affinity (Kd = 0.09 × 10(-6) M). Moreover, CgC1qDC-1 significantly enhanced the phagocytosis of oyster hemocytes towards Gram-negative bacteria Escherichia coli and V. splendidus. These results collectively indicated that CgC1qDC-1 could serve as pattern recognition receptor and opsonin in the innate immune response against invading Gram-negative bacteria.

  11. HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Yabe, T; Yamada-Okabe, T; Kasahara, S; Furuichi, Y; Nakajima, T; Ichishima, E; Arisawa, M; Yamada-Okabe, H

    1996-01-01

    We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae. PMID:8550469

  12. The coiled-coil domain containing protein Ccdc136b antagonizes maternal Wnt/β-catenin activity during zebrafish dorsoventral axial patterning.

    PubMed

    Wei, Shi; Shang, Hanqiao; Cao, Yu; Wang, Qiang

    2016-07-20

    The coiled-coil domain containing protein CCDC136 is a putative tumor suppressor and significantly down-regulated in gastric and colorectal cancer tissues. However, little is known about its biological functions during vertebrate embryo development. Zebrafish has two CCDC136 orthologs, ccdc136a and ccdc136b, but only ccdc136b is highly expressed during early embryonic development. In this study, we demonstrate that ccdc136b is required for dorsal-ventral axial patterning in zebrafish embryos. ccdc136b morphants display strongly dorsalized phenotypes. Loss- and gain-of-function experiments in zebrafish embryos and mammalian cells show that Ccdc136b is a crucial negative regulator of the Wnt/β-catenin signaling pathway, and plays a critical role in the establishment of the dorsal-ventral axis. We further find that Ccdc136b interacts with APC, promotes the binding affinity of APC with β-catenin and then facilitates the turnover of β-catenin. These results provide the first evidence that CCDC136 regulates zebrafish dorsal-ventral patterning by antagonizing Wnt/β-catenin signal transduction and suggest a potential mechanism underlying its suppressive activity in carcinogenesis.

  13. 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

  14. Characterization of strains of Leuconostoc mesenteroides by analysis of soluble whole-cell protein pattern, DNA fingerprinting and restriction of ribosomal DNA.

    PubMed

    Villani, F; Moschetti, G; Blaiotta, G; Coppola, S

    1997-05-01

    Of 215 leuconostocs isolated from field grass, natural whey cultures and water-buffalo milk, 178 were identified as Leuconostoc mesenteroides ssp. mesenteroides while 37 strains could not be identified. Biochemical characterization allowed seven groups to be defined. Representative strains of each group and different habitat and nine reference strains were selected for further analyses. Protein profiles appeared suitable for species discrimination, but did not differentiate between the three subspecies of Leuc. mesenteroides. The technique also showed some differences among equivocal strains. DNA fingerprinting for most strains of Leuc. mesenteroides ssp. mesenteroides examined showed a different restriction pattern from that of the type strain. Ribotyping was not useful for discriminating species and subspecies of the genus Leuconostoc: Leuc. mesenteroides ssp. mesenteroides and ssp. dextranicum showed the same ribopattern as Leuc. lactis while Leuc. mesenteroides ssp. cremoris exhibited a pattern distinct from all the other species examined. On the basis of ARDRA-PCR, two main groups could be distinguished: the larger group included Leuc. mesenteroides, Leuc. lactis, Leuc. pseudomesenteroides and some unidentifiable strains; the second one included Leuc. citreum, Leuc. fallax, Weissella paramesenteroides and some unidentified strains.

  15. 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.

  16. DROSOPHILA HOLOCARBOXYLASE SYNTHETASE IS A CHROMOSOMAL PROTEIN REQUIRED FOR NORMAL HISTONE BIOTINYLATION, GENE TRANSCRIPTION PATTERNS, LIFESPAN AND HEAT TOLERANCE12

    PubMed Central

    Camporeale, Gabriela; Giordano, Ennio; Rendina, Rosaria; Zempleni, Janos; Eissenberg, Joel C.

    2006-01-01

    Posttranslational modifications of histones play important roles in chromatin structure and genomic stability. Distinct lysine residues in histones are targets for covalent binding of biotin, catalyzed by holocarboxylase synthetase (HCS) and biotinidase (BTD). Histone biotinylation has been implicated in heterochromatin structures, DNA repair, and mitotic chromosome condensation. To test whether HCS and BTD deficiency alters histone biotinylation, and to characterize phenotypes associated with HCS and BTD deficiency, HCS- and BTD-deficient flies were generated by RNA interference (RNAi). Expression of HCS and BTD decreased by 65–90% in RNAi-treated flies, as judged by mRNA abundance, BTD activity, and abundance of HCS protein. Decreased expression of HCS and BTD caused decreased biotinylation of K9 and K18 in histone H3. This was associated with altered expression of 201 genes in HCS-deficient flies. Lifespan of HCS- and BTD-deficient flies decreased by up to 32% compared to wild-type controls. Heat tolerance decreased by up to 55% in HCS-deficient flies compared to controls, as judged by survival times; effects of BTD deficiency were minor. Consistent with this observation, HCS deficiency was associated with altered expression of 285 heat-responsive genes. HCS and BTD deficiency did not affect cold tolerance, suggesting stress-specific effects of chromatin remodeling by histone biotinylation. This is the first study to provide evidence that HCS-dependent histone biotinylation affects gene function and phenotype, suggesting that the complex phenotypes of HCS- and BTD-deficiency disorders may reflect chromatin structure changes. PMID:17056793

  17. Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein-specific antibodies.

    PubMed

    Höfling, Corinna; Morawski, Markus; Zeitschel, Ulrike; Zanier, Elisa R; Moschke, Katrin; Serdaroglu, Alperen; Canneva, Fabio; von Hörsten, Stephan; De Simoni, Maria-Grazia; Forloni, Gianluigi; Jäger, Carsten; Kremmer, Elisabeth; Roßner, Steffen; Lichtenthaler, Stefan F; Kuhn, Peer-Hendrik

    2016-10-01

    Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region-specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals. PMID:27470171

  18. Master Amino acid Pattern as substitute for dietary proteins during a weight-loss diet to achieve the body's nitrogen balance equilibrium with essentially no calories.

    PubMed

    Lucà-Moretti, M; Grandi, A; Lucà, E; Muratori, G; Nofroni, M G; Mucci, M P; Gambetta, P; Stimolo, R; Drago, P; Giudice, G; Tamburlin, N

    2003-01-01

    Results of this multicentric study have shown that by giving 10 g (10 tablets) of Master Amino acid Pattern (MAP) as a substitute for dietary proteins, once a day, to 114 overweight participants undergoing the American Nutrition Clinics/Overweight Management Program (ANC/OMP), the participants' nitrogen balance could be maintained in equilibrium with essentially no calories (MAP 1 g=0.04 kcal), thereby preserving the body's structural and functional proteins, eliminating excessive water retention from the interstitial compartment, and preventing the sudden weight increase after study conclusion commonly known as the yo-yo effect. Study results have shown that the use of MAP, in conjunction with the ANC/OMP, has proven to be safe and effective by preventing those adverse effects associated with a negative nitrogen balance, such as oversized or flabby tissue, stretch marks, sagging of breast tissue, increased hair loss, faded hair color, and fragile or brittle nails. Also preventing those anomalies commonly associated with weight-loss diets, such as hunger, weakness, headache caused by ketosis, constipation, or decreased libido, the use of MAP, in conjunction with the ANC/OMP, allowed for mean weight loss of 1.4 kg (3 lb) per week. PMID:14964348

  19. Master Amino acid Pattern as sole and total substitute for dietary proteins during a weight-loss diet to achieve the body's nitrogen balance equilibrium.

    PubMed

    Lucà-Moretti, M; Grandi, A; Lucà, E; Muratori, G; Nofroni, M G; Mucci, M P; Gambetta, P; Stimolo, R; Drago, P; Giudice, G; Tamburlin, N; Karbalai, M; Valente, C; Moras, G

    2003-01-01

    Results of this multicentric study have shown that by giving Master Amino acid Pattern (MAP) as a sole and total substitute of dietary proteins to 500 overweight participants undergoing the American Nutrition Clinics/Overweight Management Program (ANC/OMP), the participants' body nitrogen balance could be maintained in equilibrium with essentially no calories (MAP 1 g=0.04 kcal), thereby preserving the body's structural and functional proteins, eliminating excessive water retention from the interstitial compartment, and preventing the sudden weight increase after study conclusion commonly known as the yo-yo effect. Study results have shown that the use of MAP, in conjunction with the ANC/OMP regimen, has proven to be safe and effective by preventing those adverse effects associated with a negative nitrogen balance, such as oversized or flabby tissue, stretch marks, the sagging of breast tissue, increased hair loss, faded hair color, and fragile or brittle nails. Also prevented were those anomalies commonly associated with weight-loss diets, such as hunger, weakness, headache caused by ketosis, constipation, and decreased libido. The use of MAP in conjunction with the ANC/OMP also allowed for mean weight loss of 2.5 kg (5.5 lb) per week, achieved through reduction of excessive fat tissue and elimination of excessive water retention from the interstitial compartment. PMID:14964347

  20. Age and dark rearing bidirectionally regulate the level and laminar pattern of expression of Abelson interacting protein 2 (Abi-2): a novel candidate visual cortical plasticity gene.

    PubMed

    Yang, Cui Bo; Kiser, Paul J; Zheng, Yu Ting; Mower, George D

    2013-11-01

    Electrophysiological studies indicate that cat visual cortical critical period neuronal plasticity peaks around 5 weeks and largely disappears by 20 weeks. Dark rearing slows this time course. Normal cats are more plastic than dark-reared cats at 5 weeks, but the opposite is true at 20 weeks. Thus, a stringent criterion for identifying genes controlling neuronal plasticity is that normal and dark rearing produce opposite direction differences in expression between young and older animals. Differential display polymerase chain reaction identified Abelson interacting protein 2 (Abi-2) as a candidate plasticity gene regulated according to this criterion. Western blotting showed bidirectional regulation of Abi-2 protein levels in cats and mice that was specific to visual cortex and did not occur in frontal cortex. Immunohistochemistry indicated developmental changes in Abi-2 laminar expression in cat visual cortex. Dark rearing altered laminar expression such that at 5 weeks, dark-reared cats were similar to 1-week normally reared cats, and at 20 weeks, dark-reared cats were similar to 5-10-week normally reared animals. The effect of dark rearing on both Abi-2 expression levels and laminar expression patterns was to slow the normal developmental process, the same effect seen on physiologically assessed plasticity in visual cortex.

  1. The distribution pattern of genetic variation in the transcript isoforms of the alternatively spliced protein-coding genes in the human genome.

    PubMed

    Liu, Ting; Lin, Kui

    2015-05-01

    By enabling the transcription of multiple isoforms from the same gene locus, alternative-splicing mechanisms greatly expand the diversity of the human transcriptome and proteome. Currently, the alternatively spliced transcripts from each protein-coding gene locus in the human genome can be classified as either principal or non-principal isoforms, providing that they differ with respect to cross-species conservation or biological features. By mapping the variants from the 1000 Genomes Project onto the coding region of each isoform, an interesting pattern of the genetic variation distributions of the coding regions for these two types of transcript isoforms was revealed on a whole-genome scale: compared with the principal isoform-specific coding regions, the non-principal isoform-specific coding regions are significantly enriched in amino acid-changing variants, particularly those that have a strong impact on protein function and have higher derived allele frequencies, suggesting that non-principal isoform-specific substitutions are less likely to be related to phenotype changes or disease. The results herein can help us better understand the potential consequences of alternatively spliced products from a population perspective.

  2. Age and Dark Rearing Bidirectionally Regulate the Level and Laminar Pattern of Expression of Abelson Interacting Protein 2 (Abi-2), a Novel Candidate Visual Cortical Plasticity Gene

    PubMed Central

    Yang, Cui Bo; Kiser, Paul J.; Zheng, Yu Ting; Mower, George D.

    2013-01-01

    Electrophysiological studies indicate that cat visual cortical critical period neuronal plasticity peaks around 5 weeks and largely disappears by 20 weeks. Dark rearing slows this time course. Normal cats are more plastic than dark reared cats at 5 weeks but the opposite is true at 20 weeks. Thus, a stringent criterion for identifying genes controlling neuronal plasticity is that normal and dark rearing produce opposite direction differences in expression between young and older animals. Differential display PCR identified Abelson interacting protein 2 (Abi-2) as a candidate plasticity gene regulated according to this criterion. Western blotting showed bidirectional regulation of Abi-2 protein levels in cats and mice that was specific to visual cortex and did not occur in frontal cortex. Immunohistochemistry indicated developmental changes in Abi-2 laminar expression in cat visual cortex. Dark rearing altered laminar expression such that at 5 weeks dark reared cats were similar to 1 week normally reared cats and at 20 weeks, dark reared cats were similar to 5–10 week normally reared animals. The effect of dark rearing on both Abi-2 expression levels and laminar expression patterns was to slow the normal developmental process, the same effect seen on physiologically assessed plasticity in visual cortex. PMID:23828391

  3. Cell lineage-specific and differentiation-dependent patterns of CCAAT/enhancer binding protein alpha expression in the gut epithelium of normal and transgenic mice.

    PubMed Central

    Chandrasekaran, C; Gordon, J I

    1993-01-01

    The proliferation and differentiation programs of gut epithelial cells are expressed rapidly and perpetually along an anatomically well defined pathway. The mouse intestine thus provides an excellent in vivo model system to define the contributions of CCAAT enhancer binding protein alpha (C/EBP alpha) and related bZIP proteins to these processes. Immunocytochemical studies revealed that C/EBP alpha is produced in villus-associated enterocytes located in the duodenum and jejunum of adult mice. The protein is located in the cytoplasmic and nuclear compartments of these cells. C/EBP alpha is not detectable in proliferating and nonproliferating epithelial cells situated in small intestinal crypts nor is it evident in any gut epithelial cell lineage located in the ileum and colon. The related C/EBP beta and C/EBP delta proteins are not detectable by sensitive immunocytochemical methods in epithelial cells distributed along the duodenal-to-colonic axis. Developmental surveys indicate that C/EBP alpha is confined to postmitotic, villus-associated epithelial cells during conversion of the polyclonal intervillus epithelium to monoclonal crypts. Analyses of intestinal isografts reveal that these developmental stage-specific, lineage-specific, differentiation-dependent, and regional patterns of C/EBP alpha expression can be established and maintained in the absence of exposure to luminal contents. Transgenic mice containing nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene (I-FABP-1178 to +28) linked to the simian virus 40 large tumor antigen (T antigen) gene express T antigen in villus-associated enterocytes. This results in reentry of enterocytes into the cell cycle and a silencing of C/EBP alpha expression without an apparent effect on the accumulation of several markers of this lineage's terminal differentiation program or on gut morphogenesis. These findings indicate that there is a relationship between expression of C/EBP alpha in

  4. 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.

  5. Expression Patterns of the Wnt Pathway Inhibitors Dickkopf3 and Secreted Frizzled-Related Proteins 1 and 4 in Endometrial Endometrioid Adenocarcinoma

    PubMed Central

    Eskander, Ramez N.; Ali, Shamshad; Dellinger, Thanh; Lankes, Heather A.; Randall, Leslie M.; Ramirez, Nilsa C.; Monk, Bradley J.; Walker, Joan L.; Eisenhauer, Eric; Hoang, Bang H.

    2016-01-01

    Objective The aim of the study was to determine the differential expression patterns of the wingless-type (Wnt) pathway inhibitors Dkk3 (Dickkopf 3), SFRP1 (secreted frizzled-related protein 1), and SFRP4 in normal müllerian tissue and endometrial endometrioid adenocarcinoma specimens. Methods Messenger RNA (mRNA) and protein levels of the Wnt pathway inhibitors Dkk3, SFRP1, and SFRP4 were evaluated by real-time reverse transcription–polymerase chain reaction and Western blot analysis. A total of 87 human tissue specimens were obtained from 60 women who participated in Gynecologic Oncology Group protocol 210. Twenty-seven normal müllerian tissues, 32 early-stage, and 28 advanced-stage endometrial endometrioid cancer specimens were analyzed. Results Median age for this cohort was 60 years, with median body mass index of 32 kg/m2. There was a difference in Dkk3 protein expression between normal müllerian tissues and primary endometrial endometrioid adenocarcinoma samples (P = 0.05). There was down-regulation of Dkk3, SFRP1, and SFRP4 mRNA expression in patients with high-grade disease (P = 0.08, 0.06, and 0.05, respectfully). Furthermore, a decrease in SFRP1 and SFPR4 mRNA expression was noted in patients with a diagnosis of locoregional and distant disease recurrence. Lastly, a trend toward decreased progression-free survival in patients with low Dkk3, SFRP1, and SFRP4 mRNA expression levels was noted. Conclusions Wnt pathway inhibitor (Dkk3, sFRP1, and/or sFRP4) expression was down-regulated in patients with high-grade disease and was associated with locoregional and distant disease recurrence. Despite sample size (power) limitations, these results support previous preclinical studies and may suggest a therapeutic role for Wnt signaling in endometrial cancer. PMID:26397159

  6. 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

  7. Cartilage Oligomeric Matrix Protein Gene Multilayers Inhibit Osteogenic Differentiation and Promote Chondrogenic Differentiation of Mesenchymal Stem Cells

    PubMed Central

    Guo, Peng; Shi, Zhong-Li; Liu, An; Lin, Tiao; Bi, Fang-Gang; Shi, Ming-Min; Yan, Shi-Gui

    2014-01-01

    There are still many challenges to acquire the optimal integration of biomedical materials with the surrounding tissues. Gene coatings on the surface of biomaterials may offer an effective approach to solve the problem. In order to investigate the gene multilayers mediated differentiation of mesenchymal stem cells (MSCs), gene functionalized films of hyaluronic acid (HA) and lipid-DNA complex (LDc) encoding cartilage oligomeric matrix protein (COMP) were constructed in this study via the layer-by-layer self-assembly technique. Characterizations of the HA/DNA multilayered films indicated the successful build-up process. Cells could be directly transfected by gene films and a higher expression could be obtained with the increasing bilayer number. The multilayered films were stable for a long period and DNA could be easily released in an enzymatic condition. Real-time polymerase chain reaction (RT-PCR) assay presented significantly higher (p < 0.01) COMP expression of MSCs cultured with HA/COMP multilayered films. Compared with control groups, the osteogenic gene expression levels of MSCs with HA/COMP multilayered films were down-regulated while the chondrogenic gene expression levels were up-regulated. Similarly, the alkaline phosphatase (ALP) staining and Alizarin red S staining of MSCs with HA/COMP films were weakened while the alcian blue staining was enhanced. These results demonstrated that HA/COMP multilayered films could inhibit osteogenic differentiation and promote chondrogenic differentiation of MSCs, which might provide new insight for physiological ligament-bone healing. PMID:25380520

  8. Plant growth, metabolism and adaptation in relation to stress conditions. XXVII. Can ascorbic acid modify the adverse effects of NaCl and mannitol on amino acids, nucleic acids and protein patterns in Vicia faba seedlings?

    PubMed

    Younis, M E; Hasaneen, M N A; Kazamel, A M S

    2009-03-01

    The adverse effects of either NaCl or mannitol on amino acids, protein patterns and nucleic acids in Vicia faba seeds were investigated. The exogenous addition of 4 mM ascorbic acid to the stressing media in which the broad bean seeds were germinated in combination with either the ionic (NaCl) or osmotic (mannitol) stressor induced significant protective changes in the total amount and in the relative composition of amino acids in general and in proline, glycine, glutamic, aspartic, alanine and serine in particular. It also induced changes in nucleic acids (RNA and DNA) content. These changes occurred throughout the entire period of the experiments (12 days). Separate administration of NaCl or mannitol enhanced the occurrence of particular novel proteins that were not detected in control bean seeds (water medium). Protein banding patterns of broad bean seedlings treated with NaCl or mannitol in combination with 4 mM ascorbic acid showed different de novo protein bands, with different molecular weights, at different stages of seedlings growth, with lower levels or a nearly complete absence of the major stress proteins. The pattern of changes for amino acids and nucleic acids and the range of protein bands extracted from the variously treated broad bean seedlings indicate a positive role of ascorbic acid in the alleviation of the damage effects induced by NaCl and mannitol. The importance of this role in the stress tolerance of broad beans is discussed.

  9. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I

    PubMed Central

    Park, Seung Bum; Seronello, Scott; Mayer, Wasima; Ojcius, David M.

    2016-01-01

    Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity. PMID:27404108

  10. Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I.

    PubMed

    Park, Seung Bum; Seronello, Scott; Mayer, Wasima; Ojcius, David M

    2016-01-01

    Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNβ induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity. PMID:27404108

  11. Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2.

    PubMed

    Chaparro-Garcia, Angela; Schwizer, Simon; Sklenar, Jan; Yoshida, Kentaro; Petre, Benjamin; Bos, Jorunn I B; Schornack, Sebastian; Jones, Alexandra M E; Bozkurt, Tolga O; Kamoun, Sophien

    2015-01-01

    Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways. PMID:26348328

  12. Phytophthora infestans RXLR-WY Effector AVR3a Associates with Dynamin-Related Protein 2 Required for Endocytosis of the Plant Pattern Recognition Receptor FLS2

    PubMed Central

    Chaparro-Garcia, Angela; Schwizer, Simon; Sklenar, Jan; Yoshida, Kentaro; Petre, Benjamin; Bos, Jorunn I. B.; Schornack, Sebastian; Jones, Alexandra M. E.; Bozkurt, Tolga O.; Kamoun, Sophien

    2015-01-01

    Pathogens utilize effectors to suppress basal plant defense known as PTI (Pathogen-associated molecular pattern-triggered immunity). However, our knowledge of PTI suppression by filamentous plant pathogens, i.e. fungi and oomycetes, remains fragmentary. Previous work revealed that the co-receptor BAK1/SERK3 contributes to basal immunity against the potato pathogen Phytophthora infestans. Moreover BAK1/SERK3 is required for the cell death induced by P. infestans elicitin INF1, a protein with characteristics of PAMPs. The P. infestans host-translocated RXLR-WY effector AVR3a is known to supress INF1-mediated cell death by binding the plant E3 ligase CMPG1. In contrast, AVR3aKI-Y147del, a deletion mutant of the C-terminal tyrosine of AVR3a, fails to bind CMPG1 and does not suppress INF1-mediated cell death. Here, we studied the extent to which AVR3a and its variants perturb additional BAK1/SERK3-dependent PTI responses in N. benthamiana using the elicitor/receptor pair flg22/FLS2 as a model. We found that all tested variants of AVR3a suppress defense responses triggered by flg22 and reduce internalization of activated FLS2. Moreover, we discovered that AVR3a associates with the Dynamin-Related Protein 2 (DRP2), a plant GTPase implicated in receptor-mediated endocytosis. Interestingly, silencing of DRP2 impaired ligand-induced FLS2 internalization but did not affect internalization of the growth receptor BRI1. Our results suggest that AVR3a associates with a key cellular trafficking and membrane-remodeling complex involved in immune receptor-mediated endocytosis. We conclude that AVR3a is a multifunctional effector that can suppress BAK1/SERK3-mediated immunity through at least two different pathways. PMID:26348328

  13. A dietary pattern including nopal, chia seed, soy protein, and oat reduces serum triglycerides and glucose intolerance in patients with metabolic syndrome.

    PubMed

    Guevara-Cruz, Martha; Tovar, Armando R; Aguilar-Salinas, Carlos A; Medina-Vera, Isabel; Gil-Zenteno, Lidia; Hernández-Viveros, Isaac; López-Romero, Patricia; Ordaz-Nava, Guillermo; Canizales-Quinteros, Samuel; Guillen Pineda, Luz E; Torres, Nimbe

    2012-01-01

    Metabolic syndrome (MetS) is a health problem throughout the world and is associated with cardiovascular disease and diabetes. Thus, the purpose of the present work was to evaluate the effects of a dietary pattern (DP; soy protein, nopal, chia seed, and oat) on the biochemical variables of MetS, the AUC for glucose and insulin, glucose intolerance (GI), the relationship of the presence of certain polymorphisms related to MetS, and the response to the DP. In this randomized trial, the participants consumed their habitual diet but reduced by 500 kcal for 2 wk. They were then assigned to the placebo (P; n = 35) or DP (n = 32) group and consumed the reduced energy diet plus the P or DP beverage (235 kcal) minus the energy provided by these for 2 mo. All participants had decreases in body weight (BW), BMI, and waist circumference during the 2-mo treatment (P < 0.0001); however, only the DP group had decreases in serum TG, C-reactive protein (CRP), and AUC for insulin and GI after a glucose tolerance test. Interestingly, participants in the DP group with MetS and the ABCA1 R230C variant had a greater decrease in BW and an increase in serum adiponectin concentration after 2 mo of dietary treatment than those with the ABCA1 R230R variant. The results from this study suggest that lifestyle interventions involving specific DP for the treatment of MetS could be more effective if local foods and genetic variations of the population are considered. PMID:22090467

  14. A dietary pattern including nopal, chia seed, soy protein, and oat reduces serum triglycerides and glucose intolerance in patients with metabolic syndrome.

    PubMed

    Guevara-Cruz, Martha; Tovar, Armando R; Aguilar-Salinas, Carlos A; Medina-Vera, Isabel; Gil-Zenteno, Lidia; Hernández-Viveros, Isaac; López-Romero, Patricia; Ordaz-Nava, Guillermo; Canizales-Quinteros, Samuel; Guillen Pineda, Luz E; Torres, Nimbe

    2012-01-01

    Metabolic syndrome (MetS) is a health problem throughout the world and is associated with cardiovascular disease and diabetes. Thus, the purpose of the present work was to evaluate the effects of a dietary pattern (DP; soy protein, nopal, chia seed, and oat) on the biochemical variables of MetS, the AUC for glucose and insulin, glucose intolerance (GI), the relationship of the presence of certain polymorphisms related to MetS, and the response to the DP. In this randomized trial, the participants consumed their habitual diet but reduced by 500 kcal for 2 wk. They were then assigned to the placebo (P; n = 35) or DP (n = 32) group and consumed the reduced energy diet plus the P or DP beverage (235 kcal) minus the energy provided by these for 2 mo. All participants had decreases in body weight (BW), BMI, and waist circumference during the 2-mo treatment (P < 0.0001); however, only the DP group had decreases in serum TG, C-reactive protein (CRP), and AUC for insulin and GI after a glucose tolerance test. Interestingly, participants in the DP group with MetS and the ABCA1 R230C variant had a greater decrease in BW and an increase in serum adiponectin concentration after 2 mo of dietary treatment than those with the ABCA1 R230R variant. The results from this study suggest that lifestyle interventions involving specific DP for the treatment of MetS could be more effective if local foods and genetic variations of the population are considered.

  15. Phosphorylation pattern of the p90rsk and mitogen-activated protein kinase (MAPK) molecule: comparison of in vitro and in vivo matured porcine oocytes.

    PubMed

    Schuon, C; Ebeling, S; Meinecke, B

    2007-08-01

    The overall objective was to elucidate the phosphorylation pattern and activity of the kinase p90rsk, a substrate of mitogen-activated protein kinase (MAPK), during in vitro and in vivo maturation of pig oocytes. Cumulus-oocyte complexes were collected from slaughtered pigs and matured in vitro (0, 22, 26, 30, 34, 46 h) with and without the MEK inhibitor U0126. For in vivo maturation, gilts were stimulated with equine chorionic gonadotrophin (eCG) (600-800 IU). Maturation was induced 72 h later with hCG (500 IU). Oocytes were obtained surgically (0, 22, 30 h). The samples were submitted to electrophoresis and protein blotting analysis. Enhanced chemiluminescence was used for visualization. In vitro matured oocytes were further submitted to a commercially available radioactive kinase assay to determine kinase activity. It was shown that oocytes, as well as cumulus cells, already possess a partially phosphorylated p90rsk at the time of removal from follicles, with a further phosphorylation of the molecule occurring between 22-24 h after the initiation of culture, and in vivo maturation. The phosphorylation of p90rsk coincides with the phosphorylation of MAPK and can be prevented by U0126, indicating a MAPK-dependent phosphorylation of p90rsk. Phosphorylation of the in vivo matured oocytes occurred shown as a band of less than 200 kDa. This is presumably a molecule complex, with MAPK not being a component. Therefore, the p90rsk molecule in vivo exists as a dimer. Determination of kinase activity demonstrated decreasing enzyme activities. This led to the conclusion that the assay is not specific for p90rsk, instead measuring p70S6 kinase activities.

  16. Intake patterns and dietary associations of soya protein consumption in adults and children in the Canadian Community Health Survey, Cycle 2.2.

    PubMed

    Mudryj, Adriana N; Aukema, Harold M; Yu, Nancy

    2015-01-28

    Soya foods are one of the recommended alternatives to meat in many dietary guidelines. While this is expected to increase the intake of some nutrients, potential concerns regarding others have been raised. The purpose of the present study was to examine the prevalence and the association of soya food consumption with nutrient intakes and dietary patterns of Canadians (age ≥ 2 years). Cross-sectional data from the 2004 Canadian Community Health Survey (Cycle 2.2; n 33,218) were used to classify soya consumers and non-consumers. Soya consumers were further divided into two groups based on their soya protein intake. Sample weights were applied and logistic regression analysis was used to explore the association between nutrient intakes and soya consumption, with cultural background, sex, age and economic status being included as covariates. On any given day, 3.3% (n 1085) of Canadians consume soya foods, with females, Asian Canadians and adults with post-secondary education being more likely to be soya consumers. As a whole, adolescent and adult respondents who had consumed at least one soya food during their 24 h dietary recall had higher energy intakes, as well as increased intakes of nutrients such as protein, fibre, vitamin C, vitamin B6, naturally occurring folate, thiamin, Ca, P, Mg, PUFA, Fe and K and lowered intakes of saturated fat. These data indicate that soya food consumption is associated with improved diet quality of Canadians. However, future research is necessary to investigate the association between increased energy intake and soya consumption.

  17. 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!

  18. Curbing Workers' Comp Costs.

    ERIC Educational Resources Information Center

    Deeb, William S.

    1998-01-01

    An actuarial study revealed that Pasadena Schools had an unfunded worker's compensation liability of over $10 million and 400 open claims. Advised to implement strong cost-containment measures (an early return-to-work program) and equally strong accountability measures (strict performance guides and safe work practices), the district achieved…

  19. Collagen XII and XIV, New Partners of Cartilage Oligomeric Matrix Protein in the Skin Extracellular Matrix Suprastructure*

    PubMed Central

    Agarwal, Pallavi; Zwolanek, Daniela; Keene, Douglas R.; Schulz, Jan-Niklas; Blumbach, Katrin; Heinegård, Dick; Zaucke, Frank; Paulsson, Mats; Krieg, Thomas; Koch, Manuel; Eckes, Beate

    2012-01-01

    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. PMID:22573329

  20. New inflammatory markers for prediction of non-dipper blood pressure pattern in patients with essential hypertension: Serum YKL-40/Chitinase 3-like protein 1 levels and echocardiographic epicardial adipose tissue thickness.

    PubMed

    Bakirci, Eftal Murat; Degirmenci, Husnu; Hamur, Hikmet; Gunay, Murat; Gulhan, Barıs; Aydin, Merve; Kucuksu, Zafer; Ceyhun, Gokhan; Topal, Ergun

    2015-01-01

    The aim of the present study was to investigate whether YKL-40 levels and epicardial adipose tissue (EAT) thickness were associated with non-dipping pattern in essential hypertension (HT). Age- and sex-matched 40 dipper hypertensive patients and 40 non-dipper hypertensive patients were included in the study. Non-dippers had significantly increased EAT thickness and higher YKL-40 and high-sensitivity C-reactive protein levels than dippers. Multivariate logistic regression analysis showed that the EAT thickness and serum levels of YKL-40 and high-sensitivity C-reactive protein were independent predictors of non-dipping pattern in essential HT. In essential HT, presence of non-dipping pattern is associated with increased inflammatory response. PMID:25919569

  1. Characterization and properties of a 1,3-beta-D-glucan pattern recognition protein of Tenebrio molitor larvae that is specifically degraded by serine protease during prophenoloxidase activation.

    PubMed

    Zhang, Rong; Cho, Hae Yun; Kim, Hyun Sic; Ma, Young Gerl; Osaki, Tsukasa; Kawabata, Shun-ichiro; Söderhäll, Kenneth; Lee, Bok Luel

    2003-10-24

    Although many different pattern recognition receptors recognizing peptidoglycan and 1,3-beta-D-glucan have been identified in vertebrates and insects, the molecular mechanism of these molecules in the pattern recognition and subsequent signaling is largely unknown. To gain insights into the action mechanism of 1,3-beta-D-glucan pattern recognition protein in the insect prophenoloxidase (proPO) activation system, we purified a 53-kDa 1,3-beta-D-glucan recognition protein (Tm-GRP) to homogeneity from the hemolymph of the mealworm, Tenebrio molitor, by using a 1,3-beta-d-glucan affinity column. The purified protein specifically bound to 1,3-beta-D-glucan but not to peptidoglycan. Subsequent molecular cloning revealed that Tm-GRP contains a region with close sequence similarity to bacterial glucanases. Strikingly, two catalytically important residues in glucanases are replaced with other nonhomologous amino acids in Tm-GRP. The finding suggests that Tm-GRP has evolved from an ancestral gene of glucanases but retained only the ability to recognize 1,3-beta-D-glucan. A Western blot analysis of the protein level of endogenous Tm-GRP showed that the protein was specifically degraded following the activation of proPO with 1,3-beta-D-glucan and calcium ion. The degradation was significantly retarded by the addition of serine protease inhibitors but not by cysteine or acidic protease inhibitor. These results suggest that 1,3-beta-D-glucan pattern recognition protein is specifically degraded by serine protease(s) during proPO activation, and we propose that this degradation is an important regulatory mechanism of the activation of the proPO system.

  2. Panencephalopathic Creutzfeldt-Jakob disease with distinct pattern of prion protein deposition in a patient with D178N mutation and homozygosity for valine at codon 129 of the prion protein Gene.

    PubMed

    Marcon, Gabriella; Indaco, Antonio; Di Fede, Giuseppe; Suardi, Silvia; Finato, Nicoletta; Moretti, Valentino; Micoli, Sandro; Fociani, Paolo; Zerbi, Pietro; Pincherle, Alessandro; Redaelli, Veronica; Tagliavini, Fabrizio; Giaccone, Giorgio

    2014-03-01

    Prion diseases include sporadic, acquired and genetic forms linked to mutations of the prion protein (PrP) gene (PRNP). In subjects carrying the D178N PRNP mutation, distinct phenotypes can be observed, depending on the methionine/valine codon 129 polymorphism. We present here a 53-year-old woman with D178N mutation in the PRNP gene and homozygosity for valine at codon 129. The disease started at age 47 with memory deficits, progressive cognitive impairment and ataxia. The clinical picture slowly worsened to a state of akinetic mutism in about 2 years and the disease course was 6 years. The neuropathologic examination