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Sample records for single protein species

  1. Microencapsulation of single-cell protein from various microalgae species

    Directory of Open Access Journals (Sweden)

    Purnama Sukardi

    2015-10-01

    Full Text Available ABSTRACT The objective of the research was to evaluate nutritional values of microencapsulated diet made from single cell protein of microalgae. Complete randomized design was applied using three different types of microalgae for inclusion trials i.e. (A Nannochloropsis sp., (B Chlorella sp., and (C Spirulina sp. with five replications respectively. Microencapsulated diet was produced by a modification method based on thermal cross-linking with stable temperature. Phytoplankton was cultured in sea water for which fertilized by a modification of Walne and Guillard fertilizer. The results showed that the highest value of nutrition content was Spirulina sp. and the average composition of protein, crude lipid, carbohydrate, ash, nitrogen free extract, and water content was 34.80%, 0.30%, 18.53%, 20.09%, 26.29%, and 13.32%, respectively. Organoleptically, microcapsule showed that the color of capsule was dark green and smell fresh phytoplankton. Keywords: microcapsule, single-cell protein, thermal cross-linking, microalgae, phytoplankton  ABSTRAK Tujuan penelitian adalah mengevaluasi kandungan nutrisi pakan mikrokapsul protein sel tunggal (single cell protein yang berasal dari berbagai jenis mikroalga (fitoplankton. Rancangan percobaan yang digunakan adalah rancangan acak lengkap, dengan perlakuan inklusi mikrokapsul dari jenis fitoplankton (A Nannochloropsis sp., (B Chlorella sp., dan (C Spirulina sp., masing-masing diulang lima kali. Pembuatan mikrokapsul dilakukan dengan menggunakan modifikasi metode dasar thermal cross-linking, serta menerapkan teknik pengeringan suhu konstan. Proses pembuatan mikrokapsul protein diawali dengan kultur fitoplankton jenis Nannochloropsis sp., Chlorella sp., dan Spirulina sp. Kultur dilakukan di dalam laboratorium menggunakan media air laut dan modifikasi pupuk Walne dan Guillard. Hasil penelitian menunjukkan bahwa kandungan nutrisi tertinggi terdapat pada jenis mikrokapsul protein sel tunggal yang berasal dari

  2. Near infra-red spectroscopy quantitative modelling of bivalve protein, lipid and glycogen composition using single-species versus multi-species calibration and validation sets

    Science.gov (United States)

    Bartlett, Jill K.; Maher, William A.; Purss, Matthew B. J.

    2018-03-01

    Near infrared spectroscopy (NIRS) quantitative modelling was used to measure the protein, lipid and glycogen composition of five marine bivalve species (Saccostrea glomerata, Ostrea angasi, Crassostrea gigas, Mytilus galloprovincialis and Anadara trapezia) from multiple locations and seasons. Predictive models were produced for each component using individual species and aggregated sample populations for the three oyster species (S. glomerata, O. angasi and C. gigas) and for all five bivalve species. Whole animal tissues were freeze dried, ground to > 20 μm and scanned by NIRS. Protein, lipid and glycogen composition were determined by traditional chemical analyses and calibration models developed to allow rapid NIRS-measurement of these components in the five bivalve species. Calibration modelling was performed using wavelet selection, genetic algorithms and partial least squares analysis. Model quality was assessed using RPIQ and RMESP. For protein composition, single species model results had RPIQ values between 2.4 and 3.5 and RMSEP between 8.6 and 18%, the three oyster model had an RPIQ of 2.6 and an RMSEP of 10.8% and the five bivalve species had an RPIQ of 3.6 and RMSEP of 8.7% respectively. For lipid composition, single species models achieved RPIQ values between 2.9 and 5.3 with RMSEP between 9.1 and 11.2%, the oyster model had an RPIQ of 3.6 and RMSEP of 6.8 and the five bivalve model had an RPIQ of 5.2 and RMSEP of 6.8% respectively. For glycogen composition, the single species models had RPIQs between 3.8 and 18.9 with RMSEP between 3.5 and 9.2%, the oyster model had an RPIQ of 5.5 and RMSEP of 7.1% and the five bivalve model had an RPIQ of 4 and RMSEP of 7.6% respectively. Comparison between individual species models and aggregated models for three oyster species and five bivalve species for each component indicate that aggregating data from like species produces high quality models with robust and reliable quantitative application. The benefit of

  3. A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias

    DEFF Research Database (Denmark)

    Sergeev, Eugenia; Hansen, Anders Højgaard; Bolognini, Daniele

    2017-01-01

    that are able to block the human receptor. Docking of exemplar antagonists from two chemical series to homology models of both human and mouse Free Fatty Acid Receptor 2 suggested that a single lysine - arginine variation at the extracellular face of the receptor might provide the basis for antagonist...... selectivity and mutational swap studies confirmed this hypothesis. Extending these studies to agonist function indicated that although the lysine - arginine variation between human and mouse orthologs had limited effect on G protein-mediated signal transduction, removal of positive charge from this residue...

  4. Single transverse mode protein laser

    Science.gov (United States)

    Dogru, Itir Bakis; Min, Kyungtaek; Umar, Muhammad; Bahmani Jalali, Houman; Begar, Efe; Conkar, Deniz; Firat Karalar, Elif Nur; Kim, Sunghwan; Nizamoglu, Sedat

    2017-12-01

    Here, we report a single transverse mode distributed feedback (DFB) protein laser. The gain medium that is composed of enhanced green fluorescent protein in a silk fibroin matrix yields a waveguiding gain layer on a DFB resonator. The thin TiO2 layer on the quartz grating improves optical feedback due to the increased effective refractive index. The protein laser shows a single transverse mode lasing at the wavelength of 520 nm with the threshold level of 92.1 μJ/ mm2.

  5. Single-species microarrays and comparative transcriptomics.

    Directory of Open Access Journals (Sweden)

    Frédéric J J Chain

    Full Text Available BACKGROUND: Prefabricated expression microarrays are currently available for only a few species but methods have been proposed to extend their application to comparisons between divergent genomes. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that the hybridization intensity of genomic DNA is a poor basis on which to select unbiased probes on Affymetrix expression arrays for studies of comparative transcriptomics, and that doing so produces spurious results. We used the Affymetrix Xenopus laevis microarray to evaluate expression divergence between X. laevis, X. borealis, and their F1 hybrids. When data are analyzed with probes that interrogate only sequences with confirmed identity in both species, we recover results that differ substantially analyses that use genomic DNA hybridizations to select probes. CONCLUSIONS/SIGNIFICANCE: Our findings have implications for the experimental design of comparative expression studies that use single-species microarrays, and for our understanding of divergent expression in hybrid clawed frogs. These findings also highlight important limitations of single-species microarrays for studies of comparative transcriptomics of polyploid species.

  6. Watching Single Proteins Using Engineered Nanopores

    Science.gov (United States)

    Movileanu, Liviu

    2014-01-01

    Recent studies in the area of single-molecule detection of proteins with nanopores show a great promise in fundamental science, bionanotechnology and proteomics. In this mini-review, I discuss a comprehensive array of examinations of protein detection and characterization using protein and solid-state nanopores. These investigations demonstrate the power of the single-molecule nanopore measurements to reveal a broad range of functional, structural, biochemical and biophysical features of proteins, such as their backbone flexibility, enzymatic activity, binding affinity as well as their concentration, size and folding state. Engineered nanopores in organic materials and in inorganic membranes coupled with surface modification and protein engineering might provide a new generation of sensing devices for molecular biomedical diagnosis. PMID:24370252

  7. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    (NCS1). The NMR solution structure of NCS1, in combination with fluorescence spectroscopy and mutational analysis, suggested a novel role for the C-terminal tail in regulating conformational stability. On the single-molecule level, the C-domain folded through a partially folded intermediate state....... This knowledge-gap is partly due to our inability to unveil the details of folding mechanisms that can be buried in the ensemble-averaged output of traditional bulk methods. Single-molecule techniques have provided a perspective beyond the ensemble average and enable studying the folding trajectories of protein...... molecules in unprecedented detail. These methods can, in principle, detect rare folding or misfolding events, and ultimately lead to a reconstruction of the free energy landscape. In this thesis, the folding mechanism of both single- and double-domain proteins is unraveled using single-molecule optical...

  8. Watching single protein molecules in action

    DEFF Research Database (Denmark)

    Heiðarsson, Pétur Orri

    . This knowledge-gap is partly due to our inability to unveil the details of folding mechanisms that can be buried in the ensemble-averaged output of traditional bulk methods. Single-molecule techniques have provided a perspective beyond the ensemble average and enable studying the folding trajectories of protein...... molecules in unprecedented detail. These methods can, in principle, detect rare folding or misfolding events, and ultimately lead to a reconstruction of the free energy landscape. In this thesis, the folding mechanism of both single- and double-domain proteins is unraveled using single-molecule optical......, with transition states located almost halfway between the native and unfolded states. When pulled from the N- and C-termini, both experiments and simulations suggested that the molecule populates a transition state that resembles that observed during chemical denaturation, with respect to structure and position...

  9. Dataset of protein species from human liver

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    Stanislav Naryzhny

    2017-06-01

    Full Text Available This article contains data related to the research article entitled “Zipf׳s law in proteomics” (Naryzhny et al., 2017 [1]. The protein composition in the human liver or hepatocarcinoma (HepG2 cells extracts was estimated using a filter-aided sample preparation (FASP protocol. The protein species/proteoform composition in the human liver was determined by two-dimensional electrophoresis (2-DE followed by Electrospray Ionization Liquid Chromatography-Tandem Mass Spectrometry (ESI LC-MS/MS. In the case of two-dimensional electrophoresis (2-DE, the gel was stained with Coomassie Brilliant Blue R350, and image analysis was performed with ImageMaster 2D Platinum software (GE Healthcare. The 96 sections in the 2D gel were selected and cut for subsequent ESI LC-MS/MS and protein identification. If the same protein was detected in different sections, it was considered to exist as different protein species/proteoforms. A list of human liver proteoforms detected in this way is presented.

  10. Protein-protein interaction network-based detection of functionally similar proteins within species.

    Science.gov (United States)

    Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

    2012-07-01

    Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

  11. Species specificity in major urinary proteins by parallel evolution.

    Directory of Open Access Journals (Sweden)

    Darren W Logan

    Full Text Available Species-specific chemosignals, pheromones, regulate social behaviors such as aggression, mating, pup-suckling, territory establishment, and dominance. The identity of these cues remains mostly undetermined and few mammalian pheromones have been identified. Genetically-encoded pheromones are expected to exhibit several different mechanisms for coding 1 diversity, to enable the signaling of multiple behaviors, 2 dynamic regulation, to indicate age and dominance, and 3 species-specificity. Recently, the major urinary proteins (Mups have been shown to function themselves as genetically-encoded pheromones to regulate species-specific behavior. Mups are multiple highly related proteins expressed in combinatorial patterns that differ between individuals, gender, and age; which are sufficient to fulfill the first two criteria. We have now characterized and fully annotated the mouse Mup gene content in detail. This has enabled us to further analyze the extent of Mup coding diversity and determine their potential to encode species-specific cues.Our results show that the mouse Mup gene cluster is composed of two subgroups: an older, more divergent class of genes and pseudogenes, and a second class with high sequence identity formed by recent sequential duplications of a single gene/pseudogene pair. Previous work suggests that truncated Mup pseudogenes may encode a family of functional hexapeptides with the potential for pheromone activity. Sequence comparison, however, reveals that they have limited coding potential. Similar analyses of nine other completed genomes find Mup gene expansions in divergent lineages, including those of rat, horse and grey mouse lemur, occurring independently from a single ancestral Mup present in other placental mammals. Our findings illustrate that increasing genomic complexity of the Mup gene family is not evolutionarily isolated, but is instead a recurring mechanism of generating coding diversity consistent with a species

  12. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  13. Discriminating Microbial Species Using Protein Sequence Properties and Machine Learning

    NARCIS (Netherlands)

    Shahib, Ali Al-; Gilbert, David; Breitling, Rainer

    2007-01-01

    Much work has been done to identify species-specific proteins in sequenced genomes and hence to determine their function. We assumed that such proteins have specific physico-chemical properties that will discriminate them from proteins in other species. In this paper, we examine the validity of this

  14. Stochastic single-molecule dynamics of synaptic membrane protein domains

    Science.gov (United States)

    Kahraman, Osman; Li, Yiwei; Haselwandter, Christoph A.

    2016-09-01

    Motivated by single-molecule experiments on synaptic membrane protein domains, we use a stochastic lattice model to study protein reaction and diffusion processes in crowded membranes. We find that the stochastic reaction-diffusion dynamics of synaptic proteins provide a simple physical mechanism for collective fluctuations in synaptic domains, the molecular turnover observed at synaptic domains, key features of the single-molecule trajectories observed for synaptic proteins, and spatially inhomogeneous protein lifetimes at the cell membrane. Our results suggest that central aspects of the single-molecule and collective dynamics observed for membrane protein domains can be understood in terms of stochastic reaction-diffusion processes at the cell membrane.

  15. Global Asymptotic Stability for Discrete Single Species Population Models

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    A. Bilgin

    2017-01-01

    Full Text Available We present some basic discrete models in populations dynamics of single species with several age classes. Starting with the basic Beverton-Holt model that describes the change of single species we discuss its basic properties such as a convergence of all solutions to the equilibrium, oscillation of solutions about the equilibrium solutions, Allee’s effect, and Jillson’s effect. We consider the effect of the constant and periodic immigration and emigration on the global properties of Beverton-Holt model. We also consider the effect of the periodic environment on the global properties of Beverton-Holt model.

  16. Single species victory in a two-site, two-species model of population dispersion

    Science.gov (United States)

    Waddell, Jack; Sander, Len; Kessler, David

    2008-03-01

    We study the behavior of two species, differentiated only by their dispersal rates in an environment providing heterogeneous growth rates. Previous deterministic studies have shown that the slower-dispersal species always drives the faster species to extinction, while stochastic studies show that the opposite case can occur given small enough population and spatial heterogeneity. Other models of similar systems demonstrate the existence of an optimum dispersal rate, suggesting that distinguishing the species as faster or slower is insufficient. We here study the interface of these models for a small spatial system and determine the conditions of stability for a single species outcome.

  17. Are Ascaris lumbricoides and Ascaris suum a single species?

    Science.gov (United States)

    Leles, Daniela; Gardner, Scott L; Reinhard, Karl; Iñiguez, Alena; Araujo, Adauto

    2012-02-20

    Since the original description and naming of Ascaris lumbricoides from humans by Linnaeus in 1758 and later of Ascaris suum from pigs by Goeze 1782, these species have been considered to be valid. Four hypotheses relative to the conspecificity or lack thereof (and thus origin of these species) are possible: 1) Ascaris lumbricoides (usually infecting humans) and Ascaris suum (recorded mostly from pigs) are both valid species, with the two species originating via a speciation event from a common ancestor sometime before the domestication of pigs by humans, or 2) Ascaris lumbricoides in humans is derived directly from the species A. suum found in pigs with A. suum then existing as a persistent ancestor after formation of A. lumbricoides, or 3) Ascaris suum is derived directly from A. lumbricoides with the persistent ancestor being A. lumbricoides and A. suum being the newly derived species, and finally, 4) Ascaris lumbricoides and A. suum are the same species, this hypothesis being supported by studies showing both low morphological and low genetic divergence at several genes. We present and discuss paleoparasitological and genetic evidence that complement new data to evaluate the origin and evolution of Ascaris spp. in humans and pigs, and the uniqueness of the species in both hosts. Finally, we conclude that Ascaris lumbricoides and A. suum are a single species and that the name A. lumbricoides Linnaeus 1758 has taxonomic priority; therefore A. suum Goeze 1782 should be considered a synonym of A. lumbricoides.

  18. epidemiology of single and multiple species of helminth infections ...

    African Journals Online (AJOL)

    hi-tech

    2000-03-01

    Mar 1, 2000 ... East African Medical Journal Vol. 77 No. 3 March 2000. EPIDEMIOLOGY OF SINGLE AND MULTIPLE SPECIES OF HELMINTH INFECTIONS AMONG SCHOOL CHILDREN IN BUSIA DISTRICT, KENYA. S. Brooker, MA, Wellcome Trust Centre for the Epidemiology of Infectious Disease, University of Oxford, ...

  19. Building protein-protein interaction networks for Leishmania species through protein structural information.

    Science.gov (United States)

    Dos Santos Vasconcelos, Crhisllane Rafaele; de Lima Campos, Túlio; Rezende, Antonio Mauro

    2018-03-06

    Systematic analysis of a parasite interactome is a key approach to understand different biological processes. It makes possible to elucidate disease mechanisms, to predict protein functions and to select promising targets for drug development. Currently, several approaches for protein interaction prediction for non-model species incorporate only small fractions of the entire proteomes and their interactions. Based on this perspective, this study presents an integration of computational methodologies, protein network predictions and comparative analysis of the protozoan species Leishmania braziliensis and Leishmania infantum. These parasites cause Leishmaniasis, a worldwide distributed and neglected disease, with limited treatment options using currently available drugs. The predicted interactions were obtained from a meta-approach, applying rigid body docking tests and template-based docking on protein structures predicted by different comparative modeling techniques. In addition, we trained a machine-learning algorithm (Gradient Boosting) using docking information performed on a curated set of positive and negative protein interaction data. Our final model obtained an AUC = 0.88, with recall = 0.69, specificity = 0.88 and precision = 0.83. Using this approach, it was possible to confidently predict 681 protein structures and 6198 protein interactions for L. braziliensis, and 708 protein structures and 7391 protein interactions for L. infantum. The predicted networks were integrated to protein interaction data already available, analyzed using several topological features and used to classify proteins as essential for network stability. The present study allowed to demonstrate the importance of integrating different methodologies of interaction prediction to increase the coverage of the protein interaction of the studied protocols, besides it made available protein structures and interactions not previously reported.

  20. From single-species advice to mixed-species management: taking the next step

    DEFF Research Database (Denmark)

    Vinther, Morten; Reeves, S.A.; Patterson, K.R.

    2004-01-01

    that accounts for mixed-fishery effects, but in the short term there is a need for approaches to resolve the conflicting management advice for different species within the same fishery, and to generate catch or effort advice that accounts for the mixed-species nature of the fishery. This paper documents...... a recent approach used to address these problems. The approach takes the single-species advice for each species in the fishery as a starting point, then attempts to resolve it into consistent catch or effort advice using fleet-disaggregated catch forecasts in combination with explicitly stated management...

  1. A single molecular marker to distinguish between species of Dioscorea.

    Science.gov (United States)

    Techen, Natascha; Parveen, Iffat; Khan, Ikhlas A

    2017-03-01

    Yams are species of the genus Dioscorea (family Dioscoreaceae), which consists of approximately 630 species. The majority of the world production of yams occurs in Africa with 58.8 million t annually, but they are also produced in the Americas and Asia. The saponins in yams have been reported to possess various properties to improve health. The tuber and aerial parts of various species often share morphological similarities, which can cause problems in the proper identification of sample material. For example, the rootstocks and aerial parts of Dioscorea villosa L. share similarities with Dioscorea polystachia Turcz. Dioscorea bulbifera L. may be mistaken for Dioscorea alata L. owing to similar morphologies. Various molecular analyses have been published to help with the identification of species and varieties within the genus Dioscorea. The multi-loci or single-locus analysis has resulted in varying success, some with only a limited discrimination rate. In the present study, a single nuclear genomic region, biparentally inherited, was analyzed for its usefulness as a molecular marker for species identification and discrimination between D. bulbifera, D. villosa, D. nipponica, D. alata, D. caucasica, and D. deltoidea samples. The results of this study show that the LFY genomic region can be useful as a molecular marker to distinguish between samples.

  2. Protein quality of three different species of earthrvorms

    African Journals Online (AJOL)

    No difference in protein quality was noted between three species. of earthworms, as evaluated by net protein ... animal production industry is still limited since conventional production and harvesting methods presently being used will not suffice to produce sufficient .... Samples were defatted by ether extraction and protein ...

  3. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    Jane

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in isonitrogenous feed formulations (30% protein) in the diet of Oreochromis niloticus fingerlings for a period of 12 weeks. The control diet had fishmeal as the primary protein ...

  4. Study of sperm proteins in different mammalian species

    OpenAIRE

    Pohlová, Alžběta

    2016-01-01

    Reproduction is an essential feature of all animals and a fundamental step to produce new generations. Study of sperm proteins is crucial for understanding of the sperm-egg recognition. We searched out sperm surface proteins involving in the zona pellucida (ZP) binding and studied whether these proteins are preserved throughout mammalian species. Indirect immunofluorescent technique was used to test a panel of monoclonal antibodies prepared against boar sperm surface proteins on spermatozoa o...

  5. Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

    Science.gov (United States)

    Rose, Christopher M.; Russell, Jason D.; Ledvina, Aaron R.; McAlister, Graeme C.; Westphall, Michael S.; Griep-Raming, Jens; Schwartz, Jae C.; Coon, Joshua J.; Syka, John E. P.

    2013-06-01

    We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion-ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell's longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

  6. Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells

    Directory of Open Access Journals (Sweden)

    Spyros Darmanis

    2016-01-01

    Full Text Available Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell’s phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

  7. Protein Laboratories in Single Location | Poster

    Science.gov (United States)

    By Andrew Stephen, Timothy Veenstra, and Gordon Whiteley, Guest Writers, and Ken Michaels, Staff Writer The Laboratory of Proteomics and Analytical Technologies (LPAT), Antibody Characterization Laboratory (ACL), and Protein Chemistry Laboratory (PCL), previously located on different floors or in different buildings, are now together on the first floor of C wing in the ATRF.

  8. Single channel analysis of membrane proteins in artificial bilayer membranes.

    Science.gov (United States)

    Bartsch, Philipp; Harsman, Anke; Wagner, Richard

    2013-01-01

    The planar lipid bilayer technique is a powerful experimental approach for electrical single channel recordings of pore-forming membrane proteins in a chemically well-defined and easily modifiable environment. Here we provide a general survey of the basic materials and procedures required to set up a robust bilayer system and perform electrophysiological single channel recordings of reconstituted proteins suitable for the in-depth characterization of their functional properties.

  9. Computational identification of strain-, species- and genus-specific proteins

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    Thiagarajan Rathi

    2005-11-01

    Full Text Available Abstract Background The identification of unique proteins at different taxonomic levels has both scientific and practical value. Strain-, species- and genus-specific proteins can provide insight into the criteria that define an organism and its relationship with close relatives. Such proteins can also serve as taxon-specific diagnostic targets. Description A pipeline using a combination of computational and manual analyses of BLAST results was developed to identify strain-, species-, and genus-specific proteins and to catalog the closest sequenced relative for each protein in a proteome. Proteins encoded by a given strain are preliminarily considered to be unique if BLAST, using a comprehensive protein database, fails to retrieve (with an e-value better than 0.001 any protein not encoded by the query strain, species or genus (for strain-, species- and genus-specific proteins respectively, or if BLAST, using the best hit as the query (reverse BLAST, does not retrieve the initial query protein. Results are manually inspected for homology if the initial query is retrieved in the reverse BLAST but is not the best hit. Sequences unlikely to retrieve homologs using the default BLOSUM62 matrix (usually short sequences are re-tested using the PAM30 matrix, thereby increasing the number of retrieved homologs and increasing the stringency of the search for unique proteins. The above protocol was used to examine several food- and water-borne pathogens. We find that the reverse BLAST step filters out about 22% of proteins with homologs that would otherwise be considered unique at the genus and species levels. Analysis of the annotations of unique proteins reveals that many are remnants of prophage proteins, or may be involved in virulence. The data generated from this study can be accessed and further evaluated from the CUPID (Core and Unique Protein Identification system web site (updated semi-annually at http://pir.georgetown.edu/cupid. Conclusion CUPID

  10. Species specificity for HBsAg binding protein endonexin II

    NARCIS (Netherlands)

    deBruin, WCC; Leenders, WPJ; Moshage, H; vanHaelst, UJGM

    Background/Aims: Hepatitis B virus displays a distinct species and tissue tropism, Previously we have demonstrated that a human liver plasma membrane protein,vith a molecular weight of approximately 34 kiloDalton specifically binds to HBsAg. This protein was identified as endonexin II, a Ca2+

  11. Extraction and characterisation of protein fractions from five insect species

    NARCIS (Netherlands)

    Yi, L.; Lakemond, C.M.M.; Sagis, L.M.C.; Eisner-Schadler, V.R.; Huis, van A.; Boekel, van M.A.J.S.

    2013-01-01

    Tenebrio molitor, Zophobas morio, Alphitobius diaperinus, Acheta domesticus and Blaptica dubia were evaluated for their potential as a future protein source. Crude protein content ranged from 19% to 22% (Dumas analysis). Essential amino acid levels in all insect species were comparable with soybean

  12. Protein Expression Analyses at the Single Cell Level

    Directory of Open Access Journals (Sweden)

    Masae Ohno

    2014-09-01

    Full Text Available The central dogma of molecular biology explains how genetic information is converted into its end product, proteins, which are responsible for the phenotypic state of the cell. Along with the protein type, the phenotypic state depends on the protein copy number. Therefore, quantification of the protein expression in a single cell is critical for quantitative characterization of the phenotypic states. Protein expression is typically a dynamic and stochastic phenomenon that cannot be well described by standard experimental methods. As an alternative, fluorescence imaging is being explored for the study of protein expression, because of its high sensitivity and high throughput. Here we review key recent progresses in fluorescence imaging-based methods and discuss their application to proteome analysis at the single cell level.

  13. Evaluation of yeast single cell protein (SCP) diets on growth ...

    African Journals Online (AJOL)

    An investigation was carried out on the possibility of replacing fishmeal with graded levels of yeast single cell protein (SCP; 10, 20, 30, 40 and 50%) in ... that the 50% yeast SCP fed fish had the highest percentage of body protein (55.35%), but with a lower amount of fat at the end of the feeding trial compared to the control.

  14. PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

    African Journals Online (AJOL)

    BSN

    customary food and feed sources of protein (agriculnrre and fishery) to ocher sources like single cell protein (SCP); whose production from hydrocarbons is one ... origin is unicellular or simple multicellular organism such as bacteria, yeasts, fungi, algae. protozoa, mid even bacterinphagcs generally cultivated on substrates ...

  15. Conversion of Food waste to Single Cell Protein using Aspergillus ...

    African Journals Online (AJOL)

    ADOWIE PERE

    2018-03-13

    Mar 13, 2018 ... ABSTRACT: The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage ... as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard techniques. ..... Waste to Wealth- Value Recovery from. Agrofood.

  16. Conversion of Food waste to Single Cell Protein using Aspergillus ...

    African Journals Online (AJOL)

    The utilization of food waste into products like single cell protein is an alternative solution to global protein shortage and to alleviate pollution problems. This investigation was carried out with food wastes such as orange, pineapple, banana, watermelon and cucumber waste as growth media for A. niger using standard ...

  17. PRODt;CTION OF SINGLE CELL PROTEIN FROM BREWERY ...

    African Journals Online (AJOL)

    BSN

    The production of single cell protein (SCP) by the propagation of the yeast, Saccharomyces cerevisae ... animal feed but little or no information has been documented as per its explication for the production of single cell .... use of yeasts produced from vatious carbohydrate sources, molasses, sulphite liquors and vegetable.

  18. Reactive species formed on proteins exposed to singlet oxygen

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2004-01-01

    hydroperoxides, which can be reduced to the corresponding alcohols; other products arising from radical intermediates can also be generated, particularly in the presence of UV light and metal ions. With His side-chains, poorly characterised peroxides are also formed. Reaction with Met and Cys has been proposed...... to occur via zwitterionic peroxy intermediates. Peroxides are also generated on isolated proteins, and protein within intact cells, via(1)O(2)-mediated reactions. The peroxides formed on Trp, Tyr, and His peptides, as well as on proteins, have been shown to induce damage to other targets, with molecular...... oxidation of thiol residues an important reaction. This can result in the inactivation of cellular enzymes and the oxidation of other biological targets. Protein cross-linking and aggregation can also be induced by reactive species formed on photo-oxidised proteins, though the nature of the species...

  19. Single-species versus dual-species probiotic supplementation as an emerging therapeutic strategy for obesity.

    Science.gov (United States)

    Karimi, G; Jamaluddin, R; Mohtarrudin, N; Ahmad, Z; Khazaai, H; Parvaneh, M

    2017-10-01

    Recent studies have reported beneficial effects of specific probiotics on obesity. However, the difference in the anti-obesity effects of probiotics as single species and dual species is still uncertain. Therefore, we aimed to compare the efficacy of single and dual species of bacteria on markers of obesity in high-fat diet-induced obese rats. A total of 40 male Sprague-Dawley rats were assigned to one of five groups of varying diets as follows: standard diet, high fat diet (HFD), HFD supplemented with Lactobacillus casei strain Shirota, HFD supplemented with Bifidobacterium longum and HFD supplemented with a mixture of these two bacterial species. After 15 weeks of supplementation, the animals were examined for changes in body weight, body fat, total count of bacteria in fecal, blood serum lipid profile, leptin, adiponectin and inflammatory biomarkers. Histological analysis of the liver and adipose tissue was performed and the hepatic mRNA expression levels of genes related to lipid metabolism were measured. It was found that probiotic supplementation of either B. longum or a mixture of B. longum and LcS bacteria significantly reduced weight and triglycerides in the HFD groups. Supplementation of B. longum bacteria showed better results in terms of modulating leptin level, fat mass, adipocyte size and lipoprotein lipase expression, as well as increasing adiponectin and peroxisome proliferator-activated receptors-γ expression compared to dual species of bacteria. No significant differences were observed in the total count of fecal bacteria, glucose and inflammatory biomarker levels between supplemented groups. B. longum supplementation in obesity was more beneficial in metabolic profile changes than the mixture species. Copyright © 2017 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B

  20. Hydrophobic Interaction Chromatography for Bottom-Up Proteomics Analysis of Single Proteins and Protein Complexes.

    Science.gov (United States)

    Rackiewicz, Michal; Große-Hovest, Ludger; Alpert, Andrew J; Zarei, Mostafa; Dengjel, Jörn

    2017-06-02

    Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug-protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC-MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC-reversed-phase chromatography (RPC)-mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties.

  1. Study of total seed storage protein in indigenous Brassica species ...

    African Journals Online (AJOL)

    Genetic diversity was studied in 234 accessions of locally collected Brassica species for total seed protein content through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These accessions were collected from different locations of Pakistan. After the study of these accessions on SDS-PAGE, ...

  2. Study of total seed storage protein in indigenous Brassica species ...

    African Journals Online (AJOL)

    hope&shola

    2010-11-08

    Nov 8, 2010 ... presented in Figures 1, 2 and 3 showed close relation- ship among these studied accessions, while the diffe- .... Dendrogram showing the relationship between different protein types in indigenous brassica species based on ... Bangladesh, Japan and China. Similarly, Munazza et al. (2009) evaluated 30 ...

  3. Generation of Fluorescent Protein Fusions in Candida Species.

    Science.gov (United States)

    Gonia, Sara; Berman, Judith; Gale, Cheryl A

    2017-03-04

    Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

  4. Dynamic protein assemblies in homologous recombination with single DNA molecules

    NARCIS (Netherlands)

    van der Heijden, A.H.

    2007-01-01

    What happens when your DNA breaks? This thesis describes experimental work on the single-molecule level focusing on the interaction between DNA and DNA-repair proteins, in particular bacterial RecA and human Rad51, involved in homologous recombination. Homologous recombination and its central event

  5. Identification of animal species by protein radioimmunoassay of bone fragments and bloodstained stone tools.

    Science.gov (United States)

    Lowenstein, Jerold M; Reuther, Joshua D; Hood, Darden G; Scheuenstuhl, Gary; Gerlach, S Craig; Ubelaker, Douglas H

    2006-06-02

    In forensics and archaeology, it is important to distinguish human from animal remains and to identify animal species from fragmentary bones and bloodstains. We report blind tests in which a protein radioimmunoassay (pRIA) was used to identify the species of six bone fragments lacking morphological specificity and 43 bloodstained lithic tools, knapped experimentally and soaked in blood of known animal and human origin. The submitters of the bone fragments and the bloodstained tools each listed a number of possible species, from which the testers selected the best match with the pRIA results. All six bone fragments were correctly identified: three humans, a deer, a dog, and a cow. Forty-three tools were stained with blood from a wide variety of species including ungulates, carnivores, a fish, and a bird. On 40 of these 43, at least one species (or blood-free control) was identified correctly. Some of the tools were stained with blood of two different species. A mixture of sheep and musk ox blood was correctly identified; in several other mixtures, only a single species was detected. Two tools with human blood and one with human sweat were correctly reported as human. There was a single false positive (one of three controls reported as weakly bovine) and no false negatives. We conclude that the pRIA technique shows a high degree of accuracy in discriminating human from animal bone fragments and bloodstains and in identifying animal species.

  6. Leaf Protein Electrophoresis and Taxonomy of Species of Jatropha L. (Euphorbiaceae

    Directory of Open Access Journals (Sweden)

    Olaniran Temitope OLADIPO

    2012-08-01

    Full Text Available The systematic relationship existing among members of the all important genus Jatropha was studied using leaf protein electrophoresis. The aim was to identify possible taxonomic importance of the protein profile in the estimation and elucidation of the taxonomic affinity of the six species of Jatropha (Jatropha curcas Linn., J. podagrica Hook., J. gossypifolia Linn., J. mutifida Linn., J. tanjorensis Ellis & Saroja and J. integerrima Linn. found in Nigeria. The species were screened for total protein banding patterns using gel electrophoresis. Young leaves (0.8 g of the plants were washed with distilled water and macerated with sterile mortar and pestle in 0.8% Phosphate Buffer-Saline (PBS containing 0.4 M NaCl at pH 8.0. Results reveal that protein banding pattern was taxon specific. Generic band occurs at 8.3. The highest number of interspecific bands (4 exists between J. podagrica and J. multifida. Variations exist not only in the number of bands but also in the intensity of the bands. Sokal and Sneath coefficient of similarity ranges between 11.1-44.4 %. Single linkage Cluster Analysis (SLCA of the relative mobility values of the protein in the taxa shows partial agreement with current sub generic and sectional delimitation of the species based on morphology and anatomy of the species.

  7. Amino acid profiles and presumptive nutritional assessment of single-cell protein from certain lactobacilli.

    Science.gov (United States)

    Erdman, M D; Bergen, W G; Reddy, C A

    1977-04-01

    The amino acid profiles, modified essential amino acid (MEAA) indexes, and in vitro pepsin digestibilities were determined for single-cell protein (SCP) from certain industrially important lactobacilli. For the three parameters examined, substantial differences were seen between different Lactobacillus species and between strains with a given species. SCP from all of the lactobacilli examined appeared relatively high in MEAA indexes and pepsin digestibility. SCP from L. acidophilus 3205 and L. fermenti 3954 had the highest MEAA indexes, whereas L. bulgaricus 2217 and L. thermophilus 3863 had the highest percentage of digestible crude protein. SCP from L. plantarum strains had the lowest MEAA indexes. The essential amino acid compositions of SCP from different lactobacilli appear comparable to that of Food and Agriculture Organization reference protein and SCP from other sources.

  8. Single Protein Molecule Mapping with Magnetic Atomic Force Microscopy

    Science.gov (United States)

    Moskalenko, Andriy V.; Yarova, Polina L.; Gordeev, Sergey N.; Smirnov, Sergey V.

    2010-01-01

    Abstract Understanding the structural organization and distribution of proteins in biological cells is of fundamental importance in biomedical research. The use of conventional fluorescent microscopy for this purpose is limited due to its relatively low spatial resolution compared to the size of a single protein molecule. Atomic force microscopy (AFM), on the other hand, allows one to achieve single-protein resolution by scanning the cell surface using a specialized ligand-coated AFM tip. However, because this method relies on short-range interactions, it is limited to the detection of binding sites that are directly accessible to the AFM tip. We developed a method based on magnetic (long-range) interactions and applied it to investigate the structural organization and distribution of endothelin receptors on the surface of smooth muscle cells. Endothelin receptors were labeled with 50-nm superparamagnetic microbeads and then imaged with magnetic AFM. Considering its high spatial resolution and ability to “see” magnetically labeled proteins at a distance of up to 150 nm, this approach may become an important tool for investigating the dynamics of individual proteins both on the cell membrane and in the submembrane space. PMID:20141762

  9. Reproductive protein evolution in two cryptic species of marine chordate

    Directory of Open Access Journals (Sweden)

    Harrison Richard G

    2011-01-01

    Full Text Available Abstract Background Reproductive character displacement (RCD is a common and taxonomically widespread pattern. In marine broadcast spawning organisms, behavioral and mechanical isolation are absent and prezygotic barriers between species often operate only during the fertilization process. Such barriers are usually a consequence of differences in the way in which sperm and egg proteins interact, so RCD can be manifest as faster evolution of these proteins between species in sympatry than allopatry. Rapid evolution of these proteins often appears to be a consequence of positive (directional selection. Here, we identify a set of candidate gamete recognition proteins (GRPs in the ascidian Ciona intestinalis and showed that these GRPs evolve more rapidly than control proteins (those not involved in gamete recognition. Choosing a subset of these gamete recognition proteins that show evidence of positive selection (CIPRO37.40.1, CIPRO60.5.1, CIPRO100.7.1, we then directly test the RCD hypothesis by comparing divergence (omega and polymorphism (McDonald-Kreitman, Tajima's D, Fu and Li's D and F, Fay and Wu's H statistics in sympatric and allopatric populations of two distinct forms of C. intestinalis (Types A and B between which there are strong post-zygotic barriers. Results Candidate gamete recognition proteins from two lineages of C. intestinalis (Type A and B are evolving more rapidly than control proteins, consistent with patterns seen in insects and mammals. However, ω (dN/dS is not significantly different between the sympatric and allopatric populations, and none of the polymorphism statistics show significant differences between sympatric and allopatric populations. Conclusions Enhanced prezygotic isolation in sympatry has become a well-known feature of gamete recognition proteins in marine broadcast spawners. But in most cases the evolutionary process or processes responsible for this pattern have not been identified. Although gamete

  10. COMPARATIVE PRODUCTION OF SINGLE CELL PROTEIN FROM FISH PROTEIN ISOLATE WASTAGE AND ULTRA FILTERED CHEESE WHEY

    Directory of Open Access Journals (Sweden)

    Soroush Haghighi-Manesh

    2013-02-01

    Full Text Available Fish protein isolate wastage and ultra filtered cheese whey were used as substrates for fermentation by Kluyveromyces marxianus to produce single cell protein, under batch and aerobic condition in which pH and temperature were adjusted to 4.5 and 35°C. The produced biomass was analyzed for protein content in different periods of time during fermentation. About 82% and 75% of total protein was produced in the first 18 h of 96 h fermentation of ultra filtered cheese whey and protein isolate wastage respectively, which can be an indication of the exponential phase of the yeast growth. The results of biomass yield measurements during 96 h process also confirm this finding. Moreover, since ultra filtered cheese whey was higher in single cell protein yield, solubility, water holding capacity, water absorption and power of biological and chemical oxygen demand reduction, and also was lower in foam overrun and stability than fish protein isolate wastage, it was selected as the suitable substrate for single cell protein production.

  11. Induction of prophage lambda by chlorinated organics: Detection of some single-species/single-site carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    DeMarini, D.M.; Brooks, H.G. (Environmental Protection Agency, Research Triangle Park, NC (United States))

    1992-01-01

    Twenty-eight chlorinated organic compounds were evaluated for their ability to induce DNA damage using the Microscreen prophage-induction assay in Escherichia coli. Comparison of the performance characteristics of the prophage-induction and Salmonella assays to rodent carcinogenicity assays showed that the prophage-induction assay had a somewhat higher specificity than did the Salmonella assay (70% vs. 50%); sensitivity, concordance, and positive and negative predictivity were similar for the two microbial assays. The Microscreen prophage-induction assay failed to detect eight carcinogens, perhaps due to toxicity or other unknown factors; five of these eight carcinogens were detected by the Salmonella assay. However, the prophage-induction assay did detect six carcinogens that were not detected by the Salmonella assay, and five of these were single-species, single-site carcinogens, mostly mouse liver carcinogens. Some of these carcinogens, such as the chloroethanes, produce free radicals, which may be the basis for their carcinogenicity and ability to induce prophage. The prophage-induction (or other SOS) assay may be useful in identifying some genotoxic chlorinated carcinogens that induce DNA damage that do not revert the standard Salmonella tester strains.

  12. Evaluation of single cell protein for nutrition of farm animals

    Energy Technology Data Exchange (ETDEWEB)

    Oslage, H.J.; Schulz, E.

    1981-08-01

    For the production of microorganisms with high content of protein technologies on the basis of carbon rich substrates have been developed during the past years. Thus, signification of Single Cell Protein (SCP) for nutrition of farm animals has changed. While, in former times, yeasts were added only in small portions (1-2%) as vitamin supplementation today it is the aim to use microbial biomass as a protein component. The use of SCP as a feedstuff requires a careful physiological and toxicological evaluation as well as extensive investigations of possible use and frontiers of those products for farm animals. Topic of this work were bacteria, bred on methanol as well as yeasts, grown on alcanes and on whey/lactic acid respectively. SCP is preferently used as a feedstuff for poultry, pigs, calves and fishes. Digestibility and utilisation of protein is good till very good, for the a.m. animals, digestibility being between 75-93% and net protein utilisation (NPU) being between 60-76%. In rations of young animals (chicken, piglets and calves) contents of 5-10% SCP have been proved to be without any negative effect on acceptance, body gain, feed utilisation and mortality. For older animals SCP can be used as the only protein source beside the basic feedstuffs.

  13. Single Molecule Spectroscopy on Photosynthetic Pigment-Protein Complexes

    CERN Document Server

    Jelezko, F; Schuler, S; Thews, E; Tietz, C; Wechsler, A; Wrachtrup, J

    2001-01-01

    Single molecule spectroscopy was applied to unravel the energy transfer pathway in photosynthetic pigment-protein complexes. Detailed analysis of excitation and fluorescence emission spectra has been made for peripheral plant antenna LHC II and Photosystem I from cyanobacterium Synechococcus elongatus. Optical transitions of individual pigments were resolved under nonselective excitation of antenna chlorophylls. High-resolution fluorescence spectroscopy of individual plant antenna LHC II indicates that at low temperatures, the excitation energy is localized on the red-most Chl a pool absorbing at 680 nm. More than one pigment molecule is responsible for the fluorescence emission of the LHC II trimer. The spectral lines of single Chl a molecules absorbing at 675 nm are broadened because of the Foerster energy transfer towards the red-most pigments. Low-temperature spectroscopy on single PS I trimers indicates that two subgroups of pigments, which are present in the red antenna pool, differ by the strength of t...

  14. Single-molecule mechanics of protein-labelled DNA handles

    Directory of Open Access Journals (Sweden)

    Vivek S. Jadhav

    2016-01-01

    Full Text Available DNA handles are often used as spacers and linkers in single-molecule experiments to isolate and tether RNAs, proteins, enzymes and ribozymes, amongst other biomolecules, between surface-modified beads for nanomechanical investigations. Custom DNA handles with varying lengths and chemical end-modifications are readily and reliably synthesized en masse, enabling force spectroscopic measurements with well-defined and long-lasting mechanical characteristics under physiological conditions over a large range of applied forces. Although these chemically tagged DNA handles are widely used, their further individual modification with protein receptors is less common and would allow for additional flexibility in grabbing biomolecules for mechanical measurements. In-depth information on reliable protocols for the synthesis of these DNA–protein hybrids and on their mechanical characteristics under varying physiological conditions are lacking in literature. Here, optical tweezers are used to investigate different protein-labelled DNA handles in a microfluidic environment under different physiological conditions. Digoxigenin (DIG-dsDNA-biotin handles of varying sizes (1000, 3034 and 4056 bp were conjugated with streptavidin or neutravidin proteins. The DIG-modified ends of these hybrids were bound to surface-modified polystyrene (anti-DIG beads. Using different physiological buffers, optical force measurements showed consistent mechanical characteristics with long dissociation times. These protein-modified DNA hybrids were also interconnected in situ with other tethered biotinylated DNA molecules. Electron-multiplying CCD (EMCCD imaging control experiments revealed that quantum dot–streptavidin conjugates at the end of DNA handles remain freely accessible. The experiments presented here demonstrate that handles produced with our protein–DNA labelling procedure are excellent candidates for grasping single molecules exposing tags suitable for molecular

  15. Reconstructing the phylogeny of 21 completely sequenced arthropod species based on their motor proteins

    Directory of Open Access Journals (Sweden)

    Kollmar Martin

    2009-04-01

    Full Text Available Abstract Background Motor proteins have extensively been studied in the past and consist of large superfamilies. They are involved in diverse processes like cell division, cellular transport, neuronal transport processes, or muscle contraction, to name a few. Vertebrates contain up to 60 myosins and about the same number of kinesins that are spread over more than a dozen distinct classes. Results Here, we present the comparative genomic analysis of the motor protein repertoire of 21 completely sequenced arthropod species using the owl limpet Lottia gigantea as outgroup. Arthropods contain up to 17 myosins grouped into 13 classes. The myosins are in almost all cases clear paralogs, and thus the evolution of the arthropod myosin inventory is mainly determined by gene losses. Arthropod species contain up to 29 kinesins spread over 13 classes. In contrast to the myosins, the evolution of the arthropod kinesin inventory is not only determined by gene losses but also by many subtaxon-specific and species-specific gene duplications. All arthropods contain each of the subunits of the cytoplasmic dynein/dynactin complex. Except for the dynein light chains and the p150 dynactin subunit they contain single gene copies of the other subunits. Especially the roadblock light chain repertoire is very species-specific. Conclusion All 21 completely sequenced arthropods, including the twelve sequenced Drosophila species, contain a species-specific set of motor proteins. The phylogenetic analysis of all genes as well as the protein repertoire placed Daphnia pulex closest to the root of the Arthropoda. The louse Pediculus humanus corporis is the closest relative to Daphnia followed by the group of the honeybee Apis mellifera and the jewel wasp Nasonia vitripennis. After this group the rust-red flour beetle Tribolium castaneum and the silkworm Bombyx mori diverged very closely from the lineage leading to the Drosophila species.

  16. Reconstructing the phylogeny of 21 completely sequenced arthropod species based on their motor proteins

    Science.gov (United States)

    Odronitz, Florian; Becker, Sebastian; Kollmar, Martin

    2009-01-01

    Background Motor proteins have extensively been studied in the past and consist of large superfamilies. They are involved in diverse processes like cell division, cellular transport, neuronal transport processes, or muscle contraction, to name a few. Vertebrates contain up to 60 myosins and about the same number of kinesins that are spread over more than a dozen distinct classes. Results Here, we present the comparative genomic analysis of the motor protein repertoire of 21 completely sequenced arthropod species using the owl limpet Lottia gigantea as outgroup. Arthropods contain up to 17 myosins grouped into 13 classes. The myosins are in almost all cases clear paralogs, and thus the evolution of the arthropod myosin inventory is mainly determined by gene losses. Arthropod species contain up to 29 kinesins spread over 13 classes. In contrast to the myosins, the evolution of the arthropod kinesin inventory is not only determined by gene losses but also by many subtaxon-specific and species-specific gene duplications. All arthropods contain each of the subunits of the cytoplasmic dynein/dynactin complex. Except for the dynein light chains and the p150 dynactin subunit they contain single gene copies of the other subunits. Especially the roadblock light chain repertoire is very species-specific. Conclusion All 21 completely sequenced arthropods, including the twelve sequenced Drosophila species, contain a species-specific set of motor proteins. The phylogenetic analysis of all genes as well as the protein repertoire placed Daphnia pulex closest to the root of the Arthropoda. The louse Pediculus humanus corporis is the closest relative to Daphnia followed by the group of the honeybee Apis mellifera and the jewel wasp Nasonia vitripennis. After this group the rust-red flour beetle Tribolium castaneum and the silkworm Bombyx mori diverged very closely from the lineage leading to the Drosophila species. PMID:19383156

  17. From Protein Structure to Function via Single Crystal Optical Spectroscopy

    Directory of Open Access Journals (Sweden)

    Luca eRonda

    2015-04-01

    Full Text Available The more than 100.000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic artifacts, including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density map, thus limiting the relevance of structure determinations. Moreover, for most of these structures no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in the inference for protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5’-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms.

  18. Comparative Transcriptomic Analyses of Three Species of Placobdella (Rhynchobdellida: Glossiphoniidae) Confirms a Single Origin of Blood Feeding in Leeches.

    Science.gov (United States)

    Siddall, Mark E; Brugler, Mercer R; Kvist, Sebastian

    2016-02-01

    One of the recalcitrant questions regarding the evolutionary history of clitellate annelids involves the feeding preference of the common ancestor of extant rhynchobdellid (proboscis bearing) and arhynchobdellid (jaw bearing) leeches. Whereas early evidence, based on morphological data, pointed towards independent acquisitions of blood feeding in the 2 orders, molecular-based phylogenetic data suggest that the ancestor of modern leeches was a sanguivore. Here, we use a comparative transcriptomic approach in order to increase our understanding of the diversity of anticoagulation factors for 3 species of the genus Placobdella, for which comparative data have been lacking, and inspect these in light of archetypal anticoagulant data for both arhynchobdellid and other rhynchobdellid species. Notwithstanding the varying levels of host specificity displayed by the 3 different species of Placobdella, transcriptomic profiles with respect to anticoagulation factors were largely similar -this despite the fact that Placobdella kwetlumye only retains a single pair of salivary glands, as opposed to the 2 pairs more common in the genus. Results show that 9 different anticoagulant proteins and an additional 5 putative antihemostasis proteins are expressed in salivary secretions of the 3 species. In particular, an ortholog of the archetypal, single-copy, anticoagulant hirudin (not previously available as comparative data for rhynchobdellids) is present in at least 2 of 3 species examined, corroborating the notion of a single origin of blood feeding in the ancestral leech.

  19. Tetramethylammonium-filled protein nanopore for single-molecule analysis.

    Science.gov (United States)

    Wang, Ying; Yao, Fujun; Kang, Xiao-feng

    2015-10-06

    Nanopore technology, as the simplest and most inexpensive single-molecule tool, is being intensively developed. In nanopore stochastic sensing, KCl and NaCl have traditionally been employed as pore-filled electrolytes for recording the change of ion conductance in nanopores triggered by analyte translocation through the pore. However, some challenges limit its further advance. Here we used tetramethylammonium (TMA) chloride, instead of KCl, as a novel analysis system for nanopores. Some unique nanopore characteristics were observed: (1) The stability of the planar lipid bilayer for embedding the protein pores was elevated at least 6 times. (2) The TMA-Cl system could effectively reduce the noise of single-channel recording. (3) It was easy to control the insertion of protein pores into the lipid bilayer, and the formed single nanopore could last for a sufficiently long time. (4) TMA-Cl could be used as a DNA speed bump in the nanopore to slow DNA translocation speed. (5) The capacity of the nanopore capture of DNA (capture rate) increased significantly and simultaneously increased the translocation time of DNA in the pore. (6) The TMA-filled nanopore could discriminate between various polynucleotides.

  20. Sca1, a previously undescribed paralog from autotransporter protein-encoding genes in Rickettsia species

    Directory of Open Access Journals (Sweden)

    Raoult Didier

    2006-02-01

    , and might therefore be functional in this species. Conclusion The sca1 gene, encoding an autotransporter protein that evolves under dual evolution pressure, is the only sca-family gene to be conserved by all Rickettsia species. As such, it is a valuable identification target for these bacteria, especially because rickettsial isolates can be identified by amplification and sequencing of a discriminatory gene fragment using a single primer pair. It may also be used as a phylogenetic tool. However, its current functional status remains to be determined although it was found expressed in R. conorii.

  1. Tannin-binding salivary proteins in three captive rhinoceros species.

    Science.gov (United States)

    Clauss, Marcus; Gehrke, Janin; Hatt, Jean-Michel; Dierenfeld, Ellen S; Flach, Edmund J; Hermes, Robert; Castell, Johanna; Streich, W Juergen; Fickel, Joerns

    2005-01-01

    Tannin-binding salivary proteins (TBSP) are considered to be counter-defences acquired in the course of evolution by animals whose natural forage contains such tannins. As tannins mostly occur in browse material but not in grasses, it is assumed that grazers do not have a need for TBSP. Whereas it has been shown in several non-ungulate species that TBSP can be induced by dietary tannins, their presence or absence in ungulates has, so far, been shown to be a species-specific characteristic independent of dietary manipulations. We investigated saliva from three rhinoceros species from zoological gardens fed comparable, conventional zoo diets. As expected, saliva from white rhinoceroses (Ceratotherum simum, grazer) had lower tannin-binding capacities than that from black rhinoceroses (Diceros bicornis, browser). Surprisingly, however, Indian rhinoceroses (Rhinoceros unicornis), commonly regarded as grazers as well, displayed the highest tannin-binding capacities of the three species investigated. It is speculated that this discrepancy might be a result of an evolutionarily recent switch to a grass-dominated diet in Indian rhinoceroses, and that the black rhinoceros, which is closer related to the white rhinoceros than the Indian species, has evolved an inducible mechanism of TBSP production. In separate trials during which the tannin content of the diets of black rhinoceroses was increased by the addition of either tannic acid or quebracho, the tannin-binding capacity of black rhinoceros saliva was increased to levels within the same range as that of Indian rhinoceroses on the conventional diets. While induction trials in white and Indian rhinoceroses remain to be performed for a full understanding of salivary anti-tannin defence in rhinoceroses, these results are the first report of an induced salivary response to increased dietary tannin levels in an ungulate species.

  2. Interaction of DNA and Proteins with Single Nanopores

    Science.gov (United States)

    Kasianowicz, J. J.

    2006-03-01

    The bacterial toxins Staphylococcus aureus alpha-hemolysin and Bacillus anthracis protective antigen kill cells in part by forming ion channels in target membranes. We are using electrophysiology, molecular biology/protein biochemistry and computer modeling to study how biopolymers (e.g., single-stranded DNA and proteins) bind to and transport through these nanometer-scale pores. The results provide insight into the mechanism by which these toxins work and are the basis for several potential nanobiotechnology applications including ultra-rapid DNA sequencing, the sensitive and selective detection of a wide range of analytes and high throughput screening of therapeutic agents against several anthrax toxins. In collaboration with V.M. Stanford, M. Misakian, B. Nablo, S.E. Henrickson, NIST, EEEL, Gaithersburg, MD; T. Nguyen, R. Gussio, NCI, Ft. Detrick, MD; and K.M. Halverson, S. Bavari, R.G. Panchal, USAMRIID, Ft. Detrick, MD.

  3. A structurally based analytic model of growth and biomass dynamics in single species stands of conifers

    Science.gov (United States)

    Robin J. Tausch

    2015-01-01

    A theoretically based analytic model of plant growth in single species conifer communities based on the species fully occupying a site and fully using the site resources is introduced. Model derivations result in a single equation simultaneously describes changes over both, different site conditions (or resources available), and over time for each variable for each...

  4. Single methyl groups can act as toggle switches to specify transmembrane protein-protein interactions

    DEFF Research Database (Denmark)

    He, Li; Steinocher, Helena; Shelar, Ashish

    2017-01-01

    of leucine and isoleucine (called LIL traptamers) that specifically activate the erythropoietin receptor (EPOR) in mouse cells to confer growth factor independence. We discovered that the placement of a single side chain methyl group at specific positions in a traptamer determined whether it associated......Transmembrane domains (TMDs) engage in protein-protein interactions that regulate many cellular processes, but the rules governing the specificity of these interactions are poorly understood. To discover these principles, we analyzed 26-residue model transmembrane proteins consisting exclusively...... productively with the TMD of the human EPOR, the mouse EPOR, or both receptors. Association of the traptamers with the EPOR induced EPOR oligomerization in an orientation that stimulated receptor activity. These results highlight the high intrinsic specificity of TMD interactions, demonstrate that a single...

  5. Intra-Species Bacterial Quorum Sensing Studied at Single Cell Level in a Double Droplet Trapping System

    Directory of Open Access Journals (Sweden)

    Wilhelm T. S. Huck

    2013-05-01

    Full Text Available In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl-L-homoserine lactone (OdDHL. Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

  6. Species distribution modelling for plant communities: Stacked single species or multivariate modelling approaches?

    Science.gov (United States)

    Emilie B. Henderson; Janet L. Ohmann; Matthew J. Gregory; Heather M. Roberts; Harold S.J. Zald

    2014-01-01

    Landscape management and conservation planning require maps of vegetation composition and structure over large regions. Species distribution models (SDMs) are often used for individual species, but projects mapping multiple species are rarer. We compare maps of plant community composition assembled by stacking results from many SDMs with multivariate maps constructed...

  7. Protein Data Bank (PDB): The Single Global Macromolecular Structure Archive.

    Science.gov (United States)

    Burley, Stephen K; Berman, Helen M; Kleywegt, Gerard J; Markley, John L; Nakamura, Haruki; Velankar, Sameer

    2017-01-01

    The Protein Data Bank (PDB)--the single global repository of experimentally determined 3D structures of biological macromolecules and their complexes--was established in 1971, becoming the first open-access digital resource in the biological sciences. The PDB archive currently houses ~130,000 entries (May 2017). It is managed by the Worldwide Protein Data Bank organization (wwPDB; wwpdb.org), which includes the RCSB Protein Data Bank (RCSB PDB; rcsb.org), the Protein Data Bank Japan (PDBj; pdbj.org), the Protein Data Bank in Europe (PDBe; pdbe.org), and BioMagResBank (BMRB; www.bmrb.wisc.edu). The four wwPDB partners operate a unified global software system that enforces community-agreed data standards and supports data Deposition, Biocuration, and Validation of ~11,000 new PDB entries annually (deposit.wwpdb.org). The RCSB PDB currently acts as the archive keeper, ensuring disaster recovery of PDB data and coordinating weekly updates. wwPDB partners disseminate the same archival data from multiple FTP sites, while operating complementary websites that provide their own views of PDB data with selected value-added information and links to related data resources. At present, the PDB archives experimental data, associated metadata, and 3D-atomic level structural models derived from three well-established methods: crystallography, nuclear magnetic resonance spectroscopy (NMR), and electron microscopy (3DEM). wwPDB partners are working closely with experts in related experimental areas (small-angle scattering, chemical cross-linking/mass spectrometry, Forster energy resonance transfer or FRET, etc.) to establish a federation of data resources that will support sustainable archiving and validation of 3D structural models and experimental data derived from integrative or hybrid methods.

  8. Characterization of TPP-binding proteins in Methanococci archaeal species.

    Science.gov (United States)

    Harris, Laura K

    2016-01-01

    Acetolactate synthase (ALS) is a highly conserved protein family responsible for producing branched chain amino acids. In Methanocaldococcus jannaschii, two ALS proteins, MJ0277 and MJ0663 exist though variations in features between them are noted. Researchers are quick to examine MJ0277 homologs due to their increased function and close relationship, but few have characterized MJ0663 homologs. This study identified homologs for both MJ0277 and MJ0663 in all 15 Methanococci species with fully sequenced genomes. EggNOG database does not define four of the MJ0663 homologs, JH146_1236, WP_004591614, WP_018154400, and EHP89635. BLASTP comparisons suggest these four proteins had around 30% identity to MJ0277 homologs, close to the identity similarities between other MJ0663 homologs to the MJ0277 homologous group. ExPASY physiochemical characterization shows a statistically significant difference in molecular weight and grand average hydropathy between homologous groups. CDD-BLAST showed distinct domains between homologous groups. MJ0277 homologs had TPP_AHAS and PRL06276 while MJ0663 homologs had TPP_enzymes super family and IlvB domains instead. Multiple sequence alignment using PROMALS3D showed the MJ0277 homologs a tighter group than MJ0663 and its homologs. PHYLIP showed these homologous groups as evolutionarily distinct yet equal distance from bacterial ALS proteins of established structure. The four proteins EggNOG did not define had the same features as other MJ0663 homologs. This indicates that JH146_1236, WP_004591614, WP_018154400, and EHP89635, should be included in EggNOG database cluster arCOG02000 with the other MJ0663 homologs.

  9. Mechanisms of Formation and Structure of Chromophores of Fluorescent Proteins from Anthoza Species

    National Research Council Canada - National Science Library

    Lukyanov, Serguei A

    2005-01-01

    ...: The whole array of fluorescent proteins from Anthozoa species were described over the past few years These proteins represent the Green Fluorescent Protein-like family according to their homology...

  10. Single-molecule studies of unconventional motor protein myosin VI

    Science.gov (United States)

    Kim, HyeongJun

    Myosin VI is one of the myosin superfamily members that are actin-based molecular motors. It has received special attention due to its distinct features as compared to other myosins, such as its opposite directionality and a much larger step size than expected given the length of its "leg". This dissertation presents the author.s graduate work of several single-molecule studies on myosin VI. Special attention was paid to some of myosin VI.s tail domains that consist of proximal tail (PT), medial tail (MT), distal tail (DT) domains and cargo-binding domain (CBD). The functional form of myosin VI in cells is still under debate. Although full length myosin VI proteins in cytosolic extracts of cells were monomers from earlier studies, there are several reasons why it is now believed that myosin VI could exist as a dimer. If this is true and dimerization occurs, the next logical question would be which parts of myosin VI are dimerization regions? One model claimed that the CBD is the sole dimerization region. A competing model claimed that there must be another region that could be involved in dimerization, based on their observation that a construct without the CBD could still dimerize. Our single-molecule experiment with progressively truncated myosin VI constructs showed that the MT domain is a dimerization region, supporting the latter model. Additional single-molecule experiments and molecular dynamics (MD) simulation done with our collaborators suggest that electrostatic salt bridges formed between positive and negative amino acid residues are mainly responsible for the MT domain dimerization. After resolving this, we are left with another important question which is how myosin VI can take such a large step. Recent crystal structure showed that one of the tail domains preceding the MT domain, called the PT domain, is a three-helix bundle. The most easily conceivable way might be an unfolding of the three-helix bundle upon dimerization, allowing the protein to

  11. Single- and multi-photon ionization studies of organosulfur species

    Energy Technology Data Exchange (ETDEWEB)

    Cheung, Yu -San [Iowa State Univ., Ames, IA (United States)

    1999-02-12

    Accurate ionization energies (IE`s) for molecular species are used for prediction of chemical reactivity and are of fundamental importance to chemists. The IE of a gaseous molecule can be determined routinely in a photoionization or a photoelectron experiment. IE determinations made in conventional photoionization and photoelectron studies have uncertainties in the range of 3--100 meV (25--250 cm-1). In the past decade, the most exciting development in the field of photoionization and photoelectron spectroscopy has been the availability of high resolution, tunable ultraviolet (UV) and vacuum ultraviolet (VUV) laser sources. The laser pulsed field ionization photoelectron (PFI-PE) scheme is currently the state-of-the-art photoelectron spectroscopic technique and is capable of providing photoelectron energy resolution close to the optical resolution. The author has focused attention on the photoionization processes of some sulfur-containing species. The studies of the photoionization and photodissociation on sulfur-containing compounds [such as CS2, CH3SH, CH3SSCH3, CH3CH2SCH2CH3, HSCH2CH2SH and C4H4S (thiophene) and sulfur-containing radicals, such as HS, CS, CH3S, CH3CH2S and CH3SS], have been the major subjects in the group because sulfur is an important species contributing to air pollution in the atmosphere. The modeling of the combustion and oxidation of sulfur compounds represents important steps for the control of both the production and the elimination of sulfur-containing pollutants. Chapter 1 is a general introduction of the thesis. Chapters 2 and 6 contain five papers published in, or accepted for publication in, academic periodicals. In Chapter 7, the progress of the construction in the laboratory of a new vacuum ultraviolet laser system equipped with a reflectron mass

  12. Single-cell analysis of G-protein signal transduction.

    Science.gov (United States)

    Clister, Terri; Mehta, Sohum; Zhang, Jin

    2015-03-13

    The growing use of fluorescent biosensors to directly probe the spatiotemporal dynamics of biochemical processes in living cells has revolutionized the study of intracellular signaling. In this review, we summarize recent developments in the use of biosensors to illuminate the molecular details of G-protein-coupled receptor (GPCR) signaling pathways, which have long served as the model for our understanding of signal transduction, while also offering our perspectives on the future of this exciting field. Specifically, we highlight several ways in which biosensor-based single-cell analyses are being used to unravel many of the enduring mysteries that surround these diverse signaling pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Cross-species Virus-host Protein-Protein Interactions Inhibiting Innate Immunity

    Science.gov (United States)

    2016-07-01

    joule (J) kiloton (kt) (TNT equivalent) 4.184 × 1012 joule (J) British thermal unit (Btu) (thermochemical) 1.054 350 × 10 3 joule (J) foot-pound...Orthomyxoviridae influenza A has an extensively tracked history of host species jumps, including birds, pigs and other mammals , and humans... biology , and protein chemistry, and he presented his research studies at the HWI-UB-RPCI Summer Undergraduate Biomedical Research Day (August 1, 2014

  14. Yersinia pestis Ail: multiple roles of a single protein

    Science.gov (United States)

    Kolodziejek, Anna M.; Hovde, Carolyn J.; Minnich, Scott A.

    2012-01-01

    Yersinia pestis is one of the most virulent bacteria identified. It is the causative agent of plague—a systemic disease that has claimed millions of human lives throughout history. Y. pestis survival in insect and mammalian host species requires fine-tuning to sense and respond to varying environmental cues. Multiple Y. pestis attributes participate in this process and contribute to its pathogenicity and highly efficient transmission between hosts. These include factors inherited from its enteric predecessors; Y. enterocolitica and Y. pseudotuberculosis, as well as phenotypes acquired or lost during Y. pestis speciation. Representatives of a large Enterobacteriaceae Ail/OmpX/PagC/Lom family of outer membrane proteins (OMPs) are found in the genomes of all pathogenic Yersiniae. This review describes the current knowledge regarding the role of Ail in Y. pestis pathogenesis and virulence. The pronounced role of Ail in the following areas are discussed (1) inhibition of the bactericidal properties of complement, (2) attachment and Yersinia outer proteins (Yop) delivery to host tissue, (3) prevention of PMNL recruitment to the lymph nodes, and (4) inhibition of the inflammatory response. Finally, Ail homologs in Y. enterocolitica and Y. pseudotuberculosis are compared to illustrate differences that may have contributed to the drastic bacterial lifestyle change that shifted Y. pestis from an enteric to a vector-born systemic pathogen. PMID:22919692

  15. Yersinia pestis Ail: multiple roles of a single protein.

    Science.gov (United States)

    Kolodziejek, Anna M; Hovde, Carolyn J; Minnich, Scott A

    2012-01-01

    Yersinia pestis is one of the most virulent bacteria identified. It is the causative agent of plague-a systemic disease that has claimed millions of human lives throughout history. Y. pestis survival in insect and mammalian host species requires fine-tuning to sense and respond to varying environmental cues. Multiple Y. pestis attributes participate in this process and contribute to its pathogenicity and highly efficient transmission between hosts. These include factors inherited from its enteric predecessors; Y. enterocolitica and Y. pseudotuberculosis, as well as phenotypes acquired or lost during Y. pestis speciation. Representatives of a large Enterobacteriaceae Ail/OmpX/PagC/Lom family of outer membrane proteins (OMPs) are found in the genomes of all pathogenic Yersiniae. This review describes the current knowledge regarding the role of Ail in Y. pestis pathogenesis and virulence. The pronounced role of Ail in the following areas are discussed (1) inhibition of the bactericidal properties of complement, (2) attachment and Yersinia outer proteins (Yop) delivery to host tissue, (3) prevention of PMNL recruitment to the lymph nodes, and (4) inhibition of the inflammatory response. Finally, Ail homologs in Y. enterocolitica and Y. pseudotuberculosis are compared to illustrate differences that may have contributed to the drastic bacterial lifestyle change that shifted Y. pestis from an enteric to a vector-born systemic pathogen.

  16. The E. coli Single Protein Production (cSPP) System for Production and Structural Analysis of Membrane Proteins

    OpenAIRE

    Mao, Lili; Vaiphei, S. Thangminlal; Shimazu, Tsutomu; Schneider, William M.; Tang, Yuefeng; Mani, Rajeswari; Roth, Monica J.; Montelione, Gaetano T.; Inouye, Masayori

    2009-01-01

    At present, only 0.9% of PDB-deposited structures are of membrane proteins in spite of the fact that membrane proteins constitute approximately 30% of total proteins in most genomes from bacteria to humans. Here we address some of the major bottlenecks in the structural studies of membrane proteins and discuss the ability of the new technology, the Single-Protein Production (SPP) system, to help solve these bottlenecks.

  17. Yersinia pestis Ail: multiple roles of a single protein

    Directory of Open Access Journals (Sweden)

    Anna M Kolodziejek

    2012-08-01

    Full Text Available Yersinia pestis is one of the most virulent bacteria identified. It is the causative agent of plague – a systemic disease that has claimed millions of human lives throughout history. Y. pestis survival in insect and mammalian host species requires fine-tuning to sense and respond to varying environmental cues. Multiple Y. pestis attributes participate in this process and contribute to its pathogenicity and highly efficient transmission between hosts. These include factors inherited from its enteric predecessors; Y. enterocolitica and Y. pseudotuberculosis, as well as phenotypes acquired or lost during Y. pestis speciation. Representatives of a large Enterobacteriaceae Ail/OmpX/PagC/Lom family of outer membrane proteins (Omps are found in the genomes of all pathogenic Yersiniae. This review describes the current knowledge regarding the role of Ail in Y. pestis pathogenesis and virulence. The pronounced role of Ail in the following areas are discussed i inhibition of the bactericidal properties of complement, ii attachment and Yop delivery to host tissue iii prevention of PMNL recruitment to the lymph nodes, and iv inhibition of the inflammatory response. Finally, Ail homologs in Y. enterocolitica and Y. pseudotuberculosis are compared to illustrate differences that may have contributed to the drastic bacterial lifestyle change that shifted Y. pestis from an enteric to a vector-born systemic pathogen.

  18. Diversity of the 32-kilodalton protein gene may form a basis for species determination of potentially pathogenic mycobacterial species.

    Science.gov (United States)

    Soini, H; Viljanen, M K

    1997-03-01

    In this study, partial gene sequences of the mycobacterial 32-kDa protein gene were determined by PCR-based sequencing. A total of 50 strains representing 18 potentially pathogenic mycobacterial species were studied. In 10 cases, all three strains of the species studied were identical, and intraspecies variability was found in 6 cases. Thus, the 32-kDa protein gene may be a good target for identification of mycobacteria by PCR-based sequencing.

  19. Streptococcus mutans Protein Synthesis during Mixed-Species Biofilm Development by High-Throughput Quantitative Proteomics

    Science.gov (United States)

    Klein, Marlise I.; Xiao, Jin; Lu, Bingwen; Delahunty, Claire M.; Yates, John R.; Koo, Hyun

    2012-01-01

    Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v) was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT) approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA) and glucan-binding (gbpB) during this transition (Pmutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism), and molecular chaperones (GroEL). Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms) demonstrating fundamental differences in the matrix assembly, survival and biofilm maintenance in the presence of other

  20. Streptococcus mutans protein synthesis during mixed-species biofilm development by high-throughput quantitative proteomics.

    Directory of Open Access Journals (Sweden)

    Marlise I Klein

    Full Text Available Biofilms formed on tooth surfaces are comprised of mixed microbiota enmeshed in an extracellular matrix. Oral biofilms are constantly exposed to environmental changes, which influence the microbial composition, matrix formation and expression of virulence. Streptococcus mutans and sucrose are key modulators associated with the evolution of virulent-cariogenic biofilms. In this study, we used a high-throughput quantitative proteomics approach to examine how S. mutans produces relevant proteins that facilitate its establishment and optimal survival during mixed-species biofilms development induced by sucrose. Biofilms of S. mutans, alone or mixed with Actinomyces naeslundii and Streptococcus oralis, were initially formed onto saliva-coated hydroxyapatite surface under carbohydrate-limiting condition. Sucrose (1%, w/v was then introduced to cause environmental changes, and to induce biofilm accumulation. Multidimensional protein identification technology (MudPIT approach detected up to 60% of proteins encoded by S. mutans within biofilms. Specific proteins associated with exopolysaccharide matrix assembly, metabolic and stress adaptation processes were highly abundant as the biofilm transit from earlier to later developmental stages following sucrose introduction. Our results indicate that S. mutans within a mixed-species biofilm community increases the expression of specific genes associated with glucan synthesis and remodeling (gtfBC, dexA and glucan-binding (gbpB during this transition (P<0.05. Furthermore, S. mutans up-regulates specific adaptation mechanisms to cope with acidic environments (F1F0-ATPase system, fatty acid biosynthesis, branched chain amino acids metabolism, and molecular chaperones (GroEL. Interestingly, the protein levels and gene expression are in general augmented when S. mutans form mixed-species biofilms (vs. single-species biofilms demonstrating fundamental differences in the matrix assembly, survival and biofilm

  1. Immunotoxicity of nucleic acid reduced BioProtein - a bacterial derived single cell protein - in Wistar rats

    DEFF Research Database (Denmark)

    Mølck, Anne-marie; Poulsen, Morten; Christensen, Hanne Risager

    2002-01-01

    BioProtein is a single cell protein produced by a mixed methanotrophic and heterotrophic bacteria culture using natural gas as energy source, which has been approved for animal feed. BioProtein contains a large amount of nucleic acids making the product less suitable for human consumption, theref...

  2. Phylogeny and molecular signatures (conserved proteins and indels that are specific for the Bacteroidetes and Chlorobi species

    Directory of Open Access Journals (Sweden)

    Lorenzini Emily

    2007-05-01

    reported based on concatenated sequences for 12 conserved proteins by different methods including the character compatibility (or clique approach. The placement of Salinibacter ruber with other Bacteroidetes species was not resolved by other phylogenetic methods, but this affiliation was strongly supported by the character compatibility approach. Conclusion The molecular signatures described here provide novel tools for identifying and circumscribing species from the Bacteroidetes and Chlorobi phyla as well as some of their main groups in clear terms. These results also provide strong evidence that species from these two phyla (and also possibly Fibrobacteres are specifically related to each other and they form a single superphylum. Functional studies on these proteins and indels should aid in the discovery of novel biochemical and physiological characteristics that are unique to these groups of bacteria.

  3. The Single Cell Proteome Project - Cell-Cycle Dependent Protein Expression in Breast Cancer Cell Lines

    National Research Council Canada - National Science Library

    Dovichi, Norman J

    2005-01-01

    .... Capillary sieving electrophoresis and capillary micellar electrophoresis were used to characterize proteins in single cells in one-dimensional separations, while the two techniques were combined...

  4. Potential DNA barcodes for Melilotus species based on five single loci and their combinations.

    Directory of Open Access Journals (Sweden)

    Fan Wu

    Full Text Available Melilotus, an annual or biennial herb, belongs to the tribe Trifolieae (Leguminosae and consists of 19 species. As an important green manure crop, diverse Melilotus species have different values as feed and medicine. To identify different Melilotus species, we examined the efficiency of five candidate regions as barcodes, including the internal transcribed spacer (ITS and two chloroplast loci, rbcL and matK, and two non-coding loci, trnH-psbA and trnL-F. In total, 198 individuals from 98 accessions representing 18 Melilotus species were sequenced for these five potential barcodes. Based on inter-specific divergence, we analysed sequences and confirmed that each candidate barcode was able to identify some of the 18 species. The resolution of a single barcode and its combinations ranged from 33.33% to 88.89%. Analysis of pairwise distances showed that matK+rbcL+trnL-F+trnH-psbA+ITS (MRTPI had the greatest value and rbcL the least. Barcode gap values and similarity value analyses confirmed these trends. The results indicated that an ITS region, successfully identifying 13 of 18 species, was the most appropriate single barcode and that the combination of all five potential barcodes identified 16 of the 18 species. We conclude that MRTPI is the most effective tool for Melilotus species identification. Taking full advantage of the barcode system, a clear taxonomic relationship can be applied to identify Melilotus species and enhance their practical production.

  5. The Bifurcation and Control of a Single-Species Fish Population Logistic Model with the Invasion of Alien Species

    Directory of Open Access Journals (Sweden)

    Yi Zhang

    2014-01-01

    Full Text Available The objective of this paper is to study systematically the bifurcation and control of a single-species fish population logistic model with the invasion of alien species based on the theory of singular system and bifurcation. It regards Spartina anglica as an invasive species, which invades the fisheries and aquaculture. Firstly, the stabilities of equilibria in this model are discussed. Moreover, the sufficient conditions for existence of the trans-critical bifurcation and the singularity induced bifurcation are obtained. Secondly, the state feedback controller is designed to eliminate the unexpected singularity induced bifurcation by combining harvested effort with the purification capacity. It obviously inhibits the switch of population and makes the system stable. Finally, the numerical simulation is proposed to show the practical significance of the bifurcation and control from the biological point of view.

  6. Single-molecule detection of protein efflux from microorganisms using fluorescent single-walled carbon nanotube sensor arrays

    Science.gov (United States)

    Landry, Markita Patricia; Ando, Hiroki; Chen, Allen Y.; Cao, Jicong; Kottadiel, Vishal Isaac; Chio, Linda; Yang, Darwin; Dong, Juyao; Lu, Timothy K.; Strano, Michael S.

    2017-05-01

    A distinct advantage of nanosensor arrays is their ability to achieve ultralow detection limits in solution by proximity placement to an analyte. Here, we demonstrate label-free detection of individual proteins from Escherichia coli (bacteria) and Pichia pastoris (yeast) immobilized in a microfluidic chamber, measuring protein efflux from single organisms in real time. The array is fabricated using non-covalent conjugation of an aptamer-anchor polynucleotide sequence to near-infrared emissive single-walled carbon nanotubes, using a variable chemical spacer shown to optimize sensor response. Unlabelled RAP1 GTPase and HIV integrase proteins were selectively detected from various cell lines, via large near-infrared fluorescent turn-on responses. We show that the process of E. coli induction, protein synthesis and protein export is highly stochastic, yielding variability in protein secretion, with E. coli cells undergoing division under starved conditions producing 66% fewer secreted protein products than their non-dividing counterparts. We further demonstrate the detection of a unique protein product resulting from T7 bacteriophage infection of E. coli, illustrating that nanosensor arrays can enable real-time, single-cell analysis of a broad range of protein products from various cell types.

  7. Duplicated RGS (Regulator of G-protein signaling) proteins exhibit conserved biochemical but differential transcriptional regulation of heterotrimeric G-protein signaling in Brassica species.

    Science.gov (United States)

    Kumar, Roshan; Bisht, Naveen C

    2018-02-01

    G-alpha (Gα) and 'Regulator of G-protein Signaling (RGS)' proteins are the two key components primarily involved in regulation of heterotrimeric G-proteins signaling across phyla. Unlike Arabidopsis thaliana, our knowledge about G-protein regulation in polyploid Brassica species is sparse. In this study, we identified one Gα and two RGS genes each from three species of Brassica 'U' triangle and assessed the effects of whole genome triplication on the divergence of gene sequence and structure, protein-protein interaction, biochemical activities, and gene expression. Sequence and phylogenetic analysis revealed that the deduced Gα and RGS proteins are evolutionarily conserved across Brassica species. The duplicated RGS proteins of each Brassica species interacted with their cognate Gα but displayed varying levels of interaction strength. The Gα and the duplicated RGS proteins of Brassica species exhibited highly conserved G-protein activities when tested under in-vitro conditions. Expression analysis of the B. rapa RGS genes revealed a high degree of transcriptional differentiation across the tested tissue types and in response to various elicitors, particularly under D-glucose, salt and phytohormone treatments. Taken together, our results suggest that the RGS-mediated regulation of G-protein signaling in Brassica species is predominantly governed by stage and condition-specific expression differentiation of the duplicated RGS genes.

  8. Low Po2 conditions induce reactive oxygen species formation during contractions in single skeletal muscle fibers

    OpenAIRE

    Zuo, Li; Shiah, Amy; Roberts, William J.; Chien, Michael T.; Wagner, Peter D.; Hogan, Michael C.

    2013-01-01

    Contractions in whole skeletal muscle during hypoxia are known to generate reactive oxygen species (ROS); however, identification of real-time ROS formation within isolated single skeletal muscle fibers has been challenging. Consequently, there is no convincing evidence showing increased ROS production in intact contracting fibers under low Po2 conditions. Therefore, we hypothesized that intracellular ROS generation in single contracting skeletal myofibers increases during low Po2 compared wi...

  9. Rapeseed (Brassica napus L. as a protein plant species

    Directory of Open Access Journals (Sweden)

    Marinković Radovan

    2010-01-01

    Full Text Available Proteins of plant origin have a profound impact on human and animal lives. It is impossible to solve worldwide nutrition problem without taking into concern needs for proteins. Inadequate nutrition can only be improved by providing adequate proteins. Humans need c. 120g proteins daily, a third of which should come from meat and milk. Certain population categories, such as the sick, children, pregnant women and sportspeople are more sensitive to lack of protein. Oil crops synthesise oil, which is the basic reserve material in seed, but they also synthesise high levels of protein and can serve as protein source for human and animal nutrition. Generally speaking, protein content in seed of rapeseed at site R. Šančevi was from 19.60% (NS-L-74 to 25.93% JR-NS-36, and at site Sombor from 19.26% (NS-L-74 to 24.06% and 24.09% (NS-L-46 and cultivar Mira. Genotype NS-L-74 had the lowest protein content at both testing sites. Higher protein content was evident with spring genotypes than with winter gentypes. .

  10. Single-Molecule Study of Proteins by Biological Nanopore Sensors

    Science.gov (United States)

    Wu, Dongmei; Bi, Sheng; Zhang, Liyu; Yang, Jun

    2014-01-01

    Nanopore technology has been developed for detecting properties of proteins through monitoring of ionic current modulations as protein passes via a nanosize pore. As a real-time, sensitive, selective and stable technology, biological nanopores are of widespread concern. Here, we introduce the background of nanopore researches in the area of α-hemolysin (α-HL) nanopores in protein conformation detections and protein–ligand interactions. Moreover, several original biological nanopores are also introduced with various features and functions. PMID:25268917

  11. Single-Molecule Study of Proteins by Biological Nanopore Sensors

    Directory of Open Access Journals (Sweden)

    Dongmei Wu

    2014-09-01

    Full Text Available Nanopore technology has been developed for detecting properties of proteins through monitoring of ionic current modulations as protein passes via a nanosize pore. As a real-time, sensitive, selective and stable technology, biological nanopores are of widespread concern. Here, we introduce the background of nanopore researches in the area of α-hemolysin (α-HL nanopores in protein conformation detections and protein–ligand interactions. Moreover, several original biological nanopores are also introduced with various features and functions.

  12. Identification of species- and tissue-specific proteins using proteomic strategy

    Science.gov (United States)

    Chernukha, I. M.; Vostrikova, N. L.; Kovalev, L. I.; Shishkin, S. S.; Kovaleva, M. A.; Manukhin, Y. S.

    2017-09-01

    Proteomic technologies have proven to be very effective for detecting biochemical changes in meat products, such as changes in tissue- and species-specific proteins. In the tissues of cattle, pig, horse and camel M. longissimus dorsi both tissue- and species specific proteins were detected using two dimensional electrophoresis. Species-specific isoforms of several muscle proteins were also identified. The identified and described proteins of cattle, pig, horse and camel skeletal muscles (including mass spectra of the tryptic peptides) were added to the national free access database “Muscle organ proteomics”. This research has enabled the development of new highly sensitive technologies for meat product quality control against food fraud.

  13. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

    2015-06-05

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

    Science.gov (United States)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

    2015-01-01

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

  15. DETERMINATION O F TOTAL CELL PROTEIN PROFILES OF Streptomyces SPECIES

    OpenAIRE

    Özdemir K; Berber İ; Öğün E; Atalan M

    2013-01-01

    Present study has been conducted for finding out the total protein profile of bacterial strain Streptomyces sps by sodium dodecyl sulphate polyacrylamide gelelectrophoresis. Total 139 isolates of Streptomyces have been isolated from the soil. Amongst all isolated strain, total 20 isolates were used for getting protein profile by SDS PAGE. Amongst all isolates, 20 isolates were selected for protein profiling and these were divided in two groups. Two strains of Streptomyces i.e. S. violaceus...

  16. Nitrogen and protein contents in some aquatic plant species

    Directory of Open Access Journals (Sweden)

    Krystyna Bytniewska

    2015-01-01

    Full Text Available Nitrogen and protein contents in higher aquatic plants deriving from a natural habitat were determined. The following plants were examined: Spirodela polyrrhiza (L. Schleid., Elodea canadensis Rich., Riccia fluitans L. Total nitrogen and nitrogen of respective fractions were determined by the Kjeldahl method. Nitrogen compounds were fractionated according to Thimann et al. Protein was extracted after Fletcher and Osborne and fractionated after Osborne. It was found, that total protein content in the plants under examination constitutes 18 to 25%o of dry matter. Albumins and glutelins are the most abundant protein fractions.

  17. Localization of protein-protein interactions among three fluorescent proteins in a single living cell: three-color FRET microscopy

    Science.gov (United States)

    Sun, Yuansheng; Booker, Cynthia F.; Day, Richard N.; Periasamy, Ammasi

    2009-02-01

    Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However, the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy. Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor photobleaching FRET microscopy.

  18. Single-molecule protein sequencing through fingerprinting: computational assessment

    NARCIS (Netherlands)

    Yao, Y.; Docter, M.; Ginkel, van J.; Ridder, de D.; Joo, C.

    2015-01-01

    Proteins are vital in all biological systems as they constitute the main structural and functional components of cells. Recent advances in mass spectrometry have brought the promise of complete proteomics by helping draft the human proteome. Yet, this commonly used protein sequencing technique has

  19. Identification of intermediate species in protein-folding by ...

    Indian Academy of Sciences (India)

    TECS

    mixtures of non-interacting molecules such as fluorescent dyes, peptides and proteins. ... state, it is necessary to observe parameters which are sensitive to the ... them. Thus, such measurements offer the possibility of monitoring various regions of a protein during the transition from the U to the N form. When coupled with the ...

  20. Evidence for the robustness of protein complexes to inter-species hybridization.

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Leducq

    Full Text Available Despite the tremendous efforts devoted to the identification of genetic incompatibilities underlying hybrid sterility and inviability, little is known about the effect of inter-species hybridization at the protein interactome level. Here, we develop a screening platform for the comparison of protein-protein interactions (PPIs among closely related species and their hybrids. We examine in vivo the architecture of protein complexes in two yeast species (Saccharomyces cerevisiae and Saccharomyces kudriavzevii that diverged 5-20 million years ago and in their F1 hybrids. We focus on 24 proteins of two large complexes: the RNA polymerase II and the nuclear pore complex (NPC, which show contrasting patterns of molecular evolution. We found that, with the exception of one PPI in the NPC sub-complex, PPIs were highly conserved between species, regardless of protein divergence. Unexpectedly, we found that the architecture of the complexes in F1 hybrids could not be distinguished from that of the parental species. Our results suggest that the conservation of PPIs in hybrids likely results from the slow evolution taking place on the very few protein residues involved in the interaction or that protein complexes are inherently robust and may accommodate protein divergence up to the level that is observed among closely related species.

  1. An Investigative Alternative to Single-Species Dissection in the Introductory Biology Laboratory

    Science.gov (United States)

    Carlin, Joel L.

    2010-01-01

    Dissections of single species (e.g., fetal pig) are a common student learning activity in introductory biology courses. Such dissections demonstrate location of anatomical parts and provide dissection practice but provide less opportunity for student critical thinking, numeracy and demonstration of the scientific method. A comparative anatomy lab…

  2. Cover crops in mixtures do not use water differently than single-species plantings

    Science.gov (United States)

    Some recent statements have been made about the benefits of growing cover crops in mixtures as compared with single-species plantings of cover crops. One of those stated benefits is greatly reduced water use by cover crops grown in mixtures. The objectives of this study were to characterize soil wat...

  3. Evolution of major milk proteins in Mus musculus and Mus spretus mouse species: a genoproteomic analysis

    Directory of Open Access Journals (Sweden)

    Panthier Jean-Jacques

    2011-01-01

    Full Text Available Abstract Background Due to their high level of genotypic and phenotypic variability, Mus spretus strains were introduced in laboratories to investigate the genetic determinism of complex phenotypes including quantitative trait loci. Mus spretus diverged from Mus musculus around 2.5 million years ago and exhibits on average a single nucleotide polymorphism (SNP in every 100 base pairs when compared with any of the classical laboratory strains. A genoproteomic approach was used to assess polymorphism of the major milk proteins between SEG/Pas and C57BL/6J, two inbred strains of mice representative of Mus spretus and Mus musculus species, respectively. Results The milk protein concentration was dramatically reduced in the SEG/Pas strain by comparison with the C57BL/6J strain (34 ± 9 g/L vs. 125 ± 12 g/L, respectively. Nine major proteins were identified in both milks using RP-HPLC, bi-dimensional electrophoresis and MALDI-Tof mass spectrometry. Two caseins (β and αs1 and the whey acidic protein (WAP, showed distinct chromatographic and electrophoresis behaviours. These differences were partly explained by the occurrence of amino acid substitutions and splicing variants revealed by cDNA sequencing. A total of 34 SNPs were identified in the coding and 3'untranslated regions of the SEG/Pas Csn1s1 (11, Csn2 (7 and Wap (8 genes. In addition, a 3 nucleotide deletion leading to the loss of a serine residue at position 93 was found in the SEG/Pas Wap gene. Conclusion SNP frequencies found in three milk protein-encoding genes between Mus spretus and Mus musculus is twice the values previously reported at the whole genome level. However, the protein structure and post-translational modifications seem not to be affected by SNPs characterized in our study. Splicing mechanisms (cryptic splice site usage, exon skipping, error-prone junction sequence, already identified in casein genes from other species, likely explain the existence of multiple αs1-casein

  4. Conservation of avian diversity in the Sierra Nevada: moving beyond a single-species management focus.

    Directory of Open Access Journals (Sweden)

    Angela M White

    greater benefit to ecosystem functioning then a single-species management focus.

  5. Conservation of avian diversity in the Sierra Nevada: moving beyond a single-species management focus.

    Science.gov (United States)

    White, Angela M; Zipkin, Elise F; Manley, Patricia N; Schlesinger, Matthew D

    2013-01-01

    ecosystem functioning then a single-species management focus.

  6. Studies of Single Biomolecules, DNA Conformational Dynamics, and Protein Binding

    National Research Council Canada - National Science Library

    Hanke, Andreas

    2008-01-01

    ... may open up spontaneously due to thermal activation. By rising the ambient temperature, titration, or by external forces in single molecule setups bubbles proliferate until full denaturation of the DNA...

  7. Rapid Seafood Species Identification Using Chip-Based Capillary Electrophoresis and Protein Pattern Matching.

    Science.gov (United States)

    Walker, Calvin C; Lassitter, Cheryl L; Lynn, Shannara N; Ford, Courtney B; Rademacher, Kevin R; Ruple, Angela D; Bell, Jon W

    2017-09-01

    Authenticity is crucial to the seafood industry, as substitution and mislabeling have important economic, environmental, and food safety consequences. To address this problem, protein profiling and software algorithm techniques were developed to classify fish muscle samples by species. The method uses water-based protein extraction, chip-based microfluidic electrophoresis (Agilent 2100 Bioanalyzer) for the analysis of high abundance fish muscle proteins, and a novel data analysis method for species-specific protein pattern recognition. The method's performance in distinguishing commercially important fish from commonly reported substitutions was evaluated using sensitivity, specificity, and accuracy determinations with all three performance measures at >98% for common substitutions. This study demonstrates that uncooked seafood products of commercially important species of catfish, snapper, and grouper can be rapidly distinguished from commonly substituted species with a high level of confidence. A tiered testing approach to seafood species verification by sequentially applying a rapid screening method and DNA testing is proposed to more effectively ensure accurate product labeling.

  8. In-vitro activity of taurolidine on single species and a multispecies population associated with periodontitis.

    Science.gov (United States)

    Zollinger, Lilly; Schnyder, Simone; Nietzsche, Sandor; Sculean, Anton; Eick, Sigrun

    2015-04-01

    The antimicrobial activity of taurolidine was compared with minocycline against microbial species associated with periodontitis (four single strains and a 12-species mixture). Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs), killing as well as activities on established and forming single-species biofilms and a 12-species biofilm were determined. The MICs of taurolidine against single species were always 0.31 mg/ml, the MBCs were 0.64 mg/ml. The used mixed microbiota was less sensitive to taurolidine, MIC and the MBC was 2.5 mg/ml. The strains and the mixture were completely killed by 2.5 mg/ml taurolidine, whereas 256 μg/ml minocycline reduced the bacterial counts of the mixture by 5 log10 colony forming units (cfu). Coating the surface with 10 mg/ml taurolidine or 256 μg/ml minocycline prevented completely biofilm formation of Porphyromonas gingivalis ATCC 33277 but not of Aggregatibacter actinomycetemcomitans Y4 and the mixture. On 4.5 d old biofilms, taurolidine acted concentration dependent with a reduction by 5 log10 cfu (P. gingivalis ATCC 33277) and 7 log10 cfu (A. actinomycetemcomitans Y4) when applying 10 mg/ml. Minocycline decreased the cfu counts by 1-2 log10 cfu independent of the used concentration. The reduction of the cfu counts in the 4.5 d old multi-species biofilms was about 3 log10 cfu after application of any minocycline concentration and after using 10 mg/ml taurolidine. Taurolidine is active against species associated with periodontitis, even within biofilms. Nevertheless a complete elimination of complex biofilms by taurolidine seems to be impossible and underlines the importance of a mechanical removal of biofilms prior to application of taurolidine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Seroprofiling at the Candida albicans protein species level unveils an accurate molecular discriminator for candidemia.

    Science.gov (United States)

    Pitarch, Aida; Nombela, César; Gil, Concha

    2016-02-16

    Serum antibodies to specific Candida proteins have been reported as potential diagnostic biomarkers for candidemia. However, their diagnostic usefulness at the protein species level has hardly been examined. Using serological proteome analysis, we explored the IgG-antibody responses to Candida albicans protein species in candidemia and control patients. We found that 87 discrete protein species derived from 34 unique proteins were IgG-targets, although only 43 of them were differentially recognized by candidemia and control sera. An increase in the speciation of the immunome, connectivity and modularity of antigenic species co-recognition networks, and heterogeneity of antigenic species recognition patterns was associated with candidemia. IgG antibodies to certain discrete protein species were better predictors of candidemia than those to their corresponding proteins. A molecular discriminator delineated from the combined fingerprints of IgG antibodies to two distinct species of phosphoglycerate kinase and enolase accurately classified candidemia and control patients. These results provide new insight into the anti-Candida IgG-antibody response development in candidemia, and demonstrate that an immunoproteomic signature at the molecular level may be useful for its diagnosis. Our study further highlights the importance of defining pathogen-specific antigens at the chemical and molecular level for their potential application as immunodiagnostic reagents or even vaccine candidates.

  10. Impact of Anthropogenic Noise on Aquatic Animals: From Single Species to Community-Level Effects.

    Science.gov (United States)

    Sabet, Saeed Shafiei; Neo, Yik Yaw; Slabbekoorn, Hans

    2016-01-01

    Anthropogenic noise underwater is on the rise and may affect aquatic animals of marine and freshwater ecosystems. Many recent studies concern some sort of impact assessment of a single species. Few studies addressed the noise impact on species interactions underwater, whereas there are some studies that address community-level impact but only on land in air. Key processes such as predator-prey or competitor interactions may be affected by the masking of auditory cues, noise-related disturbance, or attentional interference. Noise-associated changes in these interactions can cause shifts in species abundance and modify communities, leading to fundamental ecosystem changes. To gain further insight into the mechanism and generality of earlier findings, we investigated the impact on both a predator and a prey species in captivity, zebrafish (Danio rerio) preying on waterfleas (Daphnia magna).

  11. Capillary Electrophoresis Single-Strand Conformational Polymorphisms as a Method to Differentiate Algal Species

    Directory of Open Access Journals (Sweden)

    Alice Jernigan

    2015-01-01

    Full Text Available Capillary electrophoresis single-strand conformational polymorphism (CE-SSCP was explored as a fast and inexpensive method to differentiate both prokaryotic (blue-green and eukaryotic (green and brown algae. A selection of two blue-green algae (Nostoc muscorum and Anabaena inaequalis, five green algae (Chlorella vulgaris, Oedogonium foveolatum, Mougeotia sp., Scenedesmus quadricauda, and Ulothrix fimbriata, and one brown algae (Ectocarpus sp. were examined and CE-SSCP electropherogram “fingerprints” were compared to each other for two variable regions of either the 16S or 18S rDNA gene. The electropherogram patterns were remarkably stable and consistent for each particular species. The patterns were unique to each species, although some common features were observed between the different types of algae. CE-SSCP could be a useful method for monitoring changes in an algae species over time as potential shifts in species occurred.

  12. Characterization of the single-stranded DNA binding protein pV(VGJΦ) of VGJΦ phage from Vibrio cholerae.

    Science.gov (United States)

    Falero, Alina; Caballero, Andy; Trigueros, Sonia; Pérez, Celso; Campos, Javier; Marrero, Karen; Fando, Rafael

    2011-09-01

    pV(VGJΦ), a single-stranded DNA binding protein of the vibriophage VGJΦ was subject to biochemical analysis. Here, we show that this protein has a general affinity for single-stranded DNA (ssDNA) as documented by Electrophoretic Mobility Shift Assay (EMSA). The apparent molecular weight of the monomer is about 12.7kDa as measured by HPLC-SEC. Moreover, isoelectrofocusing showed an isoelectric point for pV(VGJΦ) of 6.82 pH units. Size exclusion chromatography in 150mM NaCl, 50mM sodium phosphate buffer, pH 7.0 revealed a major protein species of 27.0kDa, suggesting homodimeric protein architecture. Furthermore, pV(VGJΦ) binds ssDNA at extreme temperatures and the complex was stable after extended incubation times. Upon frozen storage at -20°C for a year the protein retained its integrity, biological activity and oligomericity. On the other hand, bioinformatics analysis predicted that pV(VGJΦ) protein has a disordered C-terminal, which might be involved in its functional activity. All the aforementioned features make pV(VGJΦ) interesting for biotechnological applications. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Photoconversion of organic materials into single-cell protein

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, P.F.

    1991-12-31

    A process is described for converting organic materials (such as biomass wastes) into sterile, high-grade bacterial protein suitable for use an animal feed or human food supplements. In a preferred embodiment the process involves thermally gasifying the organic material into primarily carbon monoxide, hydrogen and nitrogen products, followed by photosynthetic bacterial assimilation of the gases into cell material, which can be high as 65% protein. The process is ideally suited for waste recycling and for food production under zero-gravity or extra-terrestrial conditions.

  14. Molecular identification of broomrape species from a single seed by High Resolution Melting analysis

    Directory of Open Access Journals (Sweden)

    Mathieu Rolland

    2016-12-01

    Full Text Available Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species (P. aegyptiaca, O. cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa from a single seed. Based on trnL and rbcL plastidial genes amplification, the designed assay successfully identifies O. cumana, O. cernua, O. crenata, O. minor, O. hederae, and O. foetida; P. ramosa and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90 %. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples. The described assay fulfils its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material.

  15. Molecular Identification of Broomrape Species from a Single Seed by High Resolution Melting Analysis.

    Science.gov (United States)

    Rolland, Mathieu; Dupuy, Aurélie; Pelleray, Aude; Delavault, Philippe

    2016-01-01

    Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species ( Phelipanche aegyptiaca , Orobanche cernua , O. crenata, O. cumana , O. foetida , O. hederae , O. minor , and P. ramosa ) from a single seed. Based on trn L and rbc L plastidial genes amplification, the designed assay successfully identifies O. cumana , O. cernua , O. crenata , O. minor , O. hederae , and O. foetida ; P. ramosa , and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90%. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples). The described assay fulfills its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material.

  16. Effects of single-stranded DNA binding proteins on primer extension by telomerase.

    Science.gov (United States)

    Cohen, Shlomit; Jacob, Eyal; Manor, Haim

    2004-08-12

    We present a biochemical analysis of the effects of three single-stranded DNA binding proteins on extension of oligonucleotide primers by the Tetrahymena telomerase. One of them, a human protein designated translin, which was shown to specifically bind the G-rich Tetrahymena and human telomeric repeats, slightly stimulated the primer extension reactions at molar ratios of translin/primer of primers, rather than by a direct interaction of this protein with telomerase. A second protein, the general human single-stranded DNA binding protein Replication Protein A (RPA), similarly affected the primer extension by telomerase, even though its mode of binding to DNA differs from that of translin. A third protein, the E. coli single-stranded DNA binding protein (SSB), whose binding to DNA is highly cooperative, caused more substantial stimulation and inhibition at the lower and the higher molar ratios of SSB/primer, respectively. Both telomere-specific and general single-stranded DNA binding proteins are found in living cells in telomeric complexes. Based on our data, we propose that these proteins may exert either stimulatory or inhibitory effects on intracellular telomerases, depending on their local concentrations. Copyright 2004 Elsevier B.V.

  17. Immunogenic proteins specific to different bird species in bird fancier's lung.

    Science.gov (United States)

    Rouzet, Adeline; Reboux, Gabriel; Rognon, Bénédicte; Barrera, Coralie; De Vuyst, Paul; Dalphin, Jean-Charles; Millon, Laurence; Roussel, Sandrine

    2014-01-01

    Bird fancier's lung (BFL) is a disease produced by exposure to avian proteins present in droppings, blooms, and serum of a variety of birds. Although serological test results are currently used to confirm clinical diagnosis of the disease, bird species specificity is poorly understood. This study aimed to contribute to a better understanding of the specificity of immunogenic proteins revealed from the droppings of three bird species. Sera from four patients with BFL and two controls without exposure were analyzed by Western blotting with antigens from droppings of two pigeon and budgerigar strains and two hen species. When the antigens from the droppings of the three bird species were compared, the profile of immunogenic proteins was different and there were similarities between strains of the same species. Only one 68-kD protein was common to pigeon and budgerigar droppings, while proteins of 200, 175, 140, 100, and 35 kD were detected as specific in one bird species. These results provide insight to further characterize these proteins, and to design new serological tests specific to different bird species. These tests may help to refine strategies of antigenic exclusion and also to allow a patient compensation in case of BFL of occupational origin.

  18. Revealing Two-State Protein-Protein Interaction of Calmodulin by Single-Molecule Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ruchuan; Hu, Dehong; Tan, Xin; Lu, H PETER.

    2006-08-09

    We report a single-molecule fluorescence resonance energy transfer (FRET) and polarization study of conformational dynamics of calmodulin (CaM) interacting with a target peptide, C28W of 28 amino-acid oligomer. The C28W peptide represents the essential binding sequence domain of the Ca-ATPase protein interacting with CaM, which is important in cellular signaling for the regulation of energy in metabolism. However, the mechanism of the CaM-C28W recognition complex formation is still unclear. The amino-terminal (N-terminal) domain of the CaM was labeled with a fluorescein-based arsenical hairpin binder (F1AsH) that enables our unambiguously probing the CaM N-terminal target-binding domain motions at a millisecond timescale without convolution of the probe-dye random motions. By analyzing the distribution of FRET efficiency between F1AsH labeled CaM and Texas Red labeled C28W and the polarization fluctuation dynamics and distributions of the CaM N-terminal domain, we reveal slow (at sub-second time scale) binding-unbinding motions of the N-terminal domain of the CaM in CaM-C28W complexes, which is a strong evidence of a two-state binding interaction of CaM-mediated cell signaling.

  19. Identification of intermediate species in protein-folding by ...

    Indian Academy of Sciences (India)

    -states, namely the native (N) and unfolded (U) forms of the protein present at any condition of the solvent, from a situation wherein intermediate state(s) could also be present. This differentiation of a two-state from a multi-state structural ...

  20. Identification of intermediate species in protein-folding by ...

    Indian Academy of Sciences (India)

    TECS

    of monitoring various regions of a protein during the transition from the U to the N form. When coupled with the maximum entropy method (MEM) of analysis of intensity decay curves,. 1,2. TR-FRET measurements can resolve heterogeneity in terms of distributions of intramolecular distances. Much has been learnt about.

  1. Zinc(II) and the single-stranded DNA binding protein of bacteriophage T4

    International Nuclear Information System (INIS)

    Gauss, P.; Krassa, K.B.; McPheeters, D.S.; Nelson, M.A.; Gold, L.

    1987-01-01

    The DNA binding domain of the gene 32 protein of the bacteriophage T4 contains a single zinc-finger sequence. The gene 32 protein is an extensively studied member of a class of proteins that bind relatively nonspecifically to single-stranded DNA. The authors have sequenced and characterized mutations in gene 32 whose defective proteins are activated by increasing the Zn(II) concentration in the growth medium. The results identify a role for the gene 32 protein in activation of T4 late transcription. Several eukaryotic proteins with zinc fingers participate in activation of transcription, and the gene 32 protein of T4 should provide a simple, well-characterized system in which genetics can be utilized to study the role of a zinc finger in nucleic acid binding and gene expression

  2. Real-time shape approximation and fingerprinting of single proteins using a nanopore

    Science.gov (United States)

    Yusko, Erik C.; Bruhn, Brandon R.; Eggenberger, Olivia M.; Houghtaling, Jared; Rollings, Ryan C.; Walsh, Nathan C.; Nandivada, Santoshi; Pindrus, Mariya; Hall, Adam R.; Sept, David; Li, Jiali; Kalonia, Devendra S.; Mayer, Michael

    2017-05-01

    Established methods for characterizing proteins typically require physical or chemical modification steps or cannot be used to examine individual molecules in solution. Ionic current measurements through electrolyte-filled nanopores can characterize single native proteins in an aqueous environment, but currently offer only limited capabilities. Here we show that the zeptolitre sensing volume of bilayer-coated solid-state nanopores can be used to determine the approximate shape, volume, charge, rotational diffusion coefficient and dipole moment of individual proteins. To do this, we developed a theory for the quantitative understanding of modulations in ionic current that arise from the rotational dynamics of single proteins as they move through the electric field inside the nanopore. The approach allows us to measure the five parameters simultaneously, and we show that they can be used to identify, characterize and quantify proteins and protein complexes with potential implications for structural biology, proteomics, biomarker detection and routine protein analysis.

  3. A Rational Engineering Strategy for Designing Protein A-Binding Camelid Single-Domain Antibodies.

    Directory of Open Access Journals (Sweden)

    Kevin A Henry

    Full Text Available Staphylococcal protein A (SpA and streptococcal protein G (SpG affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.

  4. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...... important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower...

  5. Ancient host specificity within a single species of brood parasitic bird.

    Science.gov (United States)

    Spottiswoode, Claire N; Stryjewski, Katherine Faust; Quader, Suhel; Colebrook-Robjent, John F R; Sorenson, Michael D

    2011-10-25

    Parasites that exploit multiple hosts often experience diversifying selection for host-specific adaptations. This can result in multiple strains of host specialists coexisting within a single parasitic species. A long-standing conundrum is how such sympatric host races can be maintained within a single parasitic species in the face of interbreeding among conspecifics specializing on different hosts. Striking examples are seen in certain avian brood parasites such as cuckoos, many of which show host-specific differentiation in traits such as host egg mimicry. Exploiting a Zambian egg collection amassed over several decades and supplemented by recent fieldwork, we show that the brood parasitic Greater Honeyguide Indicator indicator exhibits host-specific differentiation in both egg size and egg shape. Genetic analysis of honeyguide eggs and chicks show that two highly divergent mitochondrial DNA lineages are associated with ground- and tree-nesting hosts, respectively, indicating perfect fidelity to two mutually exclusive sets of host species for millions of years. Despite their age and apparent adaptive diversification, however, these ancient lineages are not cryptic species; a complete lack of differentiation in nuclear genes shows that mating between individuals reared by different hosts is sufficiently frequent to prevent speciation. These results indicate that host specificity is maternally inherited, that host-specific adaptation among conspecifics can be maintained without reproductive isolation, and that host specificity can be remarkably ancient in evolutionary terms.

  6. Multiple Phytophthora species associated with a single riparian ecosystem in South Africa.

    Science.gov (United States)

    Nagel, Jan H; Slippers, Bernard; Wingfield, Michael J; Gryzenhout, Marieka

    2015-01-01

    The diversity of Phytophthora spp. in rivers and riparian ecosystems has received considerable international attention, although little such research has been conducted in South Africa. This study determined the diversity of Phytophthora spp. within a single river in Gauteng province of South Africa. Samples were collected over 1 y including biweekly river baiting with Rhododendron indicum leaves. Phytophthora isolates were identified with phylogenetic analyses of sequences for the internal transcribed spacer (ITS) region of the ribosomal DNA and the mitochondrial cytochrome oxidase c subunit I (coxI) gene. Eight Phytophthora spp. were identified, including a new taxon, P. taxon Sisulu-river, and two hybrid species from Cooke's ITS clade 6. Of these, species from Clade 6 were the most abundant, including P. chlamydospora and P. lacustris. Species residing in Clade 2 also were encountered, including P. multivora, P. plurivora and P. citrophthora. The detection of eight species in this investigation of Phytophthora diversity in a single riparian river ecosystem in northern South Africa adds to the known diversity of this genus in South Africa and globally. © 2015 by The Mycological Society of America.

  7. Atomic force microscopy and spectroscopy to probe single membrane proteins in lipid bilayers.

    Science.gov (United States)

    Sapra, K Tanuj

    2013-01-01

    The atomic force microscope (AFM) has opened vast avenues hitherto inaccessible to the biological scientist. The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever stylus is aptly termed as a "lab on a tip" owing to its versatility as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples assert that the AFM can be used to study the mechanical properties and monitor processes of single proteins and single cells, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of membrane proteins. Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theoretical, and practical skills.

  8. Examining the relationship between total species richness and single island palaeo- and neo-endemics

    Science.gov (United States)

    Kallimanis, A. S.; Panitsa, M.; Bergmeier, E.; Dimopoulos, P.

    2011-03-01

    Recently, Emerson and Kolm (2005) hypothesized that diversity begets speciation (DBS hypothesis). The relationship between total species richness and single island endemic diversity (as a proportion of the total species richness of the island) has been used as evidence for the DBS hypothesis. This relationship has been documented in oceanic archipelagos, but many criticisms have been raised on whether this relationship truly supports the DBS hypothesis. In this study we tested if this hypothesis holds in the Aegean archipelago (a continental archipelago with continuous human presence over millennia). Endemism in the Aegean includes mainly neo-endemic species but also relictual populations of formerly more widespread species (i.e. palaeo-endemics). Contrary to the DBS hypothesis, we found that total species richness was not significantly correlated to single island endemics (neither neo-endemics nor palaeo-endemics) as a proportion of the island flora. Furthermore, we found that neo-endemic diversity (either as species richness or as a proportion of the islands flora) is mainly correlated to island maximum elevation, while area and isolation were less important. So if this ratio is indeed an index of speciation, then an alternative explanation might be that elevation (interpreted as a proxy for habitat heterogeneity) is the driver of speciation in our case. Palaeo-endemics, on the other hand, were present in only six of the largest islands in the Aegean and their diversity was strongly correlated only with island area, perhaps implying that larger islands support larger population sizes that buffer stochastic extinctions risks.

  9. Characterization of a mitochondrially targeted single-stranded DNA-binding protein in Arabidopsis thaliana.

    Science.gov (United States)

    Edmondson, Andrew C; Song, Daqing; Alvarez, Luis A; Wall, Melisa K; Almond, David; McClellan, David A; Maxwell, Anthony; Nielsen, Brent L

    2005-04-01

    A gene encoding a predicted mitochondrially targeted single-stranded DNA binding protein (mtSSB) was identified in the Arabidopsis thaliana genome sequence. This gene (At4g11060) codes for a protein of 201 amino acids, including a 28-residue putative mitochondrial targeting transit peptide. Protein sequence alignment shows high similarity between the mtSSB protein and single-stranded DNA binding proteins (SSB) from bacteria, including residues conserved for SSB function. Phylogenetic analysis indicates a close relationship between this protein and other mitochondrially targeted SSB proteins. The predicted targeting sequence was fused with the GFP coding region, and the organellar localization of the expressed fusion protein was determined. Specific targeting to mitochondria was observed in in-vitro import experiments and by transient expression of a GFP fusion construct in Arabidopsis leaves after microprojectile bombardment. The mature mtSSB coding region was overexpressed in Escherichia coli and the protein was purified for biochemical characterization. The purified protein binds single-stranded, but not double-stranded, DNA. MtSSB stimulates the homologous strand-exchange activity of E. coli RecA. These results indicate that mtSSB is a functional homologue of the E. coli SSB, and that it may play a role in mitochondrial DNA recombination.

  10. Nonlinearly Additive Forces in Multivalent Ligand Binding to a Single Protein Revealed with Force Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Ratto, T V; Rudd, R E; Langry, K C; Balhorn, R L; McElfresh, M W

    2005-07-15

    We present evidence of multivalent interactions between a single protein molecule and multiple carbohydrates at a pH where the protein can bind four ligands. The evidence is based not only on measurements of the force required to rupture the bonds formed between ConcanavalinA (ConA) and {alpha}-D-mannose, but also on an analysis of the polymer-extension force curves to infer the polymer architecture that binds the protein to the cantilever and the ligands to the substrate. We find that although the rupture forces for multiple carbohydrate connections to a single protein are larger than the rupture force for a single connection, they do not scale additively with increasing number. Specifically, the most common rupture forces are approximately 46, 66, and 85 pN, which we argue corresponds to 1, 2, and 3 ligands being pulled simultaneously from a single protein as corroborated by an analysis of the linkage architecture. As in our previous work polymer tethers allow us to discriminate between specific and non-specific binding. We analyze the binding configuration (i.e. serial versus parallel connections) through fitting the polymer stretching data with modified Worm-Like Chain (WLC) models that predict how the effective stiffness of the tethers is affected by multiple connections. This analysis establishes that the forces we measure are due to single proteins interacting with multiple ligands, the first force spectroscopy study that establishes single-molecule multivalent binding unambiguously.

  11. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower...... eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...

  12. COMPARISON OF STRESS PROTEINS PARTICIPATION IN ADAPTATION MECHANISMS OF BAIKALIAN AND PALEARCTIC AMPHIPOD (AMPHIPODA; CRUSTACEA SPECIES

    Directory of Open Access Journals (Sweden)

    Timofeyev M.A

    2006-03-01

    Full Text Available The aim of the present study was a study of the influence different stressful factor on syntheses and activity of the stress proteins (HSP70, sHSP and peroxidase of freshwater organism. Six freshwater amphipod species were investigated: Eulimnogammarus cyaneus (Dyb., E verrucosus (Gerstf., E vittatus (Dyb. - endemic species from Lake Baikal which were compared with Palearctic species - Gammarus lacustris Sars., G tigrinus (Sexton, Chaetogammarus ischnus (Stebbins. It was shown expression of sHSP by heat and toxic stresses for all amphipods species. Oxidative stress induced HSP70 for Palearctic species G tigrinus and C ischnus but not for baikalian species. Heat stress did not caused the increase of HSP70 level for Baikalian species of amphipods. The activity of the peroxidase was decrease by heat and toxic stresses. Oxidative stress caused the increase of peroxidase activity for Palearctic species, and the decrease for Baikalian once.

  13. Utilizing Biotinylated Proteins Expressed in Yeast to Visualize DNA–Protein Interactions at the Single-Molecule Level

    Directory of Open Access Journals (Sweden)

    Huijun Xue

    2017-10-01

    Full Text Available Much of our knowledge in conventional biochemistry has derived from bulk assays. However, many stochastic processes and transient intermediates are hidden when averaged over the ensemble. The powerful technique of single-molecule fluorescence microscopy has made great contributions to the understanding of life processes that are inaccessible when using traditional approaches. In single-molecule studies, quantum dots (Qdots have several unique advantages over other fluorescent probes, such as high brightness, extremely high photostability, and large Stokes shift, thus allowing long-time observation and improved signal-to-noise ratios. So far, however, there is no convenient way to label proteins purified from budding yeast with Qdots. Based on BirA–Avi and biotin–streptavidin systems, we have established a simple method to acquire a Qdot-labeled protein and visualize its interaction with DNA using total internal reflection fluorescence microscopy. For proof-of-concept, we chose replication protein A (RPA and origin recognition complex (ORC as the proteins of interest. Proteins were purified from budding yeast with high biotinylation efficiency and rapidly labeled with streptavidin-coated Qdots. Interactions between proteins and DNA were observed successfully at the single-molecule level.

  14. Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein

    DEFF Research Database (Denmark)

    Bendtsen, Jannick Dyrløv; Nilsson, A.S.; Lehnherr, H.

    2002-01-01

    Bacteriophage P1 encodes a single-stranded DNA-binding protein (SSB-P1), which shows 66% amino acid sequence identity to the SSB protein of the host bacterium Escherichia coli. A phylogenetic analysis indicated that the P1 ssb gene coexists with its E. coli counterpart as an independent unit...

  15. Economic Optimizing Control for Single-Cell Protein Production in a U-Loop Reactor

    DEFF Research Database (Denmark)

    Drejer, André; Ritschel, Tobias Kasper Skovborg; Jørgensen, Sten Bay

    2017-01-01

    The production of single-cell protein (SCP) in a U-loop reactor by a methanotroph is a cost efficient sustainable alternative to protein from fish meal obtained by over-fishing the oceans. SCP serves as animal feed. In this paper, we present a mathematical model that describes the dynamics of SCP...

  16. Yeast single cell protein in the diet of Oreochromis niloticus (L ...

    African Journals Online (AJOL)

    use

    Diets D10 to D50 had fish meal replaced systematically with yeast single cell protein (SCP) in the order 10, 20, 30, 40 and 50%, respectively. Trial feeding was ... Key word: microbial protein, Oreochromis niloticus, feeding, cost benefit, aquaculture. .... Supplementary Feeding for Production of Nile Tilapia, Silver Carp.

  17. See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins

    NARCIS (Netherlands)

    van Mameren, J.; Peterman, E.J.G.; Wuite, G.J.L.

    2008-01-01

    Direct visualization of DNA and proteins allows researchers to investigate DNA-protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of

  18. Enhancement of Protein and Pigment Content in Two Chlorella Species Cultivated on Industrial Process Water

    DEFF Research Database (Denmark)

    Safafar, Hamed; Uldall Nørregaard, Patrick; Ljubic, Anita

    2016-01-01

    composition of biomass. Variations in proteins, lipid, fatty acid composition, amino acids, tocopherols, and pigments were studied. Both species grew well in industrial process water. The contents of proteins were affected significantly by the growth media and cultivation duration. Microalga Chlorella...... is an environmentally friendly, sustainable bioremediation method with added value biomass production and resource valorization, since the resulting biomass also presented a good source of proteins, amino acids, and carotenoids for potential use in aquaculture feed industry....

  19. Single-cell phospho-protein analysis by flow cytometry.

    Science.gov (United States)

    Schulz, Kenneth R; Danna, Erika A; Krutzik, Peter O; Nolan, Garry P

    2012-02-01

    This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications bringing standard biochemical analysis into primary cells in order to garner valuable information about signaling events in physiologic settings. © 2012 by John Wiley & Sons, Inc.

  20. Contemporary root canal irrigants are able to disrupt and eradicate single- and dual-species biofilms.

    Science.gov (United States)

    Bryce, Graeme; O'Donnell, Darren; Ready, Derren; Ng, Yuan-Ling; Pratten, Jonathan; Gulabivala, Kishor

    2009-09-01

    Clinical/microbiological studies have consistently revealed the persistence of some bacteria after conventional root canal debridement. Although this was originally attributed to the complexity of the root canal anatomy and the difficulty of delivering antibacterial agents effectively, it has emerged that the biofilm encasement of bacterial cells may confer a further mechanism of resistance. The purpose of this study was to investigate the relative disruption and bactericidal effects of root canal irrigants on single- and dual-species biofilms of root canal isolates. Biofilms of Streptococcus sanguinis, Enterococcus faecalis, Fusobacterium nucleatum, and Porphyromonas gingivalis were grown on nitrocellulose membranes for 72 hours and immersed in NaOCl, EDTA, chlorhexidine, and iodine for 1, 5, or 10 minutes. The number of viable and nonviable bacteria disrupted from the biofilm and those remaining adherent were determined by using a viability stain in conjunction with fluorescence microscopy. Gram-negative obligate anaerobe species were more susceptible to cell removal than gram-positive facultative anaerobes. The majority of cells were disrupted after the first minute of exposure; however, the extent varied according to the agent and species. The most effective agent at disrupting biofilms was NaOCl. Iodine was generally effective at bacterial killing but not disruption. Biofilm disruption and cell viability were influenced by the species, their coassociation in dual-species biofilms, the test agent, and the duration of exposure. The effectiveness of NaOCl as an endodontic irrigant was reinforced.

  1. Substantial equivalence analysis in fruits from three Theobroma species through chemical composition and protein profiling.

    Science.gov (United States)

    Pérez-Mora, Walter; Jorrin-Novo, Jesús V; Melgarejo, Luz Marina

    2018-02-01

    Substantial equivalence studies were performed in three Theobroma spp., cacao, bicolor and grandiflorum through chemical composition analysis and protein profiling of fruit (pulp juice and seeds). Principal component analysis of sugar, organic acid, and phenol content in pulp juice revealed equivalence among the three species, with differences in some of the compounds that may result in different organoleptic properties. Proteins were extracted from seeds and pulp juice, resolved by two dimensional electrophoresis and major spots subjected to mass spectrometry analysis and identification. The protein profile, as revealed by principal component analysis, was variable among the three species in both seed and pulp, with qualitative and quantitative differences in some of protein species. The functional grouping of the identified proteins correlated with the biological role of each organ. Some of the identified proteins are of interest, being minimally discussed, including vicilin, a protease inhibitor, and a flavonol synthase/flavanone 3-hydroxylase. Theobroma grandiflorum and Theobroma bicolor are endemic Amazonian plants that are poorly traded at the local level. As close relatives of Theobroma cacao, they may provide a good alternative for human consumption and industrial purposes. In this regard, we performed equivalence studies by conducting a comparative biochemical and proteomics analysis of the fruit, pulp juice and seeds of these three species. The results indicated equivalent chemical compositions and variable protein profiles with some differences in the content of the specific compounds or protein species that may result in variable organoleptic properties between the species and can be exploited for traceability purposes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Adaptation of proteomic techniques for the identification and characterization of protein species from murine heart.

    Science.gov (United States)

    Schwab, Karima; Neumann, Boris; Scheler, Christian; Jungblut, Peter R; Theuring, Franz

    2011-07-01

    Disturbed energy metabolism with impaired fatty acid oxidation, ATP synthesis and changing levels of contractile proteins has been observed during the development and manifestation of cardiovascular diseases, with the latter showing sexual differences in terms of onset, manifestation and progress. Estrogenic compounds, such as estrogens and phytoestrogens, are known to exert beneficial effects on several cardiovascular parameters. However, global studies implying the normal, non-failing myocardium are rare. Thus, identifying and characterizing protein patterns involved in the maintenance of normal heart physiology at the protein species level will help understanding disease conditions. In this study, we performed an adapted 2-DE/MS approach in order to identify and characterize post-translational modified and truncated protein species from murine heart. Female and male animals of different age were receiving the phytoestrogen genistein and comparative analyses were performed to identify sex and genistein treatment-related effects. Selected 2-DE spots that exposed varying abundance between animal groups and identified as identical proteins were subject to multi-protease cleavage to generate an elevated sequence coverage enabling characterization of post-translational modifications and truncation loci via high-resolution MS. Several truncated, phosphorylated and acetylated species were identified for mitochondrial ATP synthase, malate dehydrogenase and trifunctional enzyme subunit alpha. However, confirmation of several of these modifications by manual spectra interpretation failed. Thus, our results warrant caution for the blind trust in software output. For the regulatory light chain of myosin, we identified an N-terminal processed species, which so far has been related to ischemic conditions only. We tried to unravel the information content of protein species separated by high-resolution 2-DE as an alternative to high-throughput proteomics, which mainly is

  3. Single-strand-conformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytophthora.

    Science.gov (United States)

    Kong, Ping; Hong, Chuanxue; Richardson, Patricia A; Gallegly, Mannon E

    2003-08-01

    Single-strand-conformation polymorphism (SSCP) of ribosomal DNA of 29 species (282 isolates) of Phytophthora was characterized in this study. Phytophthora boehmeriae, Phytophthora botryosa, Phytophthora cactorum, Phytophthora cambivora, Phytophthora capsici, Phytophthora cinnamomi, Phytophthora colocasiae, Phytophthora fragariae, Phytophthora heveae, Phytophthora hibernalis, Phytophthora ilicis, Phytophthora infestans, Phytophthora katsurae, Phytophthora lateralis, Phytophthora meadii, Phytophthora medicaginis, Phytophthora megakarya, Phytophthora nicotianae, Phytophthora palmivora, Phytophthora phaseoli, Phytophthora pseudotsugae, Phytophthora sojae, Phytophthora syringae, and Phytophthora tropicalis each showed a unique SSCP pattern. Phytophthora citricola, Phytophthora citrophthora, Phytophthora cryptogea, Phytophthora drechsleri, and Phytophthora megasperma each had more than one distinct pattern. A single-stranded DNA ladder also was developed, which facilitates comparison of SSCP patterns within and between gels. With a single DNA fingerprint, 277 isolates of Phytophthora recovered from irrigation water and plant tissues in Virginia were all correctly identified into eight species at substantially reduced time, labor, and cost. The SSCP analysis presented in this work will aid in studies on taxonomy, genetics, and ecology of the genus Phytophthora.

  4. Milk protein and cheese yield in buffalo species

    Directory of Open Access Journals (Sweden)

    Rossella Di Palo

    2010-01-01

    Full Text Available Buffalo milk samples differing significantly for cheese yield values were analysed by 2D electrophoresis in order to outline a protein profile, with specific regards to k-casein fractions. Four buffaloes, two of which showing high cheese yield and two with low cheese yield selected from a group of 135 subjects were chosen for the proteomic analyses. Six main spots in 2D gels were recognized as αs1-, αs2-, β- and k-casein, α-lactoalbumin, β-lactoglobulin. The main visible differences in the 2D gels between buffaloes with high vs. low cheese yield were found in the appearance of the four k-casein spots (spots numbers:20, 19, 16, 18 which differ in the number of phosphorilation and glycosilation. The area and the intensity of the four spots were calculated by using Melanie II (Bio-Rad software. Samples with high cheese yield showed higher value of the by-products: area x intensity of spot 16, correspondent to k-casein with one phosphorilation site, and lower values of spots 19 and 20, of k-casein with more than one phosphorilation site and glycosilated.

  5. A transdisciplinary approach to the initial validation of a single cell protein as an alternative protein source for use in aquafeeds

    Directory of Open Access Journals (Sweden)

    Michael Tlusty

    2017-04-01

    Full Text Available The human population is growing and, globally, we must meet the challenge of increased protein needs required to feed this population. Single cell proteins (SCP, when coupled to aquaculture production, offer a means to ensure future protein needs can be met without direct competition with food for people. To demonstrate a given type of SCP has potential as a protein source for use in aquaculture feed, a number of steps need to be validated including demonstrating that the SCP is accepted by the species in question, leads to equivalent survival and growth, does not result in illness or other maladies, is palatable to the consumer, is cost effective to produce and can easily be incorporated into diets using existing technology. Here we examine white shrimp (Litopenaeus vannamei growth and consumer taste preference, smallmouth grunt (Haemulon chrysargyreum growth, survival, health and gut microbiota, and Atlantic salmon (Salmo salar digestibility when fed diets that substitute the bacterium Methylobacterium extorquens at a level of 30% (grunts, 100% (shrimp, or 55% (salmon of the fishmeal in a compound feed. In each of these tests, animals performed equivalently when fed diets containing M. extorquens as when fed a standard aquaculture diet. This transdisciplinary approach is a first validation of this bacterium as a potential SCP protein substitute in aquafeeds. Given the ease to produce this SCP through an aerobic fermentation process, the broad applicability for use in aquaculture indicates the promise of M. extorquens in leading toward greater food security in the future.

  6. Single proteins that serve linked functions in intracellular and extracellular microenvironments

    Energy Technology Data Exchange (ETDEWEB)

    Radisky, Derek C.; Stallings-Mann, Melody; Hirai, Yohei; Bissell, Mina J.

    2009-06-03

    Maintenance of organ homeostasis and control of appropriate response to environmental alterations requires intimate coordination of cellular function and tissue organization. An important component of this coordination may be provided by proteins that can serve distinct, but linked, functions on both sides of the plasma membrane. Here we present a novel hypothesis in which non-classical secretion can provide a mechanism through which single proteins can integrate complex tissue functions. Single genes can exert a complex, dynamic influence through a number of different processes that act to multiply the function of the gene product(s). Alternative splicing can create many different transcripts that encode proteins of diverse, even antagonistic, function from a single gene. Posttranslational modifications can alter the stability, activity, localization, and even basic function of proteins. A protein can exist in different subcellular localizations. More recently, it has become clear that single proteins can function both inside and outside the cell. These proteins often lack defined secretory signal sequences, and transit the plasma membrane by mechanisms separate from the classical ER/Golgi secretory process. When examples of such proteins are examined individually, the multifunctionality and lack of a signal sequence are puzzling - why should a protein with a well known function in one context function in such a distinct fashion in another? We propose that one reason for a single protein to perform intracellular and extracellular roles is to coordinate organization and maintenance of a global tissue function. Here, we describe in detail three specific examples of proteins that act in this fashion, outlining their specific functions in the extracellular space and in the intracellular space, and we discuss how these functions may be linked. We present epimorphin/syntaxin-2, which may coordinate morphogenesis of secretory organs (as epimorphin) with control of

  7. Protein Signaling Networks from Single Cell Fluctuations and Information Theory Profiling

    Science.gov (United States)

    Shin, Young Shik; Remacle, F.; Fan, Rong; Hwang, Kiwook; Wei, Wei; Ahmad, Habib; Levine, R.D.; Heath, James R.

    2011-01-01

    Protein signaling networks among cells play critical roles in a host of pathophysiological processes, from inflammation to tumorigenesis. We report on an approach that integrates microfluidic cell handling, in situ protein secretion profiling, and information theory to determine an extracellular protein-signaling network and the role of perturbations. We assayed 12 proteins secreted from human macrophages that were subjected to lipopolysaccharide challenge, which emulates the macrophage-based innate immune responses against Gram-negative bacteria. We characterize the fluctuations in protein secretion of single cells, and of small cell colonies (n = 2, 3,···), as a function of colony size. Measuring the fluctuations permits a validation of the conditions required for the application of a quantitative version of the Le Chatelier's principle, as derived using information theory. This principle provides a quantitative prediction of the role of perturbations and allows a characterization of a protein-protein interaction network. PMID:21575571

  8. Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein

    NARCIS (Netherlands)

    Hink, M.A.; Griep, R.A.; Borst, J.W.; Hoek, van A.; Eppink, M.H.M.; Schots, A.; Visser, A.J.W.G.

    2000-01-01

    Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a

  9. Distribution and evolution of stable single α-helices (SAH domains in myosin motor proteins.

    Directory of Open Access Journals (Sweden)

    Dominic Simm

    Full Text Available Stable single-alpha helices (SAHs are versatile structural elements in many prokaryotic and eukaryotic proteins acting as semi-flexible linkers and constant force springs. This way SAH-domains function as part of the lever of many different myosins. Canonical myosin levers consist of one or several IQ-motifs to which light chains such as calmodulin bind. SAH-domains provide flexibility in length and stiffness to the myosin levers, and may be particularly suited for myosins working in crowded cellular environments. Although the function of the SAH-domains in human class-6 and class-10 myosins has well been characterised, the distribution of the SAH-domain in all myosin subfamilies and across the eukaryotic tree of life remained elusive. Here, we analysed the largest available myosin sequence dataset consisting of 7919 manually annotated myosin sequences from 938 species representing all major eukaryotic branches using the SAH-prediction algorithm of Waggawagga, a recently developed tool for the identification of SAH-domains. With this approach we identified SAH-domains in more than one third of the supposed 79 myosin subfamilies. Depending on the myosin class, the presence of SAH-domains can range from a few to almost all class members indicating complex patterns of independent and taxon-specific SAH-domain gain and loss.

  10. Enhanced detection of single-cell-secreted proteins using a fluorescent immunoassay on the protein-G-terminated glass substrate.

    Science.gov (United States)

    Jeong, Yoon; Lee, Kwan Hong; Park, Hansoo; Choi, Jonghoon

    2015-01-01

    We present an evaluation of protein-G-terminated glass slides that may contain a suitable substrate for aligning the orientation of antibodies to obtain better binding moiety to the target antigen. The results of the protein-G-terminated slides were compared with those obtained with epoxy-based slides to evaluate signal enhancement for human immunoglobulin G (IgG) targets, and an increase in the average fluorescence intensity was observed for the lowest measurable amount of IgG target in the assay using protein-G-terminated slides. Applying this strategy for signal amplification to single-cell assays improves the limits of detection for human IgG protein and cytokines (interleukin-2 and interferon-γ) captured from hybridomas. Our data indicate that protein-G-terminated slides have a higher binding capacity for antigens and have better spot-to-spot consistency than that of traditional epoxy-based slides. These properties would be beneficial in the detection of fine amounts of single-cell-secreted proteins, which may provide key insights into cell-cell communication and immune responses.

  11. Addressing challenges in single species assessments via a simple state-space assessment model

    DEFF Research Database (Denmark)

    Nielsen, Anders

    Single-species and age-structured fish stock assessments still remains the main tool for managing fish stocks. A simple state-space assessment model is presented as an alternative to (semi) deterministic procedures and the full parametric statistical catch at age models. It offers a solution...... to some of the key challenges of these models. Compared to the deterministic procedures it solves a list of problems originating from falsely assuming that age classified catches are known without errors and allows quantification of uncertainties of estimated quantities of interest. Compared to full...

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

    Science.gov (United States)

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

    2012-01-01

    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. PMID:22467831

  13. Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici

    Directory of Open Access Journals (Sweden)

    Lung Shiu-Cheung

    2012-03-01

    Full Text Available Abstract Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis.

  14. Geometry-induced injection dispersion in single-cell protein electrophoresis.

    Science.gov (United States)

    Pan, Qiong; Herr, Amy E

    2018-02-13

    Arrays of microwells are widely used to isolate individual cells, facilitate high throughput cytometry assays, and ensure compatibility of those assays with whole-cell imaging. Microwell geometries have recently been utilized for handling and preparation of single-cell lysate, prior to single-cell protein electrophoresis. It is in the context of single-cell electrophoresis that we investigate the interplay of microwell geometry (circular, rectangular, triangular) and transport (diffusion, electromigration) on the subsequent performance of single-cell polyacrylamide gel electrophoresis (PAGE) for protein targets. We define and measure injector-induced dispersion during PAGE, and develop a numerical model of band broadening sources, experimentally validate the numerical model, and then identify operating conditions (characterized through the Peclet number, Pe) that lead to microwell-geometry induced losses in separation performance. With analysis of mammalian cells as a case study, we sought to understand at what Pe is the PAGE separation performance adversely sensitized to the microwell geometry. In developing design rules, we find that for the microwell geometries that are the most suitable for isolation of mammalian cells and moderate mass protein targets, the Pe is usually small enough (Pe geometry on protein PAGE of single-cell lysate. In extreme cases where the largest mammalian cells are analyzed (Pe > ∼20), consideration of Pe suggests using a rectangular - and not the widely used circular - microwell geometry to maximize protein PAGE separation performance. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Single-particle electron microscopy in the study of membrane protein structure.

    Science.gov (United States)

    De Zorzi, Rita; Mi, Wei; Liao, Maofu; Walz, Thomas

    2016-02-01

    Single-particle electron microscopy (EM) provides the great advantage that protein structure can be studied without the need to grow crystals. However, due to technical limitations, this approach played only a minor role in the study of membrane protein structure. This situation has recently changed dramatically with the introduction of direct electron detection device cameras, which allow images of unprecedented quality to be recorded, also making software algorithms, such as three-dimensional classification and structure refinement, much more powerful. The enhanced potential of single-particle EM was impressively demonstrated by delivering the first long-sought atomic model of a member of the biomedically important transient receptor potential channel family. Structures of several more membrane proteins followed in short order. This review recounts the history of single-particle EM in the study of membrane proteins, describes the technical advances that now allow this approach to generate atomic models of membrane proteins and provides a brief overview of some of the membrane protein structures that have been studied by single-particle EM to date. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Building and analyzing protein interactome networks by cross-species comparisons

    Directory of Open Access Journals (Sweden)

    Blackman Barron

    2010-03-01

    Full Text Available Abstract Background A genomic catalogue of protein-protein interactions is a rich source of information, particularly for exploring the relationships between proteins. Numerous systems-wide and small-scale experiments have been conducted to identify interactions; however, our knowledge of all interactions for any one species is incomplete, and alternative means to expand these network maps is needed. We therefore took a comparative biology approach to predict protein-protein interactions across five species (human, mouse, fly, worm, and yeast and developed InterologFinder for research biologists to easily navigate this data. We also developed a confidence score for interactions based on available experimental evidence and conservation across species. Results The connectivity of the resultant networks was determined to have scale-free distribution, small-world properties, and increased local modularity, indicating that the added interactions do not disrupt our current understanding of protein network structures. We show examples of how these improved interactomes can be used to analyze a genome-scale dataset (RNAi screen and to assign new function to proteins. Predicted interactions within this dataset were tested by co-immunoprecipitation, resulting in a high rate of validation, suggesting the high quality of networks produced. Conclusions Protein-protein interactions were predicted in five species, based on orthology. An InteroScore, a score accounting for homology, number of orthologues with evidence of interactions, and number of unique observations of interactions, is given to each known and predicted interaction. Our website http://www.interologfinder.org provides research biologists intuitive access to this data.

  17. Cross-Species Virus-Host Protein-Protein Interactions Inhibiting Innate Immunity

    Science.gov (United States)

    2016-07-01

    of zoonotic viruses acquiring new host species , including humans. This news story was prompted by the emergence of the Middle Eastern Respiratory...infectious diseases are viral in nature and involve a host species jump from animals to humans. Many factors may contribute to (re-) emergence of viral...widespread morbidity and mortality, with new strains emerging from animal reservoirs possessing the potential to cause pandemics. The influenza A RNA

  18. Temperature tolerance and stress proteins as mechanisms of invasive species success.

    Directory of Open Access Journals (Sweden)

    Robyn A Zerebecki

    2011-04-01

    Full Text Available Invasive species are predicted to be more successful than natives as temperatures increase with climate change. However, few studies have examined the physiological mechanisms that theoretically underlie this differential success. Because correlative evidence suggests that invasiveness is related to the width of a species' latitudinal range, it has been assumed--but largely untested--that range width predicts breadth of habitat temperatures and physiological thermotolerances. In this study, we use empirical data from a marine community as a case study to address the hypotheses that (1 geographic temperature range attributes are related to temperature tolerance, leading to greater eurythermality in invasive species, and (2 stress protein expression is a subcellular mechanism that could contribute to differences in thermotolerance. We examined three native and six invasive species common in the subtidal epibenthic communities of California, USA. We assessed thermotolerance by exposing individuals to temperatures between 14°C and 31°C and determining the temperature lethal to 50% of individuals (LT(50 after a 24 hour exposure. We found a strong positive relationship between the LT(50 and both maximum habitat temperatures and the breadth of temperatures experience across the species' ranges. In addition, of the species in our study, invasives tended to inhabit broader habitat temperature ranges and higher maximum temperatures. Stress protein expression may contribute to these differences: the more thermotolerant, invasive species Diplosoma listerianum expressed higher levels of a 70-kDa heat-shock protein than the less thermotolerant, native Distaplia occidentalis for which levels declined sharply above the LT(50. Our data highlight differences between native and invasive species with respect to organismal and cellular temperature tolerances. Future studies should address, across a broader phylogenetic and ecosystem scope, whether this

  19. Biophysics of DNA-Protein Interactions From Single Molecules to Biological Systems

    CERN Document Server

    Williams, Mark C

    2011-01-01

    This book presents a concise overview of current research on the biophysics of DNA-protein interactions. A wide range of new and classical methods are presented by authors investigating physical mechanisms by which proteins interact with DNA. For example, several chapters address the mechanisms by which proteins search for and recognize specific binding sites on DNA, a process critical for cellular function. Single molecule methods such as force spectroscopy as well as fluorescence imaging and tracking are described in these chapters as well as other parts of the book that address the dynamics of protein-DNA interactions. Other important topics include the mechanisms by which proteins engage DNA sequences and/or alter DNA structure. These simple but important model interactions are then placed in the broader biological context with discussion of larger protein-DNA complexes . Topics include replication forks, recombination complexes, DNA repair interactions, and ultimately, methods to understand the chromatin...

  20. FORMATION OF LONG-LIVED REACTIVE SPECIES OF BLOOD SERUM PROTEINS BY THE ACTION OF HEAT

    Directory of Open Access Journals (Sweden)

    V. E. Ivanov

    2014-01-01

    Full Text Available Background: Proteins oxidized by the action of X-rays represent long-lived reactive species, which trigger the secondary generation of reactive oxygen species (ROS. A change in the hydrogen peroxide (H2O2 content induced by various physical impacts may be an important factor of the therapeutic effect and the adaptation of the organism to unfavorable environmental conditions. Moderate hyperthermia and a number of physiotherapeutic procedures leading to a local warming of tissues are widely used in medical practice. However, the biological mechanisms of their curative effect are poorly understood. The prolonged generation of H2O2 long-lived reactive protein species (LRPS after heating may be one of the mechanisms of activation of protective cellular mechanisms and thus to contribute to overcoming the disease. Aim: To investigate if the serum proteins bovine serum albumin (BSA and bovine gamma-globulin (BGG can form LRPS under moderate hyperthermia and show that heat induces LRPS, which in turn continuously generate ROS, in particular H2O2. Materials and methods: LRPS were studied by measuring the heat-induced chemiluminescence of protein solutions using a specially elaborated highly sensitive photon-counting chemiluminometer Biotoks-7 AM. The Results: Here we studied the possibility of formation of long-lived species of the blood serum proteins BSA and BGG in air-saturated solutions under the action of heat. It is shown that heat induces the generation of long-lived protein species, which in turn generate ROS (1O2, О2-•, OH•, H2O2. The formation of the long-lived reactive species of BSA and BGG with a half-life of about 4 h induced by moderate hyperthermia was revealed using the chemiluminescence of protein solutions. It was found that long-lived reactive species of BSA and BGG cause prolonged generation of H2O2. Conclusion: Thus, we found a new fundamental property of serum proteins: by the action of moderate heating, they are able to

  1. A human protein interaction network shows conservation of aging processes between human and invertebrate species.

    Directory of Open Access Journals (Sweden)

    Russell Bell

    2009-03-01

    Full Text Available We have mapped a protein interaction network of human homologs of proteins that modify longevity in invertebrate species. This network is derived from a proteome-scale human protein interaction Core Network generated through unbiased high-throughput yeast two-hybrid searches. The longevity network is composed of 175 human homologs of proteins known to confer increased longevity through loss of function in yeast, nematode, or fly, and 2,163 additional human proteins that interact with these homologs. Overall, the network consists of 3,271 binary interactions among 2,338 unique proteins. A comparison of the average node degree of the human longevity homologs with random sets of proteins in the Core Network indicates that human homologs of longevity proteins are highly connected hubs with a mean node degree of 18.8 partners. Shortest path length analysis shows that proteins in this network are significantly more connected than would be expected by chance. To examine the relationship of this network to human aging phenotypes, we compared the genes encoding longevity network proteins to genes known to be changed transcriptionally during aging in human muscle. In the case of both the longevity protein homologs and their interactors, we observed enrichments for differentially expressed genes in the network. To determine whether homologs of human longevity interacting proteins can modulate life span in invertebrates, homologs of 18 human FRAP1 interacting proteins showing significant changes in human aging muscle were tested for effects on nematode life span using RNAi. Of 18 genes tested, 33% extended life span when knocked-down in Caenorhabditis elegans. These observations indicate that a broad class of longevity genes identified in invertebrate models of aging have relevance to human aging. They also indicate that the longevity protein interaction network presented here is enriched for novel conserved longevity proteins.

  2. Are single odorous components of a predator sufficient to elicit defensive behaviors in prey species?

    Directory of Open Access Journals (Sweden)

    Raimund eApfelbach

    2015-07-01

    Full Text Available When exposed to the odor of a sympatric predator, prey animals typically display escape or defensive responses. These phenomena have been well-documented, especially in rodents, when exposed to the odor of a cat, ferret or fox. As a result of these experiments new discussions center on the following questions: 1 is a single volatile compound such as a major or a minor mixture constituent in urine or feces, emitted by the predator sufficient to cause defensive reactions in a potential prey species or 2 is a whole array of odors required to elicit a response and 3 will the relative size or escapability of the prey as compared to the predator influence responsiveness. Most predator-prey studies on this topic have been performed in the laboratory or under semi-natural conditions. Field studies could help to find answers to these questions. Australian mammals are completely naïve towards the introduced placental carnivores. That offers ideal opportunities to analyze in the field the responses of potential prey species to unknown predator odors. During the last decades researchers have accumulated an enormous amount of data exploring the effects of eutherian predator odors on native marsupial mammals. In this review, we will give a survey about the development of olfactory research, chemical signals and their influence on the behavior and - in some cases - physiology of prey species. In addition, we report on the effects of predator odor experiments performed under natural conditions in Australia. When studying all these literature we learned that data gained under controlled laboratory conditions elucidate the role of individual odors on brain structures and ultimately on a comparatively narrow range behaviors. In contrast to single odors odor arrays mimic much more the situation prey animals are confronted to in nature. Therefore, a broad range of methodology — from chemistry to ecology including anatomy, physiology and behavior — is needed to

  3. Protein secondary structure prediction for a single-sequence using hidden semi-Markov models

    Directory of Open Access Journals (Sweden)

    Borodovsky Mark

    2006-03-01

    Full Text Available Abstract Background The accuracy of protein secondary structure prediction has been improving steadily towards the 88% estimated theoretical limit. There are two types of prediction algorithms: Single-sequence prediction algorithms imply that information about other (homologous proteins is not available, while algorithms of the second type imply that information about homologous proteins is available, and use it intensively. The single-sequence algorithms could make an important contribution to studies of proteins with no detected homologs, however the accuracy of protein secondary structure prediction from a single-sequence is not as high as when the additional evolutionary information is present. Results In this paper, we further refine and extend the hidden semi-Markov model (HSMM initially considered in the BSPSS algorithm. We introduce an improved residue dependency model by considering the patterns of statistically significant amino acid correlation at structural segment borders. We also derive models that specialize on different sections of the dependency structure and incorporate them into HSMM. In addition, we implement an iterative training method to refine estimates of HSMM parameters. The three-state-per-residue accuracy and other accuracy measures of the new method, IPSSP, are shown to be comparable or better than ones for BSPSS as well as for PSIPRED, tested under the single-sequence condition. Conclusions We have shown that new dependency models and training methods bring further improvements to single-sequence protein secondary structure prediction. The results are obtained under cross-validation conditions using a dataset with no pair of sequences having significant sequence similarity. As new sequences are added to the database it is possible to augment the dependency structure and obtain even higher accuracy. Current and future advances should contribute to the improvement of function prediction for orphan proteins inscrutable

  4. Mining the transcriptomes of four commercially important shellfish species for single nucleotide polymorphisms within biomineralization genes.

    Science.gov (United States)

    Vendrami, David L J; Shah, Abhijeet; Telesca, Luca; Hoffman, Joseph I

    2016-06-01

    Transcriptional profiling not only provides insights into patterns of gene expression, but also generates sequences that can be mined for molecular markers, which in turn can be used for population genetic studies. As part of a large-scale effort to better understand how commercially important European shellfish species may respond to ocean acidification, we therefore mined the transcriptomes of four species (the Pacific oyster Crassostrea gigas, the blue mussel Mytilus edulis, the great scallop Pecten maximus and the blunt gaper Mya truncata) for single nucleotide polymorphisms (SNPs). Illumina data for C. gigas, M. edulis and P. maximus and 454 data for M. truncata were interrogated using GATK and SWAP454 respectively to identify between 8267 and 47,159 high quality SNPs per species (total=121,053 SNPs residing within 34,716 different contigs). We then annotated the transcripts containing SNPs to reveal homology to diverse genes. Finally, as oceanic pH affects the ability of organisms to incorporate calcium carbonate, we honed in on genes implicated in the biomineralization process to identify a total of 1899 SNPs in 157 genes. These provide good candidates for biomarkers with which to study patterns of selection in natural or experimental populations. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. A nonlocal and periodic reaction-diffusion-advection model of a single phytoplankton species.

    Science.gov (United States)

    Peng, Rui; Zhao, Xiao-Qiang

    2016-02-01

    In this article, we are concerned with a nonlocal reaction-diffusion-advection model which describes the evolution of a single phytoplankton species in a eutrophic vertical water column where the species relies solely on light for its metabolism. The new feature of our modeling equation lies in that the incident light intensity and the death rate are assumed to be time periodic with a common period. We first establish a threshold type result on the global dynamics of this model in terms of the basic reproduction number R0. Then we derive various characterizations of R0 with respect to the vertical turbulent diffusion rate, the sinking or buoyant rate and the water column depth, respectively, which in turn give rather precise conditions to determine whether the phytoplankton persist or become extinct. Our theoretical results not only extend the existing ones for the time-independent case, but also reveal new interesting effects of the modeling parameters and the time-periodic heterogeneous environment on persistence and extinction of the phytoplankton species, and thereby suggest important implications for phytoplankton growth control.

  6. Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

    DEFF Research Database (Denmark)

    Derouiche, Abderahmane; Petranovic, Dina; Macek, Boris

    2016-01-01

    Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA-binding pro......Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA...... assays.Results: In addition to the known tyrosine phosphorylation of SsbA on tyrosine 82, we identified a new phosphorylation site: threonine 38. The in vitro assays demonstrated that SsbA is preferentially phosphorylated by the B. subtilis Hanks-type kinase YabT, and phosphorylation of threonine 38...... leads to enhanced cooperative binding to DNA.Conclusions: Our findings contribute to the emerging picture that bacterial proteins, exemplified here by SsbA, undergo phosphorylation at multiple residues. This results in a complex regulation of cellular functions, and suggests that the complexity...

  7. Shedding light on protein folding, structural and functional dynamics by single molecule studies

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Hatzakis, Nikos

    2014-01-01

    The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean...... property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out...

  8. Identification of soybean proteins from a single cell type: The root hair

    Energy Technology Data Exchange (ETDEWEB)

    Brechenmacher, Laurent; Nguyen, Tran H.; Hixson, Kim K.; Libault, Marc; Aldrich, Joshua T.; Pasa-Tolic, Ljiljana; Stacey, Gary

    2012-11-01

    Root hairs are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean root hair cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean root hairs using an accurate mass and time tag approach, establishing the most comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome database using different proteases. Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to root hair formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and RAB proteins). Interestingly, some of these proteins appear to be specifically expressed in root hairs and constitute very good candidates for further studies to elucidate unique features of this single cell model.

  9. QAcon: single model quality assessment using protein structural and contact information with machine learning techniques.

    Science.gov (United States)

    Cao, Renzhi; Adhikari, Badri; Bhattacharya, Debswapna; Sun, Miao; Hou, Jie; Cheng, Jianlin

    2017-02-15

    Protein model quality assessment (QA) plays a very important role in protein structure prediction. It can be divided into two groups of methods: single model and consensus QA method. The consensus QA methods may fail when there is a large portion of low quality models in the model pool. In this paper, we develop a novel single-model quality assessment method QAcon utilizing structural features, physicochemical properties, and residue contact predictions. We apply residue-residue contact information predicted by two protein contact prediction methods PSICOV and DNcon to generate a new score as feature for quality assessment. This novel feature and other 11 features are used as input to train a two-layer neural network on CASP9 datasets to predict the quality of a single protein model. We blindly benchmarked our method QAcon on CASP11 dataset as the MULTICOM-CLUSTER server. Based on the evaluation, our method is ranked as one of the top single model QA methods. The good performance of the features based on contact prediction illustrates the value of using contact information in protein quality assessment. The web server and the source code of QAcon are freely available at: http://cactus.rnet.missouri.edu/QAcon. chengji@missouri.edu. Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  10. Effects of phylogenetic reconstruction method on the robustness of species delimitation using single-locus data.

    Science.gov (United States)

    Tang, Cuong Q; Humphreys, Aelys M; Fontaneto, Diego; Barraclough, Timothy G; Paradis, Emmanuel

    2014-10-01

    Coalescent-based species delimitation methods combine population genetic and phylogenetic theory to provide an objective means for delineating evolutionarily significant units of diversity. The generalised mixed Yule coalescent (GMYC) and the Poisson tree process (PTP) are methods that use ultrametric (GMYC or PTP) or non-ultrametric (PTP) gene trees as input, intended for use mostly with single-locus data such as DNA barcodes. Here, we assess how robust the GMYC and PTP are to different phylogenetic reconstruction and branch smoothing methods. We reconstruct over 400 ultrametric trees using up to 30 different combinations of phylogenetic and smoothing methods and perform over 2000 separate species delimitation analyses across 16 empirical data sets. We then assess how variable diversity estimates are, in terms of richness and identity, with respect to species delimitation, phylogenetic and smoothing methods. The PTP method generally generates diversity estimates that are more robust to different phylogenetic methods. The GMYC is more sensitive, but provides consistent estimates for BEAST trees. The lower consistency of GMYC estimates is likely a result of differences among gene trees introduced by the smoothing step. Unresolved nodes (real anomalies or methodological artefacts) affect both GMYC and PTP estimates, but have a greater effect on GMYC estimates. Branch smoothing is a difficult step and perhaps an underappreciated source of bias that may be widespread among studies of diversity and diversification. Nevertheless, careful choice of phylogenetic method does produce equivalent PTP and GMYC diversity estimates. We recommend simultaneous use of the PTP model with any model-based gene tree (e.g. RAxML) and GMYC approaches with BEAST trees for obtaining species hypotheses.

  11. Low Po2 conditions induce reactive oxygen species formation during contractions in single skeletal muscle fibers

    Science.gov (United States)

    Shiah, Amy; Roberts, William J.; Chien, Michael T.; Wagner, Peter D.; Hogan, Michael C.

    2013-01-01

    Contractions in whole skeletal muscle during hypoxia are known to generate reactive oxygen species (ROS); however, identification of real-time ROS formation within isolated single skeletal muscle fibers has been challenging. Consequently, there is no convincing evidence showing increased ROS production in intact contracting fibers under low Po2 conditions. Therefore, we hypothesized that intracellular ROS generation in single contracting skeletal myofibers increases during low Po2 compared with a value approximating normal resting Po2. Dihydrofluorescein was loaded into single frog (Xenopus) fibers, and fluorescence was used to monitor ROS using confocal microscopy. Myofibers were exposed to two maximal tetanic contractile periods (1 contraction/3 s for 2 min, separated by a 60-min rest period), each consisting of one of the following treatments: high Po2 (30 Torr), low Po2 (3–5 Torr), high Po2 with ebselen (antioxidant), or low Po2 with ebselen. Ebselen (10 μM) was administered before the designated contractile period. ROS formation during low Po2 treatment was greater than during high Po2 treatment, and ebselen decreased ROS generation in both low- and high-Po2 conditions (P Po2. Force was reduced >30% for each condition except low Po2 with ebselen, which only decreased ∼15%. We concluded that single myofibers under low Po2 conditions develop accelerated and more oxidative stress than at Po2 = 30 Torr (normal human resting Po2). Ebselen decreases ROS formation in both low and high Po2, but only mitigates skeletal muscle fatigue during reduced Po2 conditions. PMID:23576612

  12. Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

    DEFF Research Database (Denmark)

    Schindler, J.; Ye, J. Y.; Jensen, Ole Nørregaard

    2013-01-01

    proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized...... metal affinity chromatography, and TiO2 were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized...

  13. Multiple start codons and phosphorylation result in discrete Rad52 protein species

    DEFF Research Database (Denmark)

    de Mayolo, A.A.; Lisby, M.; Erdeniz, N.

    2006-01-01

    The sequence of the Saccharomyces cerevisiae RAD52 gene contains five potential translation start sites and protein-blot analysis typically detects multiple Rad52 species with different electrophoretic mobilities. Here we define the gene products encoded by RAD52. We show that the multiple Rad52 ...

  14. Crude protein electrophoresis of seeds of ten species of Solanum L ...

    African Journals Online (AJOL)

    Seeds of mature fruits of ten species of Solanum were collected from the gardens near the screen house, Botany Department, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria. Crude seed proteins were extracted from them and characterised using polyacrylamide gel electrophoresis. Inter and intra specific ...

  15. Protein markers for discrimination of meat species in raw beef, pork and poultry and their mixtures.

    Science.gov (United States)

    Kim, Gap-Don; Seo, Jin-Kyu; Yum, Hyeon-Woong; Jeong, Jin-Yeon; Yang, Han-Sul

    2017-02-15

    The purpose of this study was to find discrimination markers for four major meat species such as beef, pork, chicken and duck. Myofibrillar and sarcoplasmic proteins isolated from each meat type were analyzed by one-dimensional gel electrophoresis and some proteins were identified through LC-MS/MS analysis. We confirmed that troponin I (TnI), enolase 3, l-lactate dehydrogenase (LDH) and triose-phosphate isomerase (TPI) could be useful markers for discrimination of mammals from poultry due to their different electrophoretic mobility. Tropomyosin 1 and carbonic anhydrase 3 were observed as muscle fiber type-related proteins and these could also be markers to distinguish mammals from poultry. Species-specific peptides identified by LC-MS/MS spectra allow the identification of each species regardless of the same protein. Therefore, it is easy to discriminate between mammals and poultry by comparing the electrophoretic mobility of TnI, enolase 3, LDH, TPI and CA3, and each species could be identified through LC-MS/MS analysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Simultaneous detection of mRNA and protein in single cells using immunofluorescence-combined single-molecule RNA FISH.

    Science.gov (United States)

    Kochan, Jakub; Wawro, Mateusz; Kasza, Aneta

    2015-10-01

    Although the concept of combining immunofluorescence (IF) with single-molecule RNA fluorescence in situ hybridization (smRNA FISH) seems obvious, the specific materials used during IF and smRNA FISH make it difficult to perform these procedures simultaneously on the same specimen. Even though there are reports where IF and smRNA FISH were combined with success, these were insufficient in terms of signal intensities, staining patterns, and GFP-compatibility, and a detailed exploration of the various factors that influence IF and smRNA FISH outcome has not been published yet. Here, we report a detailed study of conditions and reagents used in classic IF and smRNA FISH that allowed us to establish an easy, robust, and GFP-compatible procedure. Our protocol enables simultaneous detection of mRNA and protein quantity as well as the subcellular distribution of these molecules in single cells by combining an RNase-free modification of the IF technique and the more recent smRNA FISH method. Using this procedure, we have shown the direct interaction of RNase MCPIP1 with IL-6 mRNA. We also demonstrate the use of our protocol in heterogeneous cell population analysis, revealing cell-to-cell differences in mRNA and protein content.

  17. Room temperature phosphorescence study on the structural flexibility of single tryptophan containing proteins

    Science.gov (United States)

    Kowalska-Baron, Agnieszka; Gałęcki, Krystian; Wysocki, Stanisław

    2015-01-01

    In this study, we have undertaken efforts to find correlation between phosphorescence lifetimes of single tryptophan containing proteins and some structural indicators of protein flexibility/rigidity, such as the degree of tryptophan burial or its exposure to solvent, protein secondary and tertiary structure of the region of localization of tryptophan as well as B factors for tryptophan residue and its immediate surroundings. Bearing in mind that, apart from effective local viscosity of the protein/solvent matrix, the other factor that concur in determining room temperature tryptophan phosphorescence (RTTP) lifetime in proteins is the extent of intramolecular quenching by His, Cys, Tyr and Trp side chains, the crystallographic structures derived from the Brookhaven Protein Data Bank were also analyzed concentrating on the presence of potentially quenching amino acid side chains in the close proximity of the indole chromophore. The obtained results indicated that, in most cases, the phosphorescence lifetimes of tryptophan containing proteins studied tend to correlate with the above mentioned structural indicators of protein rigidity/flexibility. This correlation is expected to provide guidelines for the future development of phosphorescence lifetime-based method for the prediction of structural flexibility of proteins, which is directly linked to their biological function.

  18. Series solution for continuous population models for single and interacting species by the homotopy analysis method

    Directory of Open Access Journals (Sweden)

    Magdy A. El-Tawil

    2012-07-01

    Full Text Available The homotopy analysis method (HAM is used to find approximate analytical solutions of continuous population models for single and interacting species. The homotopy analysis method contains the auxiliary parameter $hbar,$ which provides us with a simple way to adjust and control the convergence region of series solution. the solutions are compared with the numerical results obtained using NDSolve, an ordinary differential equation solver found in the Mathematica package and a good agreement is found. Also the solutions are compared with the available analytic results obtained by other methods and more accurate and convergent series solution found. The convergence region is also computed which shows the validity of the HAM solution. This method is reliable and manageable.

  19. Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy

    NARCIS (Netherlands)

    van Manen, H.J.; Lenferink, Aufrid T.M.; Otto, Cornelis

    2008-01-01

    We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C−D

  20. Single particle electron microscopy in combination with mass spectrometry to investigate novel complexes of membrane proteins

    NARCIS (Netherlands)

    Arteni, Ana A.; Nowaczyk, Marc; Lax, Julia; Rögner, Matthias; Boekema, Egbert J.; Kouril, R.; Rogner, M.

    2005-01-01

    Large data sets of molecular projections of the membrane proteins Photosystem I and Photosystem II from cyanobacteria were analyzed by single particle electron microscopy (EM). Analysis resulted in the averaging of 2D projections from the purified complexes but also in the simultaneous detection and

  1. Shedding Light on Protein Folding, Structural and Functional Dynamics by Single Molecule Studies

    Directory of Open Access Journals (Sweden)

    Krutika Bavishi

    2014-11-01

    Full Text Available The advent of advanced single molecule measurements unveiled a great wealth of dynamic information revolutionizing our understanding of protein dynamics and behavior in ways unattainable by conventional bulk assays. Equipped with the ability to record distribution of behaviors rather than the mean property of a population, single molecule measurements offer observation and quantification of the abundance, lifetime and function of multiple protein states. They also permit the direct observation of the transient and rarely populated intermediates in the energy landscape that are typically averaged out in non-synchronized ensemble measurements. Single molecule studies have thus provided novel insights about how the dynamic sampling of the free energy landscape dictates all aspects of protein behavior; from its folding to function. Here we will survey some of the state of the art contributions in deciphering mechanisms that underlie protein folding, structural and functional dynamics by single molecule fluorescence microscopy techniques. We will discuss a few selected examples highlighting the power of the emerging techniques and finally discuss the future improvements and directions.

  2. Ruminal and intestinal protein degradability of various seaweed species measured in situ in dairy cows

    DEFF Research Database (Denmark)

    Tayyab, Usama; Novoa-Garrido, Margarita; Roleda, Michael Y.

    2016-01-01

    The use of seaweeds in animal diets is not new. However, little is known about the feed value of seaweed, both in terms of chemical composition and protein digestibility, and regarding variation between species and season. In this study, eight seaweed species of the genus Acrosiphonia, Alaria......, Laminaria, Mastocarpus, Palmaria, Pelvetia, Porphyra, and Ulva were sampled in spring (March) and autumn (October and November) 2014 at the coast of Bodø in Northern Norway, and were analysed for chemical composition, in situ rumen degradability and total tract crude protein (CP) digestibility. Ash content...... for Pelvetia (90 g/kg DM). Spring samples were higher in CP than autumn samples. The effective degradability estimated at 5% rumen passage rate (ED5) of CP varied between species (P Ulva (240 g...

  3. Genome-wide single-nucleotide polymorphism data reveal cryptic species within cryptic freshwater snail species-The case of theAncylus fluviatilisspecies complex.

    Science.gov (United States)

    Weiss, Martina; Weigand, Hannah; Weigand, Alexander M; Leese, Florian

    2018-01-01

    DNA barcoding utilizes short standardized DNA sequences to identify species and is increasingly used in biodiversity assessments. The technique has unveiled an unforeseeably high number of morphologically cryptic species. However, if speciation has occurred relatively recently and rapidly, the use of single gene markers, and especially the exclusive use of mitochondrial markers, will presumably fail in delimitating species. Therefore, the true number of biological species might be even higher. One mechanism that can result in rapid speciation is hybridization of different species in combination with polyploidization, that is, allopolyploid speciation. In this study, we analyzed the population genetic structure of the polyploid freshwater snail Ancylus fluviatilis , for which allopolyploidization was postulated as a speciation mechanism. DNA barcoding has already revealed four cryptic species within A. fluviatilis (i.e., A. fluviatilis s. str., Ancylus sp. A-C), but early allozyme data even hint at the presence of additional cryptic lineages in Central Europe. We combined COI sequencing with high-resolution genome-wide SNP data (ddRAD data) to analyze the genetic structure of A. fluviatilis populations in a Central German low mountain range (Sauerland). The ddRAD data results indicate the presence of three cryptic species within A. fluviatilis s. str. occurring in sympatry and even syntopy, whereas mitochondrial sequence data only support the existence of one species, with shared haplotypes between species. Our study hence points to the limitations of DNA barcoding when dealing with organismal groups where speciation is assumed to have occurred rapidly, for example, through the process of allopolyploidization. We therefore emphasize that single marker DNA barcoding can underestimate the true species diversity and argue in strong favor of using genome-wide data for species delimitation in such groups.

  4. Two highly thermostable paralogous single-stranded DNA-binding proteins from Thermoanaerobacter tengcongensis.

    Science.gov (United States)

    Olszewski, Marcin; Mickiewicz, Małgorzata; Kur, Józef

    2008-07-01

    The thermophilic bacterium Thermoanaerobacter tengcongensis has two single-stranded DNA-binding (SSB) proteins, designated TteSSB2 and TteSSB3. In a SSB complementation assay in Escherichia coli, only TteSSB3 took over the in vivo function of EcoSSB. We have cloned the ssb genes obtained by PCR and have developed E. coli overexpression systems. The TteSSB2 and TteSSB3 consist of 153 and 150 amino acids with a calculated molecular mass of 17.29 and 16.96 kDa, respectively. They are the smallest known bacterial SSB proteins. The homology between amino acid sequences of these proteins is 40% identity and 53% similarity. They are functional as homotetramers, with each monomer encoding one single-stranded DNA binding domain (OB-fold). In fluorescence titrations with poly(dT), both proteins bind single-stranded DNA with a binding site size of about 40 nt per homotetramer. Thermostability with half-life of about 30 s at 95 degrees C makes TteSSB3 similar to the known SSB of Thermus aquaticus (TaqSSB). The TteSSB2 was fully active even after 6 h incubation at 100 degrees C. Here, we show for the first time paralogous thermostable homotetrameric SSBs, which could be an attractive alternative for known homodimeric thermostable SSB proteins in their applications for molecular biology methods and analytical purposes.

  5. Nonstructural Protein L* Species Specificity Supports a Mouse Origin for Vilyuisk Human Encephalitis Virus.

    Science.gov (United States)

    Drappier, Melissa; Opperdoes, Fred R; Michiels, Thomas

    2017-07-15

    Vilyuisk human encephalitis virus (VHEV) is a picornavirus related to Theiler's murine encephalomyelitis virus (TMEV). VHEV was isolated from human material passaged in mice. Whether this VHEV is of human or mouse origin is therefore unclear. We took advantage of the species-specific activity of the nonstructural L* protein of theiloviruses to track the origin of TMEV isolates. TMEV L* inhibits RNase L, the effector enzyme of the interferon pathway. By using coimmunoprecipitation and functional RNase L assays, the species specificity of RNase L antagonism was tested for L* from mouse (DA) and rat (RTV-1) TMEV strains as well as for VHEV. Coimmunoprecipitation and functional assay data confirmed the species specificity of L* activity and showed that L* from rat strain RTV-1 inhibited rat but not mouse or human RNase L. Next, we showed that the VHEV L* protein was phylogenetically related to L* of mouse viruses and that it failed to inhibit human RNase L but readily antagonized mouse RNase L, unambiguously showing the mouse origin of VHEV. IMPORTANCE Defining the natural host of a virus can be a thorny issue, especially when the virus was isolated only once or when the isolation story is complex. The species Theilovirus includes Theiler's murine encephalomyelitis virus (TMEV), infecting mice and rats, and Saffold virus (SAFV), infecting humans. One TMEV strain, Vilyuisk human encephalitis virus (VHEV), however, was isolated from mice that were inoculated with cerebrospinal fluid of a patient presenting with chronic encephalitis. It is therefore unclear whether VHEV was derived from the human sample or from the inoculated mouse. The L* protein encoded by TMEV inhibits RNase L, a cellular enzyme involved in innate immunity, in a species-specific manner. Using binding and functional assays, we show that this species specificity even allows discrimination between TMEV strains of mouse and of rat origins. The VHEV L* protein clearly inhibited mouse but not human RNase L

  6. Study of molasses / vinasse waste ratio for single cell protein and total microorganisms

    Directory of Open Access Journals (Sweden)

    Marcia Luciana Cazetta

    2006-02-01

    Full Text Available Different molasses/ vinasse ratio were used as substrate to investigate single cell protein and total lipids production by five microorganisms: four yeasts strains: Candida lipolytica, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, a yeast isolated from vinasse lake (denominated LLV98 and a bacterium strain, Corynebacterium glutamicum. The media utilized were: a 50% molasses and 50% vinasse; b 25% molasses and 75% vinasse and c 75% molasses and 25% vinasse. The objective of this work was to study the growth of microorganisms and also evaluate protein and lipids content in the biomass obtained from these by-products. The highest single cell protein production was obtained by S. cerevisiae, 50.35%, followed by R. mucilaginosa, 41.96%. The lowest productions were obtained by C. glutamicum. The higher total lipids productions, more than 26%, were founded in molasses plus vinasse at 50%/50% by S. cerevisiae and C. glutamicum.

  7. How much protein can the body use in a single meal for muscle-building? Implications for daily protein distribution.

    Science.gov (United States)

    Schoenfeld, Brad Jon; Aragon, Alan Albert

    2018-01-01

    Controversy exists about the maximum amount of protein that can be utilized for lean tissue-building purposes in a single meal for those involved in regimented resistance training. It has been proposed that muscle protein synthesis is maximized in young adults with an intake of ~ 20-25 g of a high-quality protein; anything above this amount is believed to be oxidized for energy or transaminated to form urea and other organic acids. However, these findings are specific to the provision of fast-digesting proteins without the addition of other macronutrients. Consumption of slower-acting protein sources, particularly when consumed in combination with other macronutrients, would delay absorption and thus conceivably enhance the utilization of the constituent amino acids. The purpose of this paper was twofold: 1) to objectively review the literature in an effort to determine an upper anabolic threshold for per-meal protein intake; 2) draw relevant conclusions based on the current data so as to elucidate guidelines for per-meal daily protein distribution to optimize lean tissue accretion. Both acute and long-term studies on the topic were evaluated and their findings placed into context with respect to per-meal utilization of protein and the associated implications to distribution of protein feedings across the course of a day. The preponderance of data indicate that while consumption of higher protein doses (> 20 g) results in greater AA oxidation, this is not the fate for all the additional ingested AAs as some are utilized for tissue-building purposes. Based on the current evidence, we conclude that to maximize anabolism one should consume protein at a target intake of 0.4 g/kg/meal across a minimum of four meals in order to reach a minimum of 1.6 g/kg/day. Using the upper daily intake of 2.2 g/kg/day reported in the literature spread out over the same four meals would necessitate a maximum of 0.55 g/kg/meal.

  8. Anaerobiosis and ethanol effects on germination, growth, and protein synthesis of five Echinochloa species

    International Nuclear Information System (INIS)

    Dybiec, L.D.; Rumpho, M.E.; Kennedy, R.A.

    1989-01-01

    Five Echinochloa species, encompassing a spectrum from flood tolerant to flood intolerant, were studied to determine the mechanisms of anaerobic germination and growth. Seeds were germinated in air or N 2 , plus 0, 1 or 3% ethanol, and germination rates and growth measurements recorded for 7 days. In air or N 2 increasing ethanol levels did not affect total germination per se, although the rate of germination was delayed in N 2 . Shoot/root lengths in air were highest for tolerant species and increased with increasing ethanol, whereas, in intolerant species, shoot/root lengths decreased with increasing ethanol. Aerobic vs. anaerobic polypeptide profiles of each of the species were compared by SDS/PAGE. For all species, the number of polypeptides decreased under anaerobiosis and several quantitative differences were apparent relative to the aerobic profile. In addition, amino acid incorporation into protein was analyzed by [ 35 S]-Met labeling of 3 day old seedlings grown in air or N 2 . Significant protein synthesis was measured in tolerant seedlings under N 2 and several polypeptides were specifically induced. These results are being compared with labeling patterns of the other semi-tolerant and intolerant Echinochloa species to determine their importance in flooding tolerance

  9. New Frontiers in NanoBiotechnology: Monitoring the Protein Function With Single Protein Resolution

    Science.gov (United States)

    2005-03-29

    Dipartimento di Fisica, Universita’ di Modena e Reggio Emilia e-mail corni.stefano@unimore.it Electron transfer proteins perform, in living cells...Center on nanoStructures and bioSystems at Surfaces (S3), Dip. di Fisica, Universita’ di Modena e Reggio Emilia Via Campi 213/A, 41100 Modena, Italy...nanowires R. Di Felice INFM-S3 - Universit6 di Modena e Reggio Emilialtaly e-mail: rosa@unimore.it Recent efforts in the field of molecular electronics

  10. Chromosomal and protein evolution in morphologically similar species of Praomys sensu lato (Rodentia, Muridae).

    Science.gov (United States)

    Qumsiyeh, M B; King, S W; Arroyo-Cabrales, J; Aggundey, I R; Schlitter, D A; Baker, R J; Morrow, K J

    1990-01-01

    Evidence of extensive chromosomal evolution in a biologically and economically important group of African murids of the Praomys/Mastomys complex was provided by examination of G- and C-band chromosomal data on P. coucha (2n = 32), P. fumatus (2n = 38), P. hildebrandti (2n = 32), P. jacksoni (2n = 28), P. misonnei (2n = 36), and P. cf. tullbergi (2n = 35). A coding system was developed for the chromosomal characters, and analyses were performed by a computer program to find the shortest tree with a minimum of 35 autosomal rearrangements (pericentric inversions, complex translocations, centric fusions, centric fissions, tandem fusions, euchromatic additions, and heterochromatic additions). The resulting phylogenetic hypothesis differs from traditionally accepted hypotheses regarding this complex group of rodents. The cytogenetic data show that 1) there is no support for the dichotomy of Mastomys/Praomys previously based on morphology, 2) the 2n = 32 species from eastern Africa (P. hildebrandti) is distinct from the 2n = 32 species from southern Africa (P. natalensis), and 3) there is a close association between P. jacksoni and P. cf. tullbergi. Polyacrylamide gel electrophoresis of liver membrane proteins demonstrated few differences in protein mobilities between species and even fewer between individuals of the same species taken from different habitats and localities in Kenya. Monoclonal antibodies produced against liver proteins of one species and tested for reactivity to other species confirmed the evolutionary similarity of species of this complex. This immunologic approach may provide a robust data set for future phylogenetic studies of muroid rodents.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    Science.gov (United States)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  12. Single-Molecule Characterization of DNA-Protein Interactions Using Nanopore Biosensors.

    Science.gov (United States)

    Squires, A H; Gilboa, T; Torfstein, C; Varongchayakul, N; Meller, A

    2017-01-01

    Detection and characterization of nucleic acid-protein interactions, particularly those involving DNA and proteins such as transcription factors, enzymes, and DNA packaging proteins, remain significant barriers to our understanding of genetic regulation. Nanopores are an extremely sensitive and versatile sensing platform for label-free detection of single biomolecules. Analyte molecules are drawn to and through a nanoscale aperture by an electrophoretic force, which acts upon their native charge while in the sensing region of the pore. When the nanopore's diameter is only slightly larger than the biopolymer's cross section (typically a few nm); the latter must translocate through the pore in a linear fashion due to the constricted geometry in this region. These features allow nanopores to interrogate protein-nucleic acids in multiple sensing modes: first, by scanning and mapping the locations of binding sites along an analyte molecule, and second, by probing the strength of the bond between a protein and nucleic acid, using the native charge of the nucleic acid to apply an electrophoretic force to the complex while the protein is geometrically prevented from passing through the nanopore. In this chapter, we describe progress toward nanopore sensing of protein-nucleic acid complexes in the context of both mapping binding sites and performing force spectroscopy to determine the strength of interactions. We conclude by reviewing the strengths and challenges of the nanopore technique in the context of studying DNA-protein interactions. © 2017 Elsevier Inc. All rights reserved.

  13. Flow-Based Single Cell Deposition for High-Throughput Screening of Protein Libraries.

    Directory of Open Access Journals (Sweden)

    Cassandra Stowe

    Full Text Available The identification and engineering of proteins having refined or novel characteristics is an important area of research in many scientific fields. Protein modelling has enabled the rational design of unique proteins, but high-throughput screening of large libraries is still required to identify proteins with potentially valuable properties. Here we report on the development and evaluation of a novel fluorescent activated cell sorting based screening platform. Single bacterial cells, expressing a protein library to be screened, are electronically sorted and deposited onto plates containing solid nutrient growth media in a dense matrix format of between 44 and 195 colonies/cm2. We show that this matrix format is readily applicable to machine interrogation (<30 seconds per plate and subsequent bioinformatic analysis (~60 seconds per plate thus enabling the high-throughput screening of the protein library. We evaluate this platform and show that bacteria containing a bioluminescent protein can be spectrally analysed using an optical imager, and a rare clone (0.5% population can successfully be identified, picked and further characterised. To further enhance this screening platform, we have developed a prototype electronic sort stream multiplexer, that when integrated into a commercial flow cytometric sorter, increases the rate of colony deposition by 89.2% to 24 colonies per second. We believe that the screening platform described here is potentially the foundation of a new generation of high-throughput screening technologies for proteins.

  14. Antifungal Effect of Arabidopsis SGT1 Proteins via Mitochondrial Reactive Oxygen Species.

    Science.gov (United States)

    Park, Seong-Cheol; Cheong, Mi Sun; Kim, Eun-Ji; Kim, Jin Hyo; Chi, Yong Hun; Jang, Mi-Kyeong

    2017-09-27

    The highly conserved SGT1 (suppressor of the G2 alleles of skp1) proteins from Arabidopsis are known to contribute to plant resistance to pathogens. While SGT1 proteins respond to fungal pathogens, their antifungal activity is not reported and the mechanism for this inhibition is not well understood. Therefore, recombinant Arabidopsis SGT1 proteins were cloned, expressed, and purified to evaluate their antifungal activity, resulting in their potent inhibition of pathogen growth. Dye-labeled proteins are localized to the cytosol of Candida albicans cells without the disruption of the cell membrane. Moreover, we showed that entry of the proteins into C. albicans cells resulted in the accumulation of reactive oxygen species (ROS) and cell death via altered mitochondrial potential. Morphological changes of C. albicans cells in the presence of proteins were visualized by scanning electron microscopy. Our data suggest that AtSGT1 proteins play a critical role in plant resistance to pathogenic fungal infection and they can be classified to a new plant antifungal protein.

  15. Multi-species call-broadcast improved detection of endangered Yuma clapper rail compared to single-species call-broadcast

    Science.gov (United States)

    Nadeau, Christopher P.; Conway, Courtney J.; Piest, Linden; Burger, William P.

    2013-01-01

    Broadcasting calls of marsh birds during point-count surveys increases their detection probability and decreases variation in the number of birds detected across replicate surveys. However, multi-species monitoring using call-broadcast may reduce these benefits if birds are reluctant to call once they hear broadcasted calls of other species. We compared a protocol that uses call-broadcast for only one species (Yuma clapper rail [Rallus longirostris yumanensis]) to a protocol that uses call-broadcast for multiple species. We detected more of each of the following species using the multi-species protocol: 25 % more pied-billed grebes, 160 % more American bitterns, 52 % more least bitterns, 388 % more California black rails, 12 % more Yuma clapper rails, 156 % more Virginia rails, 214 % more soras, and 19 % more common gallinules. Moreover, the coefficient of variation was smaller when using the multi-species protocol: 10 % smaller for pied-billed grebes, 38 % smaller for American bitterns, 19 % smaller for least bitterns, 55 % smaller for California black rails, 5 % smaller for Yuma clapper rails, 38 % smaller for Virginia rails, 44 % smaller for soras, and 8 % smaller for common gallinules. Our results suggest that multi-species monitoring approaches may be more effective and more efficient than single-species approaches even when using call-broadcast.

  16. Synthetical Analysis for Morphology, biological Species, and stable Isotopes (SAMSI) of single-cell planktonic foraminifer

    Science.gov (United States)

    Ujiie, Y.; Kimoto, K.; Ishimura, T.

    2017-12-01

    Planktonic foraminifers are widely used in the studies of paleontology and paleoceanography, because the morphology of their calcareous shells is enough highly variable to identify the morphospecies and the chemical composition of the shells reflect ambient seawater condition. Although the morphospecies were believed to represent environments associating with latitudinal temperature range of the world ocean, molecular phylogeographic studies have unveiled the presence of multiple biological species in a single morphospecies and their species-specific distributions. This implicates the actual complexity of planktonic foraminiferal ecology. Conversely, these biological species have a high potential for providing novel ecological and environmental information to us. In order to reassess the morphological and geochemical characters of biological species, the DNA extraction method with the guanidium isothiocyanate buffer was developed to preserve the calcareous shells. The present study carefully tested the physical and chemical damages of the DNA extraction process to the shells, by our novel approaches with geochemical analysis of the shells after non-destructive analysis for morphometrics on a same specimen. First, we checked the changes of the shell densities between pre- and post-DNA extraction by using the micro-focus X-ray CT (MXCT) scanning. Based on the simultaneous measurement of a sample and the standard material, we confirmed no significant changes to the shell densities through the DNA extraction process. As a next step, we compared stable oxygen and carbon isotopes among individuals of three sample sets: (1) no chemical and incubation as control, (2) incubation in the DNA extraction buffer at 65-70°C for 40 minutes as standard way, and (3) incubation in the DNA extraction buffer at 65-70°C for 120 minutes, by using the microscale isotopic analytical system (MICAL3c). Consequently, there were no significant differences among the three sample sets. These

  17. Simulation, Control and Optimization of Single Cell Protein Production in a U-Loop Reactor

    DEFF Research Database (Denmark)

    Engoulevent, Franck Guillaume; Jørgensen, John Bagterp

    2012-01-01

    In 2011, the world population passed 7 billions inhabitants. While this number witnesses the success of humankind on earth, it also rises among other things questions about food supply. Declining live stock in the wild, rising price of energy combined with climatic change give a new economic...... potential for alternative sources of protein production. Single cell protein (SCP) is protein produced by growth of micro organisms. Among these micro organisms, Methylococcus Capsulatus is particular interesting as it can grow on either methane or methanol and contains 70% protein. The U-Loop reactor...... report simulation results. In addition we design and compare dierent regulatory control systems for regulation of SCP production in the U-Loop reactor. The purpose of the regulatory control systems is to keep the process at a steady state and to reject disturbances. We design and implement such control...

  18. Epidemiological survey in single-species flocks from Poland reveals expanded genetic and antigenic diversity of small ruminant lentiviruses

    Science.gov (United States)

    Valas, Stephen; Kuźmak, Jacek

    2018-01-01

    Small ruminant lentivirus (SRLV) infections are widespread in Poland and circulation of subtypes A1, A12, A13, B1 and B2 was detected. The present work aimed at extending previous study based on the analysis of a larger number of animals from single-species flocks. Animals were selected for genetic analysis based on serological reactivity towards a range of recombinant antigens derived from Gag and Env viral proteins. Phylogenetic analysis revealed the existence of subtypes B2 and A12 in both goats and sheep and subtypes A1 and B1 in goats only. In addition, two novel subtypes, A16 and A17, were found in goats. Co-infections with strains belonging to different subtypes within A and B groups were detected in 1 sheep and 4 goats originating from four flocks. Although the reactivity of serum samples towards the recombinant antigens confirmed immunological relatedness between Gag epitopes of different subtypes and the cross-reactive nature of Gag antibodies, eleven serum samples failed to react with antigens representing all subtypes detected up-to-date in Poland, highlighting the limitations of the serological diagnosis. These data showed the complex nature of SRLV subtypes circulating in sheep and goats in Poland and the need for improving SRLV-related diagnostic capacity. PMID:29505612

  19. Network analysis and cross species comparison of protein-protein interaction networks of human, mouse and rat cytochrome P450 proteins that degrade xenobiotics.

    Science.gov (United States)

    Karthikeyan, Bagavathy Shanmugam; Akbarsha, Mohammad Abdulkader; Parthasarathy, Subbiah

    2016-06-21

    Cytochrome P450 (CYP) enzymes that degrade xenobiotics play a critical role in the metabolism and biotransformation of drugs and xenobiotics in humans as well as experimental animal models such as mouse and rat. These proteins function as a network collectively as well as independently. Though there are several reports on the organization, regulation and functionality of various CYP enzymes at the molecular level, the understanding of organization and functionality of these proteins at the holistic level remain unclear. The objective of this study is to understand the organization and functionality of xenobiotic degrading CYP enzymes of human, mouse and rat using network theory approaches and to study species differences that exist among them at the holistic level. For our analysis, a protein-protein interaction (PPI) network for CYP enzymes of human, mouse and rat was constructed using the STRING database. Topology, centrality, modularity and robustness analyses were performed for our predicted CYP PPI networks that were then validated by comparison with randomly generated network models. Network centrality analyses of CYP PPI networks reveal the central/hub proteins in the network. Modular analysis of the CYP PPI networks of human, mouse and rat resulted in functional clusters. These clusters were subjected to ontology and pathway enrichment analysis. The analyses show that the cluster of the human CYP PPI network is enriched with pathways principally related to xenobiotic/drug metabolism. Endo-xenobiotic crosstalk dominated in mouse and rat CYP PPI networks, and they were highly enriched with endogenous metabolic and signaling pathways. Thus, cross-species comparisons and analyses of human, mouse and rat CYP PPI networks gave insights about species differences that existed at the holistic level. More investigations from both reductionist and holistic perspectives can help understand CYP metabolism and species extrapolation in a much better way.

  20. Identifying the Location of a Single Protein along the DNA Strand Using Solid-State Nanopores.

    Science.gov (United States)

    Yu, Jae-Seok; Lim, Min-Cheol; Huynh, Duyen Thi Ngoc; Kim, Hyung-Jun; Kim, Hyun-Mi; Kim, Young-Rok; Kim, Ki-Bum

    2015-05-26

    Solid-state nanopore has been widely studied as an effective tool to detect and analyze small biomolecules, such as DNA, RNA, and proteins, at a single molecule level. In this study, we demonstrate a rapid identification of the location of zinc finger protein (ZFP), which is bound to a specific locus along the length of a double-stranded DNA (dsDNA) to a single protein resolution using a low noise solid-state nanopore. When ZFP labeled DNAs were driven through a nanopore by an externally applied electric field, characteristic ionic current signals arising from the passage of the DNA/ZFP complex and bare DNA were detected, which enabled us to identify the locations of ZFP binding site. We examined two DNAs with ZFP binding sites at different positions and found that the location of the additional current drop derived from the DNA/ZFP complex is well-matched with a theoretical one along the length of the DNA molecule. These results suggest that the protein binding site on DNA can be mapped or that genetic information can be read at a single molecule level using solid-state nanopores.

  1. Comparative proteome analysis of secretory proteins from pathogenic and nonpathogenic Listeria species.

    Science.gov (United States)

    Trost, Matthias; Wehmhöner, Dirk; Kärst, Uwe; Dieterich, Guido; Wehland, Jürgen; Jänsch, Lothar

    2005-04-01

    Extracellular proteins of bacterial pathogens play a crucial role in the infection of the host. Here we present the first comprehensive validation of the secretory subproteome of the Gram positive pathogen Listeria monocytogenes using predictive bioinformatic and experimental proteomic approaches. The previous original signal peptide (SP) prediction (Glaser et al., Science 2001, 294, 849-852) has been greatly improved by an in-depth analysis using seven different bioinformatic tools. Subsequent careful classification of the resulting data gives a probability dependent annotation of 121 putatively secreted proteins of which 45 are novel. Complementary proteomic analysis using both two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry and high performance liquid chromatography/electrospray ionization-mass spectrometry has identified 105 proteins in the culture supernatant of L. monocytogenes. Among these, we were able to detect all the currently known virulence factors with an SP showing the importance of this subproteome and demonstrating the reliability of the techniques used. The comparison between the L. monocytogenes wildtype and the nonpathogenic species Listeria innocua was performed to reveal proteins probably involved in pathogenicity and/or the adaptation to their respective lifestyles. In addition to the eight known virulence factors, all of which have no orthologous genes in L. innocua, eight additional proteins have been identified that exhibit the typical key feature defining the known listerial virulence factors. Further significant differences between the two species are evident in the group of cell wall and secretory proteins that warrant further study. Our investigation clearly demonstrates that the major difference between the pathogenic and nonpathogenic species, noted in the comparative genome analysis, manifests itself strongest in the secretome.

  2. Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

    DEFF Research Database (Denmark)

    Thresher, RJ; Christiansen, Gunna; Griffith, JD

    1988-01-01

    We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional Rec...... assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein...

  3. Crystal Structures and Binding Dynamics of Odorant-Binding Protein 3 from two aphid species Megoura viciae and Nasonovia ribisnigri.

    Science.gov (United States)

    Northey, Tom; Venthur, Herbert; De Biasio, Filomena; Chauviac, Francois-Xavier; Cole, Ambrose; Ribeiro, Karlos Antonio Lisboa; Grossi, Gerarda; Falabella, Patrizia; Field, Linda M; Keep, Nicholas H; Zhou, Jing-Jiang

    2016-04-22

    Aphids use chemical cues to locate hosts and find mates. The vetch aphid Megoura viciae feeds exclusively on the Fabaceae, whereas the currant-lettuce aphid Nasonovia ribisnigri alternates hosts between the Grossulariaceae and Asteraceae. Both species use alarm pheromones to warn of dangers. For N. ribisnigri this pheromone is a single component (E)-β-farnesene but M. viciae uses a mixture of (E)-β-farnesene, (-)-α-pinene, β-pinene, and limonene. Odorant-binding proteins (OBP) are believed to capture and transport such semiochemicals to their receptors. Here, we report the first aphid OBP crystal structures and examine their molecular interactions with the alarm pheromone components. Our study reveals some unique structural features: 1) the lack of an internal ligand binding site; 2) a striking groove in the surface of the proteins as a putative binding site; 3) the N-terminus rather than the C-terminus occupies the site closing off the conventional OBP pocket. The results from fluorescent binding assays, molecular docking and dynamics demonstrate that OBP3 from M. viciae can bind to all four alarm pheromone components and the differential ligand binding between these very similar OBP3s from the two aphid species is determined mainly by the direct π-π interactions between ligands and the aromatic residues of OBP3s in the binding pocket.

  4. High-affinity single-domain binding proteins with a binary-code interface.

    Science.gov (United States)

    Koide, Akiko; Gilbreth, Ryan N; Esaki, Kaori; Tereshko, Valentina; Koide, Shohei

    2007-04-17

    High degrees of sequence and conformation complexity found in natural protein interaction interfaces are generally considered essential for achieving tight and specific interactions. However, it has been demonstrated that specific antibodies can be built by using an interface with a binary code consisting of only Tyr and Ser. This surprising result might be attributed to yet undefined properties of the antibody scaffold that uniquely enhance its capacity for target binding. In this work we tested the generality of the binary-code interface by engineering binding proteins based on a single-domain scaffold. We show that Tyr/Ser binary-code interfaces consisting of only 15-20 positions within a fibronectin type III domain (FN3; 95 residues) are capable of producing specific binding proteins (termed "monobodies") with a low-nanomolar K(d). A 2.35-A x-ray crystal structure of a monobody in complex with its target, maltose-binding protein, and mutation analysis revealed dominant contributions of Tyr residues to binding as well as striking molecular mimicry of a maltose-binding protein substrate, beta-cyclodextrin, by the Tyr/Ser binary interface. This work suggests that an interaction interface with low chemical diversity but with significant conformational diversity is generally sufficient for tight and specific molecular recognition, providing fundamental insights into factors governing protein-protein interactions.

  5. Single cell protein production of Chlorella sp. using food processing waste as a cultivation medium

    Science.gov (United States)

    Putri, D.; Ulhidayati, A.; Musthofa, I. A.; Wardani, A. K.

    2018-03-01

    The aim of this study was to investigate the effect of various food processing wastes on the production of single cell protein by Chlorella sp. Three various food processing wastes i.e. tofu waste, tempeh waste and cheese whey waste were used as cultivation medium for Chlorella sp. growth. Sea water was used as a control of cultivation medium. The addition of waste into cultivation medium was 10%, 20%, 30%, 40%, and 50%. The result showed that the highest yield of cell mass and protein content was found in 50% tofu waste cultivation medium was 47.8 × 106 cell/ml with protein content was 52.24%. The 50% tofu waste medium showed improved cell yield as nearly as 30% than tempeh waste medium. The yield of biomass and protein content when 30% tempeh waste was used as cultivation medium was 37.1 × 106 cell/ml and 52%, respectively. Thus, food processing waste especially tofu waste would be a promising candidate for cultivation medium for single cell production from Chlorella sp. Moreover, the utilization of waste can reduce environmental pollution and increase protein supply for food supplement or animal feed.

  6. Two states or not two states: Single-molecule folding studies of protein L

    Science.gov (United States)

    Aviram, Haim Yuval; Pirchi, Menahem; Barak, Yoav; Riven, Inbal; Haran, Gilad

    2018-03-01

    Experimental tools of increasing sophistication have been employed in recent years to study protein folding and misfolding. Folding is considered a complex process, and one way to address it is by studying small proteins, which seemingly possess a simple energy landscape with essentially only two stable states, either folded or unfolded. The B1-IgG binding domain of protein L (PL) is considered a model two-state folder, based on measurements using a wide range of experimental techniques. We applied single-molecule fluorescence resonance energy transfer (FRET) spectroscopy in conjunction with a hidden Markov model analysis to fully characterize the energy landscape of PL and to extract the kinetic properties of individual molecules of the protein. Surprisingly, our studies revealed the existence of a third state, hidden under the two-state behavior of PL due to its small population, ˜7%. We propose that this minority intermediate involves partial unfolding of the two C-terminal β strands of PL. Our work demonstrates that single-molecule FRET spectroscopy can be a powerful tool for a comprehensive description of the folding dynamics of proteins, capable of detecting and characterizing relatively rare metastable states that are difficult to observe in ensemble studies.

  7. Nano-bio-optomechanics: nanoaperture tweezers probe single nanoparticles, proteins, and their interactions

    Science.gov (United States)

    Gordon, Reuven

    2015-09-01

    Nanoparticles in the single digit nanometer range can be easily isolated and studied with low optical powers using nanoaperture tweezers. We have studied individual proteins and their interactions with small molecules, DNA and antibodies. Recently, using the fluctuations of the trapped object, we have pioneered a new way to "listen" to the vibrations of nanoparticles in the 100 GHz - 1 THz range; the approach is called extraordinary acoustic Raman (EAR). EAR gives unprecedented low frequency spectra of individual proteins in solution, allowing for identification and analysis, as well as probing their role in biological functions. We have also used EAR to study the elastic properties, shape and size of various individual nanoparticles.

  8. Differential role of eDNA, proteins, and polysaccharides in cell-cell and cell-substrate adhesion by three Staphylococcus species

    DEFF Research Database (Denmark)

    Meyer, Rikke Louise; Okshevsky, Mira Ursula; Zeng, Guanghong

    valuable for designing new approaches to biofilm prevention. In this study, we combine microfluidic flow-cell studies with single-cell analyses to understand how polysaccharides, extracellular DNA (eDNA), and proteins contribute individually and in concert to mediate bacterial adhesion and aggregation...... on abiotic surfaces. We quantified initial adhesion, cell aggregation, and single-cell adhesion forces of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus xylosus in the presence and absence of DNase, dispersin, or subtilisin, which cleave extracellular DNA, polysaccharides and proteins...... affected by DNase and dispersin treatments, hence eDNA and polysaccharides were essential for cell-cell interactions. We showed that proteins, polysaccharides and eDNA contribute differently to the adhesion of three Staphylcococcus species, underlining the need to either tailor biofilm prevention...

  9. Preparation of Disease-Related Protein Assemblies for Single Particle Electron Microscopy.

    Science.gov (United States)

    Cameron Varano, A; Harafuji, Naoe; Dearnaley, William; Guay-Woodford, Lisa; Kelly, Deborah F

    2017-01-01

    Electron microscopy (EM) is a rapidly growing area of structural biology that permits us to decode biological assemblies at the nanoscale. To examine biological materials for single particle EM analysis, purified assemblies must be obtained using biochemical separation techniques. Here, we describe effective methodologies for isolating histidine (his)-tagged protein assemblies from the nucleus of disease-relevant cell lines. We further demonstrate how isolated assemblies are visualized using single particle EM techniques and provide representative results for each step in the process.

  10. 3D structure determination of protein using TEM single particle analysis.

    Science.gov (United States)

    Sato, Chikara; Mio, Kazuhiro; Kawata, Masaaki; Ogura, Toshihiko

    2014-11-01

    Proteins play important roles in cell functions such as enzymes, cell trafficking, neurotransmission, muscle contraction and hormone secretion. However, some proteins are very difficult to be crystallized and their structures are undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Among them, electron microscopy based single particle reconstruction (SPA) technique is a computer-aided structure determination method. This method reconstructs the 3D structure from projection images of dispersed protein. A large number of two-dimensional particle images are picked up from EM films, aligned and classified to generate 2D averages, and used to reconstruct the 3D structure by assigning the Euler angle of each 2D average. Due to the necessity of elaborate collaboration between the classical biology and the innovative information technology including parallel computing, scientists needed to break unseen barriers to get a start of this analysis. However, recent progresses in electron microscopes, mathematical algorithms, and computational abilities greatly reduced the height of barriers and expanded targets that are considered to be primarily addressable using single particle analysis. Membrane proteins are one of these targets to which the single particle analysis is successfully applied for the understanding of their 3D structures. For this purpose, we have developed various SPA methods [1-5] and applied them to different proteins [6-8].Here, we introduce reconstructed proteins, and discuss the availability of this technique. The intramembrane-cleaving proteases (I-CLiPs) that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in biological conditions. I-CLiPs have been classified into three groups: serine-, aspartyl- and metalloprotease

  11. Brief communication: Is variation in the cranial capacity of the Dmanisi sample too high to be from a single species?

    Science.gov (United States)

    Lee, Sang-Hee

    2005-07-01

    This study uses data resampling to test the null hypothesis that the degree of variation in the cranial capacity of the Dmanisi hominid sample is within the range variation of a single species. The statistical significance of the variation in the Dmanisi sample is examined using simulated distributions based on comparative samples of modern humans, chimpanzees, and gorillas. Results show that it is unlikely to find the maximum difference observed in the Dmanisi sample in distributions of female-female pairs from comparative single-species samples. Given that two sexes are represented, the difference in the Dmanisi sample is not enough to reject the null hypothesis of a single species. Results of this study suggest no compelling reason to invoke multiple taxa to explain variation in the cranial capacity of the Dmanisi hominids. (c) 2004 Wiley-Liss, Inc

  12. Distribution and biological role of the oligopeptide-binding protein (OppA) in Xanthomonas species.

    Science.gov (United States)

    Oshiro, Elisa E; Tavares, Milene B; Suzuki, Celso F; Pimenta, Daniel C; Angeli, Claudia B; de Oliveira, Julio C F; Ferro, Maria I T; Ferreira, Luis C S; Ferreira, Rita C C

    2010-04-01

    In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA) of the major bacterial oligopeptide uptake system (Opp), in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis) and Rangpur lime (Citrus limonia). Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis.

  13. Distribution and biological role of the oligopeptide-binding protein (OppA in Xanthomonas species

    Directory of Open Access Journals (Sweden)

    Elisa E. Oshiro

    2010-01-01

    Full Text Available In this study we investigated the prevalence of the oppA gene, encoding the oligopeptide binding protein (OppA of the major bacterial oligopeptide uptake system (Opp, in different species of the genus Xanthomonas. The oppA gene was detected in two Xanthomonas axonopodis strains among eight tested Xanthomonas species. The generation of an isogenic oppA-knockout derivative of the Xac 306 strain, showed that the OppA protein neither plays a relevant role in oligopeptide uptake nor contributes to the infectivity and multiplication of the bacterial strain in leaves of sweet orange (Citrus sinensis and Rangpur lime (Citrus limonia. Taken together these results suggest that the oppA gene has a recent evolutionary history in the genus and does not contribute in the physiology or pathogenesis of X. axonopodis.

  14. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L. (UW-MED); (UCB)

    2015-04-22

    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  15. The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex

    Directory of Open Access Journals (Sweden)

    Gribble Kristin E

    2012-08-01

    Full Text Available Abstract Background Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP, a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. Results Isolates of the B. plicatilis species complex have 1–4 copies of mmr-b, each composed of 2–9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. Conclusions Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving

  16. Can we predict performance and spatial structure of two-species mixtures using only single species information from monocultures?

    Czech Academy of Sciences Publication Activity Database

    Yu, F.-H.; Herben, Tomáš; Wildová, Radka; Hershock, C.; Goldberg, D. E.

    2012-01-01

    Roč. 234, Sp. iss. (2012), s. 31-37 ISSN 0304-3800 R&D Projects: GA ČR GA206/09/1471 Institutional research plan: CEZ:AV0Z60050516 Institutional support: RVO:67985939 Keywords : model * mixture * species Subject RIV: EF - Botanics Impact factor: 2.069, year: 2012

  17. Reconciling single-species TACs in the North Sea demersal fisheries using the Fcube mixed-fisheries advice framework

    DEFF Research Database (Denmark)

    Ulrich, Clara; Reeves, Stuart A.; Vermard, Youen

    2011-01-01

    be exhausted before the TAC of another, leading to catches of valuable fish that cannot be landed legally. This important issue is, however, usually not quantified and not accounted for in traditional management advice. A simple approach using traditional catch and effort information was developed, estimating...... in the North Sea and shaped into the advice framework. The substantial overquota catches of North Sea cod likely under the current fisheries regimes are quantified, and it is estimated that the single-species management targets for North Sea cod cannot be achieved unless substantial reductions in TACs of all......Single-species management is a cause of discarding in mixed fisheries, because individual management objectives may not be consistent with each other and the species are caught simultaneously in relatively unselective fishing operations. As such, the total allowable catch (TAC) of one species may...

  18. Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding.

    Science.gov (United States)

    Sengupta, Ishita; Udgaonkar, Jayant B

    2017-12-01

    The folding and aggregation of proteins has been studied extensively, using multiple probes. To facilitate such experiments, introduction of spectroscopically-active moieties in to the protein of interest is often necessary. This is commonly achieved by specifically labelling cysteine residues in the protein, which are either present naturally or introduced artificially by site-directed mutagenesis. In the case of the recombinant prion protein, which is normally expressed in inclusion bodies, the presence of the native disulfide bond complicates the correct refolding of single cysteine-containing mutant variants of the protein. To overcome this major bottleneck, a simple purification strategy for single tryptophan, single cysteine-containing mutant variants of the mouse prion protein is presented, with yields comparable to that of the wild type protein. The protein(s) obtained by this method are correctly folded, with a single reduced cysteine, and the native disulfide bond between residues C178 and C213 intact. The β-sheet rich oligomers formed from these mutant variant protein(s) are identical to the wild type protein oligomer. The current strategy facilitates sample preparation for a number of high resolution spectroscopic measurements for the prion protein, which specifically require thiol labelling. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Sulfur single-wavelength anomalous diffraction crystal structure of a pheromone-binding protein from the honeybee Apis mellifera L.

    Science.gov (United States)

    Lartigue, Audrey; Gruez, Arnaud; Briand, Loïc; Blon, Florence; Bézirard, Valérie; Walsh, Martin; Pernollet, Jean-Claude; Tegoni, Mariella; Cambillau, Christian

    2004-02-06

    Pheromone binding proteins (PBPs) are small helical proteins ( approximately 13-17 kDa) present in several sensory organs from moth and other insect species. They are involved in the transport of pheromones from the sensillar lymph to the olfactory receptors. We report here the crystal structure of a PBP (Amel-ASP1) originating from the honey-bee (Apis mellifera) antennae and expressed as recombinant protein in the yeast Pichia pastoris. Crystals of Amel-ASP1 were obtained at pH 5.5 using the nano-drops technique of crystallization with a novel optimization procedure, and the structure was solved initially with the single-wavelength anomalous diffraction technique using sulfur anomalous dispersion. The structure of Amel-ASP1 has been refined at 1.6-A resolution. Its fold is roughly similar to that of other PBP/odorant binding proteins, presenting six helices and three disulfide bridges. Contrary to the PBPs from Bombyx mori (Sandler, B. H., Nikonova, L., Leal, W. S., and Clardy, J. (2000) Chem. Biol. 7, 143-151) and Leucophea maderae (Lartigue, A., Gruez, A., Spinelli, S., Riviere, S., Brossut, R., Tegoni, M., and Cambillau, C. (2003) J. Biol. Chem. 278, 30213-30218), the extended C terminus folds into the protein and forms a wall of the internal hydrophobic cavity. Its backbone groups establish two hydrogen bonds with a serendipitous ligand, n-butyl-benzene-sulfonamide, an additive used in plastics. This mode of binding might, however, mimic that used by one of the pheromonal blend components and illustrates the binding versatility of PBPs.

  20. Production of single cell protein (SCP) from food and agricultural waste by using Saccharomyces cerevisiae.

    Science.gov (United States)

    Gervasi, Teresa; Pellizzeri, Vito; Calabrese, Giorgio; Di Bella, Giuseppa; Cicero, Nicola; Dugo, Giacomo

    2018-03-01

    Food waste is the single-largest component of the waste stream, in order to protect and safeguard the public health, useful and innovative recycling methods are investigated. The conversion of food wastes in value-added products is becoming a more economically viable and interesting practice. Food waste, collected in the distribution sector and citrus industries, was characterised for its potential as a raw material to use in fermentation processes. In this study, the production of single-cell protein (SCP) using food waste as a substrate was investigated. The purpose of this study has been to produce SCP from mixtures of food waste using Saccharomyces cerevisiae. The main fermentation test was carried out using a 25 l bioreactor. The utilisation of food waste can allow us to not only to reduce environmental pollution, but also to obtain value-added products such as protein supply for animal feed.

  1. Genetics of single-cell protein abundance variation in large yeast populations

    Science.gov (United States)

    Albert, Frank W.; Treusch, Sebastian; Shockley, Arthur H.; Bloom, Joshua S.; Kruglyak, Leonid

    2014-02-01

    Variation among individuals arises in part from differences in DNA sequences, but the genetic basis for variation in most traits, including common diseases, remains only partly understood. Many DNA variants influence phenotypes by altering the expression level of one or several genes. The effects of such variants can be detected as expression quantitative trait loci (eQTL). Traditional eQTL mapping requires large-scale genotype and gene expression data for each individual in the study sample, which limits sample sizes to hundreds of individuals in both humans and model organisms and reduces statistical power. Consequently, many eQTL are probably missed, especially those with smaller effects. Furthermore, most studies use messenger RNA rather than protein abundance as the measure of gene expression. Studies that have used mass-spectrometry proteomics reported unexpected differences between eQTL and protein QTL (pQTL) for the same genes, but these studies have been even more limited in scope. Here we introduce a powerful method for identifying genetic loci that influence protein expression in the yeast Saccharomyces cerevisiae. We measure single-cell protein abundance through the use of green fluorescent protein tags in very large populations of genetically variable cells, and use pooled sequencing to compare allele frequencies across the genome in thousands of individuals with high versus low protein abundance. We applied this method to 160 genes and detected many more loci per gene than previous studies. We also observed closer correspondence between loci that influence protein abundance and loci that influence mRNA abundance of a given gene. Most loci that we detected were clustered in `hotspots' that influence multiple proteins, and some hotspots were found to influence more than half of the proteins that we examined. The variants that underlie these hotspots have profound effects on the gene regulatory network and provide insights into genetic variation in cell

  2. Identification of two-step chemical mechanisms using small temperature oscillations and a single tagged species.

    Science.gov (United States)

    Closa, F; Gosse, C; Jullien, L; Lemarchand, A

    2015-05-07

    In order to identify two-step chemical mechanisms, we propose a method based on a small temperature modulation and on the analysis of the concentration oscillations of a single tagged species involved in the first step. The thermokinetic parameters of the first reaction step are first determined. Then, we build test functions that are constant only if the chemical system actually possesses some assumed two-step mechanism. Next, if the test functions plotted using experimental data are actually even, the mechanism is attributed and the obtained constant values provide the rate constants and enthalpy of reaction of the second step. The advantage of the protocol is to use the first step as a probe reaction to reveal the dynamics of the second step, which can hence be relieved of any tagging. The protocol is anticipated to apply to many mechanisms of biological relevance. As far as ligand binding is considered, our approach can address receptor conformational changes or dimerization as well as competition with or modulation by a second partner. The method can also be used to screen libraries of untagged compounds, relying on a tracer whose concentration can be spectroscopically monitored.

  3. Single and dual drug selection for transgenes following bombardment of Caenorhabditis species.

    Science.gov (United States)

    Semple, Jennifer I; Lehner, Ben

    2014-08-01

    The use of drugs and drug resistance genes is a powerful method to select for the presence of a transgene. Unlike methods that require the complementation of a genetic mutation, this system can be used on any genetic background. Drug selection does not require extensive manipulation or costly equipment, yet it is very rapid and can achieve extremely high efficiency, selecting a small number of transgenic worms from among millions of non-transgenic worms. Introducing integrated transgenes into Caenorhabditis elegans by microparticle bombardment represents just such a challenge. Here we describe in detail the protocol we have developed for dual-drug selection in liquid with puromycin and G418 which works well in a variety of Caenorhabditis species. We also show that single drug selection with only puromycin or only G418 is effective in C. elegans. The growing number of drug selection markers that have been adapted to C. elegans are an important addition to the genetic toolkit at our disposal. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. A deep-sea agglutinated foraminifer tube constructed with planktonic foraminifer shells of a single species

    Science.gov (United States)

    Pearson, Paul N.; Expedition 363 Shipboard Scientific Party, IODP

    2018-01-01

    Agglutinated foraminifera are marine protists that show apparently complex behaviour in constructing their shells, involving selecting suitable sedimentary grains from their environment, manipulating them in three dimensions, and cementing them precisely into position. Here we illustrate a striking and previously undescribed example of complex organisation in fragments of a tube-like foraminifer (questionably assigned to Rhabdammina) from 1466 m water depth on the northwest Australian margin. The tube is constructed from well-cemented siliciclastic grains which form a matrix into which hundreds of planktonic foraminifer shells are regularly spaced in apparently helical bands. These shells are of a single species, Turborotalita clarkei, which has been selected to the exclusion of all other bioclasts. The majority of shells are set horizontally in the matrix with the umbilical side upward. This mode of construction, as is the case with other agglutinated tests, seems to require either an extraordinarily selective trial-and-error process at the site of cementation or an active sensory and decision-making system within the cell.

  5. Inter- and intra-specific differences in serum proteins of different species and subspecies of zebras.

    Science.gov (United States)

    Stratil, A; Cízová, D; Gábrisová, E; Pokorný, R

    1992-11-01

    1. Serum proteins of Equus grevyi, E. zebra hartmannae, E. burchelli boehmi, E. b. chapmanni and E. b. antiquorum were studied using starch-gel electrophoresis, 1-D polyacrylamide-gel electrophoresis, inhibitions of trypsin and chymotrypsin, immunoblotting, and specific staining for esterase. 2. Clear species-specific patterns were observed in albumin, transferrin, and for E. grevyi in protease inhibitor-1. Specific esterase was detected only in E. z. hartmannae. 3. Protein polymorphism was found in all studied species: E. grevyi--transferrin; E. z. hartmannae--protease inhibitor-1; E. b. boehmi--albumin, GC, transferrin, protease inhibitor-1, protease inhibitor-T; E. b. chapmanni--albumin, GC, transferrin, protease inhibitor-1; E. b. antiquorum--GC, transferrin, protease inhibitor-1. 4. Phenotype patterns of the polymorphic proteins were indicative of simple codominant inheritance. Further studies of polymorphism of protease inhibitor-2 and variability of protease inhibitor-X are needed. 5. alpha 1B glycoprotein in all zebra species was monomorphic. 6. The main transferrin components and alpha 1B glycoprotein of zebra (E. b. boehmi) were characterized for terminal sialic acid content.

  6. Studies on Nodulation, Biochemical Analysis and Protein Profiles of Rhizobium Isolated From Indigofera species

    Directory of Open Access Journals (Sweden)

    Kumari, B. S.

    2010-01-01

    Full Text Available Nodulation characteristics in five species of Indigofera viz., I .trita, I. linnaei, I. astragalina, I. parviflora and I. viscosa was studied at regular intervals on the plants raised in garden soil. Among the species studied, highest average number of nodules per plant of 23 with maximum sized nodules of 8.0 mm diameter was observed in I. astragalina. Biochemical analysis of root nodules of I. astragalina revealed that the leghaemoglobin content of nodules and nitrogen content of root, shoot, leaves and nodules were gradually increased up to 60 DAS, and then decreased with increase in age. Rhizobium isolates of five species of Indigofera were isolated and screened for enzymatic activities and total cellular protein profiles. All the five isolates showed nitrate reductase, citrase, tryptophanase and catalase activity while much variation was observed for enzymes like gelatinase, urease, caseinase, lipase, amylase, lysine decarboxylase and protease activities. Among the isolates studied, only the isolate from I. viscosa has the ability to solubilize the insoluble tricalcium phosphate. All the Rhizobium isolates exhibit similarity in protein content, except the isolate from I. viscosa which showed one additional protein band.

  7. Development of Microfluidic Systems Enabling High-Throughput Single-Cell Protein Characterization

    OpenAIRE

    Fan, Beiyuan; Li, Xiufeng; Chen, Deyong; Peng, Hongshang; Wang, Junbo; Chen, Jian

    2016-01-01

    This article reviews recent developments in microfluidic systems enabling high-throughput characterization of single-cell proteins. Four key perspectives of microfluidic platforms are included in this review: (1) microfluidic fluorescent flow cytometry; (2) droplet based microfluidic flow cytometry; (3) large-array micro wells (microengraving); and (4) large-array micro chambers (barcode microchips). We examine the advantages and limitations of each technique and discuss future research oppor...

  8. Comparative Analysis of Single-Species and Polybacterial Wound Biofilms Using a Quantitative, In Vivo, Rabbit Ear Model

    Science.gov (United States)

    2012-08-08

    biofilm behavior of mixed-species cultures with dental and periodontal pathogens. PLoS One 5(10): 131–135. 47. Ma H, Bryers JD (2010) Non-invasive method...Comparative Analysis of Single-Species and Polybacterial Wound Biofilms Using a Quantitative, In Vivo, Rabbit Ear Model Akhil K. Seth1*, Matthew R...Northwestern University, Chicago, Illinois, United States of America, 2 Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical

  9. Structure Based Thermostability Prediction Models for Protein Single Point Mutations with Machine Learning Tools.

    Directory of Open Access Journals (Sweden)

    Lei Jia

    Full Text Available Thermostability issue of protein point mutations is a common occurrence in protein engineering. An application which predicts the thermostability of mutants can be helpful for guiding decision making process in protein design via mutagenesis. An in silico point mutation scanning method is frequently used to find "hot spots" in proteins for focused mutagenesis. ProTherm (http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html is a public database that consists of thousands of protein mutants' experimentally measured thermostability. Two data sets based on two differently measured thermostability properties of protein single point mutations, namely the unfolding free energy change (ddG and melting temperature change (dTm were obtained from this database. Folding free energy change calculation from Rosetta, structural information of the point mutations as well as amino acid physical properties were obtained for building thermostability prediction models with informatics modeling tools. Five supervised machine learning methods (support vector machine, random forests, artificial neural network, naïve Bayes classifier, K nearest neighbor and partial least squares regression are used for building the prediction models. Binary and ternary classifications as well as regression models were built and evaluated. Data set redundancy and balancing, the reverse mutations technique, feature selection, and comparison to other published methods were discussed. Rosetta calculated folding free energy change ranked as the most influential features in all prediction models. Other descriptors also made significant contributions to increasing the accuracy of the prediction models.

  10. Quantification of functional dynamics of membrane proteins reconstituted in nanodiscs membranes by single turnover functional readout

    DEFF Research Database (Denmark)

    Moses, Matias Emil; Hedegård, Per; Hatzakis, Nikos

    2016-01-01

    Single-molecule measurements are emerging as a powerful tool to study the individual behavior of biomolecules, revolutionizing our understanding of biological processes. Their ability to measure the distribution of behaviors, instead of the average behavior, allows the direct observation and quan......Single-molecule measurements are emerging as a powerful tool to study the individual behavior of biomolecules, revolutionizing our understanding of biological processes. Their ability to measure the distribution of behaviors, instead of the average behavior, allows the direct observation...... and quantification of the activity, abundance, and lifetime of multiple states and transient intermediates in the energy landscape that are typically averaged out in nonsynchronized ensemble measurements. Studying the function of membrane proteins at the single-molecule level remains a formidable challenge......, and to date there is limited number of available functional assays. In this chapter, we describe in detail our recently developed methodology to reconstitute membrane proteins such as the integral membrane protein cytochrome P450 oxidoreductase on membrane systems such as Nanodiscs and study their functional...

  11. Biotic stress protein markers of Aquilaria sp. for gaharu species identification in Malaysia

    International Nuclear Information System (INIS)

    Azhar Mohamad; Abdul Rahim Harun

    2012-01-01

    Gaharu trees (Aquilaria) is in danger of extinction in the wild due to illegal logging. Its resin (Gaharu) is used for the production of highly valued incense throughout Asia. In Aquilaria sp. systemic induction of defense genes in response to mechanical wounding in nature is regulated by an 18-amino-acid peptide signal protein called systemin. This protein is produced in response to the natural stress at the vicinity of the wound and is also influenced by its genetic background. As the protein can be differentiated by its locality, the protein expressed is also found to be significantly different which, in turn, can be used for identification of this plant species. In this work, A. malaccensis and A. hirta were evaluated based on the targeted genes related to systemin. Targeted gene refers to specific sequence in genomic DNA. Sequence mining from public databases is part of the crucial process in getting the specific genes. The sequences will go through alignment step to identify conserved region prior to primer design. The primers were used in Polymerase Chain Reaction (PCR) techniques to amplify the conserved regions. It was found that both samples can be differentiated. This would be useful for plant breeders, trader and planter in ensuring authentic planting materials. This paper will describe the use of targeted genes primers as markers in identifying the Aquilaria species. (author)

  12. Enhancement of Protein and Pigment Content in Two Chlorella Species Cultivated on Industrial Process Water

    Directory of Open Access Journals (Sweden)

    Hamed Safafar

    2016-12-01

    Full Text Available Chlorella pyrenoidosa and Chlorella vulgaris were cultivated in pre-gasified industrial process water with high concentration of ammonia representing effluent from a local biogas plant. The study aimed to investigate the effects of growth media and cultivation duration on the nutritional composition of biomass. Variations in proteins, lipid, fatty acid composition, amino acids, tocopherols, and pigments were studied. Both species grew well in industrial process water. The contents of proteins were affected significantly by the growth media and cultivation duration. Microalga Chlorella pyrenoidosa produced the highest concentrations of protein (65.2% ± 1.30% DW while Chlorella vulgaris accumulated extremely high concentrations of lutein and chlorophylls (7.14 ± 0.66 mg/g DW and 32.4 ± 1.77 mg/g DW, respectively. Cultivation of Chlorella species in industrial process water is an environmentally friendly, sustainable bioremediation method with added value biomass production and resource valorization, since the resulting biomass also presented a good source of proteins, amino acids, and carotenoids for potential use in aquaculture feed industry.

  13. Quantification of storage proteins during seed imbibition of native species from the brazilian caatinga vegetation

    Directory of Open Access Journals (Sweden)

    Fabrício Francisco Santos da Silva

    2016-08-01

    Full Text Available Poincianella pyramidalis (Fabaceae, Schinopsis brasiliensis (Anacardiaceae and Sideroxylon obtusifolium (Sapotaceae are native species of the Caatinga vegetation from northeastern Brazil and have both biological importance and potential economic uses. Little is known about the water uptake and degradation of storage proteins during seed germination of these species. The aim of this study was to evaluate the imbibition and quantify the amount of storage proteins during seed germination of P. pyramidalis, S. brasiliensis and S. obtusifolium. Two lots of S. obtusifolium seeds with different vigour were used. Four replicates of 20 seeds of P. pyramidalis, S. brasiliensis and S. obtusifolium, were sown onto gerboxes with blotting paper soaked in distilled water and incubated during 72, 200 and 624 hours. Before and after imbibition seeds were weighed and frozen at until the sequential extraction and analysis of the seed storage proteins. Based on our results, we conclude that seed germination of P. pyramidalis, S. brasiliensis and S. obtusifolium has a well-defined triphasic imbibition. All storage proteins content of P. pyramidalis and S. brasiliensis seeds degraded along with the seed imbibition. Likewise, the content of albumins, globulins and glutelins decreased as S. obtusifolium seeds absorbed water.

  14. In vivo synthesized 34S enriched amino acid standards for species specific isotope dilution of proteins

    DEFF Research Database (Denmark)

    Hermann, Gerrit; Moller, Laura Hyrup; Gammelgaard, Bente

    2016-01-01

    with the concept of species specific isotope dilution analysis (IDA). The method relies on the determination of the two sulfur containing amino acids, cysteine and methionine by sulfur speciation analysis and is hence applicable to any protein containing sulfur. In vivo synthesis using 34S as sulfur source...... (ICP-MS) combined to anion exchange showed that very high concentrated spike material could be produced with [small mu ]mol amounts of proteinogenic sulfur containing amino acids per g cell dry weight. An enrichment of 34S to 96.3 +/- 0.4% (n = 3) and 98.5 +/- 0.4% (n = 3) for cysteic acid......A generic quantification approach was introduced addressing the characterization of protein standards while fulfilling the principles of metrology. Traceable absolute quantification was achieved combining a proven biochemical method, i.e. protein hydrolysis followed by amino acid quantification...

  15. Protein residue linking in a single spectrum for magic-angle spinning NMR assignment

    Energy Technology Data Exchange (ETDEWEB)

    Andreas, Loren B.; Stanek, Jan; Marchand, Tanguy Le; Bertarello, Andrea; Paepe, Diane Cala-De; Lalli, Daniela; Krejčíková, Magdaléna; Doyen, Camille; Öster, Carl [Université de Lyon, Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1) (France); Knott, Benno; Wegner, Sebastian; Engelke, Frank [Bruker Biospin (Germany); Felli, Isabella C.; Pierattelli, Roberta [University of Florence, Department of Chemistry “Ugo Schiff“and Magnetic Resonance Center (CERM) (Italy); Dixon, Nicholas E. [University of Wollongong, School of Chemistry (Australia); Emsley, Lyndon; Herrmann, Torsten; Pintacuda, Guido, E-mail: guido.pintacuda@ens-lyon.fr [Université de Lyon, Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1) (France)

    2015-07-15

    Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.

  16. Single Nucleotide Variants in the Protein C Pathway and Mortality in Dialysis Patients

    Science.gov (United States)

    Ocak, Gürbey; Drechsler, Christiane; Vossen, Carla Y.; Vos, Hans L.; Rosendaal, Frits R.; Reitsma, Pieter H.; Hoffmann, Michael M.; März, Winfried; Ouwehand, Willem H.; Krediet, Raymond T.; Boeschoten, Elisabeth W.; Dekker, Friedo W.; Wanner, Christoph; Verduijn, Marion

    2014-01-01

    Background The protein C pathway plays an important role in the maintenance of endothelial barrier function and in the inflammatory and coagulant processes that are characteristic of patients on dialysis. We investigated whether common single nucleotide variants (SNV) in genes encoding protein C pathway components were associated with all-cause 5 years mortality risk in dialysis patients. Methods Single nucleotides variants in the factor V gene (F5 rs6025; factor V Leiden), the thrombomodulin gene (THBD rs1042580), the protein C gene (PROC rs1799808 and 1799809) and the endothelial protein C receptor gene (PROCR rs867186, rs2069951, and rs2069952) were genotyped in 1070 dialysis patients from the NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort) and in 1243 dialysis patients from the German 4D cohort. Results Factor V Leiden was associated with a 1.5-fold (95% CI 1.1–1.9) increased 5-year all-cause mortality risk and carriers of the AG/GG genotypes of the PROC rs1799809 had a 1.2-fold (95% CI 1.0–1.4) increased 5-year all-cause mortality risk. The other SNVs in THBD, PROC, and PROCR were not associated with 5-years mortality. Conclusion Our study suggests that factor V Leiden and PROC rs1799809 contributes to an increased mortality risk in dialysis patients. PMID:24816905

  17. Mechanosensing of DNA bending in a single specific protein-DNA complex

    Science.gov (United States)

    Le, Shimin; Chen, Hu; Cong, Peiwen; Lin, Jie; Dröge, Peter; Yan, Jie

    2013-12-01

    Many crucial biological processes are regulated by mechanical stimuli. Here, we report new findings that pico-Newton forces can drastically affect the stability of the site-specific DNA binding of a single transcription factor, the E. coli integration host factor (IHF), by stretching a short ~150 nm DNA containing a single IHF binding site. Dynamic binding and unbinding of single IHF were recorded and analyzed for the force-dependent stability of the IHF-DNA complex. Our results demonstrate that the IHF-DNA interaction is fine tuned by force in different salt concentration and temperature over physiological ranges, indicating that, besides other physiological factors, force may play equally important role in transcription regulation. These findings have broad implications with regard to general mechanosensitivity of site-specific DNA bending proteins.

  18. The binding of in vitro synthesized adenovirus DNA binding protein to single-stranded DNA is stimulated by zinc ions

    NARCIS (Netherlands)

    Vos, H.L.; Lee, F.M. van der; Sussenbach, J.S.

    1988-01-01

    We have synthesized wild type DNA binding protein (DBP) of adenovirus type 5 (Ad5) and several truncated forms of this protein by a combination of in vitro transcription and translation. The proteins obtained were tested for binding to a single-stranded DNA-cellulose column. It could be shown that

  19. Discontinuous genetic variation among mesophilic Naegleria isolates: further evidence that N. gruberi is not a single species.

    Science.gov (United States)

    Robinson, B S; Christy, P; Hayes, S J; Dobson, P J

    1992-01-01

    Naegleria isolates which are currently placed in the type species N. gruberi display great genetic, physiological and morphological heterogeneity. There are two possible interpretations of the nature of this species--that N. gruberi is a species complex or that it is a single continuously variable species. To distinguish between these alternatives, allelic states were determined for 33 loci in 74 new isolates selected to represent wide geographic sources and diverse temperature limits for growth. The results were compared with data for culture collection strains of N. gruberi and other species in the genus. The isolates formed a discontinuous series of clusters, separated by genetic distances similar to those separating the better-characterised taxa N. fowleri, N. lovaniensis, N. jadini, N. australiensis australiensis and N. australiensis italica. Culture collection strains assigned to N. gruberi fell into six distinct clusters, while other clusters were not represented by reference strains. The data are most consistent with the interpretation that N. gruberi is a group of several distinct species, each equivalent to the recently described species in the genus. Naegleria andersoni andersoni and N. andersoni jamiesoni also formed two distinct clusters, equivalent to species. Characteristics temperature limits for growth show that the mesophilic species are ecological as well as genetic entities.

  20. Single cell cytometry of protein function in RNAi treated cells and in native populations

    Directory of Open Access Journals (Sweden)

    Hill Andrew

    2008-08-01

    Full Text Available Abstract Background High Content Screening has been shown to improve results of RNAi and other perturbations, however significant intra-sample heterogeneity is common and can complicate some analyses. Single cell cytometry can extract important information from subpopulations within these samples. Such approaches are important for immune cells analyzed by flow cytometry, but have not been broadly available for adherent cells that are critical to the study of solid-tumor cancers and other disease models. Results We have directly quantitated the effect of resolving RNAi treatments at the single cell level in experimental systems for both exogenous and endogenous targets. Analyzing the effect of an siRNA that targets GFP at the single cell level permits a stronger measure of the absolute function of the siRNA by gating to eliminate background levels of GFP intensities. Extending these methods to endogenous proteins, we have shown that well-level results of the knockdown of PTEN results in an increase in phospho-S6 levels, but at the single cell level, the correlation reveals the role of other inputs into the pathway. In a third example, reduction of STAT3 levels by siRNA causes an accumulation of cells in the G1 phase of the cell cycle, but does not induce apoptosis or necrosis when compared to control cells that express the same levels of STAT3. In a final example, the effect of reduced p53 levels on increased adriamycin sensitivity for colon carcinoma cells was demonstrated at the whole-well level using siRNA knockdown and in control and untreated cells at the single cell level. Conclusion We find that single cell analysis methods are generally applicable to a wide range of experiments in adherent cells using technology that is becoming increasingly available to most laboratories. It is well-suited to emerging models of signaling dysfunction, such as oncogene addition and oncogenic shock. Single cell cytometry can demonstrate effects on cell

  1. Single particle 3D reconstruction for 2D crystal images of membrane proteins.

    Science.gov (United States)

    Scherer, Sebastian; Arheit, Marcel; Kowal, Julia; Zeng, Xiangyan; Stahlberg, Henning

    2014-03-01

    In cases where ultra-flat cryo-preparations of well-ordered two-dimensional (2D) crystals are available, electron crystallography is a powerful method for the determination of the high-resolution structures of membrane and soluble proteins. However, crystal unbending and Fourier-filtering methods in electron crystallography three-dimensional (3D) image processing are generally limited in their performance for 2D crystals that are badly ordered or non-flat. Here we present a single particle image processing approach, which is implemented as an extension of the 2D crystallographic pipeline realized in the 2dx software package, for the determination of high-resolution 3D structures of membrane proteins. The algorithm presented, addresses the low single-to-noise ratio (SNR) of 2D crystal images by exploiting neighborhood correlation between adjacent proteins in the 2D crystal. Compared with conventional single particle processing for randomly oriented particles, the computational costs are greatly reduced due to the crystal-induced limited search space, which allows a much finer search space compared to classical single particle processing. To reduce the considerable computational costs, our software features a hybrid parallelization scheme for multi-CPU clusters and computer with high-end graphic processing units (GPUs). We successfully apply the new refinement method to the structure of the potassium channel MloK1. The calculated 3D reconstruction shows more structural details and contains less noise than the map obtained by conventional Fourier-filtering based processing of the same 2D crystal images. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  2. Ultra-fast optical manipulation of single proteins binding to the actin cytoskeleton

    Science.gov (United States)

    Capitanio, Marco; Gardini, Lucia; Pavone, Francesco Saverio

    2014-02-01

    In the last decade, forces and mechanical stresses acting on biological systems are emerging as regulatory factors essential for cell life. Emerging evidences indicate that factors such as applied forces or the rigidity of the extracellular matrix (ECM) determine the shape and function of cells and organisms1. Classically, the regulation of biological systems is described through a series of biochemical signals and enzymatic reactions, which direct the processes and cell fate. However, mechanotransduction, i.e. the conversion of mechanical forces into biochemical and biomolecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. We recently developed an in vitro system that allows the investigation of force-dependence of the interaction of proteins binding the actin cytoskeleton, at the single molecule level. Our system displays a delay of only ~10 μs between formation of the molecular bond and application of the force and is capable of detecting interactions as short as 100 μs. Our assay allows direct measurements of load-dependence of lifetimes of single molecular bonds and conformational changes of single proteins and molecular motors. We demonstrate our technique on molecular motors, using myosin II from fast skeletal muscle and on protein-DNA interaction, specifically on Lactose repressor (LacI). The apparatus is stabilized to less than 1 nm with both passive and active stabilization, allowing resolving specific binding regions along the actin filament and DNA molecule. Our technique extends single-molecule force-clamp spectroscopy to molecular complexes that have been inaccessible up to now, opening new perspectives for the investigation of the effects of forces on biological processes.

  3. MIR and FIR Analysis of Inorganic Species in a Single Data Acquisition

    Science.gov (United States)

    Wang, Peng; Shilov, Sergey

    2017-06-01

    The extension of the mid IR towards the far IR spectral range below 400 \\wn is of great interest for molecular vibrational analysis for inorganic and organometallic chemistry, for geological, pharmaceutical, and physical applications, polymorph screening and crystallinity analysis as well as for matrix isolation spectroscopy. In these cases, the additional far infrared region offers insight to low energy vibrations which are observable only there. This includes inorganic species, lattice vibrations or intermolecular vibrations in the ordered solid state. The spectral range of a FTIR spectrometer is defined by the major optical components such as the source, beamsplitter, and detector. The globar source covers a broad spectral range from 8000 to 20 \\wn. However a bottle neck exists with respect to the beamsplitter and detector. To extend the spectral range further into the far IR and THz spectral ranges, one or more additional far IR beam splitters and detectors have been previously required. Two new optic components have been incorporated in a spectrometer to achieve coverage of both the mid and far infrared in a single scan: a wide range MIR-FIR beam splitter and the wide range DLaTGS detector that utilizes a diamond window. The use of a standard SiC IR source with these components yields a spectral range of 6000 down to 50 \\wn in one step for all types of transmittance, reflectance and ATR measurements. Utilizing the external water cooled mercury arc high power lamp the spectral range can be ultimately extended down to 10 \\wn. Examples of application will include emission in MIR-THz range, identification of pigments, additives in polymers, and polymorphism studies.

  4. Vibrational and structural investigation of SOUL protein single crystals by using micro-Raman spectroscopy

    Science.gov (United States)

    Rossi, Barbara; Giarola, Marco; Mariotto, Gino; Ambrosi, Emmanuele; Monaco, Hugo L.

    2010-05-01

    Protein SOUL is a new member of the recently discovered putative heme-binding protein family called SOUL/HEBP and, to date, no structural information exists for this protein. Here, micro-Raman spectroscopy is used to study the vibrational properties of single crystals obtained from recombinant protein SOUL by means of two different optimization routes. This spectroscopic approach offers the valuable advantage of the in-situ collection of experimental data from protein crystals, placed onto a hanging-drop plate, under the same conditions used to grow the crystals. By focusing on the regions of amides I and III bands, some secondary structure characteristic features have been recognized. Moreover, some side-chain marker bands were observed in the Raman spectra of SOUL crystals and the unambiguous assignment of these peaks inferred by comparing the experimental Raman spectra of pure amino acids and their Raman intensities computed using quantum chemical calculations. Our comparative analysis allows to get a deeper understanding of the side-chain environments and of the interactions involving these specific amino acids in the two different SOUL crystals.

  5. Single-Molecule Analysis of Protein Large-Amplitude Conformational Transitions

    Science.gov (United States)

    Yang, Haw

    2011-03-01

    Proteins have evolved to harness thermal fluctuations, rather than frustrated by them, to carry out chemical transformations and mechanical work. What are, then, the operation and design principles of protein machines? To frame the problem in a tractable way, several basic questions have been formulated to guide the experimental design: (a) How many conformational states can a protein sample on the functionally important timescale? (b) What are the inter-conversion rates between states? (c) How do ligand binding or interactions with other proteins modulate the motions? (d) What are the structural basis of flexibility and its underlying molecular mechanics? Guided by this framework, we have studied protein tyrosine phosphatase B, PtpB, from M. tuberculosis (a virulence factor of tuberculosis and a potential drug target) and adenylate kinase, AK, from E. coli (a ubiquitous energy-balancing enzyme in cells). These domain movements have been followed in real time on their respective catalytic timescales using high-resolution single-molecule Förster resonance energy transfer (FRET) spectroscopy. It is shown quantitatively that both PtpB and AK are capable of dynamically sampling two distinct states that correlate well with those observed by x-ray crystallography. Integrating these microscopic dynamics into macroscopic kinetics allows us to place the experimentally measured free-energy landscape in the context of enzymatic turnovers.

  6. Chlorella species as hosts for genetic engineering and expression of heterologous proteins: Progress, challenge and perspective.

    Science.gov (United States)

    Yang, Bo; Liu, Jin; Jiang, Yue; Chen, Feng

    2016-10-01

    The species of Chlorella represent a highly specialized group of green microalgae that can produce high levels of protein. Many Chlorella strains can grow rapidly and achieve high cell density under controlled conditions and are thus considered to be promising protein sources. Many advances in the genetic engineering of Chlorella have occurred in recent years, with significant developments in successful expression of heterologous proteins for various applications. Nevertheless, a lot of obstacles remain to be addressed, and a sophisticated and stable Chlorella expression system has yet to emerge. This review provides a brief summary of current knowledge on Chlorella and an overview of recent progress in the genetic engineering of Chlorella, and highlights the advances in the development of a genetic toolbox of Chlorella for heterologous protein expression. Research directions to further exploit the Chlorella expression system with respect to both challenges and perspectives are also discussed. This paper serves as a comprehensive literature review for the Chlorella community and will provide valuable insights into future exploration of Chlorella as a promising host for heterologous protein expression. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Thermal adaptation analyzed by comparison of protein sequences from mesophilic and extremely thermophilic Methanococcus species

    Science.gov (United States)

    Haney, P. J.; Badger, J. H.; Buldak, G. L.; Reich, C. I.; Woese, C. R.; Olsen, G. J.

    1999-01-01

    The genome sequence of the extremely thermophilic archaeon Methanococcus jannaschii provides a wealth of data on proteins from a thermophile. In this paper, sequences of 115 proteins from M. jannaschii are compared with their homologs from mesophilic Methanococcus species. Although the growth temperatures of the mesophiles are about 50 degrees C below that of M. jannaschii, their genomic G+C contents are nearly identical. The properties most correlated with the proteins of the thermophile include higher residue volume, higher residue hydrophobicity, more charged amino acids (especially Glu, Arg, and Lys), and fewer uncharged polar residues (Ser, Thr, Asn, and Gln). These are recurring themes, with all trends applying to 83-92% of the proteins for which complete sequences were available. Nearly all of the amino acid replacements most significantly correlated with the temperature change are the same relatively conservative changes observed in all proteins, but in the case of the mesophile/thermophile comparison there is a directional bias. We identify 26 specific pairs of amino acids with a statistically significant (P < 0.01) preferred direction of replacement.

  8. Plasmonic photocatalyst-like fluorescent proteins for generating reactive oxygen species

    Science.gov (United States)

    Leem, Jung Woo; Kim, Seong-Ryul; Choi, Kwang-Ho; Kim, Young L.

    2018-03-01

    The recent advances in photocatalysis have opened a variety of new possibilities for energy and biomedical applications. In particular, plasmonic photocatalysis using hybridization of semiconductor materials and metal nanoparticles has recently facilitated the rapid progress in enhancing photocatalytic efficiency under visible or solar light. One critical underlying aspect of photocatalysis is that it generates and releases reactive oxygen species (ROS) as intermediate or final products upon light excitation or activation. Although plasmonic photocatalysis overcomes the limitation of UV irradiation, synthesized metal/semiconductor nanomaterial photocatalysts often bring up biohazardous and environmental issues. In this respect, this review article is centered in identifying natural photosensitizing organic materials that can generate similar types of ROS as those of plasmonic photocatalysis. In particular, we propose the idea of plasmonic photocatalyst-like fluorescent proteins for ROS generation under visible light irradiation. We recapitulate fluorescent proteins that have Type I and Type II photosensitization properties in a comparable manner to plasmonic photocatalysis. Plasmonic photocatalysis and protein photosensitization have not yet been compared systemically in terms of ROS photogeneration under visible light, although the phototoxicity and cytotoxicity of some fluorescent proteins are well recognized. A comprehensive understanding of plasmonic photocatalyst-like fluorescent proteins and their potential advantages will lead us to explore new environmental, biomedical, and defense applications.

  9. Two Positive Periodic Solutions for a Neutral Delay Model of Single-Species Population Growth with Harvesting

    OpenAIRE

    Fang, Hui

    2012-01-01

    By coincidence degree theory for k-set-contractive mapping, this paper establishes a new criterion for the existence of at least two positive periodic solutions for a neutral delay model of single-species population growth with harvesting. An example is given to illustrate the effectiveness of the result.

  10. Two Positive Periodic Solutions for a Neutral Delay Model of Single-Species Population Growth with Harvesting

    Directory of Open Access Journals (Sweden)

    Hui Fang

    2012-01-01

    Full Text Available By coincidence degree theory for k-set-contractive mapping, this paper establishes a new criterion for the existence of at least two positive periodic solutions for a neutral delay model of single-species population growth with harvesting. An example is given to illustrate the effectiveness of the result.

  11. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    NARCIS (Netherlands)

    Quevedo, Beatrice; Giertsen, Elin; Zijnge, Vincent; Luethi-Schaller, Helga; Guggenheim, Bernhard; Thurnheer, Thomas; Gmuer, Rudolf

    2011-01-01

    Background: The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to

  12. Use of a Cellulase-Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose †

    Science.gov (United States)

    Hitchner, E. V.; Leatherwood, J. M.

    1980-01-01

    A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30°C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet. PMID:16345511

  13. Characterization of the C-protein from posterior latissimus dorsi muscle of the adult chicken: heterogeneity within a single sarcomere

    OpenAIRE

    1983-01-01

    Specific isoforms of myofibrillar proteins are expressed in different muscles and in various fiber types within a single muscle. We have isolated and characterized monoclonal antibodies against C-proteins from slow tonic (anterior latissimus dorsi, ALD) and fast twitch (pectoralis major) muscles of the chicken. Although the antibody against "fast" C-protein (MF-1) did not bind to the "slow" isoform and the antibody to the "slow" C-protein (ALD-66) did not bind to the "fast" isoform, we observ...

  14. Identification of proteins involved in the adhesionof Candida species to different medical devices.

    Science.gov (United States)

    Núñez-Beltrán, Arianna; López-Romero, Everardo; Cuéllar-Cruz, Mayra

    2017-06-01

    Adhesion is the first step for Candida species to form biofilms on medical devices implanted in the human host. Both the physicochemical nature of the biomaterial and cell wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true that some CWP have been identified in vitro, little is known about the CWP of pathogenic species of Candida involved in adhesion. On this background, we considered it important to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C. parapsilosis in adhesion to different medical devices. Our results indicate that the four species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C. parapsilosis to adher to silicone-made implants. Results presented here suggest that these CWP participate in the initial event of adhesion and are probably followed by other proteins that covalently bind to the biomaterial thus providing conditions for biofilm formation and eventually the onset of infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Stimulated synthesis of plasma protein species in Q fever and endotoxemia

    Energy Technology Data Exchange (ETDEWEB)

    Picking, W.D.; Ershadi, M.; Hackstadt, T.; Paretsky, D.

    1987-05-01

    Q fever stimulates hepatic transcription and translation. Products of stimulated transcription have been identified, but not of translation. Protein (Pr) synthesis and rPr S6 phosphorylation correlated. The authors now report stimulated synthesis of plasma Pr species in early febrile responses to Q fever and Coxiella burnetii lipopolysaccharide (LPS). Guinea pigs received 400 g LPS intraperitoneally and 7 hr later 250 Ci L-(TVS)met, then sacrificed 3 hr later. Plasma Pr sp act (cpm/mg Pr) increased 2.3X over controls (N). Exptl plasma Pr PAGE autorads showed intensified Pr bands at M/sub r/ 55K. Guinea pigs infected with C. burnetii (Inf) received 400 Ci (TVS)met 84 hr p.i. and were sacrificed 3 hr later. Inf plasma Pr 1D-PAGE showed bands at 55K similar to that found with LPS, with lower albumin concn. Coomassie stain and autorads of 2-D PAGEs showed intensified or new acidic peptide species in Inf plasma. PAGE autorads in vitro translations using liver mRNA and ribosomes showed major species in Inf systems at 49K (4+) and 62K (2+) compared to N. The data indicate acute phase protein induction by LPS or rickettsial infection.

  16. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    Science.gov (United States)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  17. Suite of three protein crystallography beamlines with single superconducting bend magnet as the source

    Energy Technology Data Exchange (ETDEWEB)

    MacDowell, Alastair A.; Celestre, Richard S.; Howells, Malcolm; McKinney, Wayne; Krupnick, James; Cambie, Daniella; Domning, Edward E; Duarte, Robert M.; Kelez, Nicholas; Plate, David W.; Cork, Carl W.; Earnest, Thomas N.; Dickert, Jeffery; Meigs, George; Ralston, Corie; Holton, James M.; Alber, Thomas; Berger, James M.; Agard, David A.; Padmore, Howard A.

    2004-08-01

    At the Advanced Light Source (ALS), three protein crystallography (PX) beamlines have been built that use as a source one of the three 6 Tesla single pole superconducting bending magnets (superbends) that were recently installed in the ring. The use of such single pole superconducting bend magnets enables the development of a hard x-ray program on a relatively low energy 1.9 GeV ring without taking up insertion device straight sections. The source is of relatively low power, but due to the small electron beam emittance, it has high brightness. X-ray optics are required to preserve the brightness and to match the illumination requirements for protein crystallography. This was achieved by means of a collimating premirror bent to a plane parabola, a double crystal monochromator followed by a toroidal mirror that focuses in the horizontal direction with a 2:1 demagnification. This optical arrangement partially balances aberrations from the collimating and toroidal mirrors such that a tight focused spot size is achieved. The optical properties of the beamline are an excellent match to those required by the small protein crystals that are typically measured. The design and performance of these new beamlines are described.

  18. Suite of three protein crystallography beamlines with single superconducting bend magnet as the source

    International Nuclear Information System (INIS)

    MacDowell, Alastair A.; Celestre, Richard S.; Howells, Malcolm; McKinney, Wayne; Krupnick, James; Cambie, Daniella; Domning, Edward E; Duarte, Robert M.; Kelez, Nicholas; Plate, David W.; Cork, Carl W.; Earnest, Thomas N.; Dickert, Jeffery; Meigs, George; Ralston, Corie; Holton, James M.; Alber, Thomas; Berger, James M.; Agard, David A.; Padmore, Howard A.

    2004-01-01

    At the Advanced Light Source (ALS), three protein crystallography (PX) beamlines have been built that use as a source one of the three 6 Tesla single pole superconducting bending magnets (superbends) that were recently installed in the ring. The use of such single pole superconducting bend magnets enables the development of a hard x-ray program on a relatively low energy 1.9 GeV ring without taking up insertion device straight sections. The source is of relatively low power, but due to the small electron beam emittance, it has high brightness. X-ray optics are required to preserve the brightness and to match the illumination requirements for protein crystallography. This was achieved by means of a collimating premirror bent to a plane parabola, a double crystal monochromator followed by a toroidal mirror that focuses in the horizontal direction with a 2:1 demagnification. This optical arrangement partially balances aberrations from the collimating and toroidal mirrors such that a tight focused spot size is achieved. The optical properties of the beamline are an excellent match to those required by the small protein crystals that are typically measured. The design and performance of these new beamlines are described

  19. Detection of isolated protein-bound metal ions by single-particle cryo-STEM.

    Science.gov (United States)

    Elad, Nadav; Bellapadrona, Giuliano; Houben, Lothar; Sagi, Irit; Elbaum, Michael

    2017-10-17

    Metal ions play essential roles in many aspects of biological chemistry. Detecting their presence and location in proteins and cells is important for understanding biological function. Conventional structural methods such as X-ray crystallography and cryo-transmission electron microscopy can identify metal atoms on protein only if the protein structure is solved to atomic resolution. We demonstrate here the detection of isolated atoms of Zn and Fe on ferritin, using cryogenic annular dark-field scanning transmission electron microscopy (cryo-STEM) coupled with single-particle 3D reconstructions. Zn atoms are found in a pattern that matches precisely their location at the ferroxidase sites determined earlier by X-ray crystallography. By contrast, the Fe distribution is smeared along an arc corresponding to the proposed path from the ferroxidase sites to the mineral nucleation sites along the twofold axes. In this case the single-particle reconstruction is interpreted as a probability distribution function based on the average of individual locations. These results establish conditions for detection of isolated metal atoms in the broader context of electron cryo-microscopy and tomography.

  20. Single nucleotide polymorphism barcoding of cytochrome c oxidase I sequences for discriminating 17 species of Columbidae by decision tree algorithm.

    Science.gov (United States)

    Yang, Cheng-Hong; Wu, Kuo-Chuan; Dahms, Hans-Uwe; Chuang, Li-Yeh; Chang, Hsueh-Wei

    2017-07-01

    DNA barcodes are widely used in taxonomy, systematics, species identification, food safety, and forensic science. Most of the conventional DNA barcode sequences contain the whole information of a given barcoding gene. Most of the sequence information does not vary and is uninformative for a given group of taxa within a monophylum. We suggest here a method that reduces the amount of noninformative nucleotides in a given barcoding sequence of a major taxon, like the prokaryotes, or eukaryotic animals, plants, or fungi. The actual differences in genetic sequences, called single nucleotide polymorphism (SNP) genotyping, provide a tool for developing a rapid, reliable, and high-throughput assay for the discrimination between known species. Here, we investigated SNPs as robust markers of genetic variation for identifying different pigeon species based on available cytochrome c oxidase I (COI) data. We propose here a decision tree-based SNP barcoding (DTSB) algorithm where SNP patterns are selected from the DNA barcoding sequence of several evolutionarily related species in order to identify a single species with pigeons as an example. This approach can make use of any established barcoding system. We here firstly used as an example the mitochondrial gene COI information of 17 pigeon species (Columbidae, Aves) using DTSB after sequence trimming and alignment. SNPs were chosen which followed the rule of decision tree and species-specific SNP barcodes. The shortest barcode of about 11 bp was then generated for discriminating 17 pigeon species using the DTSB method. This method provides a sequence alignment and tree decision approach to parsimoniously assign a unique and shortest SNP barcode for any known species of a chosen monophyletic taxon where a barcoding sequence is available.

  1. Highly sensitive immunoassay of protein molecules based on single nanoparticle fluorescence detection in a nanowell

    Science.gov (United States)

    Han, Jin-Hee; Kim, Hee-Joo; Lakshmana, Sudheendra; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2011-03-01

    A nanoarray based-single molecule detection system was developed for detecting proteins with extremely high sensitivity. The nanoarray was able to effectively trap nanoparticles conjugated with biological sample into nanowells by integrating with an electrophoretic particle entrapment system (EPES). The nanoarray/EPES is superior to other biosensor using immunoassays in terms of saving the amounts of biological solution and enhancing kinetics of antibody binding due to reduced steric hindrance from the neighboring biological molecules. The nanoarray patterned onto a layer of PMMA and LOL on conductive and transparent indium tin oxide (ITO)-glass slide by using e-beam lithography. The suspension of 500 nm-fluorescent (green emission)-carboxylated polystyrene (PS) particles coated with protein-A followed by BDE 47 polyclonal antibody was added to the chip that was connected to the positive voltage. The droplet was covered by another ITO-coated-glass slide and connected to a ground terminal. After trapping the particles into the nanowells, the solution of different concentrations of anti-rabbit- IgG labeled with Alexa 532 was added for an immunoassay. A single molecule detection system could quantify the anti-rabbit IgG down to atto-mole level by counting photons emitted from the fluorescent dye bound to a single nanoparticle in a nanowell.

  2. Protein structural model selection by combining consensus and single scoring methods.

    Directory of Open Access Journals (Sweden)

    Zhiquan He

    Full Text Available Quality assessment (QA for predicted protein structural models is an important and challenging research problem in protein structure prediction. Consensus Global Distance Test (CGDT methods assess each decoy (predicted structural model based on its structural similarity to all others in a decoy set and has been proved to work well when good decoys are in a majority cluster. Scoring functions evaluate each single decoy based on its structural properties. Both methods have their merits and limitations. In this paper, we present a novel method called PWCom, which consists of two neural networks sequentially to combine CGDT and single model scoring methods such as RW, DDFire and OPUS-Ca. Specifically, for every pair of decoys, the difference of the corresponding feature vectors is input to the first neural network which enables one to predict whether the decoy-pair are significantly different in terms of their GDT scores to the native. If yes, the second neural network is used to decide which one of the two is closer to the native structure. The quality score for each decoy in the pool is based on the number of winning times during the pairwise comparisons. Test results on three benchmark datasets from different model generation methods showed that PWCom significantly improves over consensus GDT and single scoring methods. The QA server (MUFOLD-Server applying this method in CASP 10 QA category was ranked the second place in terms of Pearson and Spearman correlation performance.

  3. Molecular analysis of the freshwater prawn Macrobrachium olfersii (Decapoda, Palaemonidae) supports the existence of a single species throughout its distribution.

    Science.gov (United States)

    Rossi, Natália; Mantelatto, Fernando Luis

    2013-01-01

    Macrobrachium olfersii is an amphidromous freshwater prawn, widespread along the eastern coasts of the Americas. This species shows great morphological modifications during ontogenesis, and several studies have verified the existence of a wide intraspecific variation. Because of this condition, the species is often misidentified, and several synonyms have been documented. To elucidate these aspects, individuals of M. olfersii from different populations along its range of distribution were investigated. The taxonomic limit was established, and the degree of genetic variability of this species was described. We extracted DNA from 53 specimens of M. olfersii, M. americanum, M. digueti and M. faustinum, which resulted in 84 new sequences (22 of 16S mtDNA, 45 of Cythocrome Oxidase I (COI) mtDNA, and 17 of Histone (H3) nDNA). Sequences of three genes (single and concatenated) from these species were used in the Maximum Likelihood and Bayesian Inference phylogenetic analyses and COI sequences from M. olfersii were used in population analysis. The genetic variation was evaluated through the alignment of 554 bp from the 16S, 638 bp from the COI, and 338 bp from the H3. The rates of genetic divergence among populations were lower at the intraspecific level. This was confirmed by the haplotype net, which showed a continuous gene flow among populations. Although a wide distribution and high morphological intraspecific variation often suggest the existence of more than one species, genetic similarity of Caribbean and Brazilian populations of M. olfersii supported them as a single species.

  4. Molecular analysis of the freshwater prawn Macrobrachium olfersii (Decapoda, Palaemonidae supports the existence of a single species throughout its distribution.

    Directory of Open Access Journals (Sweden)

    Natália Rossi

    Full Text Available Macrobrachium olfersii is an amphidromous freshwater prawn, widespread along the eastern coasts of the Americas. This species shows great morphological modifications during ontogenesis, and several studies have verified the existence of a wide intraspecific variation. Because of this condition, the species is often misidentified, and several synonyms have been documented. To elucidate these aspects, individuals of M. olfersii from different populations along its range of distribution were investigated. The taxonomic limit was established, and the degree of genetic variability of this species was described. We extracted DNA from 53 specimens of M. olfersii, M. americanum, M. digueti and M. faustinum, which resulted in 84 new sequences (22 of 16S mtDNA, 45 of Cythocrome Oxidase I (COI mtDNA, and 17 of Histone (H3 nDNA. Sequences of three genes (single and concatenated from these species were used in the Maximum Likelihood and Bayesian Inference phylogenetic analyses and COI sequences from M. olfersii were used in population analysis. The genetic variation was evaluated through the alignment of 554 bp from the 16S, 638 bp from the COI, and 338 bp from the H3. The rates of genetic divergence among populations were lower at the intraspecific level. This was confirmed by the haplotype net, which showed a continuous gene flow among populations. Although a wide distribution and high morphological intraspecific variation often suggest the existence of more than one species, genetic similarity of Caribbean and Brazilian populations of M. olfersii supported them as a single species.

  5. Life in extreme environments: single molecule force spectroscopy as a tool to explore proteins from extremophilic organisms.

    Science.gov (United States)

    Tych, Katarzyna M; Hoffmann, Toni; Batchelor, Matthew; Hughes, Megan L; Kendrick, Katherine E; Walsh, Danielle L; Wilson, Michael; Brockwell, David J; Dougan, Lorna

    2015-04-01

    Extremophiles are organisms which survive and thrive in extreme environments. The proteins from extremophilic single-celled organisms have received considerable attention as they are structurally stable and functionally active under extreme physical and chemical conditions. In this short article, we provide an introduction to extremophiles, the structural adaptations of proteins from extremophilic organisms and the exploitation of these proteins in industrial applications. We provide a review of recent developments which have utilized single molecule force spectroscopy to mechanically manipulate proteins from extremophilic organisms and the information which has been gained about their stability, flexibility and underlying energy landscapes.

  6. Mare’s milk: composition and protein fraction in comparison with different milk species

    Directory of Open Access Journals (Sweden)

    Krešimir Kuterovac

    2011-06-01

    Full Text Available The usage of the mare’s milk as functional food especial for children intolerant to cow’s milk, with neurodermitis, allergies and similar disorders desiring to improve the quality of life is fiercely debated for last decades but there were no scientific studies to suggest such use of mare’s milk based on scientific research. The objectives of this study were to determine similarities of mare’s milk in comparison with milk of ruminants (cattle, sheep and goat and human milk in terms of milk composition and protein fraction as whey proteins, caseins and micelles size. All differences were discussed regarding usage of mare’s milk in human diet and compared to milk which is usually used in human nutrition. Regarding composition, the mare’s milk is similar to human milk in of crude protein, salt and lactose content, but it has significantly lower content of fat. Fractions of main proteins are similar between human and mare’s milk, except nitrogen casein (casein N which has twice lower content in human than in mare’s milk. Content of casein N from all ruminants’ milk differ much more. Just for true whey N and non-protein nitrogen (NPN similar content as human and mare’s milk has also goat milk. The casein content is the lowest in human milk; this content is three times greater in mare’s milk and six to seven times greater in goat’s and cow’s milk, while in sheep’s milk it is more than 10 times grater. In many components and fractions mare’s milk is more similar to human milk than milk of ruminants. A detail comparison of protein fraction shows quite large differences between milk of different species. More study and clinical research are needed that can recommend usage of mare’s milk in human diet as functional food on scientific bases.

  7. Functional mapping of cell surface proteins with localized stimulation of single cells

    Science.gov (United States)

    Sun, Bingyun; Chiu, Daniel T.

    2003-11-01

    This paper describes the development of using individual micro and nano meter-sized vesicles as delivery vessels to functionally map the distribution of cell surface proteins at the level of single cells. The formation of different sizes of vesicles from tens of nanometers to a few micrometers in diameter that contain the desired molecules is addressed. An optical trap is used to manipulate the loaded vesicle to specific cell morphology of interest, and a pulsed UV laser is used to photo-release the stimuli onto the cell membrane. Carbachol activated cellular calcium flux, upon binding to muscarinic acetylcholine receptors, is studied by this method, and the potential of using this method for the functional mapping of localized proteins on the cell surface membrane is discussed.

  8. Energy transduction and signal averaging of fluctuating electric fields by a single protein ion channel.

    Science.gov (United States)

    Verdia-Baguena, C; Gomez, V; Cervera, J; Ramirez, P; Mafe, S

    2016-12-21

    We demonstrate the electrical rectification and signal averaging of fluctuating signals using a biological nanostructure in aqueous solution: a single protein ion channel inserted in the lipid bilayer characteristic of cell membranes. The conversion of oscillating, zero time-average potentials into directional currents permits charging of a load capacitor to significant steady-state voltages within a few minutes in the case of the outer membrane porin F (OmpF) protein, a bacterial channel of Escherichia coli. The experiments and simulations show signal averaging effects at a more fundamental level than the traditional cell and tissue scales, which are characterized by ensembles of many ion channels operating simultaneously. The results also suggest signal transduction schemes with bio-electronic interfaces and ionic circuits where soft matter nanodiodes can be coupled to conventional electronic elements.

  9. Evolution of Helicobacter: Acquisition by Gastric Species of Two Histidine-Rich Proteins Essential for Colonization.

    Directory of Open Access Journals (Sweden)

    Daniel Vinella

    2015-12-01

    Full Text Available Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2 play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn

  10. Protein-fold recognition using an improved single-source K diverse shortest paths algorithm.

    Science.gov (United States)

    Lhota, John; Xie, Lei

    2016-04-01

    Protein structure prediction, when construed as a fold recognition problem, is one of the most important applications of similarity search in bioinformatics. A new protein-fold recognition method is reported which combines a single-source K diverse shortest path (SSKDSP) algorithm with Enrichment of Network Topological Similarity (ENTS) algorithm to search a graphic feature space generated using sequence similarity and structural similarity metrics. A modified, more efficient SSKDSP algorithm is developed to improve the performance of graph searching. The new implementation of the SSKDSP algorithm empirically requires 82% less memory and 61% less time than the current implementation, allowing for the analysis of larger, denser graphs. Furthermore, the statistical significance of fold ranking generated from SSKDSP is assessed using ENTS. The reported ENTS-SSKDSP algorithm outperforms original ENTS that uses random walk with restart for the graph search as well as other state-of-the-art protein structure prediction algorithms HHSearch and Sparks-X, as evaluated by a benchmark of 600 query proteins. The reported methods may easily be extended to other similarity search problems in bioinformatics and chemoinformatics. The SSKDSP software is available at http://compsci.hunter.cuny.edu/~leixie/sskdsp.html. © 2016 Wiley Periodicals, Inc.

  11. Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

    International Nuclear Information System (INIS)

    Neto, J.L. Siqueira; Lira, C.B.B.; Giardini, M.A.; Khater, L.; Perez, A.M.; Peroni, L.A.; Reis, J.R.R. dos; Freitas-Junior, L.H.; Ramos, C.H.I.; Cano, M.I.N.

    2007-01-01

    Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres

  12. Computational exploration of single-protein mechanics by steered molecular dynamics

    Energy Technology Data Exchange (ETDEWEB)

    Sotomayor, Marcos [Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio (United States)

    2015-12-31

    Hair cell mechanotransduction happens in tens of microseconds, involves forces of a few picoNewtons, and is mediated by nanometer-scale molecular conformational changes. As proteins involved in this process become identified and their high resolution structures become available, multiple tools are being used to explore their “single-molecule responses” to force. Optical tweezers and atomic force microscopy offer exquisite force and extension resolution, but cannot reach the high loading rates expected for high frequency auditory stimuli. Molecular dynamics (MD) simulations can reach these fast time scales, and also provide a unique view of the molecular events underlying protein mechanics, but its predictions must be experimentally verified. Thus a combination of simulations and experiments might be appropriate to study the molecular mechanics of hearing. Here I review the basics of MD simulations and the different methods used to apply force and study protein mechanics in silico. Simulations of tip link proteins are used to illustrate the advantages and limitations of this method.

  13. Comparative genomic analyses of transport proteins encoded within the genomes of Leptospira species.

    Science.gov (United States)

    Buyuktimkin, Bora; Saier, Milton H

    2015-11-01

    Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity was accompanied by progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Pathogenic Leptospira species acquire factor H and vitronectin via the surface protein LcpA.

    Science.gov (United States)

    da Silva, Ludmila Bezerra; Miragaia, Lidia Dos Santos; Breda, Leandro Carvalho Dantas; Abe, Cecilia Mari; Schmidt, Mariana Costa Braga; Moro, Ana Maria; Monaris, Denize; Conde, Jonas Nascimento; Józsi, Mihály; Isaac, Lourdes; Abreu, Patrícia Antônia Estima; Barbosa, Angela Silva

    2015-03-01

    Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  15. Comparative analyses of transport proteins encoded within the genomes of Leptospira species.

    Science.gov (United States)

    Buyuktimkin, Bora; Saier, Milton H

    2016-09-01

    Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, Leptospira interrogans serovar Lai str. 56601 and Leptospira borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, Leptospira biflexa serovar Patoc str. 'Patoc 1 (Ames)'. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they all have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity arose in Leptospira correlating to progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. Copyright © 2016. Published by Elsevier Ltd.

  16. Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species.

    Directory of Open Access Journals (Sweden)

    Erin Shanker

    2016-12-01

    Full Text Available Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS mediated by peptide pheromones. One such system, ComRS, consists of a peptide (ComS that is processed (XIP, secreted, and later imported into the cytoplasm, where it binds and activates the transcription factor ComR. ComR then engages in a positive feedback loop for the expression of ComS and the alternative sigma-factor SigX. Although ComRS are present in the majority of Streptococcus species, the sequence of both ComS/XIP and ComR diverge significantly, suggesting a mechanism for species-specific communication. To study possible cross-talk between streptococcal species in the regulation of competence, and to explore in detail the molecular interaction between ComR and XIP we undertook an interdisciplinary approach. We developed a 'test-bed' assay to measure the activity of different ComR proteins in response to cognate and heterologous XIP peptides in vivo, revealing distinct ComR classes of strict, intermediate, and promiscuous specificity among species. We then solved an X-ray crystal structure of ComR from S. suis to further understand the interaction with XIP and to search for structural features in ComR proteins that may explain XIP recognition. Using the structure as a guide, we probed the apo conformation of the XIP-binding pocket by site-directed mutagenesis, both in test-bed cultures and biochemically in vitro. In alignments with ComR proteins from other species, we find that the pocket is lined by a variable and a conserved face, where residues of the conserved face contribute to ligand binding and the variable face discriminate among XIP

  17. Stepwise bending of DNA by a single TATA box binding protein

    DEFF Research Database (Denmark)

    Tolic-Nørrelykke, Simon F; Rasmussen, Mette B; Pavone, Francesco S

    2006-01-01

    The TATA-box binding protein (TBP) is required by all three eucaryotic RNA polymerases for the initiation of transcription from most promoters. TBP recognizes, binds to, and bends promoter sequences called "TATA-boxes" in the DNA. We present results from the study of individual Saccharomyces...... cerevisiae TBPs interacting with single DNA molecules containing a TATA-box. Using video microscopy, we observed the Brownian motion of the beads tethered by short surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the DNA changes and the amplitude of Brownian motion of the tehtered...

  18. Probing Protein Multidimensional Conformational Fluctuations by Single-Molecule Multiparameter Photon Stamping Spectroscopy

    Science.gov (United States)

    2015-01-01

    Conformational motions of proteins are highly dynamic and intrinsically complex. To capture the temporal and spatial complexity of conformational motions and further to understand their roles in protein functions, an attempt is made to probe multidimensional conformational dynamics of proteins besides the typical one-dimensional FRET coordinate or the projected conformational motions on the one-dimensional FRET coordinate. T4 lysozyme hinge-bending motions between two domains along α-helix have been probed by single-molecule FRET. Nevertheless, the domain motions of T4 lysozyme are rather complex involving multiple coupled nuclear coordinates and most likely contain motions besides hinge-bending. It is highly likely that the multiple dimensional protein conformational motions beyond the typical enzymatic hinged-bending motions have profound impact on overall enzymatic functions. In this report, we have developed a single-molecule multiparameter photon stamping spectroscopy integrating fluorescence anisotropy, FRET, and fluorescence lifetime. This spectroscopic approach enables simultaneous observations of both FRET-related site-to-site conformational dynamics and molecular rotational (or orientational) motions of individual Cy3-Cy5 labeled T4 lysozyme molecules. We have further observed wide-distributed rotational flexibility along orientation coordinates by recording fluorescence anisotropy and simultaneously identified multiple intermediate conformational states along FRET coordinate by monitoring time-dependent donor lifetime, presenting a whole picture of multidimensional conformational dynamics in the process of T4 lysozyme open-close hinge-bending enzymatic turnover motions under enzymatic reaction conditions. By analyzing the autocorrelation functions of both lifetime and anisotropy trajectories, we have also observed the dynamic and static inhomogeneity of T4 lysozyme multidimensional conformational fluctuation dynamics, providing a fundamental

  19. Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines

    Directory of Open Access Journals (Sweden)

    Rahmberg Andrew R

    2011-05-01

    Full Text Available Abstract Background Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. Results Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. Conclusions Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent TH1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

  20. SVMQA: support-vector-machine-based protein single-model quality assessment.

    Science.gov (United States)

    Manavalan, Balachandran; Lee, Jooyoung

    2017-08-15

    The accurate ranking of predicted structural models and selecting the best model from a given candidate pool remain as open problems in the field of structural bioinformatics. The quality assessment (QA) methods used to address these problems can be grouped into two categories: consensus methods and single-model methods. Consensus methods in general perform better and attain higher correlation between predicted and true quality measures. However, these methods frequently fail to generate proper quality scores for native-like structures which are distinct from the rest of the pool. Conversely, single-model methods do not suffer from this drawback and are better suited for real-life applications where many models from various sources may not be readily available. In this study, we developed a support-vector-machine-based single-model global quality assessment (SVMQA) method. For a given protein model, the SVMQA method predicts TM-score and GDT_TS score based on a feature vector containing statistical potential energy terms and consistency-based terms between the actual structural features (extracted from the three-dimensional coordinates) and predicted values (from primary sequence). We trained SVMQA using CASP8, CASP9 and CASP10 targets and determined the machine parameters by 10-fold cross-validation. We evaluated the performance of our SVMQA method on various benchmarking datasets. Results show that SVMQA outperformed the existing best single-model QA methods both in ranking provided protein models and in selecting the best model from the pool. According to the CASP12 assessment, SVMQA was the best method in selecting good-quality models from decoys in terms of GDTloss. SVMQA method can be freely downloaded from http://lee.kias.re.kr/SVMQA/SVMQA_eval.tar.gz. jlee@kias.re.kr. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  1. Species-specific serine-threonine protein kinase Pkb2 of Bifidobacterium longum subsp. longum: Genetic environment and substrate specificity.

    Science.gov (United States)

    Nezametdinova, V Z; Mavletova, D A; Alekseeva, M G; Chekalina, M S; Zakharevich, N V; Danilenko, V N

    2018-03-10

    The objective of this study was to determine for phosphorylated substrates of the species-specific serine-threonine protein kinase (STPK) Pkb2 from Bifidobacterium longum subsp. longum GT15. Two approaches were employed: analyses of phosphorylated membrane vesicles protein spectra following kinase reactions and analyses of the genes surrounding pkb2. A bioinformatics analysis of the genes surrounding pkb2 found a species-specific gene cluster PFNA in the genomes of 34 different bifidobacterial species. The identified cluster consisted of 5-8 genes depending on the species. The first five genes are characteristic for all considered species. These are the following genes encoding serine-threonine protein kinase (pkb2), fibronectin type III domain-containing protein (fn3), AAA-ATPase (aaa-atp), hypothetical protein with DUF58 domain (duf58) and transglutaminase (tgm). The sixth (protein phosphatase, prpC), seventh (hypothetical protein, BLGT_RS02790), and eighth (FHA domain-containing protein, fha) genes are included in this cluster, but they are not found in all species. The operon organization of the PFNA gene cluster was confirmed with transcriptional analysis. AAA-ATPase, which is encoded by a gene of the PFNA gene cluster, was found to be a substrate of the STPK Pkb2. Fourteen AAA-ATPase sites (seven serine, six threonine, and one tyrosine) phosphorylated by STPK Pkb2 were revealed. Analysis of the spectra of phosphorylated membrane vesicles proteins allowed us to identify eleven proteins that were considered as possible Pkb2 substrates. They belong to several functional classes: proteins involved in transcription and translation; proteins of the F1-domain of the FoF1-ATPase; ABC-transporters; molecular chaperone GroEL; and glutamine synthase, GlnA1. All identified proteins were considered moonlighting proteins. Three out of 11 proteins (glutamine synthetase GlnA1 and FoF1-ATPase alpha and beta subunits) were selected for further in vitro phosphorylation assays

  2. Effects of the daily consumption of protein enriched bread and protein enriched drinking yoghurt on the total protein intake in older adults in a rehabilitation centre: a single blind randomised controlled trial.

    Science.gov (United States)

    van Til, A J; Naumann, E; Cox-Claessens, I J H M; Kremer, S; Boelsma, E; de van der Schueren, M A E

    2015-05-01

    To investigate the effects of protein enriched bread and drinking yoghurt, substituting regular products, on the total protein intake and the distribution of protein intake over the day in older adults. A single blind randomised controlled trial. Rehabilitation centre. Older adults (≥ 55 years) admitted to a rehabilitation centre after hospital discharge (n=34). Participants received a high protein diet (protein enriched bread and protein enriched drinking yoghurt; n=17) or a regular diet (regular bread and regular drinking yoghurt; n=17) for three consecutive weeks. Total protein intake and protein intake per meal, measured twice weekly over a three weeks period (six measurements per participant). Compared with controls, patients who received the protein enriched products had a significantly higher protein intake (115.3 g/d vs 72.5 g/d, Pconsumption of protein enriched products improves protein distribution over the day.

  3. Single aromatic residue location alters nucleic acid binding and chaperone function of FIV nucleocapsid protein

    Science.gov (United States)

    Wu, Hao; Wang, Wei; Naiyer, Nada; Fichtenbaum, Eric; Qualley, Dominic F.; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    Feline immunodeficiency virus (FIV) is a retrovirus that infects domestic cats, and is an excellent animal model for human immunodeficiency virus type 1 (HIV-1) pathogenesis. The nucleocapsid (NC) protein is critical for replication in both retroviruses. FIV NC has several structural features that differ from HIV-1 NC. While both NC proteins have a single conserved aromatic residue in each of the two zinc fingers, the aromatic residue on the second finger of FIV NC is located on the opposite C-terminal side relative to its location in HIV-1 NC. In addition, whereas HIV-1 NC has a highly charged cationic N-terminal tail and a relatively short C-terminal extension, the opposite is true for FIV NC. To probe the impact of these differences on the nucleic acid (NA) binding and chaperone properties of FIV NC, we carried out ensemble and single-molecule assays with wild-type (WT) and mutant proteins. The ensemble studies show that FIV NC binding to DNA is strongly electrostatic, with a higher effective charge than that observed for HIV-1 NC. The C-terminal basic domain contributes significantly to the NA binding capability of FIV NC. In addition, the non-electrostatic component of DNA binding is much weaker for FIV NC than for HIV-1 NC. Mutation of both aromatic residues in the zinc fingers to Ala (F12A/W44A) further increases the effective charge of FIV NC and reduces its non-electrostatic binding affinity. Interestingly, switching the location of the C-terminal aromatic residue to mimic the HIV-1 NC sequence (N31W/W44A) reduces the effective charge of FIV NC and increases its non-electrostatic binding affinity to values similar to HIV-1 NC. Consistent with the results of these ensemble studies, single-molecule DNA stretching studies show that while WT FIV NC has reduced stacking capability relative to HIV-1 NC, the aromatic switch mutant recovers the ability to intercalate between the DNA bases. Our results demonstrate that altering the position of a single aromatic

  4. Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence.

    Science.gov (United States)

    Meghraoui, Alaeddine; Schiavolin, Lionel; Allaoui, Abdelmounaaïm

    2014-07-01

    Infection of colonic epithelial cells by Shigella is associated with the type III secretion system, which serves as a molecular syringe to inject effectors into host cells. This system includes an extracellular needle used as a conduit for secreted proteins. Two of these proteins, IpaB and IpaD, dock at the needle tip to control secretion and are also involved in the insertion of a translocation pore into host cell membrane allowing effector delivery. To better understand the function of IpaD, we substituted thirteen residues conserved among homologous proteins in other bacterial species. Generated variants were tested for their ability to surface expose IpaB and IpaD, to control secretion, to insert the translocation pore, and to invade host cells. In addition to a first group of seven ipaD variants that behaved similarly to the wild-type strain, we identified a second group with mutations V314D and I319D that deregulated secretion of all effectors, but remained fully invasive. Moreover, we identified a third group with mutations Y153A, T161D, Q165L and Y276A, that exhibited increased levels of translocators secretion, pore formation, and cell entry. Altogether, our results offer a better understanding of the role of IpaD in the control of Shigella virulence. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. Solar light-induced production of reactive oxygen species by single walled carbon nanotubes in water

    Science.gov (United States)

    Photosensitizing processes of engineered nanomaterials (ENMs) which include photo-induced production of reactive oxygen species (ROS) convert light energy into oxidizing chemical energy that mediates transformations of nanomaterials. The oxidative stress associated with ROS may p...

  6. Tackling Bet v 1 and associated food allergies with a single hybrid protein.

    Science.gov (United States)

    Hofer, Heidi; Asam, Claudia; Hauser, Michael; Nagl, Birgit; Laimer, Josef; Himly, Martin; Briza, Peter; Ebner, Christof; Lang, Roland; Hawranek, Thomas; Bohle, Barbara; Lackner, Peter; Ferreira, Fátima; Wallner, Michael

    2017-08-01

    Allergy vaccines should be easily applicable, safe, and efficacious. For Bet v 1-mediated birch pollen and associated food allergies, a single wild-type allergen does not provide a complete solution. We aimed to combine immunologically relevant epitopes of Bet v 1 and the 2 clinically most important related food allergens from apple and hazelnut to a single hybrid protein, termed MBC4. After identification of T cell epitope-containing parts on each of the 3 parental allergens, the hybrid molecule was designed to cover relevant epitopes and evaluated in silico. Thereby a mutation was introduced into the hybrid sequence, which should alter the secondary structure without compromising the immunogenic properties of the molecule. MBC4 and the parental allergens were purified to homogeneity. Analyses of secondary structure elements revealed substantial changes rendering the hybrid de facto nonreactive with patients' serum IgE. Nevertheless, the protein was monomeric in solution. MBC4 was able to activate T-cell lines from donors with birch pollen allergy and from mice immunized with the parental allergens. Moreover, on immunization of mice and rabbits, MBC4 induced cross-reactive IgG antibodies, which were able to block the binding of human serum IgE. Directed epitope rearrangements combined with a knowledge-based structural modification resulted in a protein unable to bind IgE from allergic patients. Still, properties to activate specific T cells or induce blocking antibodies were conserved. This suggests that MBC4 is a suitable vaccine candidate for the simultaneous treatment of Bet v 1 and associated food allergies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  7. Anti-botulism single-shot vaccine using chitosan for protein encapsulation by simple coacervation.

    Science.gov (United States)

    Sari, Roger S; de Almeida, Anna Christina; Cangussu, Alex S R; Jorge, Edson V; Mozzer, Otto D; Santos, Hércules Otacílio; Quintilio, Wagner; Brandi, Igor Viana; Andrade, Viviane Aguiar; Miguel, Angelo Samir M; Sobrinho Santos, Eliane M

    2016-12-01

    The aim of the present study was to compare the potency and safety of vaccines against Clostridium botulinum (C. botulinum) type C and D formulated with chitosan as controlled release matrix and vaccines formulated in conventional manner using aluminum hydroxide. Parameters were established for the development of chitosan microspheres, using simple coacervation to standardize the use of this polymer in protein encapsulation for vaccine formulation. To formulate a single shot vaccine inactivated antigens of C. botulinum type C and D were used with original toxin titles equal to 5.2 and 6.2 log LD50/ml, respectively. For each antigen a chitosan based solution of 50 mL was prepared. Control vaccines were formulated by mixing toxoid type C and D with aluminum hydroxide [25% Al(OH) 3 , pH 6.3]. The toxoid sterility, innocuity and potency of vaccines were evaluated as stipulated by MAPA-BRASIL according to ministerial directive no. 23. Encapsulation efficiency of BSA in chitosan was 32.5-40.37%, while that the encapsulation efficiency to toxoid type C was 41,03% (1.94 mg/mL) and of the toxoid type D was 32.30% (1.82 mg/mL). The single shot vaccine formulated using chitosan for protein encapsulation through simple coacervation showed potency and safety similar to conventional vaccine currently used in Brazilian livestock (10 and 2 IU/mL against C. botulinum type C and D, respectively). The present work suggests that our single shot vaccine would be a good option as a cattle vaccine against these C. botulinum type C and D. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Nucleotide excision repair at the single-molecule level : analysis of the E. coli UvrA protein

    NARCIS (Netherlands)

    Wagner, Koen

    2011-01-01

    In this thesis, the characteristics of the Escherichia coli UvrA protein were analyzed with microscopy techniques that allow detection of protein complexes at the single-molecule level. Together with UvrB and UvrC, UvrA catalyzes the excision of damaged DNA from the bacterial genome. This DNA repair

  9. Kinetic Ductility and Force-Spike Resistance of Proteins from Single-Molecule Force Spectroscopy.

    Science.gov (United States)

    Cossio, Pilar; Hummer, Gerhard; Szabo, Attila

    2016-08-23

    Ductile materials can absorb spikes in mechanical force, whereas brittle ones fail catastrophically. Here we develop a theory to quantify the kinetic ductility of single molecules from force spectroscopy experiments, relating force-spike resistance to the differential responses of the intact protein and the unfolding transition state to an applied mechanical force. We introduce a class of unistable one-dimensional potential surfaces that encompass previous models as special cases and continuously cover the entire range from ductile to brittle. Compact analytic expressions for force-dependent rates and rupture-force distributions allow us to analyze force-clamp and force-ramp pulling experiments. We find that the force-transmitting protein domains of filamin and titin are kinetically ductile when pulled from their two termini, making them resistant to force spikes. For the mechanostable muscle protein titin, a highly ductile model reconciles data over 10 orders of magnitude in force loading rate from experiment and simulation. Copyright © 2016 Biophysical Society. All rights reserved.

  10. High-throughput single-molecule force spectroscopy for membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios [M E Mueller Institute for Structural Biology, Biozentrum of the University of Basel, CH-4056 Basel (Switzerland); Ratera, Merce; Palacin, Manuel [Institute for Research in Biomedicine, Barcelona Science Park, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona and Centro de Investigacion Biomedica en Red de Enfermedades Raras, E-08028 Barcelona (Spain); Bippes, Christian A; Mueller, Daniel J [BioTechnology Center, Technical University, Tatzberg 47, D-01307 Dresden (Germany)], E-mail: andreas.engel@unibas.ch, E-mail: dimitrios.fotiadis@mci.unibe.ch

    2008-09-24

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether {approx}400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with {approx}200 (AdiC) and {approx}400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

  11. High-throughput single-molecule force spectroscopy for membrane proteins

    International Nuclear Information System (INIS)

    Bosshart, Patrick D; Casagrande, Fabio; Frederix, Patrick L T M; Engel, Andreas; Fotiadis, Dimitrios; Ratera, Merce; Palacin, Manuel; Bippes, Christian A; Mueller, Daniel J

    2008-01-01

    Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ∼400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ∼200 (AdiC) and ∼400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications

  12. Prolamin proteins alteration in durum wheat by species of the genus Eurygaster and Aelia (Insecta, Hemiptera)

    International Nuclear Information System (INIS)

    Salis, L.; Goula, M.; Valero, J.; Gordun, E.

    2010-01-01

    Wheat bugs are widely distributed in various areas of Europe, Asia and North Africa. Species belonging to the genus Eurygaster and Aelia pierce wheat kernels affecting protein quality. The effects of these insects feeding activity have been studied mainly in bread wheat (Triticum aestivum L.). This study provides information on the degradation of prolamin proteins (glutenins and gliadins) of bug-damaged durum wheat (Triticum turgidum L. var durum) in six cultivars grown in Sardinia (Italy). Samples of whole flour mixture of 70% sound wheat and 30% damaged wheat were hydrated and incubated at two temperatures (45 and 4 degree centigrade), for different periods of time (0, 1 and 3 h). Glutenin and gliadin content was analysed using free zone capillary electrophoresis. The presence of bug-damaged kernels had influence on the quality of durum wheat proteins. Glutenins were rapidly degraded independently to incubation temperature. Gliadin degradation, however, took place with dependence on temperature and incubation time. Therefore glutenin degradation was possibly not due solely to the activity of proteolytic enzymes but also to some other as yet unknown factor linked to wheat bugs feeding activity. (Author) 35 refs.

  13. Prolamin proteins alteration in durum wheat by species of the genus Eurygaster and Aelia (Insecta, Hemiptera)

    Energy Technology Data Exchange (ETDEWEB)

    Salis, L.; Goula, M.; Valero, J.; Gordun, E.

    2010-07-01

    Wheat bugs are widely distributed in various areas of Europe, Asia and North Africa. Species belonging to the genus Eurygaster and Aelia pierce wheat kernels affecting protein quality. The effects of these insects feeding activity have been studied mainly in bread wheat (Triticum aestivum L.). This study provides information on the degradation of prolamin proteins (glutenins and gliadins) of bug-damaged durum wheat (Triticum turgidum L. var durum) in six cultivars grown in Sardinia (Italy). Samples of whole flour mixture of 70% sound wheat and 30% damaged wheat were hydrated and incubated at two temperatures (45 and 4 degree centigrade), for different periods of time (0, 1 and 3 h). Glutenin and gliadin content was analysed using free zone capillary electrophoresis. The presence of bug-damaged kernels had influence on the quality of durum wheat proteins. Glutenins were rapidly degraded independently to incubation temperature. Gliadin degradation, however, took place with dependence on temperature and incubation time. Therefore glutenin degradation was possibly not due solely to the activity of proteolytic enzymes but also to some other as yet unknown factor linked to wheat bugs feeding activity. (Author) 35 refs.

  14. Monitoring single membrane protein dynamics in a liposome manipulated in solution by the ABELtrap

    Science.gov (United States)

    Rendler, T.; Renz, M.; Hammann, E.; Ernst, S.; Zarrabi, N.; Börsch, M.

    2011-02-01

    FoF1-ATP synthase is the essential membrane enzyme maintaining the cellular level of adenosine triphosphate (ATP) and comprises two rotary motors. We measure subunit rotation in FoF1-ATP synthase by intramolecular Foerster resonance energy transfer (FRET) between two fluorophores at the rotor and at the stator of the enzyme. Confocal FRET measurements of freely diffusing single enzymes in lipid vesicles are limited to hundreds of milliseconds by the transit times through the laser focus. We evaluate two different methods to trap the enzyme inside the confocal volume in order to extend the observation times. Monte Carlo simulations show that optical tweezers with low laser power are not suitable for lipid vesicles with a diameter of 130 nm. A. E. Cohen (Harvard) and W. E. Moerner (Stanford) have recently developed an Anti-Brownian electrokinetic trap (ABELtrap) which is capable to apparently immobilize single molecules, proteins, viruses or vesicles in solution. Trapping of fluorescent particles is achieved by applying a real time, position-dependent feedback to four electrodes in a microfluidic device. The standard deviation from a given target position in the ABELtrap is smaller than 200 nm. We develop a combination of the ABELtrap with confocal FRET measurements to monitor single membrane enzyme dynamics by FRET for more than 10 seconds in solution.

  15. Inactivation of Spores of Bacillus Species by Wet Heat: Studies on Single Spores Using Laser Tweezers Taman Spectroscopy

    Science.gov (United States)

    2013-02-01

    13/2011 22.00 Keren K. Griffiths, Jingqiao Zhang, Ann E. Cowan, Ji Yu, Peter Setlow. Germination proteins in the inner membrane of dormant Bacillus...that this technique can be used to rapidly identify single airborne particles or bacteria collected on a slide and to monitor germination dynamics of...the environment of dipicolinic acid in the core of superdormant spores is different from that in dormant spores [J. Bacteriol., 191, 5584 (2009

  16. Tannin, protein contents and fatty acid compositions of the seeds of several Vicia L. species from Turkey

    OpenAIRE

    Kökten, Kağan; Koçak, Alpaslan; Bağci, Eyüp; Akçura, Mevlüt; Çelik, Sait

    2010-01-01

    The seedoils of six Vicia species (Leguminosae) were investigated for their protein, tannin contents and fatty acid compositions. The protein contents of the seeds were found to be between 21.87%-31.33%. The tannin contents of the seeds were found to be between 0.13%-1.07%. The fatty acid compositions of these six different species were determined by the GC of the methyl esters of their fatty acids. The oilseeds of Vicia species contain palmitic and stear...

  17. Streptococcus pneumoniae Proteins AmiA, AliA, and AliB Bind Peptides Found in Ribosomal Proteins of Other Bacterial Species

    Directory of Open Access Journals (Sweden)

    Fauzy Nasher

    2018-01-01

    Full Text Available The nasopharynx is frequently colonized by both commensal and pathogenic bacteria including Streptococcus pneumoniae (pneumococcus. Pneumococcus is an important pathogen responsible for bacterial meningitis and community acquired pneumonia but is also commonly an asymptomatic colonizer of the nasopharynx. Understanding interactions between microbes may provide insights into pathogenesis. Here, we investigated the ability of the three oligopeptide-binding proteins AmiA, AliA, and AliB of an ATP-binding cassette transporter of pneumococcus to detect short peptides found in other bacterial species. We found three possible peptide ligands for AmiA and four each for AliA and AliB of which two for each protein matched ribosomal proteins of other bacterial species. Using synthetic peptides we confirmed the following binding: AmiA binds peptide AKTIKITQTR, matching 50S ribosomal subunit protein L30, AliA binds peptide FNEMQPIVDRQ, matching 30S ribosomal protein S20, and AliB binds peptide AIQSEKARKHN, matching 30S ribosomal protein S20, without excluding the possibility of binding of the other peptides. These Ami–AliA/AliB peptide ligands are found in multiple species in the class of Gammaproteobacteria which includes common colonizers of the nostrils and nasopharynx. Binding such peptides may enable pneumococcus to detect and respond to neighboring species in its environment and is a potential mechanism for interspecies communication and environmental surveillance.

  18. A Chitin-binding Protein Purified from Moringa oleifera Seeds Presents Anticandidal Activity by Increasing Cell Membrane Permeability and Reactive Oxygen Species Production

    Directory of Open Access Journals (Sweden)

    João X.S. Neto

    2017-06-01

    Full Text Available Candida species are opportunistic pathogens that infect immunocompromised and/or immunosuppressed patients, particularly in hospital facilities, that besides representing a significant threat to health increase the risk of mortality. Apart from echinocandins and triazoles, which are well tolerated, most of the antifungal drugs used for candidiasis treatment can cause side effects and lead to the development of resistant strains. A promising alternative to the conventional treatments is the use of plant proteins. M. oleifera Lam. is a plant with valuable medicinal properties, including antimicrobial activity. This work aimed to purify a chitin-binding protein from M. oleifera seeds and to evaluate its antifungal properties against Candida species. The purified protein, named Mo-CBP2, represented about 0.2% of the total seed protein and appeared as a single band on native PAGE. By mass spectrometry, Mo-CBP2 presented 13,309 Da. However, by SDS-PAGE, Mo-CBP2 migrated as a single band with an apparent molecular mass of 23,400 Da. Tricine-SDS-PAGE of Mo-CBP2 under reduced conditions revealed two protein bands with apparent molecular masses of 7,900 and 4,600 Da. Altogether, these results suggest that Mo-CBP2 exists in different oligomeric forms. Moreover, Mo-CBP2 is a basic glycoprotein (pI 10.9 with 4.1% (m/m sugar and it did not display hemagglutinating and hemolytic activities upon rabbit and human erythrocytes. A comparative analysis of the sequence of triptic peptides from Mo-CBP2 in solution, after LC-ESI-MS/MS, revealed similarity with other M. oleifera proteins, as the 2S albumin Mo-CBP3 and flocculating proteins, and 2S albumins from different species. Mo-CBP2 possesses in vitro antifungal activity against Candida albicans, C. parapsilosis, C. krusei, and C. tropicalis, with MIC50 and MIC90 values ranging between 9.45–37.90 and 155.84–260.29 μM, respectively. In addition, Mo-CBP2 (18.90 μM increased the cell membrane permeabilization

  19. Effects of the daily consumption of protein enriched bread and protein enriched drinking yoghurt on the total protein intake in older adults in a rehabilitation centre: A single blind randomised controlled trial

    NARCIS (Netherlands)

    van Til, A.J.; Naumann, E.; Cox-Claessens, I.J.H.M.; Kremer, S.; Boelsma, E.; van Bokhorst-de van der Schueren, M.A.E.

    2015-01-01

    Objectives: To investigate the effects of protein enriched bread and drinking yoghurt, substituting regular products, on the total protein intake and the distribution of protein intake over the day in older adults.Design: A single blind randomised controlled trial.Setting: Rehabilitation

  20. Effect of the daily consumption of protein enriched bread and protein enriched drinking yoghurt on the total protein intake in older adults in a rehabilitation centre: a single blind randomised controlled trial

    NARCIS (Netherlands)

    Til, van A.J.; Naumann, E.; Cox-Claessens, I.J.H.M.; Kremer, S.; Boelsma, E.; Schueren, van der D.E.

    2015-01-01

    Objectives To investigate the effects of protein enriched bread and drinking yoghurt, substituting regular products, on the total protein intake and the distribution of protein intake over the day in older adults. Design A single blind randomised controlled trial. Setting Rehabilitation centre.

  1. Comparative toxicity of a brominated flame retardant (tetrabromobisphenol A) on microalgae with single and multi-species bioassays.

    Science.gov (United States)

    Debenest, Timothée; Petit, Anne-Nöelle; Gagné, François; Kohli, Mohan; Nguyen, Nien; Blaise, Christian

    2011-09-01

    The potential threat of emerging chemicals to the aquatic flora is a major issue. The purpose of the study was to develop a multispecies microalgae test in order to determine the impact of species interactions on the cytoxicity of an emergent toxic contaminant: the tetrabromobisphenol A (TBBPA). Single and multi-species tests were thus performed to study the effects of this flame retardant on two microalgae (Pseudokirchneriella subcapitata and Nitzschia palea) commonly observed in freshwater. A synthetic medium was designed to allow the growth of both species. The algae were exposed to 1.8, 4.8, 9.2, 12.9 and 16.5 μM of TBBPA for 72 h. After staining with fluorescein diacetate (FDA), viable cells of each alga species were analyzed by flow cytometry based on chlorophyll autofluorescence and intracellular esterase activity. Density and abundance of viable cells were assessed to follow the population growth and the cell viability. In TBBPA treated samples, the growth of the two microalgae was significantly inhibited at the three highest concentrations (9.2, 12.9 and 16.5 μM) in the two tests. At the end of the experiment (t=72 h), the cell viability was also significantly smaller at these concentrations. The decreases of growth rate and viable cell abundance in TBBPA treated populations of N. palea were significantly higher in multi-species test in comparison with the single-species test. No significant differences were noticed between the two tests for P. subcapitata populations exposed to TBBPA. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  2. NMR structure of the single QALGGH zinc finger domain from the Arabidopsis thaliana SUPERMAN protein.

    Science.gov (United States)

    Isernia, Carla; Bucci, Enrico; Leone, Marilisa; Zaccaro, Laura; Di Lello, Paola; Digilio, Giuseppe; Esposito, Sabrina; Saviano, Michele; Di Blasio, Benedetto; Pedone, Carlo; Pedone, Paolo V; Fattorusso, Roberto

    2003-03-03

    Zinc finger domains of the classical type represent the most abundant DNA binding domains in eukaryotic transcription factors. Plant proteins contain from one to four zinc finger domains, which are characterized by high conservation of the sequence QALGGH, shown to be critical for DNA-binding activity. The Arabidopsis thaliana SUPERMAN protein, which contains a single QALGGH zinc finger, is necessary for proper spatial development of reproductive floral tissues and has been shown to specifically bind to DNA. Here, we report the synthesis and UV and NMR spectroscopic structural characterization of a 37 amino acid SUPERMAN region complexed to a Zn(2+) ion (Zn-SUP37) and present the first high-resolution structure of a classical zinc finger domain from a plant protein. The NMR structure of the SUPERMAN zinc finger domain consists of a very well-defined betabetaalpha motif, typical of all other Cys(2)-His(2) zinc fingers structurally characterized. As a consequence, the highly conserved QALGGH sequence is located at the N terminus of the alpha helix. This region of the domain of animal zinc finger proteins consists of hypervariable residues that are responsible for recognizing the DNA bases. Therefore, we propose a peculiar DNA recognition code for the QALGGH zinc finger domain that includes all or some of the amino acid residues at positions -1, 2, and 3 (numbered relative to the N terminus of the helix) and possibly others at the C-terminal end of the recognition helix. This study further confirms that the zinc finger domain, though very simple, is an extremely versatile DNA binding motif.

  3. Genus delineation of Chlamydiales by analysis of the percentage of conserved proteins justifies the reunifying of the genera Chlamydia and Chlamydophila into one single genus Chlamydia.

    Science.gov (United States)

    Pannekoek, Yvonne; Qi-Long, Qin; Zhang, Yu-Zhong; van der Ende, Arie

    2016-08-01

    Many studies have aimed to set up boundaries for the classification and definition of prokaryotic genus and species classification; however, studies that focused on genus-level genomic differences for existing taxonomy systems are limited. Recently, a novel method was described for prokaryotic genus delineation using the percentage of conserved proteins (POCP) between two strains to estimate their evolutionary and phenotypic distance (Qin et al. A proposed genus boundary for the prokaryotes based on genomic insights. J Bacteriol 2014; 196: :2210-5). Here, we extended the POCP analysis of the order Chlamydiales and pairwise compared all currently recognized species and candidate species of the family Chlamydiaceae as well as some species from other families. Using the taxonomy advised by the International Committee on Systematics of Prokaryotes, subcommittee on the taxonomy of the Chlamydiae, POCP analysis revealed that all pairwise comparisons of species from different families resulted in values lower than 50%, the proposed threshold for genus boundary. In contrast, all interspecies pairwise comparisons of species from the single genus within the family Chlamydiaceae resulted in POCP values higher than 70%. We conclude that the recommended genus classification of the family Chlamydiaceae is rational and that POCP analyses can provide a robust genomic index for the taxonomy of members of the order Chlamydiales in terms of genus demarcation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Fitting hidden Markov models of protein domains to a target species: application to Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Terrapon Nicolas

    2012-05-01

    Full Text Available Abstract Background Hidden Markov Models (HMMs are a powerful tool for protein domain identification. The Pfam database notably provides a large collection of HMMs which are widely used for the annotation of proteins in new sequenced organisms. In Pfam, each domain family is represented by a curated multiple sequence alignment from which a profile HMM is built. In spite of their high specificity, HMMs may lack sensitivity when searching for domains in divergent organisms. This is particularly the case for species with a biased amino-acid composition, such as P. falciparum, the main causal agent of human malaria. In this context, fitting HMMs to the specificities of the target proteome can help identify additional domains. Results Using P. falciparum as an example, we compare approaches that have been proposed for this problem, and present two alternative methods. Because previous attempts strongly rely on known domain occurrences in the target species or its close relatives, they mainly improve the detection of domains which belong to already identified families. Our methods learn global correction rules that adjust amino-acid distributions associated with the match states of HMMs. These rules are applied to all match states of the whole HMM library, thus enabling the detection of domains from previously absent families. Additionally, we propose a procedure to estimate the proportion of false positives among the newly discovered domains. Starting with the Pfam standard library, we build several new libraries with the different HMM-fitting approaches. These libraries are first used to detect new domain occurrences with low E-values. Second, by applying the Co-Occurrence Domain Discovery (CODD procedure we have recently proposed, the libraries are further used to identify likely occurrences among potential domains with higher E-values. Conclusion We show that the new approaches allow identification of several domain families previously absent in

  5. Deorphanization and target validation of cross-tick species conserved novel Amblyomma americanum tick saliva protein.

    Science.gov (United States)

    Mulenga, Albert; Kim, Tae Kwon; Ibelli, Adriana Mércia Guaratini

    2013-05-01

    We previously identified a cross-tick species conserved tick feeding stimuli responsive Amblyomma americanum (Aam) AV422 gene. This study demonstrates that AamAV422 belongs to a novel group of arthropod proteins that is characterized by 14 cysteine amino acid residues: C(23)-X7/9-C(33)-X23/24-C(58)-X8-C(67)-X7-C(75)-X23-C(99)-X15-C(115)-X10-C(126)-X24/25/33-C(150)C(151)-X7-C(159)-X8-C(168)-X23/24-C(192)-X9/10-C(202) predicted to form seven disulfide bonds. We show that AamAV422 protein is a ubiquitously expressed protein that is injected into the host within the first 24h of the tick attaching onto the host as revealed by Western blotting analyses of recombinant (r)AamAV422, tick saliva and dissected tick organ protein extracts using antibodies to 24 and 48 h tick saliva proteins. Native AamAV422 is apparently involved with mediating tick anti-hemostasis and anti-complement functions in that rAamAV422 delayed plasma clotting time in a dose responsive manner by up to ≈ 160 s, prevented platelet aggregation by up to ≈ 16% and caused ≈ 24% reduction in production of terminal complement complexes. Target validation analysis revealed that rAamAV422 is a potential candidate for a cocktail or multivalent tick vaccine preparation in that RNA interference (RNAi)-mediated silencing of AamAV422 mRNA caused a statistically significant (≈ 44%) reduction in tick engorgement weights, which is proxy for amounts of ingested blood. We speculate that AamAV422 is a potential target antigen for development of the highly desired universal tick vaccine in that consistent with high conservation among ticks, antibodies to 24h Ixodes scapularis tick saliva proteins specifically bound rAamAV422. We discuss data in this study in the context of advancing the biology of tick feeding physiology and discovery of potential target antigens for tick vaccine development. Copyright © 2013 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

  6. Eimeria Species and Genetic Background Influence the Serum Protein Profile of Broilers with Coccidiosis

    Science.gov (United States)

    Gilbert, Elizabeth R.; Cox, Chasity M.; Williams, Patricia M.; McElroy, Audrey P.; Dalloul, Rami A.; Ray, W. Keith; Barri, Adriana; Emmerson, Derek A.; Wong, Eric A.; Webb, Kenneth E.

    2011-01-01

    Background Coccidiosis is an intestinal disease caused by protozoal parasites of the genus Eimeria. Despite the advent of anti-coccidial drugs and vaccines, the disease continues to result in substantial annual economic losses to the poultry industry. There is still much unknown about the host response to infection and to date there are no reports of protein profiles in the blood of Eimeria-infected animals. The objective of this study was to evaluate the serum proteome of two genetic lines of broiler chickens after infection with one of three species of Eimeria. Methodology/Principal Findings Birds from lines A and B were either not infected or inoculated with sporulated oocysts from one of the three Eimeria strains at 15 d post-hatch. At 21 d (6 d post-infection), whole blood was collected and lesion scoring was performed. Serum was harvested and used for 2-dimensional gel electrophoresis. A total of 1,266 spots were quantitatively assessed by densitometry. Protein spots showing a significant effect of coccidia strain and/or broiler genetic line on density at P<0.05−0.01 (250 spots), P<0.01−0.001 (248 spots), and P<0.001 (314 spots) were excised and analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Proteins were identified in 172 spots. A total of 46 different proteins were identified. Of the spots with a corresponding protein identification, 57 showed a main effect of coccidia infection and/or 2-way interaction of coccidia infection×broiler genetic line at P<0.001. Conclusions/Significance Several of the metabolic enzymes identified in this study are potential candidates for early diagnostic markers of E. acervulina infection including malate dehydrogenase 2, NADH dehydrogenase 1 alpha subcomplex 9, and an ATP synthase. These proteins were detected only in Line A birds that were inoculated with E. acervulina. Results from this study provide a basic framework for future research aimed at uncovering the complex

  7. Design of custom oligonucleotide microarrays for single species or interspecies hybrids using Array Oligo Selector.

    Science.gov (United States)

    Caudy, Amy A

    2011-01-01

    New technologies for DNA sequencing have made it feasible to determine the genome sequence of any organism of interest. This sequence is the resource required to create tools for downstream studies, including DNA microarrays. A number of vendors can produce DNA microarrays containing customer-specified sequences, allowing investigators to design and order arrays customized for any species of interest. Freely available, user-friendly computer programs are available for designing microarray probes. These design programs can be used to create probes that distinguish between two related genomes, allowing investigation of gene expression or gene representation in intra- or interspecies hybrids or in samples containing DNA from multiple species.

  8. Evaluation of canine adverse food reactions by patch testing with single proteins, single carbohydrates and commercial foods.

    Science.gov (United States)

    Johansen, Cornelia; Mariani, Claire; Mueller, Ralf S

    2017-10-01

    Adverse food reaction (AFR) is an important differential diagnosis for the pruritic dog. It is usually diagnosed by feeding an elimination diet with a novel protein and carbohydrate source for eight weeks followed by subsequent food provocation. A previous study demonstrated that patch testing dogs with foods had a high sensitivity and negative predictability for selection of elimination diet ingredients. The aim of this study was to investigate patch testing with proteins, carbohydrates and dry commercial dog food in dogs to determine whether there was value in patch testing to aid the diagnosis of canine adverse food reaction. Twenty five privately owned dogs, with confirmed AFR, underwent provocation trials with selected food antigens and patch testing. For proteins, carbohydrates and dry dog food the sensitivity of patch testing was 100%, 70% and 22.2%, respectively; the negative predictive values of patch testing were 100%, 79% and 72%. The positive predictive values of patch testing for proteins and carbohydrates were 75% and 74%, respectively. This study confirmed that patch testing may be useful for the selection of a suitable protein source for an elimination diet in dogs with suspected AFR, but not as a diagnostic tool for canine AFR. Results for proteins are more reliable than for carbohydrates and the majority of positive patch test reactions were observed with raw protein. Patch testing with commercial dog food does not seem to be useful. © 2017 ESVD and ACVD.

  9. An in vitro tag-and-modify protein sample generation method for single-molecule fluorescence resonance energy transfer.

    Science.gov (United States)

    Hamadani, Kambiz M; Howe, Jesse; Jensen, Madeleine K; Wu, Peng; Cate, Jamie H D; Marqusee, Susan

    2017-09-22

    Biomolecular systems exhibit many dynamic and biologically relevant properties, such as conformational fluctuations, multistep catalysis, transient interactions, folding, and allosteric structural transitions. These properties are challenging to detect and engineer using standard ensemble-based techniques. To address this drawback, single-molecule methods offer a way to access conformational distributions, transient states, and asynchronous dynamics inaccessible to these standard techniques. Fluorescence-based single-molecule approaches are parallelizable and compatible with multiplexed detection; to date, however, they have remained limited to serial screens of small protein libraries. This stems from the current absence of methods for generating either individual dual-labeled protein samples at high throughputs or protein libraries compatible with multiplexed screening platforms. Here, we demonstrate that by combining purified and reconstituted in vitro translation, quantitative unnatural amino acid incorporation via AUG codon reassignment, and copper-catalyzed azide-alkyne cycloaddition, we can overcome these challenges for target proteins that are, or can be, methionine-depleted. We present an in vitro parallelizable approach that does not require laborious target-specific purification to generate dual-labeled proteins and ribosome-nascent chain libraries suitable for single-molecule FRET-based conformational phenotyping. We demonstrate the power of this approach by tracking the effects of mutations, C-terminal extensions, and ribosomal tethering on the structure and stability of three protein model systems: barnase, spectrin, and T4 lysozyme. Importantly, dual-labeled ribosome-nascent chain libraries enable single-molecule co-localization of genotypes with phenotypes, are well suited for multiplexed single-molecule screening of protein libraries, and should enable the in vitro directed evolution of proteins with designer single-molecule conformational

  10. New insights into metal interactions with the prion protein: EXAFS analysis and structure calculations of copper binding to a single octarepeat from the prion protein.

    Science.gov (United States)

    McDonald, Alex; Pushie, M Jake; Millhauser, Glenn L; George, Graham N

    2013-11-07

    Copper coordination to the prion protein (PrP) has garnered considerable interest for almost 20 years, due in part to the possibility that this interaction may be part of the normal function of PrP. The most characterized form of copper binding to PrP has been Cu(2+) interaction with the conserved tandem repeats in the N-terminal domain of PrP, termed the octarepeats, with many studies focusing on single and multiple repeats of PHGGGWGQ. Extended X-ray absorption fine structure (EXAFS) spectroscopy has been used in several previous instances to characterize the solution structure of Cu(2+) binding into the peptide backbone in the HGGG portion of the octarepeats. All previous EXAFS studies, however, have benefitted from crystallographic structure information for [Cu(II) (Ac-HGGGW-NH2)(-2H)] but have not conclusively demonstrated that the complex EXAFS spectrum represents the same coordination environment for Cu(2+) bound to the peptide backbone. Density functional structure calculations as well as full multiple scattering EXAFS curve fitting analysis are brought to bear on the predominant coordination mode for Cu(2+) with the Ac-PHGGGWGQ-NH2 peptide at physiological pH, under high Cu(2+) occupancy conditions. In addition to the structure calculations, which provide a thermodynamic link to structural information, methods are also presented for extensive deconvolution of the EXAFS spectrum. We demonstrate how the EXAFS data can be analyzed to extract the maximum structural information and arrive at a structural model that is significantly improved over previous EXAFS characterizations. The EXAFS spectrum for the chemically reduced form of copper binding to the Ac-PHGGGWGQ-NH2 peptide is presented, which is best modeled as a linear two-coordinate species with a single His imidazole ligand and a water molecule. The extent of in situ photoreduction of the copper center during standard data collection is also presented, and EXAFS curve fitting of the photoreduced species

  11. A single dominant Ganoderma species is responsible for root rot of ...

    African Journals Online (AJOL)

    Ganoderma root rot is the most serious disease affecting commercially planted Acacia mangium in plantations in Indonesia. Numerous Ganoderma spp. have been recorded from diseased trees of this species and to a lesser extent Eucalyptus, causing confusion regarding the primary cause of the disease. In this study, a ...

  12. Beyond the single species climate envelope: A multifaceted approach to mapping climate change vulnerability

    Science.gov (United States)

    Christopher S. Balzotti; Stanley G. Kitchen; Clinton McCarthy

    2016-01-01

    Federal land management agencies and conservation organizations have begun incorporating climate change vulnerability assessments (CCVAs) as an important component in the management and conservation of landscapes. It is often a challenge to translate that knowledge into management plans and actions, even when research infers species risk. Predictive maps can...

  13. Affinity chromatography revealed insights into unique functionality of two 14-3-3 protein species in developing maize kernels.

    Science.gov (United States)

    Dou, Yao; Liu, Xiangguo; Yin, Yuejia; Han, Siping; Lu, Yang; Liu, Yang; Hao, Dongyun

    2015-01-30

    The 14-3-3 proteins are a group of regulatory proteins of divergent functions in plants. However, little is known about their roles in maize kernel development. Using publically available gene expression profiling data, we found that two 14-3-3 species genes, zmgf14-4 and zmgf14-6, exhibited prominent expression profiles over other 14-3-3 protein genes during maize kernel development. More than 5000 transcripts of these two genes were identified accounting for about 1/10 of the total transcripts of genes correlating to maize kernel development. We constructed a proteomics pipeline based on the affinity chromatography, in combination with 2-DE and LC-MS/MS technologies to identify the specific client proteins of the two proteins for their functional characterization. Consequently, we identified 77 specific client proteins from the developing kernels of the inbred maize B73. More than 60% of the client proteins were commonly affinity-identified by the two 14-3-3 species and are predicted to be implicated in the fundamental functions of metabolism, protein destination and storage. In addition, we found ZmGF14-4 specifically bound to the disease- or defense-relating proteins, whilst ZmGF14-6 tended to interact with the proteins involving metabolism and cell structure. Our findings provide primary insights into the functional roles of 14-3-3 proteins in maize kernel development. Maize kernel development is a complicated physiological process for its importance in both genetics and cereal breeding. 14-3-3 proteins form a multi-gene family participating in regulations of developmental processes in plants. However, the correlation between this protein family and maize kernel development has hardly been studied. We have for the first time found 12 14-3-3 protein genes from maize genome and studied in silico the gene transcription profiling of these genes. Comparative studies revealed that maize kernel development aroused a great number of gene expression, among which 14

  14. Comparative study to develop a single method for retrieving wide class of recombinant proteins from classical inclusion bodies.

    Science.gov (United States)

    Padhiar, Arshad Ahmed; Chanda, Warren; Joseph, Thomson Patrick; Guo, Xuefang; Liu, Min; Sha, Li; Batool, Samana; Gao, Yifan; Zhang, Wei; Huang, Min; Zhong, Mintao

    2018-03-01

    The formation of inclusion bodies (IBs) is considered as an Achilles heel of heterologous protein expression in bacterial hosts. Wide array of techniques has been developed to recover biochemically challenging proteins from IBs. However, acquiring the active state even from the same protein family was found to be an independent of single established method. Here, we present a new strategy for the recovery of wide sub-classes of recombinant protein from harsh IBs. We found that numerous methods and their combinations for reducing IB formation and producing soluble proteins were not effective, if the inclusion bodies were harsh in nature. On the other hand, different practices with mild solubilization buffers were able to solubilize IBs completely, yet the recovery of active protein requires large screening of refolding buffers. With the integration of previously reported mild solubilization techniques, we proposed an improved method, which comprised low sarkosyl concentration, ranging from 0.05 to 0.1% coupled with slow freezing (- 1 °C/min) and fast thaw (room temperature), resulting in greater solubility and the integrity of solubilized protein. Dilution method was employed with single buffer to restore activity for every sub-class of recombinant protein. Results showed that the recovered protein's activity was significantly higher compared with traditional solubilization/refolding approach. Solubilization of IBs by the described method was proved milder in nature, which restored native-like conformation of proteins within IBs.

  15. Network single-walled carbon nanotube biosensors for fast and highly sensitive detection of proteins.

    Science.gov (United States)

    Hu, Pingán; Zhang, Jia; Wen, Zhenzhong; Zhang, Can

    2011-08-19

    Detection of proteins is powerfully assayed in the diagnosis of diseases. A strategy for the development of an ultrahigh sensitivity biosensor based on a network single-walled carbon nanotube (SWNT) field-effect transistor (FET) has been demonstrated. Metallic SWNTs (m-SWNTs) in the network nanotube FET were selectively removed or cut via a carefully controlled procedure of electrical break-down (BD), and left non-conducting m-SWNTs which magnified the Schottky barrier (SB) area. This nanotube FET exhibited ultrahigh sensitivity and fast response to biomolecules. The lowest detection limit of 0.5 pM was achieved by exploiting streptavidin (SA) or a biotin/SA pair as the research model, and BD-treated nanotube biosensors had a 2 × 10(4)-fold lower minimum detectable concentration than the device without BD treatment. The response time is in the range of 0.3-3 min.

  16. Co-cultivation of Green Microalgae and Methanotrophic Bacteria for Single Cell Protein Production from Wastewater

    DEFF Research Database (Denmark)

    Rasouli, Zahra; Valverde Pérez, Borja; D'Este, Martina

    2017-01-01

    microalgae – as a means to recover nutrients from industrial wastewater and upcycle them to feed grade single cell protein. Results demonstrated that both algae and bacteria could remove or assimilate most of the organic carbon present in the wastewater. However, their growth stopped before nutrients...... and substrates in the gas phase (i.e., methane and oxygen for methanotrophs and carbon dioxide for algae) were depleted. Likely, algal growth was light limited and stopped after organic carbon was consumed, whilst growth of methanotrophic bacteria could be limited by trace elements (e.g., copper). Nevertheless......Conventional water treatment technologies remove nutrients via resource intensive processes. However, new approaches for residual nutrient recycling are needed to provide food to the increasing world population. This work explores the use of microbial biomass – methane oxidizing bacteria and green...

  17. Single-tree water use and water-use efficiencies of selected indigenous and introduced species in the Southern Cape region of South Africa

    CSIR Research Space (South Africa)

    Mapeto, P

    2017-03-01

    Full Text Available indigenous tree species can provide an additional low water-use form of forestry. Single-tree water use and water-use efficiencies of three indigenous species (Ilex mitis, Ocotea bullata and Podocarpus latifolius) and one introduced species (Pinus radiata...

  18. The Number, Organization, and Size of Polymorphic Membrane Protein Coding Sequences as well as the Most Conserved Pmp Protein Differ within and across Chlamydia Species.

    Science.gov (United States)

    Van Lent, Sarah; Creasy, Heather Huot; Myers, Garry S A; Vanrompay, Daisy

    2016-01-01

    Variation is a central trait of the polymorphic membrane protein (Pmp) family. The number of pmp coding sequences differs between Chlamydia species, but it is unknown whether the number of pmp coding sequences is constant within a Chlamydia species. The level of conservation of the Pmp proteins has previously only been determined for Chlamydia trachomatis. As different Pmp proteins might be indispensible for the pathogenesis of different Chlamydia species, this study investigated the conservation of Pmp proteins both within and across C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci. The pmp coding sequences were annotated in 16 C. trachomatis, 6 C. pneumoniae, 2 C. abortus, and 16 C. psittaci genomes. The number and organization of polymorphic membrane coding sequences differed within and across the analyzed Chlamydia species. The length of coding sequences of pmpA,pmpB, and pmpH was conserved among all analyzed genomes, while the length of pmpE/F and pmpG, and remarkably also of the subtype pmpD, differed among the analyzed genomes. PmpD, PmpA, PmpH, and PmpA were the most conserved Pmp in C. trachomatis,C. pneumoniae,C. abortus, and C. psittaci, respectively. PmpB was the most conserved Pmp across the 4 analyzed Chlamydia species. © 2016 S. Karger AG, Basel.

  19. Interaction of amidated single-walled carbon nanotubes with protein by multiple spectroscopic methods

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lili [China Pharmaceutical University, Nanjing 210009 (China); The Nursing College of Pingdingshan University, Pingdingshan 467000 (China); Lin, Rui [Yancheng Health Vocational and Technical College, Yancheng 224005 (China); He, Hua, E-mail: dochehua@163.com [China Pharmaceutical University, Nanjing 210009 (China); Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, Nanjing 210009 (China); Sun, Meiling, E-mail: sml-nir@sohu.com [China Pharmaceutical University, Nanjing 210009 (China); Jiang, Li; Gao, Mengmeng [China Pharmaceutical University, Nanjing 210009 (China)

    2014-01-15

    The aim of this work was to investigate the detailed interaction between BSA and amidated single walled carbon nanotubes (e-SWNTs) in vitro. Ethylenediamine (EDA) was successfully linked on the surface of single-walled carbon nanotubes (SWNTs) via acylation to improve their dispersion and to introduce active groups. Bovine serum albumin (BSA) was selected as the template protein to inspect the interaction of e-SWNTs with protein. Decreases in fluorescence intensity of BSA induced by e-SWNTs demonstrated the occurrence of interaction between BSA and e-SWNTs. Quenching parameters and different absorption spectra for e-SWNTs–BSA show that the quenching effect of e-SWNTs was static quenching. Hydrophobic force had a leading contribution to the binding roles of BSA on e-SWNTs, which was confirmed by positive enthalpy change and entropy change. The interference of Na{sup +} with the quenching effect of e-SWNTs authenticated that electrostatic force existed in the interactive process simultaneously. The hydrophobicity of amino acid residues markedly increased with the addition of e-SWNTs viewed from UV spectra of BSA. The content of α-helix structure in BSA decreased by 6.8% due to the addition of e-SWNTs, indicating that e-SWNTs have an effect on the secondary conformation of BSA. -- Highlights: • The interaction between e-SWNTs and BSA was investigated by multiple spectroscopic methods. • Quenching mechanism was static quenching. • Changes in structure of BSA were inspected by synchronous fluorescence, UV–vis and CD spectrum.

  20. The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

    Science.gov (United States)

    Van Damme, E J; Goossens, K; Smeets, K; Van Leuven, F; Verhaert, P; Peumans, W J

    1995-01-01

    A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin. PMID:7770523

  1. The major tuber storage protein of araceae species is a lectin. Characterization and molecular cloning of the lectin from Arum maculatum L.

    Science.gov (United States)

    Van Damme, E J; Goossens, K; Smeets, K; Van Leuven, F; Verhaert, P; Peumans, W J

    1995-04-01

    A new lectin was purified from tubers of Arum maculatum L. by affinity chromatography on immobilized asialofetuin. Although this lectin is also retained on mannose-Sepharose 4B, under the appropriate conditions free mannose is a poor inhibitor of its agglutination activity. Pure preparations of the Arum lectin apparently yielded a single polypeptide band of approximately 12 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, N-terminal sequencing of the purified protein combined with molecular cloning of the lectin have shown that the lectin is composed of two different 12-kD lectin subunits that are synthesized on a single large precursor translated from an mRNA of approximately 1400 nucleotides. Lectins with similar properties were also isolated from the Araceae species Colocasia esculenta (L.) Schott, Xanthosoma sagittifolium (L.) Schott, and Dieffenbachia sequina Schott. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration of the different Araceae lectins have shown that they are tetrameric proteins composed of lectin subunits of 12 to 14 kD. Interestingly, these lectins are the most prominent proteins in the tuber tissue. Evidence is presented that a previously described major storage protein of Colocasia tubers corresponds to the lectin.

  2. Diversity Within the O-linked Protein Glycosylation Systems of Acinetobacter Species

    DEFF Research Database (Denmark)

    Scott, N. E.; Kinsella, R. L.; Edwards, A. V. G.

    2014-01-01

    The opportunistic human pathogen Acinetobacter baumannii is a concern to health care systems worldwide because of its persistence in clinical settings and the growing frequency of multiple drug resistant infections. To combat this threat, it is necessary to understand factors associated...... with disease and environmental persistence of A. baumannii. Recently, it was shown that a single biosynthetic pathway was responsible for the generation of capsule polysaccharide and O-linked protein glycosylation. Because of the requirement of these carbohydrates for virulence and the non-template driven...... nature of glycan biogenesis we investigated the composition, diversity, and properties of the Acinetobacter glycoproteome. Utilizing global and targeted mass spectrometry methods, we examined 15 strains and found extensive glycan diversity in the O-linked glycoproteome of Acinetobacter. Comparison...

  3. The Escherichia coli BtuE protein functions as a resistance determinant against reactive oxygen species.

    Directory of Open Access Journals (Sweden)

    Felipe A Arenas

    2011-01-01

    Full Text Available This work shows that the recently described Escherichia coli BtuE peroxidase protects the bacterium against oxidative stress that is generated by tellurite and by other reactive oxygen species elicitors (ROS. Cells lacking btuE (ΔbtuE displayed higher sensitivity to K(2TeO(3 and other oxidative stress-generating agents than did the isogenic, parental, wild-type strain. They also exhibited increased levels of cytoplasmic reactive oxygen species, oxidized proteins, thiobarbituric acid reactive substances, and lipoperoxides. E. coli ΔbtuE that was exposed to tellurite or H(2O(2 did not show growth changes relative to wild type cells either in aerobic or anaerobic conditions. Nevertheless, the elimination of btuE from cells deficient in catalases/peroxidases (Hpx(- resulted in impaired growth and resistance to these toxicants only in aerobic conditions, suggesting that BtuE is involved in the defense against oxidative damage. Genetic complementation of E. coli ΔbtuE restored toxicant resistance to levels exhibited by the wild type strain. As expected, btuE overexpression resulted in decreased amounts of oxidative damage products as well as in lower transcriptional levels of the oxidative stress-induced genes ibpA, soxS and katG.

  4. Methods of detection, species identification and quantification of processed animal proteins in feedingstuffs

    Directory of Open Access Journals (Sweden)

    Fumière O.

    2009-01-01

    Full Text Available The ban of processed animal proteins (PAPs in feed for farmed animals led to a significant reduction of the number of bovine spongiform encephalopathy cases. Presently, optical microscopy remains the only reference method for the detection of PAPs to be applied for official control as required by Commission Directive 2003/126/EC. The legislation also foresees that other methods may be applied in addition to classical microscopy, if – for instance – they provide more information about the origin of the animal constituents. Therefore, alternative and complementary techniques were developed as such or in combination. The most promising ones seem to be PCR (Polymerase Chain Reaction, near infrared microscopy and imaging, as well as immunology. Within the framework of a PAP ban regardless of its species origin (total feed ban, most of the studies were mainly focused on the ability of the techniques to detect the presence of PAPs at 0.1% (mass percentage of constituents of animal origin in feed as indicated as limit of detection in the official method protocol. A possible modification of the legislation requires that the techniques are also able to determine their species origin and to quantify them. The present paper gives a state of the art of the different methods.

  5. Plasmonic welded single walled carbon nanotubes on monolayer graphene for sensing target protein

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jangheon; Kim, Soohyun [Department of Mechanical Engineering, Korea Advanced Institute of Science and Technology, 373-1 Guseong, Yuseong, Daejeon 305-806 (Korea, Republic of); Kim, Gi Gyu; Jung, Wonsuk, E-mail: wonsuk81@wku.ac.kr [Department of Mechanical and Automotive Engineering, Wonkwang University, Iksan, Jeonbuk 570-749 (Korea, Republic of)

    2016-05-16

    We developed plasmonic welded single walled carbon nanotubes (SWCNTs) on monolayer graphene as a biosensor to detect target antigen molecules, fc fusion protein without any treatment to generate binder groups for linker and antibody. This plasmonic welding induces atomic networks between SWCNTs as junctions containing carboxylic groups and improves the electrical sensitivity of a SWCNTs and the graphene membrane to detect target protein. We investigated generation of the atomic networks between SWCNTs by field-emission scanning electron microscopy and atomic force microscopy after plasmonic welding process. We compared the intensity ratios of D to G peaks from the Raman spectra and electrical sheet resistance of welded SWCNTs with the results of normal SWCNTs, which decreased from 0.115 to 0.086 and from 10.5 to 4.12, respectively. Additionally, we measured the drain current via source/drain voltage after binding of the antigen to the antibody molecules. This electrical sensitivity of the welded SWCNTs was 1.55 times larger than normal SWCNTs.

  6. Growth kinetics of protein single crystals in the gel acupuncture technique

    Science.gov (United States)

    García-Ruiz, Juan Manuel; Moreno, Abel

    1997-07-01

    The growth of single crystals of tetragonal HEW lysozyme and thaumatin I into glass capillaries was monitored by time lapse video-microscopy. The crystals were obtained by unidirectional transport of the precipitating agent through capillaries of internal diameter ranging from 0.2 to 1.2 mm, using the gel acupuncture technique. For crystals growing from true protein solutions, the measured average growth rates varies with capillary diameter from 1.7 to 3.7 Å/s for thaumatin and from 2.8 to 22 Å/s for lysozyme. The measured average growth rates for crystals growing into gelled protein solutions were 1.8 Å/s for thaumatin and 2.5 Å/s for lysozyme. The trend in the variation of the growth rate with time is similar and suggests that, for capillaries with internal radius lower than 0.8 mm, diffusion dominates the global mass transport control. However, the existence of convection rolls near the crystal-solution interface and close to zones with high density gradient cannot be discarded.

  7. Quantification and imaging of HER2 protein using nanocrystals conjugated with single-domain antibodies

    International Nuclear Information System (INIS)

    Glukhov, S; Berestovoy, M; Nabiev, I; Sukhanova, A; Chames, P; Baty, D

    2017-01-01

    This study dealt with quantification and imaging of human epidermal growth factor receptor 2 (HER2), an important prognostic marker for cancer diagnosis and treatment, using specific quantum-dot-based conjugates. Fluorescent inorganic nanocrystals or quantum dots (QDs) are extremely highly resistant to photobleaching and have a high emission quantum yield and a continuous range of emission spectra, from the ultraviolet to the infrared regions. Ultrasmall nanoprobes consisting of highly affine anti-HER2 single-domain antibodies (sdAbs or 'nanobodies') conjugated with QDs in a strictly oriented manner have been designed. QDs with a fluorescence peak maxima at wavelengths of 562 nm, 569 nm, 570 nm or in the near-infrared region were used. Here, we present our results of ISA quantification of HER2 protein, in situ imaging of HER2 protein on the surface of HER2-positive SK-BR-3 cells in immunohistochemical experiments, and counting of stained with anti-HER2 conjugates HER2-positive SK-BR-3 cells in their mixture with unstained cells of the same culture in flow cytometry experiments. The data demonstrate that the anti-HER2 QD–sdAb conjugates obtained are highly specific and sensitive and could be used in numerous applications for advanced integrated diagnosis. (paper)

  8. Centromere-associated protein-E is essential for the mammalian mitotic checkpoint to prevent aneuploidy due to single chromosome loss

    OpenAIRE

    Weaver, Beth A.A.; Bonday, Zahid Q.; Putkey, Frances R.; Kops, Geert J.P.L.; Silk, Alain D.; Cleveland, Don W.

    2003-01-01

    Centromere-associated protein-E (CENP-E) is an essential mitotic kinesin that is required for efficient, stable microtubule capture at kinetochores. It also directly binds to BubR1, a kinetochore-associated kinase implicated in the mitotic checkpoint, the major cell cycle control pathway in which unattached kinetochores prevent anaphase onset. Here, we show that single unattached kinetochores depleted of CENP-E cannot block entry into anaphase, resulting in aneuploidy in 25% of divisions in p...

  9. Coupled aggregation of mitochondrial single-strand DNA-binding protein tagged with Eos fluorescent protein visualizes synchronized activity of mitochondrial nucleoids

    Czech Academy of Sciences Publication Activity Database

    Olejár, Tomáš; Pajuelo-Reguera, David; Alán, Lukáš; Dlasková, Andrea; Ježek, Petr

    2015-01-01

    Roč. 12, č. 4 (2015), s. 5185-5190 ISSN 1791-2997 R&D Projects: GA ČR(CZ) GAP302/10/0346; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : mitochondrial nucleoid * single- strand ed DNA -binding protein * photoconvertible fluorescent protein Eos Subject RIV: EA - Cell Biology Impact factor: 1.559, year: 2015

  10. Single-molecule resolution of G protein-coupled receptor (GPCR) complexes.

    Science.gov (United States)

    Jonas, Kim C; Huhtaniemi, Ilpo; Hanyaloglu, Aylin C

    2016-01-01

    The organization of G protein-coupled receptors (GPCRs) into dimers and higher-order oligomers has provided a potential mechanistic system in defining complex GPCR responses. Despite being studied for nearly 20 years it has, and still is, been an area of controversy. Although technology has developed to quantitatively measure these associations in real time, identify the structural interfaces and even systems to understand the physiological significance of di/oligomerization, key questions remain outstanding including the role of each individual complex from the monomer to the higher-order oligomer, in their native system. Recently, single-molecule microscopy approaches have provided the tools to directly visualize individual GPCRs in dimers and oligomers, though as with any technological development each have their advantages and limitations. This chapter will describe these recent developments in single-molecule fluorescent microscopy, focusing on our recent application of super-resolution imaging of the GPCR for the luteinizing hormone/chorionic gonadotropin to quantify GPCR monomers and formation of protomers in to dimers and distinct oligomeric forms. We present our approach, considerations, strategy, and challenges to visualize this receptor beyond the light diffraction limit via photoactivated localization microscopy with photoactivatable dyes. The addition of super-resolution approaches to the GPCR "nano-tool kit" will pave the way for novel avenues to answer outstanding questions regarding the existence and significance of these complexes to GPCR signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Measuring the force of single protein molecule detachment from surfaces with AFM.

    Science.gov (United States)

    Tsapikouni, Theodora S; Missirlis, Yannis F

    2010-01-01

    Atomic force microscopy (AFM) was used to measure the non-specific detachment force of single fibrinogen molecules from glass surfaces. The identification of single unbinding events was based on the characteristics of the parabolic curves, recorded during the stretching of protein molecules. Fibrinogen molecules were covalently bound to Si(3)N(4) AFM tips, previously modified with 3-aminopropyl-dimethyl-ethoxysilane, through a homobifunctional poly(ethylene glycol) linker bearing two hydroxysulfosuccinimide esters. The most probable detachment force was found to be 210 pN, when the tip was retracting with a velocity of 1400 nm/s, while the distribution of the detachment distances indicated that the fibrinogen chain can be elongated beyond the length of the physical conformation before detachment. The dependence of the most probable detachment force on the loading rate was examined and the dynamics of fibrinogen binding to the surface were found amenable to the simple expression of the Bell-Evans theory. The theory's expansion, however, by incorporating the concept of the rupture of parallel residue-surface bonds could only describe the detachment of fibrinogen for a small number of such bonds. Finally, the mathematical expression of the Worm-Like Chain model was used to fit the stretching curves before rupture and two interpretations are suggested for the description of the AFM curves with multiple detachment events.

  12. Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Charina L.; Claridge, Shelley A.; Garner, Ethan C.; Alivisatos, A. Paul; Mullins, R. Dyche

    2008-07-15

    To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. Wefound that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmidssegregating as a unit.

  13. Development and regulation of single- and multi-species Candida albicans biofilms

    Science.gov (United States)

    Lohse, Matthew B.; Gulati, Megha; Johnson, Alexander D.; Nobile, Clarissa J.

    2017-01-01

    Candida albicans is among the most prevalent fungal species of the human microbiota and asymptomatically colonizes healthy individuals. However, it is also an opportunistic pathogen that can cause severe, and often fatal, bloodstream infections. The medical impact of C. albicans typically depends on its ability to form biofilms, which are closely packed communities of cells that attach to surfaces, such as tissues and implanted medical devices. In this Review, we provide an overview of the processes involved in the formation of C. albicans biofilms and discuss the core transcriptional network that regulates biofilm development. We also consider some of the advantages that biofilms provide to C. albicans in comparison with planktonic growth and explore polymicrobial biofilms that are formed by C. albicans and certain bacterial species. PMID:29062072

  14. Mapping Regional Distribution of a Single Tree Species: Whitebark Pine in the Greater Yellowstone Ecosystem

    Directory of Open Access Journals (Sweden)

    Charles C. Schwartz

    2008-08-01

    Full Text Available Moderate resolution satellite imagery traditionally has been thought to be inadequate for mapping vegetation at the species level. This has made comprehensive mapping of regional distributions of sensitive species, such as whitebark pine, either impractical or extremely time consuming. We sought to determine whether using a combination of moderate resolution satellite imagery (Landsat Enhanced Thematic Mapper Plus, extensive stand data collected by land management agencies for other purposes, and modern statistical classification techniques (boosted classification trees could result in successful mapping of whitebark pine. Overall classification accuracies exceeded 90%, with similar individual class accuracies. Accuracies on a localized basis varied based on elevation. Accuracies also varied among administrative units, although we were not able to determine whether these differences related to inherent spatial variations or differences in the quality of available reference data.

  15. Specific leaf mass, fresh: dry weight ratio, sugar and protein contents in species of Lamiaceae from different light environments.

    Science.gov (United States)

    Castrillo, M; Vizcaino, D; Moreno, E; Latorraca, Z

    2005-01-01

    Samples from eleven species of Lamiaceae were collected from different light environments in Venezuela for laboratory analysis. The studied species were: Plectranthus scutellarioides (Ps), Scutellaria purpurascens (Sp), Hyptis pectinata (Hp)), H. sinuata (Hs). Leonorus japonicus (Lj), Plecthranthus amboinicus (Pa) Ocimum hasilicum (Ocb), O. campechianum (Occ) Origanum majorana (Orm), Rosmarinus officinali, (Ro) and Salvia officinalis (So). Protein and soluble sugar contents per unit of area were measured, Specific Leaf Mass (SLM) and fresh:dry weight (FW/DW) ratios were calculated. The higher values for soluble sugars contents were present in sun species: Lj, Pa, Ocb, Occ, Orm, Ro and So; the lower values were obtained in low light species: Ps, Sp, Hp, Hs. The values of protein content do not show any clear trend or difference between sun and shade environments. The lowest values for the fresh weight: dry weight ratio are observed in sun species with the exception of Lj and Pa, while the highest value is observed in Pa, a succulent plant. The higher values of specific leaf mass (SLM) (Kg DMm(-2)) are observed in sun plants. The two way ANOVA revealed that there were significant differences among species and between sun and low light environments for sugar content and FW:DW ratio. while SLM was significant for environments but no significant for species, and not significant for protein for both species and environments. The soluble sugar content, FW:DW ratio and SLM values obtained in this work, show a clear separation between sun and shade plants. The sugar content and FW:DW ratio are distinctive within the species, and the light environment affected sugar content. FW:DW ratio and SLM. These species may he shade-tolerant and able to survive in sunny environments. Perhaps these species originated in shaded environments and have been adapting to sunny habitats.

  16. [Study of the adsorption behaviors of plasma proteins on the single-walled carbon nanotubes nonwoven].

    Science.gov (United States)

    Meng, Jie; Song, Li; Meng, Jie; Kong, Hua; Wang, Chaoying; Guo, Xiaotian; Xu, Haiyan; Xie, Sishen

    2007-02-01

    Single walled carbon nanotubes (SWNT) have attracted increasing research interests for the purpose of biomedical application because they provide not only nanostructured topography, but also chemical composition of pure carbon atoms, as well as ultra high strength and excellent flexibility. Regarding the interactions of nanomaterials to biological systems, non-specific adsorption of plasma proteins is one of the most important issues to be concerned, which plays a crucial role that would determine how biological systems response to the biomaterials. Motivated by application of SWNT materials in biomedical fields, in this study, the adsorption behaviors of plasma proteins on the surface of SWNT nonwoven, prepared directly by floating chemical vapor observation and energy deposition method were investigated by means of scanning electron microscope (SEM), dispersive X-ray (EDX) analysis and ELISA. Results indicated the SWNT non-woven showed a clear adsorption preference of fibrinogen over albumin. There was no human serum albumin detected using above analysis methods on the SWNT nonwoven even incubated in the albumin solution of 4 mg/ml. While more than 0.15 microg of human fibrinogen was detected by ELISA on the SWNT nonwoven with area of 40 mm x 40 mm incubated in the fibrinogen solution of 5 microg/ml. In addition, IgG of sheep-anti-human serum fibrinogen exhibited strong nonspecific adsorption on the surface of SWNT nonwoven. The adsorption behaviors are different significantly from those of other carbon materials and conventional biomaterials. The unique interaction of SWNT nonwoven to plasma proteins is of significance to further studies of blood cells responses.

  17. Design of a single-chain multi-enzyme fusion protein establishing the polyhydroxybutyrate biosynthesis pathway.

    Science.gov (United States)

    Mullaney, Jane A; Rehm, Bernd H A

    2010-05-03

    Polyhydroxyalkanoates are biodegradable biocompatible polymers naturally produced by various bacteria and archaea. Biotechnological production in transgenic plants has already been demonstrated with efficient polyhydroxybutyrate production requiring targeting of the enzymes to the chloroplasts. Three enzymes are required to establish the polyhydroxybutyrate biosynthesis pathway in non-naturally producing microorganisms or plants. To facilitate production of biopolyesters in plants, a gene encoding a translational fusion of the polyhydroxybutyrate biosynthesis enzymes PhaA (beta-ketothiolase), PhaB (acetoacetyl-CoA reductase) and PhaC (PHA synthase) was constructed. Escherichia coli harboring a plasmid encoding this fusion protein (PhaA-PhaB-PhaC) under control of the lac promoter accumulated polyhydroxybutyrate contributing to 0.4% (w/w) of cellular dry weight. Insertion of an extended linker between PhaA and PhaB increased polyhydroxybutyrate accumulation to 3.9% (w/w) of cellular dry weight. Introduction of a second plasmid encoding PhaA and PhaB restored polyhydroxybutyrate accumulation to wildtype levels of about 35% (w/w) of cellular dry weight suggesting that the functions of PhaA and/or PhaB were limiting factors. Deletion of PhaA in trans led to significantly reduced polyhydroxybutyrate production suggesting that the PhaA activity in the fusion protein is reduced. This study showed that a single-chain translational fusion protein comprising the three enzymes essential for polyhydroxybutyrate synthesis can be engineered which will strongly facilitate the establishment of recombinant polyhydroxybutyrate production organisms particularly requiring targeting to sub-cellular compartments such as the chloroplasts in plants. 2010 Elsevier B.V. All rights reserved.

  18. Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

    Science.gov (United States)

    Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-01-01

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

  19. Protein crystal structure from non-oriented, single-axis sparse X-ray data

    Directory of Open Access Journals (Sweden)

    Jennifer L. Wierman

    2016-01-01

    Full Text Available X-ray free-electron lasers (XFELs have inspired the development of serial femtosecond crystallography (SFX as a method to solve the structure of proteins. SFX datasets are collected from a sequence of protein microcrystals injected across ultrashort X-ray pulses. The idea behind SFX is that diffraction from the intense, ultrashort X-ray pulses leaves the crystal before the crystal is obliterated by the effects of the X-ray pulse. The success of SFX at XFELs has catalyzed interest in analogous experiments at synchrotron-radiation (SR sources, where data are collected from many small crystals and the ultrashort pulses are replaced by exposure times that are kept short enough to avoid significant crystal damage. The diffraction signal from each short exposure is so `sparse' in recorded photons that the process of recording the crystal intensity is itself a reconstruction problem. Using the EMC algorithm, a successful reconstruction is demonstrated here in a sparsity regime where there are no Bragg peaks that conventionally would serve to determine the orientation of the crystal in each exposure. In this proof-of-principle experiment, a hen egg-white lysozyme (HEWL crystal rotating about a single axis was illuminated by an X-ray beam from an X-ray generator to simulate the diffraction patterns of microcrystals from synchrotron radiation. Millions of these sparse frames, typically containing only ∼200 photons per frame, were recorded using a fast-framing detector. It is shown that reconstruction of three-dimensional diffraction intensity is possible using the EMC algorithm, even with these extremely sparse frames and without knowledge of the rotation angle. Further, the reconstructed intensity can be phased and refined to solve the protein structure using traditional crystallographic software. This suggests that synchrotron-based serial crystallography of micrometre-sized crystals can be practical with the aid of the EMC algorithm even in cases

  20. Separate origins of ice-binding proteins in antarctic chlamydomonas species.

    Science.gov (United States)

    Raymond, James A; Morgan-Kiss, Rachael

    2013-01-01

    The green alga Chlamydomonas raudensis is an important primary producer in a number of ice-covered lakes and ponds in Antarctica. A C. raudensis isolate (UWO241) from Lake Bonney in the McMurdo Dry Valleys, like many other Antarctic algae, was found to secrete ice-binding proteins (IBPs), which appear to be essential for survival in icy environments. The IBPs of several Antarctic algae (diatoms, a prymesiophyte, and a prasinophyte) are similar to each other (here designated as type I IBPs) and have been proposed to have bacterial origins. Other IBPs (type II IBPs) that bear no resemblance to type I IBPs, have been found in the Antarctic Chlamydomonas sp. CCMP681, a putative snow alga, raising the possibility that chlamydomonad IBPs developed separately from the IBPs of other algae. To test this idea, we obtained the IBP sequences of C. raudensis UWO241 by sequencing the transcriptome. A large number of transcripts revealed no sequences resembling type II IBPs. Instead, many isoforms resembling type I IBPs were found, and these most closely matched a hypothetical protein from the bacterium Stigmatella aurantiaca. The sequences were confirmed to encode IBPs by the activity of a recombinant protein and by the matching of predicted and observed isoelectric points and molecular weights. Furthermore, a mesophilic sister species, C. raudensis SAG49.72, showed no ice-binding activity or PCR products from UWO241 IBP primers. These results confirm that algal IBPs are required for survival in icy habitats and demonstrate that they have diverse origins that are unrelated to the taxonomic positions of the algae. Last, we show that the C. raudensis UWO241 IBPs can change the structure of ice in a way that could increase the survivability of cells trapped in the ice.

  1. Environmental genomics reveals a single species ecosystem deep within the Earth

    Energy Technology Data Exchange (ETDEWEB)

    Chivian, Dylan; Brodie, Eoin L.; Alm, Eric J.; Culley, David E.; Dehal, Paramvir S.; DeSantis, Todd Z.; Gihring, Thomas M.; Lapidus, Alla; Lin, Li-Hung; Lowry, Stephen R.; Moser, Duane P.; Richardson, Paul; Southam, Gordon; Wanger, Greg; Pratt, Lisa M.; Andersen, Gary L.; Hazen, Terry C.; Brockman, Fred J.; Arkin, Adam P.; Onstott, Tullis C.

    2008-09-17

    DNA from low biodiversity fracture water collected at 2.8 km depth in a South African gold mine was sequenced and assembled into a single, complete genome. This bacterium, Candidatus Desulforudis audaxviator, comprises>99.9percent of the microorganisms inhabiting the fluid phase of this particular fracture. Its genome indicates a motile, sporulating, sulfate reducing, chemoautotrophic thermophile that can fix its own nitrogen and carbon using machinery shared with archaea. Candidatus Desulforudis audaxviator is capable of an independent lifestyle well suited to long-term isolation from the photosphere deep within Earth?s crust, and offers the first example of a natural ecosystem that appears to have its biological component entirely encoded within a single genome.

  2. Conditional independence mapping of DIGE data reveals PDIA3 protein species as key nodes associated with muscle aerobic capacity.

    Science.gov (United States)

    Burniston, Jatin G; Kenyani, Jenna; Gray, Donna; Guadagnin, Eleonora; Jarman, Ian H; Cobley, James N; Cuthbertson, Daniel J; Chen, Yi-Wen; Wastling, Jonathan M; Lisboa, Paulo J; Koch, Lauren G; Britton, Steven L

    2014-06-25

    Profiling of protein species is important because gene polymorphisms, splice variations and post-translational modifications may combine and give rise to multiple protein species that have different effects on cellular function. Two-dimensional gel electrophoresis is one of the most robust methods for differential analysis of protein species, but bioinformatic interrogation is challenging because the consequences of changes in the abundance of individual protein species on cell function are unknown and cannot be predicted. We conducted DIGE of soleus muscle from male and female rats artificially selected as either high- or low-capacity runners (HCR and LCR, respectively). In total 696 protein species were resolved and LC-MS/MS identified proteins in 337 spots. Forty protein species were differentially (P<0.05, FDR<10%) expressed between HCR and LCR and conditional independence mapping found distinct networks within these data, which brought insight beyond that achieved by functional annotation. Protein disulphide isomerase A3 emerged as a key node segregating with differences in aerobic capacity and unsupervised bibliometric analysis highlighted further links to signal transducer and activator of transcription 3, which were confirmed by western blotting. Thus, conditional independence mapping is a useful technique for interrogating DIGE data that is capable of highlighting latent features. Quantitative proteome profiling revealed that there is little or no sexual dimorphism in the skeletal muscle response to artificial selection on running capacity. Instead we found that noncanonical STAT3 signalling may be associated with low exercise capacity and skeletal muscle insulin resistance. Importantly, this discovery was made using unsupervised multivariate association mapping and bibliometric network analyses. This allowed our interpretation of the findings to be guided by patterns within the data rather than our preconceptions about which proteins or processes are of

  3. The Arabidopsis SUPERMAN protein is able to specifically bind DNA through its single Cys2-His2 zinc finger motif.

    Science.gov (United States)

    Dathan, Nina; Zaccaro, Laura; Esposito, Sabrina; Isernia, Carla; Omichinski, James G; Riccio, Andrea; Pedone, Carlo; Di Blasio, Benedetto; Fattorusso, Roberto; Pedone, Paolo V

    2002-11-15

    The Arabidopsis SUPERMAN (SUP) gene has been shown to be important in maintaining the boundary between stamens and carpels, and is presumed to act by regulating cell proliferation. In this work, we show that the SUP protein, which contains a single Cys2-His2 zinc finger domain including the QALGGH sequence, highly conserved in the plant zinc finger proteins, binds DNA. Using a series of deletion mutants, it was determined that the minimal domain required for specific DNA binding (residues 15-78) includes the single zinc finger and two basic regions located on either side of this motif. Furthermore, amino acid substitutions in the zinc finger or in the basic regions, including a mutation that knocks out the function of the SUP protein in vivo (glycine 63 to aspartate), have been found to abolish the activity of the SUP DNA-binding domain. These results strongly suggest that the SUP protein functions in vivo by acting as a DNA-binding protein, likely involved in transcriptional regulation. The association of both an N-terminal and a C-terminal basic region with a single Cys2-His2 zinc finger represents a novel DNA-binding motif suggesting that the mechanism of DNA recognition adopted by the SUP protein is different from that described so far in other zinc finger proteins.

  4. Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

    Science.gov (United States)

    Koh, Eunjung; Song, Ha Jeong; Kwon, Na Young; Kim, Gi Won; Lee, Kwang Ho; Jo, Soyeon; Park, Sujin; Park, Jihyun; Park, Eun Kyeong; Hwang, Seung Yong

    2017-06-01

    Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).

  5. Amino acid sequences of predicted proteins and their annotation for 95 organism species. - Gclust Server | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Gclust Server Amino acid sequences of predicted proteins and their annotation for 95 organis...m species. Data detail Data name Amino acid sequences of predicted proteins and their annotation for 95 organi...d proteins and their annotation for 95 organism species. The data are given in a ..._seq#en Data acquisition method Protein sequences of a total of 95 organism speci...quences of predicted proteins and their annotation for 95 organism species. - Gclust Server | LSDB Archive ...

  6. The effect of selected plant extracts on the development of single-species dental biofilms

    International Nuclear Information System (INIS)

    Rahim, Z.H.; Shaikh, S.; Razak, A.; Ismail, W.N.H.W.

    2014-01-01

    To determine the effect of a mixture of plant extracts on the adherence and retention of bacteria in dental biofilm. Study Design: Experimental study. Place and Duration of Study:Department of Oral Biology, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia, from December 2009 to December 2011. Methodology: For determination of adhering ability, experimental pellicle was first treated with the Plant Extracts Mixture (PEM) before inoculating it with individual bacterial species ( S. mitis / S. sanguinis / S. mutans). For the determination of retention ability, the procedure was repeated with the experimental pellicle being inoculated first with the individual bacterial species and then treating it with the PEM. These two experiments were repeated with deionized distilled water (negative control) and Thymol (0.64%) (positive control). The bacterial populations in biofilms for the two experiments were expressed as Colony Forming Unit (CFU) / mL x 10/sup 4/ and the corresponding values were expressed as mean +- SD. Results: The effect of the Plant Extracts Mixture (PEM) for the two experiments was compared with that of Thymol and deionized distilled water. It was shown that there is a reduced adherence of bacteria to PEM-treated and Thymol (0.064%) treated experimental pellicle compared with the negative control (p < 0.001). It was also found that the retention of bacteria in both treated biofilms is also lower than that of negative control (p = 0.001). Conclusion: Plant Extracts Mixture (PEM) may influence the development of dental biofilm by affecting the adhering and retention capacities of the bacterial species in the dental biofilms. (author)

  7. Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

    DEFF Research Database (Denmark)

    Thresher, RJ; Christiansen, Gunna; Griffith, JD

    1988-01-01

    We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional Rec......M in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein...... assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein...

  8. Real-Time Visualization of Active Species in a Single-Site Metal–Organic Framework Photocatalyst

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Sizhuo [Department of Chemistry, Marquette University, Milwaukee, Wisconsin 53201, United States; Pattengale, Brian [Department of Chemistry, Marquette University, Milwaukee, Wisconsin 53201, United States; Lee, Sungsik [X-ray Science Division, Argonne National Laboratory, Argonne, Illinois 60349, United States; Huang, Jier [Department of Chemistry, Marquette University, Milwaukee, Wisconsin 53201, United States

    2018-02-06

    In this work, we report a new single-site photocatalyst (Co-Ru-UIO- 67(bpy)) based on a metal-organic framework platform with incorporated molecular photosensitizer and catalyst. We show that this catalyst not only demonstrates exceptional activity for light-driven H2 production but also can be recycled without loss of activity. Using the combination of optical transient absorption spectroscopy and in situ X-ray absorption spectroscopy, we not only captured the key CoI intermediate species formed after ultrafast charge transfer from the incorporated photosensitizer but also identified the rate-limiting step in the catalytic cycle, providing insight into the catalysis mechanism of these single-site metal-organic framework photocatalysts.

  9. Evolutionary rate heterogeneity between multi- and single-interface hubs across human housekeeping and tissue-specific protein interaction network: Insights from proteins' and its partners' properties.

    Science.gov (United States)

    Biswas, Kakali; Acharya, Debarun; Podder, Soumita; Ghosh, Tapash Chandra

    2017-12-02

    Integrating gene expression into protein-protein interaction network (PPIN) leads to the construction of tissue-specific (TS) and housekeeping (HK) sub-networks, with distinctive TS- and HK-hubs. All such hub proteins are divided into multi-interface (MI) hubs and single-interface (SI) hubs, where MI hubs evolve slower than SI hubs. Here we explored the evolutionary rate difference between MI and SI proteins within TS- and HK-PPIN and observed that this difference is present only in TS, but not in HK-class. Next, we explored whether proteins' own properties or its partners' properties are more influential in such evolutionary discrepancy. Statistical analyses revealed that this evolutionary rate correlates negatively with protein's own properties like expression level, miRNA count, conformational diversity and functional properties and with its partners' properties like protein disorder and tissue expression similarity. Moreover, partial correlation and regression analysis revealed that both proteins' and its partners' properties have independent effects on protein evolutionary rate. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Role of the major antigenic membrane protein in phytoplasma transmission by two insect vector species.

    Science.gov (United States)

    Rashidi, Mahnaz; Galetto, Luciana; Bosco, Domenico; Bulgarelli, Andrea; Vallino, Marta; Veratti, Flavio; Marzachì, Cristina

    2015-09-30

    and abdominal microinjection protocols were developed to address the two barriers separately. The in vivo interactions between Amp of 'Candidatus Phytoplasma asteris' Chrysanthemum yellows strain (CYP) and vector proteins were studied by evaluating their effects on phytoplasma transmission by Euscelidius variegatus and Macrosteles quadripunctulatus leafhoppers. An internalization assay was developed, consisting of dissected salivary glands from healthy E. variegatus exposed to phytoplasma suspension alone or together with anti-Amp antibody. To visualize possible differences among treatments in localization/presence of CYP cells, the organs were observed in confocal microscopy. Pre-feeding of E. variegatus and M. quadripunctulatus on anti-Amp antibody resulted in a significant decrease of acquisition efficiencies in both species. Inoculation efficiency of microinjected E. variegatus with CYP suspension and anti-Amp antibody was significantly reduced compared to that of the control with phytoplasma suspension only. The possibility that this was due to reduced infection efficiency or antibody-mediated inhibition of phytoplasma multiplication was ruled out. These results provided the first indirect proof of the role of Amp in the transmission process. Protocols were developed to assess the in vivo role of the phytoplasma native major antigenic membrane protein in two phases of the vector transmission process: movement through the midgut epithelium and colonization of the salivary glands. These methods will be useful also to characterize other phytoplasma-vector combinations. Results indicated for the first time that native CYP Amp is involved in vivo in specific crossing of the gut epithelium and salivary gland colonization during early phases of vector infection.

  11. Graphene/graphene oxide and their derivatives in the separation/isolation and preconcentration of protein species: A review

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xuwei; Hai, Xin; Wang, Jianhua, E-mail: jianhuajrz@mail.neu.edu.cn

    2016-05-30

    The distinctive/unique electrical, chemical and optical properties make graphene/graphene oxide-based materials popular in the field of analytical chemistry. Its large surface offers excellent capacity to anchor target analyte, making it an powerful sorbent in the adsorption and preconcentration of trace level analyte of interest in the field of sample preparation. The large delocalized π-electron system of graphene framework provides strong affinity to species containing aromatic rings, such as proteins, and the abundant active sites on its surface offers the chance to modulate adsorption tendency towards specific protein via functional modification/decoration. This review provides an overview of the current research on graphene/graphene oxide-based materials as attractive and powerful adsorption media in the separation/isolation and preconcentration of protein species from biological sample matrixes. These practices are aiming at providing protein sample of high purity for further investigations and applications, or to achieve certain extent of enrichment prior to quantitative assay. In addition, the challenges and future perspectives in the related research fields have been discussed. - Highlights: • This review provides an updated overview of G/GO-materials in the separation, isolation/preconcentration of protein species. • The strategies to improve protein extraction performance by G/GO-materials are summarized. • The challenges and future perspectives in the related research fields are discussed.

  12. Chasing the hare - Evaluating the phylogenetic utility of a nuclear single copy gene region at and below species level within the species rich group Peperomia (Piperaceae

    Directory of Open Access Journals (Sweden)

    Naumann Julia

    2011-12-01

    Full Text Available Abstract Background The rapidly increasing number of available plant genomes opens up almost unlimited prospects for biology in general and molecular phylogenetics in particular. A recent study took advantage of this data and identified a set of nuclear genes that occur in single copy in multiple sequenced angiosperms. The present study is the first to apply genomic sequence of one of these low copy genes, agt1, as a phylogenetic marker for species-level phylogenetics. Its utility is compared to the performance of several coding and non-coding chloroplast loci that have been suggested as most applicable for this taxonomic level. As a model group, we chose Tildenia, a subgenus of Peperomia (Piperaceae, one of the largest plant genera. Relationships are particularly difficult to resolve within these species rich groups due to low levels of polymorphisms and fast or recent radiation. Therefore, Tildenia is a perfect test case for applying new phylogenetic tools. Results We show that the nuclear marker agt1, and in particular the agt1 introns, provide a significantly increased phylogenetic signal compared to chloroplast markers commonly used for low level phylogenetics. 25% of aligned characters from agt1 intron sequence are parsimony informative. In comparison, the introns and spacer of several common chloroplast markers (trnK intron, trnK-psbA spacer, ndhF-rpl32 spacer, rpl32-trnL spacer, psbA-trnH spacer provide less than 10% parsimony informative characters. The agt1 dataset provides a deeper resolution than the chloroplast markers in Tildenia. Conclusions Single (or very low copy nuclear genes are of immense value in plant phylogenetics. Compared to other nuclear genes that are members of gene families of all sizes, lab effort, such as cloning, can be kept to a minimum. They also provide regions with different phylogenetic content deriving from coding and non-coding parts of different length. Thus, they can be applied to a wide range of

  13. Albumin Adsorption: Inferences of Protein Interactions Measured by Sedimentation both Between Species and Induced by Denaturing

    OpenAIRE

    McKeon, Kristin Dianne

    2008-01-01

    Biological development and progression are managed by a diverse macromolecular group called proteins. Protein structure results from a complex folding process that leads to a final active form. This protein state is susceptible to changes in the surrounding environment and an incorrect structure can be produced. Changes in the protein conformation can lead to the formation of protein aggregates. Adsorption of proteins onto surfaces is utilized in many research analyses, but is capable of irre...

  14. Electronic transport in single-helical protein molecules: Effects of multiple charge conduction pathways and helical symmetry

    Energy Technology Data Exchange (ETDEWEB)

    Kundu, Sourav, E-mail: sourav.kunduphy@gmail.com; Karmakar, S.N.

    2016-07-15

    We propose a tight-binding model to investigate electronic transport properties of single helical protein molecules incorporating both the helical symmetry and the possibility of multiple charge transfer pathways. Our study reveals that due to existence of both the multiple charge transfer pathways and helical symmetry, the transport properties are quite rigid under influence of environmental fluctuations which indicates that these biomolecules can serve as better alternatives in nanoelectronic devices than its other biological counterparts e.g., single-stranded DNA.

  15. Compact halo-ligand-conjugated quantum dots for multicolored single-molecule imaging of overcrowding GPCR proteins on cell membranes.

    Science.gov (United States)

    Komatsuzaki, Akihito; Ohyanagi, Tatsuya; Tsukasaki, Yoshikazu; Miyanaga, Yukihiro; Ueda, Masahiro; Jin, Takashi

    2015-03-25

    To detect single molecules within the optical diffraction limit (< ca. 200 nm), a multicolored imaging technique is developed using Halo-ligand conjugated quantum dots (Halo-QDs; <6 nm in diameter). Using three types of Halo-QDs, multicolored single-molecule fluorescence imaging of GPCR proteins in Dictyostelium cells is achieved. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Fructose 1,6-Bisphosphate Aldolase, a Novel Immunogenic Surface Protein on Listeria Species.

    Science.gov (United States)

    Mendonça, Marcelo; Moreira, Gustavo Marçal Schmidt Garcia; Conceição, Fabricio Rochedo; Hust, Michael; Mendonça, Karla Sequeira; Moreira, Ângela Nunes; França, Rodrigo Correa; da Silva, Wladimir Padilha; Bhunia, Arun K; Aleixo, José Antonio G

    2016-01-01

    Listeria monocytogenes is a ubiquitous food-borne pathogen, and its presence in food or production facilities highlights the importance of surveillance. Increased understanding of the surface exposed antigens on Listeria would provide potential diagnostic and therapeutic targets. In the present work, using mass spectrometry and genetic cloning, we show that fructose-1,6-bisphosphate aldolase (FBA) class II in Listeria species is the antigen target of the previously described mAb-3F8. Western and dot blot assays confirmed that the mAb-3F8 could distinguish all tested Listeria species from close-related bacteria. Localization studies indicated that FBA is present in every fraction of Listeria cells, including supernatant and the cell wall, setting Listeria spp. as one of the few bacteria described to have this protein on their cell surface. Epitope mapping using ORFeome display and a peptide membrane revealed a 14-amino acid peptide as the potential mAb-3F8 epitope. The target epitope in FBA allowed distinguishing Listeria spp. from closely-related bacteria, and was identified as part of the active site in the dimeric enzyme. However, its function in cell surface seems not to be host cell adhesion-related. Western and dot blot assays further demonstrated that mAb-3F8 together with anti-InlA mAb-2D12 could differentiate pathogenic from non-pathogenic Listeria isolated from artificially contaminated cheese. In summary, we report FBA as a novel immunogenic surface target useful for the detection of Listeria genus.

  17. Matrix Gla protein species and risk of cardiovascular events in type 2 diabetic patients.

    Science.gov (United States)

    Dalmeijer, Geertje W; van der Schouw, Yvonne T; Magdeleyns, Elke J; Vermeer, Cees; Verschuren, W M Monique; Boer, Jolanda M A; Beulens, Joline W J

    2013-11-01

    To investigate the relationship of circulating matrix Gla protein (MGP) species with incident cardiovascular disease (CVD) or coronary heart disease (CHD) in type 2 diabetic patients. EPIC-NL is a prospective cohort study among 40,011 Dutch men and women. At baseline (1993-1997), 518 participants were known to have type 2 diabetes. MGP levels were measured by ELISA techniques in baseline plasma samples. The incidence of fatal and nonfatal CVD and CVD subtypes-CHD, peripheral arterial disease (PAD), heart failure, and stroke-were obtained by linkage to national registers. Cox proportional hazard models were used to calculate hazard ratios (HRs), adjusted for sex, waist-to-hip ratio, physical activity, and history of CVD. During a median 11.2 years of follow-up, 160 cases of CVD were documented. Higher circulating desphospho-uncarboxylated MGP (dp-ucMGP) levels were significantly associated with higher risk of CVD, with an HR per SD (HRSD) of 1.21 (95% CI 1.06-1.38), PAD (HRSD 1.32 [95% CI 1.07-1.65]), and heart failure (HRSD 1.75 [95% CI 1.42-2.17]) after adjustment. Higher circulating dp-ucMGP levels were not related to risk of CHD (HRSD 1.12 [95% CI 0.94-1.34]) or stroke (HRSD 1.05 [95% CI 0.73-1.49]). Circulating desphospho-carboxylated MGP and circulating total-uncarboxylated MGP levels were not associated with CVD or CVD subtypes. High dp-ucMGP levels were associated with increased CVD risk among type 2 diabetic patients, especially with the subtypes PAD and heart failure, while other MGP species were not related to CVD risk. These results suggest that a poor vitamin K status is associated with increased CVD risk.

  18. Revisiting the single cell protein application of Cupriavidus necator H16 and recovering bioplastic granules simultaneously.

    Science.gov (United States)

    Kunasundari, Balakrishnan; Murugaiyah, Vikneswaran; Kaur, Gurjeet; Maurer, Frans H J; Sudesh, Kumar

    2013-01-01

    Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha) was famous as a potential single cell protein (SCP) in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB) storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals.

  19. Characterization and quantification of proteins secreted by single human embryos prior to implantation.

    Science.gov (United States)

    Poli, Maurizio; Ori, Alessandro; Child, Tim; Jaroudi, Souraya; Spath, Katharina; Beck, Martin; Wells, Dagan

    2015-11-01

    The use of in vitro fertilization (IVF) has revolutionized the treatment of infertility and is now responsible for 1-5% of all births in industrialized countries. During IVF, it is typical for patients to generate multiple embryos. However, only a small proportion of them possess the genetic and metabolic requirements needed in order to produce a healthy pregnancy. The identification of the embryo with the greatest developmental capacity represents a major challenge for fertility clinics. Current methods for the assessment of embryo competence are proven inefficient, and the inadvertent transfer of non-viable embryos is the principal reason why most IVF treatments (approximately two-thirds) end in failure. In this study, we investigate how the application of proteomic measurements could improve success rates in clinical embryology. We describe a procedure that allows the identification and quantification of proteins of embryonic origin, present in attomole concentrations in the blastocoel, the enclosed fluid-filled cavity that forms within 5-day-old human embryos. By using targeted proteomics, we demonstrate the feasibility of quantifying multiple proteins in samples derived from single blastocoels and that such measurements correlate with aspects of embryo viability, such as chromosomal (ploidy) status. This study illustrates the potential of high-sensitivity proteomics to measure clinically relevant biomarkers in minute samples and, more specifically, suggests that key aspects of embryo competence could be measured using a proteomic-based strategy, with negligible risk of harm to the living embryo. Our work paves the way for the development of "next-generation" embryo competence assessment strategies, based on functional proteomics. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  20. Revisiting the single cell protein application of Cupriavidus necator H16 and recovering bioplastic granules simultaneously.

    Directory of Open Access Journals (Sweden)

    Balakrishnan Kunasundari

    Full Text Available Cupriavidus necator H16 (formerly known as Hydrogenomonas eutropha was famous as a potential single cell protein (SCP in the 1970s. The drawback however was the undesirably efficient accumulation of non-nutritive polyhydroxybutyrate (PHB storage compound in the cytoplasm of this bacterium. Eventually, competition from soy-based protein resulted in SCP not receiving much attention. Nevertheless, C. necator H16 remained in the limelight as a producer of PHB, which is a material that resembles commodity plastics such as polypropylene. PHB is a 100% biobased and biodegradable polyester. Although tremendous achievements have been attained in the past 3 decades in the efficient production of PHB, this bioplastic is still costly. One of the main problems has been the recovery of PHB from the cell cytoplasm. In this study, we showed for the first time that kilogram quantities of PHB can be easily recovered in the laboratory without the use of any solvents and chemicals, just by using the cells as SCP. In addition, the present study also demonstrated the safety and tolerability of animal model used, Sprague Dawley given lyophilized cells of C. necator H16. The test animals readily produced fecal pellets that were whitish in color, as would be expected of PHB granules. The pellets were determined to contain about 82-97 wt% PHB and possessed molecular mass of around 930 kg/mol. The PHB granules recovered biologically possessed similar molecular mass compared to chloroform extracted PHB [950 kg/mol]. This method now allows the production and purification of substantial quantities of PHB for various experimental trials. The method reported here is easy, does not require expensive instrumentation, scalable and does not involve extensive use of solvents and strong chemicals.

  1. Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

    Science.gov (United States)

    Kwon, Kwang-Chul; Verma, Dheeraj; Jin, Shuangxia; Singh, Nameirakpam D; Daniell, Henry

    2013-01-01

    Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

  2. Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

    Directory of Open Access Journals (Sweden)

    Kwang-Chul Kwon

    Full Text Available Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress or paraquat (abiotic stress, GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide, which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These

  3. Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species during Biotic and Abiotic Stress

    Science.gov (United States)

    Singh, Nameirakpam D.; Daniell, Henry

    2013-01-01

    Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

  4. Single-Particle Momentum Distributions of Efimov States in Mixed-Species Systems

    DEFF Research Database (Denmark)

    T. Yamashita, M.; F. Bellotti, F.; Frederico, T.

    2013-01-01

    to derive formulas for the scaling factor of the Efimov spectrum for any mass ratio assuming either that two or three of the two-body subsystems have a bound state at zero energy. We consider the single-particle momentum distribution analytically and numerically and analyse the tail of the momentum......We solve the three-body bound state problem in three dimensions for mass imbalanced systems of two identical bosons and a third particle in the universal limit where the interactions are assumed to be of zero-range. The system displays the Efimov effect and we use the momentum-space wave equation...... distribution to obtain the three-body contact parameter. Our finding demonstrate that the functional form of the three-body contact term depends on the mass ratio and we obtain an analytic expression for this behavior. To exemplify our results, we consider mixtures of Lithium with either two Caesium or Rubium...

  5. Quantitative diagnosis of HER2 protein expressing breast cancer by single-particle quantum dot imaging.

    Science.gov (United States)

    Miyashita, Minoru; Gonda, Kohsuke; Tada, Hiroshi; Watanabe, Mika; Kitamura, Narufumi; Kamei, Takashi; Sasano, Hironobu; Ishida, Takanori; Ohuchi, Noriaki

    2016-10-01

    Overexpression of HER2 is one of the major causes of breast cancer, and therefore precise diagnosis of its protein expression level is important. However, current methods estimating the HER2-expression level are insufficient due to problem with the lack of quantification. This might result in a gap between diagnostics and therapeutics targeting HER2. Therefore, a new effective diagnostic method is needed. We developed a new immunohistochemical (IHC) technique with quantum dots (QD)-conjugated trastuzumab using single-particle imaging to quantitatively measure the HER2 expression level. Tissues from 37 breast cancer patients with available detailed clinical information were tested by IHC with QDs (IHC-QD) and the correlation with IHC with 3,3'-diaminobenzidine (DAB), fluorescence in situ hybridization (FISH), and IHC-QD was examined. The number of QD-conjugated trastuzumab particles binding specifically to a cancer cell was precisely calculated as the IHC-QD score. The IHC-QD score in 37 cases was correlated proportionally with the score of HER2 gene copy number as assessed by FISH (R = 0.83). When HER2 positivity was judged to be positive, the IHC-QD score with our cut-off level was exactly concordant with the FISH score with a cut-off value of 2.0. Furthermore, IHC-QDs score and time to progression (TTP) of trastuzumab therapy were well correlated in HER2-positive cases (R = 0.69). Conversely, the correlation between FISH score and TTP was not observed. We developed a precisely quantitative IHC method using trastuzumab-conjugated QDs and single-particle imaging analysis and propose the possibility of using IHC-QDs score as a predictive factor for trastuzumab therapy. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  6. Self-assembling protein arrays on DNA chips by auto-labeling fusion proteins with a single DNA address

    NARCIS (Netherlands)

    Jongsma, M.A.; Litjens, R.H.G.M.

    2006-01-01

    The high-throughput deposition of recombinant proteins on chips, beads or biosensor devices would be greatly facilitated by the implementation of self-assembly concepts. DNA-directed immobilization via conjugation of proteins to an oligonucleotide would be preeminently suited for this purpose. Here,

  7. Protein analysis and gene expression indicate differential vulnerability of Iberian fish species under a climate change scenario

    Science.gov (United States)

    Moreno, João M.; Repolho, Tiago; Athanasiadis, Alekos; Rosa, Rui; Almeida-Val, Vera M. F.; Coelho, Maria M.

    2017-01-01

    Current knowledge on the biological responses of freshwater fish under projected scenarios of climate change remains limited. Here, we examine differences in the protein configuration of two endemic Iberian freshwater fish species, Squalius carolitertii and the critically endangered S. torgalensis that inhabit in the Atlantic-type northern and in the Mediterranean-type southwestern regions, respectively. We performed protein structure modeling of fourteen genes linked to protein folding, energy metabolism, circadian rhythms and immune responses. Structural differences in proteins between the two species were found for HSC70, FKBP52, HIF1α and GPB1. For S. torgalensis, besides structural differences, we found higher thermostability for two proteins (HSP90 and GBP1), which can be advantageous in a warmer environment. Additionally, we investigated how these species might respond to projected scenarios of 3° climate change warming, acidification (ΔpH = -0.4), and their combined effects. Significant changes in gene expression were observed in response to all treatments, particularly under the combined warming and acidification. While S. carolitertii presented changes in gene expression for multiple proteins related to folding (hsp90aa1, hsc70, fkbp4 and stip1), only one such gene was altered in S. torgalensis (stip1). However, S. torgalensis showed a greater capacity for energy production under both the acidification and combined scenarios by increasing cs gene expression and maintaining ldha gene expression in muscle. Overall, these findings suggest that S. torgalensis is better prepared to cope with projected climate change. Worryingly, under the simulated scenarios, disturbances to circadian rhythm and immune system genes (cry1aa, per1a and gbp1) raise concerns for the persistence of both species, highlighting the need to consider multi-stressor effects when evaluating climate change impacts upon fish. This work also highlights that assessments of the potential of

  8. Human serum albumin adsorption on TiO2 from single protein solutions and from plasma.

    Science.gov (United States)

    Sousa, S R; Moradas-Ferreira, P; Saramago, B; Melo, L Viseu; Barbosa, M A

    2004-10-26

    In the present work, the adsorption of human serum albumin (HSA) on commercially pure titanium with a titanium oxide layer formed in a H(2)O(2) solution (TiO(2) cp) and on TiO(2) sputtered on Si (TiO(2) sp) was analyzed. Adsorption isotherms, kinetic studies, and work of adhesion determinations were carried out. HSA exchangeability was also evaluated. Surface characterization was performed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and wettability studies. The two TiO(2) surfaces have very distinct roughnesses, the TiO(2) sp having a mean R(a) value 14 times smaller than the one of TiO(2) cp. XPS analysis revealed consistent peaks representative of TiO(2) on sputtered samples as well as on Ti cp substrate after 48 h of H(2)O(2) immersion. Nitrogen was observed as soon as protein was present, while sulfur, present in disulfide bonds in HSA, was observed for concentrations of protein higher than 0.30 mg/mL. The work of adhesion was determined from contact angle measurements. As expected from the surface free energy values, the work of adhesion of HSA solution is higher for the TiO(2) cp substrate, the more hydrophilic one, and lower for the TiO(2) sp substrate, the more hydrophobic one. The work of adhesion between plasma and the substrates assumed even higher values for the TiO(2) cp surface, indicating a greater interaction between the surface and the complex protein solutions. Adsorption studies by radiolabeling of albumin ((125)I-HSA) suggest that rapid HSA adsorption takes place on both surfaces, reaching a maximum value after approximately 60 min of incubation. For the higher HSA concentrations in solution, a multilayer coverage was observed on both substrates. After the adsorption step from single HSA solutions, the exchangeability of adsorbed HSA molecules by HSA in solution was evaluated. The HSA molecules adsorbed on TiO(2) sp seem to be more easily exchanged by HSA itself than those adsorbed on TiO(2) cp after 24 h. In

  9. Mechanical unfolding of proteins: reduction to a single-reaction coordinate unfolding potential, and an application of the Jarzynski Relation

    Science.gov (United States)

    Olmsted, Peter; West, Daniel; Paci, Emanuele

    2007-03-01

    Single molecule force spectroscopy (AFM, optical tweezers, etc) has revolutionized the study of many biopolymers, including DNA, RNA, and proteins. In this talk I will discuss recent work on modelling of mechanical unfolding of proteins, as often probed by AFM. I will address two issues in obtaining a coarse-grained description of protein unfolding: how to project the entire energy landscape onto an effective one dimensional unfolding potential, and how to apply the Jarzynski Relation to extract equilibrium free energies from nonequilibrium unfolding experiments.

  10. Exploring abiotic stress on asynchronous protein metabolism in single kernels of wheat studied by NMR spectroscopy and chemometrics

    DEFF Research Database (Denmark)

    Winning, H.; Viereck, N.; Wollenweber, B.

    2009-01-01

    at terminal spikelet, during grain-filling or at both stages. Principal component trajectories of the total protein content and the protein fractions of flour as well as the H-1 NMR spectra of single wheat kernels, wheat flour, and wheat methanol extracts were analysed to elucidate the metabolic development...... indicating that protein metabolism is influenced by multiple drought events, the H-1 NMR spectra of the methanol extracts of flour from mature grains revealed that the amount of fumaric acid is particularly sensitive to water deficits....

  11. A Single Species of Clostridium Subcluster XIVa Decreased in Ulcerative Colitis Patients.

    Science.gov (United States)

    Takeshita, Kozue; Mizuno, Shinta; Mikami, Yohei; Sujino, Tomohisa; Saigusa, Keiichiro; Matsuoka, Katsuyoshi; Naganuma, Makoto; Sato, Tadashi; Takada, Toshihiko; Tsuji, Hirokazu; Kushiro, Akira; Nomoto, Koji; Kanai, Takanori

    2016-12-01

    Imbalance of the intestinal microbiota is associated with gastrointestinal disease and autoimmune disease and metabolic syndrome. Analysis of the intestinal microbiota has recently progressed, and the association with inflammatory bowel disease has been reported at the species level. Such findings suggest that the recovery of homeostasis in the intestinal microbiota could cure inflammatory bowel disease. We aimed to search new probiotic candidates for inflammatory bowel disease through translational research by analysis of ulcerative colitis (UC) patients' intestinal microbiota and clarify the effects of them on inflammation. Here, we focused on Fusicatenibacter saccharivorans, which belongs to Clostridium subcluster XIVa and was successfully isolated and cultured in 2013. We analyzed the association of F. saccharivorans to UC patients' activity and inflammation for the first time. Feces from UC patients and healthy controls were analyzed by 16S ribosomal RNA gene sequences. F. saccharivorans was administered to murine colitis model. Colitic lamina propria mononuclear cells from UC patients and mice were stimulated with F. saccharivorans. The whole fecal bacteria in active UC patients were less than that in quiescent UC patients. Furthermore, F. saccharivorans was decreased in active UC patients and increased in quiescent. The administration of F. saccharivorans improved murine colitis. F. saccharivorans induced interleukin 10 production by lamina propria mononuclear cells from not only colitis model mice but also UC patients. F. saccharivorans decreased in correlation to UC activity and suppresses intestinal inflammation. These results suggest that F. saccharivorans could lead to a novel UC treatment.

  12. Single amino acid loss in the dystrophin protein associated with a mild clinical phenotype.

    Science.gov (United States)

    Pons, Roser; Kekou, Kyriaki; Gkika, Artemis; Papadimas, George; Vogiatzakis, Nikolaos; Svingou, Maria; Papadopooulos, Constantinos; Nikas, Ioanis; Dinopoulos, Argirios; Youroukos, Sotiris; Kanavakis, Emmanouel

    2017-01-01

    The dystrophinopathies include a spectrum of muscle diseases caused by mutations in the dystrophin (DMD) gene. The clinical phenotype ranges from severe Duchenne muscular dystrophy to a mild phenotype with elevated creatine kinase (CK). Clinical and molecular assessment of 7 patients carrying a single amino acid loss in the dystrophin protein (p.His1690del) caused by a c.5068_5070delCAC tri-nucleotide deletion in exon 36 of the DMD gene. All patients were asymptomatic or oligosymptomatic and had elevated CK levels. Febrile illness, but not exercise, induced muscle symptoms in some patients. None had evidence of cardiomyopathy. Analysis of the short tandem repeat (STR)45 locus and sequencing of exon 36 of the DMD gene indicates that c.5068_5070delCAC is a founder mutation. The c.5068_5070delCAC locus in the DMD gene is associated with a very mild phenotype. Further study is needed to evaluate disease progression in these patients. Muscle Nerve 55: 46-50, 2017. © 2016 Wiley Periodicals, Inc.

  13. A mathematical model of T lymphocyte calcium dynamics derived from single transmembrane protein properties

    Directory of Open Access Journals (Sweden)

    Christine Dorothee Schmeitz

    2013-09-01

    Full Text Available Fate decision processes of T lymphocytes are crucial for health and disease. Whether a T lymphocyte is activated, divides, gets anergic or initiates apoptosis depends on extracellular triggers and intracellular signalling. Free cytosolic calcium dynamics plays an important role in this context. The relative contributions of store-derived calcium entry and calcium entry from extracellular space to T lymphocyte activation are still a matter of debate. Here we develop a quantitative mathematical model of T lymphocyte calcium dynamics in order to establish a tool which allows to disentangle cause-effect relationships between ion fluxes and observed calcium time courses. The model is based on single transmembrane protein characteristics which have been determined in independent experiments. This reduces the number of unknown parameters in the model to a minimum and ensures the predictive power of the model. Simulation results are subsequently used for an analysis of whole cell calcium dynamics measured under various experimental conditions. The model accounts for a variety of these conditions, which supports the suitability of the modelling approach. The simulation results suggest a model in which calcium dynamics dominantly relies on the opening of channels in calcium stores while calcium entry through calcium-release activated channels (CRAC is more associated with the maintenance of the T lymphocyte calcium levels and prevents the cell from calcium depletion. Our findings indicate that CRAC guarantees a long-term stable calcium level which is required for cell survival and sustained calcium enhancement.

  14. Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species.

    Science.gov (United States)

    Symons, F M; Murray, P J; Ji, H; Simpson, R J; Osborn, A H; Cappai, R; Handman, E

    1994-09-01

    Antibodies raised against a Leishmania major recombinant promastigote surface antigen 2 (PSA-2) fragment recognized three major polypeptides of approximate M(r) 96,000, 80,000 and 50,000 in promastigotes of three Israeli isolates of L. major including the cloned line LRC-L137-V121, but detected a different array of polypeptides in other L. major isolates. The pattern was different both in number of polypeptides detected and their molecular weight. The antibodies to L. major PSA-2 also recognized polypeptides in L. tropica, L. donovani and very weakly in L. mexicana promastigotes and in Crithidia lucilliae. The number and size of the polypeptides was different in each species. In addition to the membrane-bound PSA-2 polypeptides we identified water-soluble forms of PSA-2 released in promastigote culture supernatants. Peptide maps of the various L. major PSA-2 membrane polypeptides showed they were different from each other. N-terminal amino acid sequence of the three polypeptides expressed by L. major showed they are similar but distinct, consistent with being members of a polymorphic family. Because of the extensive sequence similarity between the PSA-2 genes it has been difficult to assign protein products to individual genes. As a first step towards solving this problem, we have transfected into L. mexicana a genomic clone of a L. major PSA-2 gene and shown that it produces a M(r) 35,000 polypeptide recognized by monoclonal and polyclonal antibodies to L. major PSA-2.

  15. Nonencapsulated Streptococcus pneumoniae causes otitis media during single-species infection and during polymicrobial infection with nontypeable Haemophilus influenzae.

    Science.gov (United States)

    Murrah, Kyle A; Pang, Bing; Richardson, Stephen; Perez, Antonia; Reimche, Jennifer; King, Lauren; Wren, John; Swords, W Edward

    2015-07-01

    Streptococcus pneumoniae strains lacking capsular polysaccharide have been increasingly reported in carriage and disease contexts. Since most cases of otitis media involve more than one bacterial species, we aimed to determine the capacity of a nonencapsulated S. pneumoniae clinical isolate to induce disease in the context of a single-species infection and as a polymicrobial infection with nontypeable Haemophilus influenzae. Using the chinchilla model of otitis media, we found that nonencapsulated S. pneumoniae colonizes the nasopharynx following intranasal inoculation, but does not readily ascend into the middle ear. However, when we inoculated nonencapsulated S. pneumoniae directly into the middle ear, the bacteria persisted for two weeks post-inoculation and induced symptoms consistent with chronic otitis media. During coinfection with nontypeable H. influenzae, both species persisted for one week and induced polymicrobial otitis media. We also observed that nontypeable H. influenzae conferred passive protection from killing by amoxicillin upon S. pneumoniae from within polymicrobial biofilms in vitro. Therefore, based on these results, we conclude that nonencapsulated pneumococci are a potential causative agent of chronic/recurrent otitis media, and can also cause mutualistic infection with other opportunists, which could complicate treatment outcomes. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Effect of seed treatments on the chemical composition and properties of two amaranth species: starch and protein

    NARCIS (Netherlands)

    Gamel, T.H.; Linssen, J.P.H.; Mesallem, A.S.; Damir, A.A.; Shekib, L.A.

    2005-01-01

    The seeds of two Amaranth species were studied. The starch contents were 543 and 623 g kg-1 while crude protein contents were 154 and 169 g kg-1 for Amaranthus caudatus and Amaranthus cruentus seeds, respectively. The effect of several treatments, including cooking, popping and germination and flour

  17. Detection of eight different tospovirus species by a monoclonal antibody against the common epitope of NSs protein

    NARCIS (Netherlands)

    Chen, T.C.; Lu, Y.Y.; Kang, Y.C.; Li, J.T.; Yeh, Y.C.; Kormelink, R.J.M.; Yeh, S.D.

    2008-01-01

    Rabbit antisera against the nucleocapsid protein (NP) have been commonly used for detection of tospoviruses and classification into serogroups or serotypes. Mouse monoclonal antibodies (MAbs) with high specificity to the NPs have also been widely used to identify tospovirus species. Recently, a

  18. Analysis of the hybrid proline-rich protein families from seven plant species suggests rapid diversification of their sequences and expression patterns

    Directory of Open Access Journals (Sweden)

    Fischer Lukáš

    2007-11-01

    Full Text Available Abstract Background Plant hybrid proline-rich proteins (HyPRPs are putative cell wall proteins consisting, usually, of a repetitive proline-rich (PR N-terminal domain and a conserved eight-cysteine motif (8 CM C-terminal domain. Understanding the evolutionary dynamics of HyPRPs might provide not only insight into their so far elusive function, but also a model for other large protein families in plants. Results We have performed a phylogenetic analysis of HyPRPs from seven plant species, including representatives of gymnosperms and both monocot and dicot angiosperms. Every species studied possesses a large family of 14–52 HyPRPs. Angiosperm HyPRPs exhibit signs of recent major diversification involving, at least in Arabidopsis and rice, several independent tandem gene multiplications. A distinct subfamily of relatively well-conserved C-type HyPRPs, often with long hydrophobic PR domains, has been identified. In most of gymnosperm (pine HyPRPs, diversity appears within the C-type group while angiosperms have only a few of well-conserved C-type representatives. Atypical (glycine-rich or extremely short N-terminal domains apparently evolved independently in multiple lineages of the HyPRP family, possibly via inversion or loss of sequences encoding proline-rich domains. Expression profiles of potato and Arabidopsis HyPRP genes exhibit instances of both overlapping and complementary organ distribution. The diversified non-C-type HyPRP genes from recently amplified chromosomal clusters in Arabidopsis often share their specialized expression profiles. C-type genes have broader expression patterns in both species (potato and Arabidopsis, although orthologous genes exhibit some differences. Conclusion HyPRPs represent a dynamically evolving protein family apparently unique to seed plants. We suggest that ancestral HyPRPs with long proline-rich domains produced the current diversity through ongoing gene duplications accompanied by shortening

  19. A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

    Directory of Open Access Journals (Sweden)

    Patrizia Marinelli

    2018-04-01

    Full Text Available Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states. Keywords: Protein oxidation, Protein misfolding, Protein aggregation, Oxidative stress, Post-translational modification

  20. A rapid method for selecting suitable animal species for studying pathogen interactions with plasma protein ligands in vivo.

    Science.gov (United States)

    Naudin, Clément; Schumski, Ariane; Salo-Ahen, Outi M H; Herwald, Heiko; Smeds, Emanuel

    2017-05-01

    Species tropism constitutes a serious problem for developing relevant animal models of infection. Human pathogens can express virulence factors that show specific selectivity to human proteins, while their affinity for orthologs from other species can vary significantly. Suitable animal species must be used to analyse whether virulence factors are potential targets for drug development. We developed an assay that rapidly predicts applicable animal species for studying virulence factors binding plasma proteins. We used two well-characterized Staphylococcus aureus proteins, SSL7 and Efb, to develop an ELISA-based inhibition assay using plasma from different animal species. The interaction between SSL7 and human C5 and the binding of Efb to human fibrinogen and human C3 was studied. Affinity experiments and Western blot analyses were used to validate the assay. Human, monkey and cat plasma interfered with binding of SSL7 to human C5. Binding of Efb to human fibrinogen was blocked in human, monkey, gerbil and pig plasma, while human, monkey, gerbil, rabbit, cat and guinea pig plasma inhibited the binding of Efb to human C3. These results emphasize the importance of choosing correct animal models, and thus, our approach is a rapid and cost-effective method that can be used to prevent unnecessary animal experiments. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  1. Intracellular formation of α-synuclein oligomers and the effect of heat shock protein 70 characterized by confocal single particle spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Levin, Johannes [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); German Center for Neurodegenerative Diseases – DZNE, Site Munich, Feodor-Lynen-Str. 17, 81377 Munich (Germany); Hillmer, Andreas S. [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany); Högen, Tobias [Department of Neurology, Ludwig-Maximilians-University, Marchioninistr. 15, 81377 Munich (Germany); McLean, Pamela J. [Department of Neuroscience, Mayo Clinic, Jacksonville, FL 32224 (United States); Giese, Armin, E-mail: armin.giese@med.uni-muenchen.de [Center for Neuropathology and Prion Research, Ludwig-Maximilians-University, Feodor-Lynen-Str. 23, 81377 Munich (Germany)

    2016-08-12

    Synucleinopathies such as dementia with Lewy bodies or Parkinson’s disease are characterized by intracellular deposition of pathologically aggregated α-synuclein. The details of the molecular pathogenesis of PD and especially the conditions that lead to intracellular aggregation of α-synuclein and the role of these aggregates in cell death remain unknown. In cell free in vitro systems considerable knowledge about the aggregation processes has been gathered. In comparison, the knowledge about these aggregation processes in cells is far behind. In cells α-synuclein aggregates can be toxic. However, the crucial particle species responsible for decisive steps in pathogenesis such as seeding a continuing aggregation process and triggering cell death remain to be identified. In order to understand the complex nature of intracellular α-synuclein aggregate formation, we analyzed fluorescent particles formed by venus and α-synuclein-venus fusion proteins and α-synuclein-hemi-venus fusion proteins derived from gently lyzed cells. With these techniques we were able to identify and characterize α-synuclein oligomers formed in cells. Especially the use of α-synuclein-hemi-venus fusion proteins enabled us to identify very small α-synuclein oligomers with high sensitivity. Furthermore, we were able to study the molecular effect of heat shock protein 70, which is known to inhibit α-synuclein aggregation in cells. Heat shock protein 70 does not only influence the size of α-synuclein oligomers, but also their quantity. In summary, this approach based on fluorescence single particle spectroscopy, that is suited for high throughput measurements, can be used to detect and characterize intracellularly formed α-synuclein aggregates and characterize the effect of molecules that interfere with α-synuclein aggregate formation. - Highlights: • Single particle spectroscopy detects intracellular formed α-synuclein aggregates. • Fusion proteins allow detection of protein

  2. The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins

    DEFF Research Database (Denmark)

    Farver, O; Skov, L K; van de Kamp, M

    1992-01-01

    An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To fu...

  3. Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

    NARCIS (Netherlands)

    Dijkman, Ronald; Jebbink, Maarten F.; Wilbrink, Berry; Pyrc, Krzysztof; Zaaijer, Hans L.; Minor, Philip D.; Franklin, Sally; Berkhout, Ben; Thiel, Volker; van der Hoek, Lia

    2006-01-01

    BACKGROUND: The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV) 229E. The prototype virus has a

  4. Reversible G Protein βγ9 Distribution-Based Assay Reveals Molecular Underpinnings in Subcellular, Single-Cell, and Multicellular GPCR and G Protein Activity.

    Science.gov (United States)

    Senarath, Kanishka; Ratnayake, Kasun; Siripurapu, Praneeth; Payton, John L; Karunarathne, Ajith

    2016-12-06

    Current assays to measure the activation of G protein coupled receptors (GPCRs) and G proteins are time-consuming, indirect, and expensive. Therefore, an efficient method which directly measures the ability of a ligand to govern GPCR-G protein interactions can help to understand the molecular underpinnings of the associated signaling. A live cell imaging-based approach is presented here to directly measure ligand-induced GPCR and G protein activity in real time. The number of active GPCRs governs G protein heterotrimer (αβγ) dissociation, thereby controlling the concentration of free βγ subunits. The described γ9 assay measures the GPCR activation-induced extent of the reversible βγ9 subunit exchange between the plasma membrane (PM) and internal membranes (IMs). Confocal microscopy-based γ9 assay quantitatively determines the concentration dependency of ligands on GPCR activation. Demonstrating the high-throughput screening (HTS) adaptability, the γ9 assay performed using an imaging plate reader measures the ligand-induced GPCR activation. This suggests that the γ9 assay can be employed to screen libraries of compounds for their ability to activate GPCRs. Together with subcellular optogenetics, the spatiotemporal sensitivity of the γ9 assay permits experimental determination of the limits of spatially restricted activation of GPCRs and G proteins in subcellular regions of single cells. This assay works effectively for GPCRs coupled to αi/o and αs heterotrimers, including light-sensitive GPCRs. In addition, computational modeling of experimental data from the assay is used to decipher intricate molecular details of the GPCR-G protein activation process. Overall, the γ9 assay provides a robust strategy for quantitative as well as qualitative determination of GPCR and G protein function on a single-cell, multicell, and subcellular level. This assay not only provides information about the inner workings of the signaling pathway, but it also strengthens

  5. Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles.

    Science.gov (United States)

    Remenant, Benoît; de Cambiaire, Jean-Charles; Cellier, Gilles; Jacobs, Jonathan M; Mangenot, Sophie; Barbe, Valérie; Lajus, Aurélie; Vallenet, David; Medigue, Claudine; Fegan, Mark; Allen, Caitilyn; Prior, Philippe

    2011-01-01

    The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical

  6. Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles.

    Directory of Open Access Journals (Sweden)

    Benoît Remenant

    Full Text Available The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB. All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence

  7. Cluster based on sequence comparison of homologous proteins of 95 organism species - Gclust Server | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Gclust Server Cluster based on sequence comparison of homologous proteins of 95 organism spe...cies Data detail Data name Cluster based on sequence comparison of homologous proteins of 95 organism specie...ta, and heuristic estimation of a similarity threshold for homologs of each protein by entropy-optimized organi...no acid sequences of predicted proteins and their annotation for 95 organism species . Data analysis method ...y threshold for homologs of each protein by entropy-optimized organism count method ( Bioinformatics 2009 Ma

  8. Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein.

    Directory of Open Access Journals (Sweden)

    Marcos T Oliveira

    2010-10-01

    Full Text Available Biochemical studies of the mitochondrial DNA (mtDNA replisome demonstrate that the mtDNA polymerase and the mtDNA helicase are stimulated by the mitochondrial single-stranded DNA-binding protein (mtSSB. Unlike Escherichia coli SSB, bacteriophage T7 gp2.5 and bacteriophage T4 gp32, mtSSBs lack a long, negatively charged C-terminal tail. Furthermore, additional residues at the N-terminus (notwithstanding the mitochondrial presequence are present in the sequence of species across the animal kingdom. We sought to analyze the functional importance of the N- and C-terminal regions of the human mtSSB in the context of mtDNA replication. We produced the mature wild-type human mtSSB and three terminal deletion variants, and examined their physical and biochemical properties. We demonstrate that the recombinant proteins adopt a tetrameric form, and bind single-stranded DNA with similar affinities. They also stimulate similarly the DNA unwinding activity of the human mtDNA helicase (up to 8-fold. Notably, we find that unlike the high level of stimulation that we observed previously in the Drosophila system, stimulation of DNA synthesis catalyzed by human mtDNA polymerase is only moderate, and occurs over a narrow range of salt concentrations. Interestingly, each of the deletion variants of human mtSSB stimulates DNA synthesis at a higher level than the wild-type protein, indicating that the termini modulate negatively functional interactions with the mitochondrial replicase. We discuss our findings in the context of species-specific components of the mtDNA replisome, and in comparison with various prokaryotic DNA replication machineries.

  9. Effects of Nitrogen Addition on Leaf Decomposition of Single-Species and Litter Mixture in Pinus tabulaeformis Forests

    Directory of Open Access Journals (Sweden)

    Jinsong Wang

    2015-12-01

    Full Text Available The litter decomposition process is closely correlated with nutrient cycling and the maintenance of soil fertility in the forest ecosystem. In particular, the intense environmental concern about atmospheric nitrogen (N deposition requires a better understanding of its influence on the litter decomposition process. This study examines the responses of single-species litter and litter mixture decomposition processes to N addition in Chinese pine (Pinus tabulaeformis Carr. ecosystems. Chinese pine litter, Mongolian oak (Quercus mongolica Fisch. ex Ledeb. litter, and a pine–oak mixture were selected from a plantation and a natural forest of Chinese pine. Four N addition treatments, i.e., control (N0: 0 kg N ha−1·year−1, low-N (N1: 5 kg N ha−1·year−1, medium-N (N2: 10 kg N ha−1·year−1, and high-N (N3: 15 kg N ha−1·year−1, were applied starting May 2010. In the plantation, N addition significantly stimulated the decomposition of the Chinese pine litter. In the natural forest, N addition had variable effects on the decomposition of single-species litter and the litter mixture. A stimulatory effect of the high-N treatment on the Chinese pine litter decomposition could be attributed to a decrease in the substrate C:N ratio. However, an opposite effect was found for the Mongolian oak litter decomposition. The stimulating effect of N addition on the Chinese pine litter may offset the suppressive effect on the Mongolian oak litter, resulting in a neutral effect on the litter mixture. These results suggest that the different responses in decomposition of single-species litter and the litter mixture to N addition are mainly attributed to litter chemical composition. Further investigations are required to characterize the effect of long-term high-level N addition on the litter decomposition as N deposition is likely to increase rapidly in the region where this study was conducted.

  10. A Flp-nick system to study repair of a single protein-bound nick in vivo

    DEFF Research Database (Denmark)

    Nielsen, Ida; Bentsen, Iben Bach; Lisby, Michael

    2009-01-01

    recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I-DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill......We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I-DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp...... cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address....

  11. A simple SDS-PAGE protein pattern from pitcher secretions as a new tool to distinguish Nepenthes species (Nepenthaceae).

    Science.gov (United States)

    Biteau, Flore; Nisse, Estelle; Miguel, Sissi; Hannewald, Paul; Bazile, Vincent; Gaume, Laurence; Mignard, Benoit; Hehn, Alain; Bourgaud, Frederic

    2013-12-01

    Carnivorous plants have always fascinated scientists because these plants are able to attract, capture, and digest animal prey using their remarkable traps that contain digestive secretions. Nepenthes is one of the largest genera of carnivorous plants, with 120 species described thus far. Despite an outstanding diversity of trap designs, many species are often confused with each other and remain difficult to classify because they resemble pitchers or of the occurrence of interspecific hybrids. Here, we propose a new method to easily distinguish Nepenthes species based on a SDS PAGE protein pattern analysis of their pitcher secretions. Intraspecific comparisons were performed among specimens growing in different environmental conditions to ascertain the robustness of this method. Our results show that, at the juvenile stage and in the absence of prey in the pitcher, an examined species is characterized by a specific and stable profile, whatever the environmental conditions. The method we describe here can be used as a reliable tool to easily distinguish between Nepenthes species and to help with potential identification based on the species-specific protein pattern of their pitcher secretions, which is complementary to the monograph information.

  12. Verification of Single-Peptide Protein Identifications by the Application of Complementary Database Search Algorithms

    National Research Council Canada - National Science Library

    Rohrbough, James G; Breci, Linda; Merchant, Nirav; Miller, Susan; Haynes, Paul A

    2005-01-01

    .... One such technique, known as the Multi-Dimensional Protein Identification Technique, or MudPIT, involves the use of computer search algorithms that automate the process of identifying proteins...

  13. Interaction of bacteriophage T4 and T7 single-stranded DNA-binding proteins with DNA

    International Nuclear Information System (INIS)

    Shokri, Leila; Williams, Mark C; Rouzina, Ioulia

    2009-01-01

    Bacteriophages T4 and T7 are well-studied model replication systems, which have allowed researchers to determine the roles of many proteins central to DNA replication, recombination and repair. Here we summarize and discuss the results from two recently developed single-molecule methods to determine the salt-dependent DNA-binding kinetics and thermodynamics of the single-stranded DNA (ssDNA)-binding proteins (SSBs) from these systems. We use these methods to characterize both the equilibrium double-stranded DNA (dsDNA) and ssDNA binding of the SSBs T4 gene 32 protein (gp32) and T7 gene 2.5 protein (gp2.5). Despite the overall two-orders-of-magnitude weaker binding of gp2.5 to both forms of DNA, we find that both proteins exhibit four-orders-of-magnitude preferential binding to ssDNA relative to dsDNA. This strong preferential ssDNA binding as well as the weak dsDNA binding is essential for the ability of both proteins to search dsDNA in one dimension to find available ssDNA-binding sites at the replication fork

  14. Site-Specific SERS Assay for Survivin Protein Dimer: From Ensemble Experiments to Correlative Single-Particle Imaging.

    Science.gov (United States)

    Wissler, Jörg; Bäcker, Sandra; Feis, Alessandro; Knauer, Shirley K; Schlücker, Sebastian

    2017-08-01

    An assay for Survivin, a small dimeric protein which functions as modulator of apoptosis and cell division and serves as a promising diagnostic biomarker for different types of cancer, is presented. The assay is based on switching on surface-enhanced Raman scattering (SERS) upon incubation of the Survivin protein dimer with Raman reporter-labeled gold nanoparticles (AuNP). Site-specificity is achieved by complexation of nickel-chelated N-nitrilo-triacetic acid (Ni-NTA) anchors on the particle surface by multiple histidines (His 6 -tag) attached to each C-terminus of the centrosymmetric protein dimer. Correlative single-particle analysis using light sheet laser microscopy enables the simultaneous observation of both elastic and inelastic light scattering from the same sample volume. Thereby, the SERS-inactive AuNP-protein monomers can be directly discriminated from the SERS-active AuNP-protein dimers/oligomers. This information, i.e. the percentage of SERS-active AuNP in colloidal suspension, is not accessible from conventional SERS experiments due to ensemble averaging. The presented correlative single-particle approach paves the way for quantitative site-specific SERS assays in which site-specific protein recognition by small chemical and in particular supramolecular ligands can be tested. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Bioaccumulation of Cry1Ab protein from an herbivore reduces anti-oxidant enzyme activities in two spider species.

    Directory of Open Access Journals (Sweden)

    Ji Zhou

    Full Text Available Cry proteins are expressed in rice lines for lepidopteran pest control. These proteins can be transferred from transgenic rice plants to non-target arthropods, including planthoppers and then to a predatory spider. Movement of Cry proteins through food webs may reduce fitness of non-target arthropods, although recent publications indicated no serious changes in non-target populations. Nonetheless, Cry protein intoxication influences gene expression in Cry-sensitive insects. We posed the hypothesis that Cry protein intoxication influences enzyme activities in spiders acting in tri-trophic food webs. Here we report on the outcomes of experiments designed to test our hypothesis with two spider species. We demonstrated that the movement of CryAb protein from Drosophila culture medium into fruit flies maintained on the CryAb containing medium and from the flies to the spiders Ummeliata insecticeps and Pardosa pseudoannulata. We also show that the activities of three key metabolic enzymes, acetylcholine esterase (AchE, glutathione peroxidase (GSH-Px, and superoxide dismutase (SOD were significantly influenced in the spiders after feeding on Cry1Ab-containing fruit flies. We infer from these data that Cry proteins originating in transgenic crops impacts non-target arthropods at the physiological and biochemical levels, which may be one mechanism of Cry protein-related reductions in fitness of non-target beneficial predators.

  16. Dynamic Evolution of Nitric Oxide Detoxifying Flavohemoglobins, a Family of Single-Protein Metabolic Modules in Bacteria and Eukaryotes.

    Science.gov (United States)

    Wisecaver, Jennifer H; Alexander, William G; King, Sean B; Hittinger, Chris Todd; Rokas, Antonis

    2016-08-01

    Due to their functional independence, proteins that comprise standalone metabolic units, which we name single-protein metabolic modules, may be particularly prone to gene duplication (GD) and horizontal gene transfer (HGT). Flavohemoglobins (flavoHbs) are prime examples of single-protein metabolic modules, detoxifying nitric oxide (NO), a ubiquitous toxin whose antimicrobial properties many life forms exploit, to nitrate, a common source of nitrogen for organisms. FlavoHbs appear widespread in bacteria and have been identified in a handful of microbial eukaryotes, but how the distribution of this ecologically and biomedically important protein family evolved remains unknown. Reconstruction of the evolutionary history of 3,318 flavoHb protein sequences covering the family's known diversity showed evidence of recurrent HGT at multiple evolutionary scales including intrabacterial HGT, as well as HGT from bacteria to eukaryotes. One of the most striking examples of HGT is the acquisition of a flavoHb by the dandruff- and eczema-causing fungus Malassezia from Corynebacterium Actinobacteria, a transfer that growth experiments show is capable of mediating NO resistance in fungi. Other flavoHbs arose via GD; for example, many filamentous fungi possess two flavoHbs that are differentially targeted to the cytosol and mitochondria, likely conferring protection against external and internal sources of NO, respectively. Because single-protein metabolic modules such as flavoHb function independently, readily undergo GD and HGT, and are frequently involved in organismal defense and competition, we suggest that they represent "plug-and-play" proteins for ecological arms races. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Non-uniform binding of single-stranded DNA binding proteins to hybrids of single-stranded DNA and single-walled carbon nanotubes observed by atomic force microscopy in air and in liquid

    Energy Technology Data Exchange (ETDEWEB)

    Umemura, Kazuo, E-mail: meicun2006@163.com; Ishizaka, Kei; Nii, Daisuke; Izumi, Katsuki

    2016-12-01

    Highlights: • Conjugates of protein, DNA, and SWNTs were observed by AFM in liquid. • Non-uniform binding of proteins was visualized in liquid. • Thickness of DNA molecules on SWNT surfaces was well characterized in liquid. - Abstract: Using atomic force spectroscopy (AFM), we observed hybrids of single-stranded DNA (ssDNA) and single-walled carbon nanotubes (SWNTs) with or without protein molecules in air and in an aqueous solution. This is the first report of ssDNA–SWNT hybrids with proteins in solution analyzed by AFM. In the absence of protein, the height of the ssDNA–SWNT hybrids was 1.1 ± 0.3 nm and 2.4 ± 0.6 nm in air and liquid, respectively, suggesting that the ssDNA molecules adopted a flexible structure on the SWNT surface. In the presence of single-stranded DNA binding (SSB) proteins, the heights of the hybrids in air and liquid increased to 6.4 ± 3.1 nm and 10.0 ± 4.5 nm, respectively. The AFM images clearly showed binding of the SSB proteins to the ssDNA–SWNT hybrids. The morphology of the SSB–ssDNA–SWNT hybrids was non-uniform, particularly in aqueous solution. The variance of hybrid height was quantitatively estimated by cross-section analysis along the long-axis of each hybrid. The SSB–ssDNA–SWNT hybrids showed much larger variance than the ssDNA–SWNT hybrids.

  18. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

    International Nuclear Information System (INIS)

    Kim, Yoon Sik; Seo, Hyun Wook; Jung, Guhung

    2015-01-01

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H 2 O 2 and GSH modulate HBV capsid assembly. • H 2 O 2 facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H 2 O 2 and GSH induce conformation change of Hsp90

  19. Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yoon Sik, E-mail: yumshak@naver.com; Seo, Hyun Wook, E-mail: suruk@naver.com; Jung, Guhung, E-mail: drjung@snu.ac.kr

    2015-02-13

    Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study that Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.

  20. Singly protonated dehydronorcantharidin silver coordination polymer induces apoptosis of lung cancer cells via reactive oxygen species-mediated mitochondrial pathway.

    Science.gov (United States)

    Li, Senpeng; Zhang, Shuo; Jin, Xing; Tan, Xuejie; Lou, Jianfang; Zhang, Xiumei; Zhao, Yunxue

    2014-10-30

    Silver complexes have been shown to possess antimicrobial and anticancer properties. Ag-SP-DNC, a novel silver and singly protonated dehydronorcantharidin complex, was synthesized in our previous study. In this study, we offer evidence that Ag-SP-DNC elicits a reactive oxygen species (ROS)-mediated mitochondrial apoptosis in lung cancer cells. Ag-SP-DNC inhibited the growth of A549 cells by inducing G2/M phase cell cycle arrest and apoptosis. Ag-SP-DNC induced apoptosis was associated with the levels of intracellular ROS. The further study revealed that Ag-SP-DNC disrupted the mitochondrial membrane potential, induced the caspase-3 activation and led to the translocation of apoptosis inducing factor and endonucleaseG to the nucleus. These findings have important implications for the development of silver complexes for anticancer applications. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. Single chain variable fragment displaying M13 phage library functionalized magnetic microsphere-based protein equalizer for human serum protein analysis.

    Science.gov (United States)

    Zhu, Guijie; Zhao, Peng; Deng, Nan; Tao, Dingyin; Sun, Liangliang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2012-09-18

    Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.

  2. A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

    DEFF Research Database (Denmark)

    Greibe, Eva Holm; Fedosov, Sergey; Sorensen, Boe S

    2012-01-01

    Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin......-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases......, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding...

  3. Fabrication of enzyme-degradable and size-controlled protein nanowires using single particle nano-fabrication technique

    Science.gov (United States)

    Omichi, Masaaki; Asano, Atsushi; Tsukuda, Satoshi; Takano, Katsuyoshi; Sugimoto, Masaki; Saeki, Akinori; Sakamaki, Daisuke; Onoda, Akira; Hayashi, Takashi; Seki, Shu

    2014-04-01

    Protein nanowires exhibiting specific biological activities hold promise for interacting with living cells and controlling and predicting biological responses such as apoptosis, endocytosis and cell adhesion. Here we report the result of the interaction of a single high-energy charged particle with protein molecules, giving size-controlled protein nanowires with an ultra-high aspect ratio of over 1,000. Degradation of the human serum albumin nanowires was examined using trypsin. The biotinylated human serum albumin nanowires bound avidin, demonstrating the high affinity of the nanowires. Human serum albumin-avidin hybrid nanowires were also fabricated from a solid state mixture and exhibited good mechanical strength in phosphate-buffered saline. The biotinylated human serum albumin nanowires can be transformed into nanowires exhibiting a biological function such as avidin-biotinyl interactions and peroxidase activity. The present technique is a versatile platform for functionalizing the surface of any protein molecule with an extremely large surface area.

  4. Strain typing of Zygosaccharomyces yeast species using a single molecular method based on polymorphism of the intergenic spacer region (IGS).

    Science.gov (United States)

    Wrent, Petra; Rivas, Eva-María; Peinado, José M; de Silóniz, María-Isabel

    2010-08-15

    Unlike previously reported methods that need a combination of several typing techniques, we have developed a single method for strain typing of the Zygosaccharomyces bailii, Z. mellis and Z. rouxii spoilage species. Strains belonging to other species have also been included for comparison. We have demonstrated that the IGS-PCR RFLP method has a high discriminative power. Considering the three endonucleases used in this work, we have obtained a variability of 100% for Z. mellis and Z. rouxii strains and up to 70% for Z. bailii. We have also detected two misidentified Z. mellis strains (CBS 711 and CBS 7412) which have RFLP patterns with a set of bands characteristic of Z. rouxii strains. Sequencing of 26S rDNA D1/D2 domains and the 5.8-ITS rDNA region confirmed these strains as Z. rouxii. The method also groups three certified hybrid strains of Zygosaccharomyces in a separate cluster. Copyright 2010 Elsevier B.V. All rights reserved.

  5. Emendation of the family Chlamydiaceae: proposal of a single genus, Chlamydia, to include all currently recognized species.

    Science.gov (United States)

    Sachse, Konrad; Bavoil, Patrik M; Kaltenboeck, Bernhard; Stephens, Richard S; Kuo, Cho-Chou; Rosselló-Móra, Ramon; Horn, Matthias

    2015-03-01

    The family Chlamydiaceae (order Chlamydiales, phylum Chlamydiae) comprises important, obligate intracellular bacterial pathogens of humans and animals. Subdivision of the family into the two genera Chlamydia and Chlamydophila has been discussed controversially during the past decade. Here, we have revisited the current classification in the light of recent genomic data and in the context of the unique biological properties of these microorganisms. We conclude that neither generally used 16S rRNA sequence identity cut-off values nor parameters based on genomic similarity consistently separate the two genera. Notably, no easily recognizable phenotype such as host preference or tissue tropism is available that would support a subdivision. In addition, the genus Chlamydophila is currently not well accepted and not used by a majority of research groups in the field. Therefore, we propose the classification of all 11 currently recognized Chlamydiaceae species in a single genus, the genus Chlamydia. Finally, we provide emended descriptions of the family Chlamydiaceae, the genus Chlamydia, as well as the species Chlamydia abortus, Chlamydia caviae and Chlamydia felis. Copyright © 2015 Elsevier GmbH. All rights reserved.

  6. Estimating protein-protein interaction affinity in single living cells using Förster resonance energy transfer measurements

    DEFF Research Database (Denmark)

    Jensen, Jens Ledet; Raarup, Merete Krog; Rubak, Ege

    Using Förster resonance energy transfer (FRET) images we study the possibility of estimating the equilibrium dissociation constant Kd and the intrinsic FRET efficiency Em from single cells. We model the measurement uncertainty in the acquired images and use the method of total least squares...

  7. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    Science.gov (United States)

    2011-01-01

    Background The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH) probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. Results As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. Conclusions Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of oral microbiology, as many of

  8. Phylogenetic group- and species-specific oligonucleotide probes for single-cell detection of lactic acid bacteria in oral biofilms

    Directory of Open Access Journals (Sweden)

    Thurnheer Thomas

    2011-01-01

    Full Text Available Abstract Background The purpose of this study was to design and evaluate fluorescent in situ hybridization (FISH probes for the single-cell detection and enumeration of lactic acid bacteria, in particular organisms belonging to the major phylogenetic groups and species of oral lactobacilli and to Abiotrophia/Granulicatella. Results As lactobacilli are known for notorious resistance to probe penetration, probe-specific assay protocols were experimentally developed to provide maximum cell wall permeability, probe accessibility, hybridization stringency, and fluorescence intensity. The new assays were then applied in a pilot study to three biofilm samples harvested from variably demineralized bovine enamel discs that had been carried in situ for 10 days by different volunteers. Best probe penetration and fluorescent labeling of reference strains were obtained after combined lysozyme and achromopeptidase treatment followed by exposure to lipase. Hybridization stringency had to be established strictly for each probe. Thereafter all probes showed the expected specificity with reference strains and labeled the anticipated morphotypes in dental plaques. Applied to in situ grown biofilms the set of probes detected only Lactobacillus fermentum and bacteria of the Lactobacillus casei group. The most cariogenic biofilm contained two orders of magnitude higher L. fermentum cell numbers than the other biofilms. Abiotrophia/Granulicatella and streptococci from the mitis group were found in all samples at high levels, whereas Streptococcus mutans was detected in only one sample in very low numbers. Conclusions Application of these new group- and species-specific FISH probes to oral biofilm-forming lactic acid bacteria will allow a clearer understanding of the supragingival biome, its spatial architecture and of structure-function relationships implicated during plaque homeostasis and caries development. The probes should prove of value far beyond the field of

  9. Mixing state of particles with secondary species by single particle aerosol mass spectrometer in an atmospheric pollution event

    Science.gov (United States)

    Xu, Lingling; Chen, Jinsheng

    2016-04-01

    Single particle aerosol mass spectrometer (SPAMS) was used to characterize size distribution, chemical composition, and mixing state of particles in an atmospheric pollution event during 20 Oct. - 5 Nov., 2015 in Xiamen, Southeast China. A total of 533,012 particle mass spectra were obtained and clustered into six groups, comprising of industry metal (4.5%), dust particles (2.6%), carbonaceous species (70.7%), K-Rich particles (20.7%), seasalt (0.6%) and other particles (0.9%). Carbonaceous species were further divided into EC (70.6%), OC (28.5%), and mixed ECOC (0.9%). There were 61.7%, 58.3%, 4.0%, and 14.6% of particles internally mixed with sulfate, nitrate, ammonium and C2H3O, respectively, indicating that these particles had undergone significant aging processing. Sulfate was preferentially mixed with carbonaceous particles, while nitrate tended to mix with metal-containing and dust particles. Compared to clear days, the fractions of EC-, metal- and dust particles remarkably increased, while the fraction of OC-containing particles decreased in pollution days. The mixing state of particles, excepted for OC-containing particles with secondary species was much stronger in pollution days than that in clear days, which revealed the significant influence of secondary particles in atmospheric pollution. The different activity of OC-containing particles might be related to their much smaller aerodynamic diameter. These results could improve our understanding of aerosol characteristics and could be helpful to further investigate the atmospheric process of particles.

  10. Functionalization of single-walled carbon nanotubes with protein by click chemistry as sensing platform for sensitized electrochemical immunoassay

    International Nuclear Information System (INIS)

    Qi Honglan; Ling Chen; Huang Ru; Qiu Xiaoying; Shangguan Li; Gao Qiang; Zhang Chengxiao

    2012-01-01

    Highlights: ► Single-walled carbon nanotubes were functionalized with protein by click chemistry. ► The SWNTs conjugated with protein showed excellent dispersion in water and kept good bioacitvity. ► A competitive electrochemical immunoassay for the determination of anti-IgG was developed with high sensitivity and good stability. - Abstract: The application of the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition to the functionalization of single-walled carbon nanotubes (SWNTs) with the protein and the use of the artificial SWNTs as a sensing platform for sensitive immunoassay were reported. Covalent functionalization of azide decorated SWNTs with alkyne modified protein was firstly accomplished by the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition. FT-IR spectroscopy, Raman spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy and transmission electron micrograph were used to characterize the protein-functionalized SWNTs. It was found that the SWNTs conjugated with the proteins showed excellent dispersion in water and kept good bioacitivity when immunoglobulin (IgG) and horseradish peroxidase (HRP) were chosen as model proteins. As a proof-of-concept, IgG-functionalized SWNTs were immobilized onto the surface of a glassy carbon electrode by simple casting method as immunosensing platform and a sensitive competitive electrochemical immunoassay was developed for the determination of anti-immunoglobulin (anti-IgG) using HRP as enzyme label. The fabrication of the immunosensor were characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the redox probe [Fe(CN) 6 ] 3−/4− . The SWNTs as immobilization platform showed better sensitizing effect, a detection limit of 30 pg mL −1 (S/N = 3) was obtained for anti-IgG. The proposed strategy provided a stable immobilization method and sensitized recognition platform for analytes. This work demonstrated that the click coupling of SWNTs with protein was an effective

  11. On-line separation of native proteins by two-dimensional liquid chromatography using a single column.

    Science.gov (United States)

    Geng, Xindu; Ke, Congyu; Chen, Gang; Liu, Peng; Wang, Fei; Zhang, Huiqiang; Sun, Xuan

    2009-04-17

    This paper reports the on-line separation of native (N) proteins by two-dimensional liquid chromatography (2D-LC) using a single column with one phase (called 2D column). The 2D column exhibits excellent resolution, selectivity, and retention of proteins in the N state and functions in two retention modes--hydrophobic interaction chromatography (HIC) and weak-cation exchange chromatography (WCX). We describe a new approach to on-line buffer exchange and collection of fractions from the first retention mode and their quantitative re-injection into the same column, followed by re-separation in the second retention mode. Thus, liquid chromatography in a closed system and in an on-line manner could be successfully carried out. This method was termed on-line protein separation by 2D-LC using only a single column (on-line 2D-LC-1C). The applicability of this method was experimentally demonstrated using standard proteins and a human serum sample. The total hypothetical maximum possible peak capacity n(c,total) and total sample peak capacity n(c,total)(*) of the 2D column were 329 and 199, respectively. By comparison against several popular commercially available columns, it was found that the 2D column had not only comparable resolution and better selectivity but also some unique characteristics. This 2D-LC-1C method could be applied to the fast purification of intact proteins in the N state, such protein drugs from natural products, and recombinant proteins and also for the fast pre-fractionation of intact proteins in the "top-down" MS strategy in proteomics.

  12. Molecular characterization and phylogenetic analysis of three odorant binding protein gene transcripts in Dendrolimus species (Lepidoptera: Lasiocampidae).

    Science.gov (United States)

    Zhang, Su-Fang; Zhang, Zhen; Kong, Xiang-Bo; Wang, Hong-Bin

    2014-10-01

    Pine caterpillar moths, Dendrolimus spp. (Lepidoptera: Lasiocampidae), are serious economic pest of pines. Previously, phylogenetic analyses of Dendrolimus using different methods yielded inconsistent results. The chemosensory systems of insects may play fundamental roles in promoting speciation. Odorant-binding proteins (OBPs) participate in the first step of odor detection. Studying the evolution of OBPs in closely related species may help us to identify their role in speciation. We identified three OBPs - one pheromone-binding protein and two general odorant-binding proteins - from male antennae of four Dendrolimus species, D. superans (Butler), D. punctatus (Walker), D. kikuchii Matsumura, and D. houi Lajonquiere, the olfactory recognition systems of which had not been previously investigated. We analyzed their molecular characteristics and compared their sequences to those of OBPs in D. tabulaeformis Tsai et Liu. Ka/Ks ratio analyses among the five Dendrolimus species indicate that PBP1 genes experienced more evolutionary pressure than the GOBPs. Phylogenetic relationships of PBP1 and GOBP1 both indicated that D. houi was the basal species, then branched D. kikuchii, while D. tabulaeformis, D. punctatus, and D. superans evolved more recently. These relationships are consistent with the changes in sex pheromone components of these five species. Dendrolimus tabulaeformis and D. punctatus are closely related sister species. However, the distances among GOBP2 sequences in the five Dendrolimus were very short, and the relationships of D. houi and D. kikuchii could not be resolved. Integrating our results with those of previous studies, we hypothesized that D. kikuchii, D. punctatus and D. superans evolved from the basal ancestor because of sex pheromone mutations and environmental pressure. © 2013 Institute of Zoology, Chinese Academy of Sciences.

  13. Single nucleotide polymorphism analysis of ubiquitin extension protein genes (ubq) of gossypium arboreum and gossypium herbaceum in comparison with arabidopsis thaliana

    International Nuclear Information System (INIS)

    Shaheen, T.; Zafar, Y.; Rahman, M.

    2014-01-01

    Single nucleotide polymorphism analysis is an expedient way to study polymorphisms at genomic level. In the present study we have explored Ubiquitin extension protein gene of G. arboreum (A2) and G. herbaceum (A1) of cotton which is a multiple copy gene. We have found SNPs at 16 positions in 200 bp region within A genome of cotton indicating frequency of SNPs 1/13 bp. Both sequences from cotton have shown maximum similarity with UBQ5 and UBQ6 of Arabidopsis thaliana. Sequence obtained from G. arboreum has shown SNPs at 28 positions in comparison with each UBQ5 and UBQ6 of Arabidopsis thaliana while sequence obtained from G. herbaceum has shown SNPs at 31 positions in comparison with each UBQ5 and UBQ6 of Arabidopsis thaliana. In conclusion although during pace of evolution ubiquitin extension protein genes of both A genome species have got some mutations from nature but still most of their sequence is similar. Single nucleotide polymorphism study can prove a vital tool to identify gene type in case of Multicopy genes. (author)

  14. Effect of single-point sequence alterations on the aggregationpropensity of a model protein

    Energy Technology Data Exchange (ETDEWEB)

    Bratko, Dusan; Cellmer, Troy; Prausnitz, John M.; Blanch, Harvey W.

    2005-10-07

    Sequences of contemporary proteins are believed to have evolved through process that optimized their overall fitness including their resistance to deleterious aggregation. Biotechnological processing may expose therapeutic proteins to conditions that are much more conducive to aggregation than those encountered in a cellular environment. An important task of protein engineering is to identify alternative sequences that would protect proteins when processed at high concentrations without altering their native structure associated with specific biological function. Our computational studies exploit parallel tempering simulations of coarse-grained model proteins to demonstrate that isolated amino-acid residue substitutions can result in significant changes in the aggregation resistance of the protein in a crowded environment while retaining protein structure in isolation. A thermodynamic analysis of protein clusters subject to competing processes of folding and association shows that moderate mutations can produce effects similar to those caused by changes in system conditions, including temperature, concentration, and solvent composition that affect the aggregation propensity. The range of conditions where a protein can resist aggregation can therefore be tuned by sequence alterations although the protein generally may retain its generic ability for aggregation.

  15. Effects of sediment-spiked lufenuron on benthic macroinvertebrates in outdoor microcosms and single-species toxicity tests.

    Science.gov (United States)

    Brock, T C M; Bas, D A; Belgers, J D M; Bibbe, L; Boerwinkel, M-C; Crum, S J H; Diepens, N J; Kraak, M H S; Vonk, J A; Roessink, I

    2016-08-01

    Sediment ecotoxicity studies were conducted with lufenuron to (i) complement the results of a water-spiked mesocosm experiment with this lipophilic benzoylurea insecticide, (ii) to explore the predictive value of laboratory single-species tests for population and community-level responses of benthic macroinvertebrates, and (iii) to calibrate the tier-1 effect assessment procedure for sediment organisms. For this purpose the concentration-response relationships for macroinvertebrates between sediment-spiked microcosms and those of 28-d sediment-spiked single-species toxicity tests with Chironomus riparius, Hyalella azteca and Lumbriculus variegatus were compared. Lufenuron persisted in the sediment of the microcosms. On average, 87.7% of the initial lufenuron concentration could still be detected in the sediment after 12 weeks. Overall, benthic insects and crustaceans showed treatment-related declines and oligochaetes treatment-related increases. The lowest population-level NOEC in the microcosms was 0.79μg lufenuron/g organic carbon in dry sediment (μg a.s./g OC) for Tanytarsini, Chironomini and Dero sp. Multivariate analysis of the responses of benthic macroinvertebrates revealed a community-level NOEC of 0.79μg a.s./g OC. The treatment-related responses observed in the microcosms are in accordance with the results of the 28-d laboratory toxicity tests. These tests showed that the insect C. riparius and the crustacean H. azteca were approximately two orders of magnitude more sensitive than the oligochaete L. variegatus. In our laboratory tests, using field-collected sediment, the lowest 28-d EC10 (0.49μg a.s./g OC) was observed for C. riparius (endpoint survival), while for the standard OECD test with this species, using artificial sediment, a NOEC of 2.35μg a.s./g OC (endpoint emergence) is reported. In this particular case, the sediment tier-1 effect assessment using the chronic EC10 (field-collected sediment) or chronic NOEC (artificial sediment) of C

  16. A first-principles model of early evolution: emergence of gene families, species, and preferred protein folds.

    Directory of Open Access Journals (Sweden)

    Konstantin B Zeldovich

    2007-07-01

    Full Text Available In this work we develop a microscopic physical model of early evolution where phenotype--organism life expectancy--is directly related to genotype--the stability of its proteins in their native conformations-which can be determined exactly in the model. Simulating the model on a computer, we consistently observe the "Big Bang" scenario whereby exponential population growth ensues as soon as favorable sequence-structure combinations (precursors of stable proteins are discovered. Upon that, random diversity of the structural space abruptly collapses into a small set of preferred proteins. We observe that protein folds remain stable and abundant in the population at timescales much greater than mutation or organism lifetime, and the distribution of the lifetimes of dominant folds in a population approximately follows a power law. The separation of evolutionary timescales between discovery of new folds and generation of new sequences gives rise to emergence of protein families and superfamilies whose sizes are power-law distributed, closely matching the same distributions for real proteins. On the population level we observe emergence of species--subpopulations that carry similar genomes. Further, we present a simple theory that relates stability of evolving proteins to the sizes of emerging genomes. Together, these results provide a microscopic first-principles picture of how first-gene families developed in the course of early evolution.

  17. Fluorescence of the single tryptophan of cutinase: temperature and pH effect on protein conformation and dynamics.

    Science.gov (United States)

    Martinho, J M G; Santos, A M; Fedorov, A; Baptista, R P; Taipa, M A; Cabral, J M S

    2003-07-01

    The cutinase from Fusarium solani pisi is an enzyme with a single L-tryptophan (Trp) involved in a hydrogen bond with an alanine (Ala) residue and located close to a cystine formed by a disulfide bridge between two cysteine (Cys) residues. The Cys strongly quenches the fluorescence of Trp by both static and dynamic quenching mechanisms. The Trp fluorescence intensity increases by about fourfold on protein melting because of the disruption of the Ala-Trp hydrogen bond that releases the Trp from the vicinity of the cystine residue. The Trp forms charge-transfer complexes with the disulfide bridge, which is disrupted by UV light irradiation of the protein. This results in a 10-fold increase of the Trp fluorescence quantum yield because of the suppression of the static quenching by the cystine residue. The Trp fluorescence anisotropy decays are similar to those in other proteins and were interpreted in terms of the wobbling-in-cone model. The long relaxation time is attributed to the Brownian rotational correlation time of the protein as a whole below the protein-melting temperature and to protein-backbone dynamics above it. The short relaxation time is related to the local motion of the Trp, whose mobility increases on protein denaturation.

  18. Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

    Energy Technology Data Exchange (ETDEWEB)

    Mao, Lili; Inoue, Koichi [Robert Wood Johnson Medical School, Department of Biochemistry, Center for Advanced Biotechnology and Medicine (United States); Tao, Yisong [Columbia University, Department of Chemistry (United States); Montelione, Gaetano T. [Robert Wood Johnson Medical School, Department of Biochemistry, Center for Advanced Biotechnology and Medicine (United States); McDermott, Ann E. [Columbia University, Department of Chemistry (United States); Inouye, Masayori, E-mail: inouye@umdnj.edu [Robert Wood Johnson Medical School, Department of Biochemistry, Center for Advanced Biotechnology and Medicine (United States)

    2011-02-15

    Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that {sup 13}C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that {sup 13}C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

  19. Suppression of phospholipid biosynthesis by cerulenin in the condensed Single-Protein-Production (cSPP) system

    International Nuclear Information System (INIS)

    Mao, Lili; Inoue, Koichi; Tao, Yisong; Montelione, Gaetano T.; McDermott, Ann E.; Inouye, Masayori

    2011-01-01

    Using the single-protein-production (SPP) system, a protein of interest can be exclusively produced in high yield from its ACA-less gene in Escherichia coli expressing MazF, an ACA-specific mRNA interferase. It is thus feasible to study a membrane protein by solid-state NMR (SSNMR) directly in natural membrane fractions. In developing isotope-enrichment methods, we observed that 13 C was also incorporated into phospholipids, generating spurious signals in SSNMR spectra. Notable, with the SPP system a protein can be produced in total absence of cell growth caused by antibiotics. Here, we demonstrate that cerulenin, an inhibitor of phospholipid biosynthesis, can suppress isotope incorporation in the lipids without affecting membrane protein yield in the SPP system. SSNMR analysis of ATP synthase subunit c, an E. coli inner membrane protein, produced by the SPP method using cerulenin revealed that 13 C resonance signals from phospholipid were markedly reduced, while signals for the isotope-enriched protein were clearly present.

  20. Effects of sediment-spiked lufenuron on benthic macroinvertebrates in outdoor microcosms and single-species toxicity tests

    Energy Technology Data Exchange (ETDEWEB)

    Brock, T.C.M., E-mail: theo.brock@wur.nl [Alterra, Wageningen University and Research Centre, P.O. Box 47, 6700 AA Wageningen (Netherlands); Bas, D.A. [Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (Netherlands); Belgers, J.D.M. [Alterra, Wageningen University and Research Centre, P.O. Box 47, 6700 AA Wageningen (Netherlands); Bibbe, L. [Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (Netherlands); Boerwinkel, M-C.; Crum, S.J.H. [Alterra, Wageningen University and Research Centre, P.O. Box 47, 6700 AA Wageningen (Netherlands); Diepens, N.J. [Department of Aquatic Ecology and Water Quality Management, Wageningen University, P.O. Box 47, 6700 AA Wageningen (Netherlands); Kraak, M.H.S.; Vonk, J.A. [Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam (Netherlands); Roessink, I. [Alterra, Wageningen University and Research Centre, P.O. Box 47, 6700 AA Wageningen (Netherlands)

    2016-08-15

    Highlights: • In outdoor microcosms constructed with lufenuron-spiked sediment we observed that this insecticide persistent in the sediment compartment. • Sediment exposure to lufenuron caused population-level declines (insects and crustaceans) and increases (mainly oligochaete worms) of benthic invertebrates. • The direct and indirect effects observed in the microcosms were supported by results of sediment-spiked single species tests with Chironomus riparius, Hyalella azteca and Lumbriculus variegatus. • The tier-1 effect assessment procedure for sediment organisms recommended by the European Food Safety Authority is protective for the treatment-related responses observed in the microcosm test. - Abstract: Sediment ecotoxicity studies were conducted with lufenuron to (i) complement the results of a water-spiked mesocosm experiment with this lipophilic benzoylurea insecticide, (ii) to explore the predictive value of laboratory single-species tests for population and community-level responses of benthic macroinvertebrates, and (iii) to calibrate the tier-1 effect assessment procedure for sediment organisms. For this purpose the concentration-response relationships for macroinvertebrates between sediment-spiked microcosms and those of 28-d sediment-spiked single-species toxicity tests with Chironomus riparius, Hyalella azteca and Lumbriculus variegatus were compared. Lufenuron persisted in the sediment of the microcosms. On average, 87.7% of the initial lufenuron concentration could still be detected in the sediment after 12 weeks. Overall, benthic insects and crustaceans showed treatment-related declines and oligochaetes treatment-related increases. The lowest population-level NOEC in the microcosms was 0.79 μg lufenuron/g organic carbon in dry sediment (μg a.s./g OC) for Tanytarsini, Chironomini and Dero sp. Multivariate analysis of the responses of benthic macroinvertebrates revealed a community-level NOEC of 0.79 μg a.s./g OC. The treatment

  1. Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein.

    Science.gov (United States)

    Arpino, James A J; Rizkallah, Pierre J; Jones, D Dafydd

    2014-08-01

    Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFP(D190Δ) containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFP(A227Δ) revealed that a `flipping' mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.

  2. Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell-cell interaction

    Directory of Open Access Journals (Sweden)

    Minsuk eKwak

    2013-02-01

    Full Text Available Secreted proteins including cytokines, chemokines and growth factors represent important functional regulators mediating a range of cellular behavior and cell-cell paracrine/autocrine signaling, e.g. in the immunological system, tumor microenvironment or stem cell niche. Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically-identical cell population can give rise to diverse phenotypic differences. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this Perspective Article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer.

  3. Optimal conditions for decorating outer surface of single-walled carbon nanotubes with RecA proteins

    Science.gov (United States)

    Oura, Shusuke; Umemura, Kazuo

    2016-03-01

    In this study, we estimated the optimal reaction conditions for decorating the outer surface of single-walled carbon nanotubes (SWNTs) with RecA proteins by comparison with hybrids of RecA and single-stranded DNA (ssDNA). To react SWNTs with RecA proteins, we first prepared ssDNA-SWNT hybrids. The heights of the ssDNA-SWNT hybrids increased as the amount of RecA used in the reaction increased, as determined from atomic force microscopy images. We further confirmed the increasing adsorption of RecA proteins onto ssDNA on SWNT surfaces by agarose gel electrophoresis. These results suggest that the combination of RecA proteins and ssDNA-SWNT hybrids forms RecA-ssDNA-SWNT hybrids. We also successfully controlled the amount of RecA adsorbed on the ssDNA-SWNT hybrids. Our results thus indicate the optimized reaction conditions for decorating the outer surface of SWNTs with RecA proteins, which is the key to the development of novel biosensors and nanomaterial-based bioelectronics.

  4. Induction and evaluation of mutations for improved protein production in certain species of yeasts in the Philippines

    International Nuclear Information System (INIS)

    Borromeo, J.D.

    1976-02-01

    The species of yeasts included in the studies are Saccharomyces cerevisiae, Rhodeterula rubra, Rhodeterula pilimane and those isolated from fruits such as citrus, papaya and banana. Part of the project involved induction of sporulation to obtain haploid cells for crossing to produce stable disploids exhibiting improved protein production. Although S. cerevisiae produce less protein than Rhodeterula, it produces ascesperes which are haploid cells. These haploid cells can be used to obtain stable diploids with the desirable characteristics by crossing cultures. Rhodeterula, a fungus that does not produce ascesperes will be subjected to certain adverse conditions to induce, hopefully, sperulation

  5. Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

    Science.gov (United States)

    Gopaul, Krishna K; Sells, Jessica; Lee, Robin; Beckstrom-Sternberg, Stephen M; Foster, Jeffrey T; Whatmore, Adrian M

    2014-12-11

    The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

  6. Single Particle Tracking to Characterize the Mechanism of Pore Formation by Pore Forming Proteins

    OpenAIRE

    Subburaj, Yamunadevi

    2014-01-01

    Pore formation is a common natural mechanism occurring in large number of organisms where proteins are involved as toxins, effectors in immune response or apoptosis. Despite intense research, the structural and dynamic aspects of oligomerization and membrane permeabilization by pore forming proteins remains poorly understood. In this work we have aimed to provide a better understanding on dynamics, oligomerization and pore forming process of two proteins; a) Equinatoxin II, b) Bax (Bcl2 famil...

  7. A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response. | Office of Cancer Genomics

    Science.gov (United States)

    Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis.

  8. Imatinib (Gleevec@) conformations observed in single crystals, protein-Imatinib co-crystals and molecular dynamics: Implications for drug selectivity

    Science.gov (United States)

    Golzarroshan, B.; Siddegowda, M. S.; Li, Hong qi; Yathirajan, H. S.; Narayana, B.; Rathore, R. S.

    2012-06-01

    Structure and dynamics of the Leukemia drug, Imatinib, were examined using X-ray crystallography and molecular dynamics studies. Comparison of conformations observed in single crystals with several reported co-crystals of protein-drug complexes suggests existence of two conserved conformations of Imatinib, extended and compact (or folded), corresponding to two binding modes of interaction with the receptor. Furthermore, these conformations are conserved throughout a dynamics simulation. The present study attempts to draw a parallel on conformations and binding patterns of interactions, obtained from small-molecule single-crystal and macromolecule co-crystal studies, and provides structural insights for understanding the high selectivity of this drug molecule.

  9. Validation of a single biopsy approach and bolus protein feeding to determine myofibrillar protein synthesis in stable isotope tracer studies in humans

    Directory of Open Access Journals (Sweden)

    Baker Steven K

    2011-03-01

    Full Text Available Abstract Background Minimizing the number of muscle biopsies has important methodological implications and minimizes subject discomfort during a stable isotope amino acid infusion. We aimed to determine the reliability of obtaining a single muscle biopsy for the calculation of muscle protein fractional synthetic rate (FSR as well as the amount of incorporation time necessary to obtain that biopsy after initiating a stable isotope infusion (Study 1. The calculation of muscle protein FSR requires tracer steady-state during the stable isotope infusion. Therefore, a second aim was to examine if steady-state conditions are compromised in the precursor pools (plasma free or muscle intracellular [IC] after ingestion of a tracer enriched protein drink and after resistance exercise (Study 2. Methods Sixteen men (23 ± 3 years; BMI = 23.8 ± 2.2 kg/m2, means ± SD were randomized to perform Study 1 or Study 2 (n = 8, per study. Subjects received a primed, constant infusion of L-[ring-13C6]phenylalanine coupled with muscle biopsies of the vastus lateralis to measure rates of myofibrillar protein synthesis (MPS. Subjects in Study 2 were fed 25 g of whey protein immediately after an acute bout of unilateral resistance exercise. Results There was no difference (P = 0.3 in rates of MPS determined using the steady-state precursor-product equation and determination of tracer incorporation between sequential biopsies 150 min apart or using plasma protein as the baseline enrichment, provided the infusion length was sufficient (230 ± 0.3 min. We also found that adding a modest amount of tracer (4% enriched, calculated based on the measured phenylalanine content of the protein (3.5% in the drink, did not compromise steady-state conditions (slope of the enrichment curve not different from zero in the plasma free or, more importantly, the IC pool (both P > 0.05. Conclusions These data demonstrate that the single biopsy approach yields comparable rates of muscle

  10. Validation of a single biopsy approach and bolus protein feeding to determine myofibrillar protein synthesis in stable isotope tracer studies in humans.

    Science.gov (United States)

    Burd, Nicholas A; West, Daniel Wd; Rerecich, Tracy; Prior, Todd; Baker, Steven K; Phillips, Stuart M

    2011-03-09

    Minimizing the number of muscle biopsies has important methodological implications and minimizes subject discomfort during a stable isotope amino acid infusion. We aimed to determine the reliability of obtaining a single muscle biopsy for the calculation of muscle protein fractional synthetic rate (FSR) as well as the amount of incorporation time necessary to obtain that biopsy after initiating a stable isotope infusion (Study 1). The calculation of muscle protein FSR requires tracer steady-state during the stable isotope infusion. Therefore, a second aim was to examine if steady-state conditions are compromised in the precursor pools (plasma free or muscle intracellular [IC]) after ingestion of a tracer enriched protein drink and after resistance exercise (Study 2). Sixteen men (23 ± 3 years; BMI = 23.8 ± 2.2 kg/m2, means ± SD) were randomized to perform Study 1 or Study 2 (n = 8, per study). Subjects received a primed, constant infusion of L-[ring-13C6]phenylalanine coupled with muscle biopsies of the vastus lateralis to measure rates of myofibrillar protein synthesis (MPS). Subjects in Study 2 were fed 25 g of whey protein immediately after an acute bout of unilateral resistance exercise. There was no difference (P = 0.3) in rates of MPS determined using the steady-state precursor-product equation and determination of tracer incorporation between sequential biopsies 150 min apart or using plasma protein as the baseline enrichment, provided the infusion length was sufficient (230 ± 0.3 min). We also found that adding a modest amount of tracer (4% enriched), calculated based on the measured phenylalanine content of the protein (3.5%) in the drink, did not compromise steady-state conditions (slope of the enrichment curve not different from zero) in the plasma free or, more importantly, the IC pool (both P > 0.05). These data demonstrate that the single biopsy approach yields comparable rates of muscle protein synthesis, provided a longer incorporation time is

  11. Intrinsic structural differences in the N-terminal segment of pulmonary surfactant protein SP-C from different species

    DEFF Research Database (Denmark)

    Plasencia, I; Rivas, L; Casals, C

    2001-01-01

    Predictive studies suggest that the known sequences of the N-terminal segment of surfactant protein SP-C from animal species have an intrinsic tendency to form beta-turns, but there are important differences on the probable location of these motifs in different SP-C species. Our hypothesis...... is that intrinsic structural determinants of the sequence of the N-terminal region of SP-C could define conformation, acylation and perhaps surface properties of the mature protein. To test this hypothesis we have synthesized peptides corresponding to the 13-residue N-terminal sequence of porcine and canine SP......-C, and studied their structural behaviour in solution and in phospholipid bilayers and monolayers. In these peptides, leucine at position 1 of both sequences has been replaced by tryptophan in order to allow their study by fluorescence spectroscopy. Far-u.v. circular dichroism spectra of the peptides in aqueous...

  12. Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

    Science.gov (United States)

    Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

    2010-10-01

    Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

  13. Control of transmembrane protein diffusion within the postsynaptic density assessed by simultaneous single-molecule tracking and localization microscopy

    Directory of Open Access Journals (Sweden)

    Thomas A Blanpied

    2016-07-01

    Full Text Available Postsynaptic transmembrane proteins are critical elements of synapses, mediating trans-cellular contact, sensitivity to neurotransmitters and other signaling molecules, and flux of Ca and other ions. Positioning and mobility of each member of this large class of proteins is critical to their individual function at the synapse. One critical example is that the position of glutamate receptors within the postsynaptic density (PSD strongly modulates their function by aligning or misaligning them with sites of presynaptic vesicle fusion. In addition, the regulated ability of receptors to move in or out of the synapse is critical for activity-dependent plasticity. However, factors that control receptor mobility within the boundaries of the synapse are not well understood. Notably, PSD scaffold molecules accumulate in domains much smaller than the synapse. Within these nanodomains, the density of proteins is considerably higher than that of the synapse as a whole, so high that steric hindrance is expected to reduce receptor mobility substantially. However, while numerical modeling has demonstrated several features of how the varying protein density across the face of a single PSD may modulate receptor motion, there is little experimental information about the extent of this influence. To address this critical aspect of synaptic organizational dynamics, we performed single-molecule tracking of transmembrane proteins using uPAINT over PSDs whose internal structure was simultaneously resolved using PALM. The results provide important experimental confirmation that PSD scaffold density protein strongly influences the mobility of transmembrane proteins. Tracking a protein with a cytosolic domain that does not bind PSD-95 still was slowed in regions of high PSD-95 density, suggesting that crowding by scaffold molecules and perhaps other proteins is sufficient to stabilize receptors even in the absence of binding. Because numerous proteins thought to be

  14. Measuring titratable alkalinity by single versus double endpoint titration: An evaluation in two cyprinodont species and implications for characterizing net H+ flux in aquatic organisms.

    Science.gov (United States)

    Brix, Kevin V; Wood, Chris M; Grosell, Martin

    2013-01-01

    In this study, Na(+) uptake and acid-base balance in the euryhaline pupfish Cyprinodon variegatus variegatus were characterized when fish were exposed to pH 4.5 freshwater (7mM Na(+)). Similar to the related cyprinodont, Fundulus heteroclitus, Na(+) uptake was significantly inhibited when exposed to low pH water. However, it initially appeared that C. v. variegatus increased apparent net acid excretion at low pH relative to circumneutral pH. This result is opposite to previous observations for F. heteroclitus under similar conditions where fish were observed to switch from apparent net H(+) excretion at circumneutral pH to apparent net H(+) uptake at low pH. Further investigation revealed disparate observations between these studies were the result of using double endpoint titrations to measure titratable alkalinity fluxes in the current study, while the earlier study utilized single endpoint titrations to measure these fluxes (i.e.,. Cyprinodon acid-base transport is qualitatively similar to Fundulus when characterized using single endpoint titrations). This led to a comparative investigation of these two methods. We hypothesized that either the single endpoint methodology was being influenced by a change in the buffer capacity of the water (e.g., mucus being released by the fish) at low pH, or the double endpoint methodology was not properly accounting for ammonia flux by the fish. A series of follow-up experiments indicated that buffer capacity of the water did not change significantly, that excretion of protein (a surrogate for mucus) was actually reduced at low pH, and that the double endpoint methodology does not properly account for NH(3) excretion by fish under low pH conditions. As a result, it overestimates net H(+) excretion during low pH exposure. After applying the maximum possible correction for this error (i.e., assuming that all ammonia is excreted as NH(3)), the double endpoint methodology indicates that net H(+) transport was reduced to

  15. Role of reactive oxygen species and Bcl-2 family proteins in TNF-α-induced apoptosis of lymphocytes.

    Science.gov (United States)

    Ryazanceva, N V; Novickiy, V V; Zhukova, O B; Biktasova, A K; Chechina, O E; Sazonova, E V; Belkina, M V; Chasovskih, N Yu; Khaitova, Z K

    2010-08-01

    We studied the in vitro apoptosis-inducing effect of recombinant TNF-α (rTNF-α) on blood lymphocytes from healthy donors. rTNF-α-induced apoptosis was accompanied by an increase in the number of cells with low mitochondrial transmembrane potential, increased intracellular content of reactive oxygen species, reduced content of Bcl-2, Bcl-xL, and Bax proteins, and elevated Bad content. The molecular mechanisms of these changes are discussed.

  16. Efficient Fluorescence Based Protein Chip using Pseudo 3D Single-Walled Carbon Nanotube Film

    Science.gov (United States)

    2006-05-22

    due to its size and common biochemical nature as a protein, but also because of the complexity in pre- and post -treatment steps, such as protecting...by the software provided by ImaGene ®. Cross reactivity was also investigated by immersing a substrate containing the same arrays of probe proteins

  17. Single-molecule analysis of ligand efficacy in beta(2)AR-G-protein activation

    DEFF Research Database (Denmark)

    Gregorio, G. Glenn; Masureel, Matthieu; Hilger, Daniel

    2017-01-01

    G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiology and disease intervention, yet the molecular mechanisms responsible for ligand-dependent signalling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail ...

  18. Single protein omission reconstitution studies of tetracycline binding to the 30S subunit of Escherichia coli ribosomes

    International Nuclear Information System (INIS)

    Buck, M.; Cooperman, B.S.

    1990-01-01

    In previous work the authors showed that on photolysis of Escherichia coli ribosomes in the presence of [ 3 H]tetracycline (TC) the major protein labeled is S7, and they presented strong evidence that such labeling takes place from a high-affinity site related to the inhibitory action of TC. In this work they use single protein omission reconstitution (SPORE) experiments to identify those proteins that are important for high-affinity TC binding to the 30S subunit, as measured by both cosedimentation and filter binding assays. With respect to both sedimentation coefficients and relative Phe-tRNA Phe binding, the properties of the SPORE particles they obtain parallel very closely those measured earlier, with the exception of the SPORE particle lacking S13. A total of five proteins, S3, S7, S8, S14, and S19, are shown to be important for TC binding, with the largest effects seen on omission of proteins S7 and S14. Determination of the protein compositions of the corresponding SPORE particles demonstrates that the observed effects are, for the most part, directly attributable to the omission of the given protein rather than reflecting an indirect effect of omitting one protein on the uptake of another. A large body of evidence supports the notion that four of these proteins, S3, S7, S14, and S19, are included, along with 16S rRNA bases 920-1,396, in one of the major domains of the 30S subunit. The results support the conclusion that the structure of this domain is important for the binding of TC and that, within this domain, TC binds directly to S7

  19. Structure of a second crystal form of Bence-Jones protein Loc: Strikingly different domain associations in two crystal forms of a single protein

    International Nuclear Information System (INIS)

    Schiffer, M.; Ainsworth, C.; Xu, Z.B.; Carperos, W.; Olsen, K.; Solomon, A.; Stevens, F.J.; Chang, C.H.

    1989-01-01

    The authors have determined the structure of the immunoglobulin light-chain dimer Loc in a second crystal form that was grown from distilled water. The crystal structure was determined to 2.8-angstrom resolution; the R factor is 0.22. The two variable domains are related by local 2-fold axes and form an antigen binding pocket. The variable domain-variable domain interaction observed in this crystal form differs from the one exhibited by the protein when crystallized from ammonium sulfate in which the two variable domains formed a protrusion. The structure attained in the distilled water crystals is similar to, but not identical with, the one observed for the Mcg light-chain dimer in crystals grown from ammonium sulfate. Thus, two strikingly different structures were attained by this multisubunit protein in crystals grown under two different, commonly used, crystallization techniques. The quaternary interactions exhibited by the protein in the two crystal forms are sufficiently different to suggest fundamentally different interpretations of the structural basis for the function of this protein. This observation may have general implications regarding the use of single crystallographic determinations for detailed identification of structural and functional relationships. On the other hand, proteins whose structures can be altered by manipulation of crystallization conditions may provide useful systems for study of fundamental structural chemistry

  20. Assessment of nutritional value of single-cell protein from waste-activated sludge as a protein supplement in poultry feed.

    Science.gov (United States)

    Nkhalambayausi-Chirwa, Evans M; Lebitso, Moses T

    2012-12-01

    The amount of protein wasted through sludge in Gauteng, South Africa, amounts to 95 000 metric tonne/yr, with the order of magnitude of the national protein requirement of approximately 145 000 metric tonne/yr. Waste-activated sludge (WAS) from wastewater treatment plants (WWTPs) that treat domestic wastewater contains protein in a ratio of 2:1 against fishmeal. This protein source has not been utilized because of the high content of toxic heavy metals and other potential carcinogenic pollutants in the sludge. In this study, a pretreatment method of modified aqua regia dilute acid wash was used to lower the metal content by approximately 60%. However, this resulted in a 33% loss of amino acids in the acid-washed WAS. A feed substitution test in poultry with different fishmeal-sludge ratios (0%, 25%, 50%, 75%, and 100% WAS as percent substitution of fishmeal) showed no impact of sludge single-cell protein (SCP) on mortality rate. However, sludge substitution in the feed yielded weight gains and cost savings up to 46%.

  1. Protein hydrogen exchange measured at single-residue resolution by electron transfer dissociation mass spectrometry

    DEFF Research Database (Denmark)

    Rand, Kasper D; Zehl, Martin; Jensen, Ole Nørregaard

    2009-01-01

    Because of unparalleled sensitivity and tolerance to protein size, mass spectrometry (MS) has become a popular method for measuring the solution hydrogen (1H/2H) exchange (HX) of biologically relevant protein states. While incorporated deuterium can be localized to different regions by pepsin...... proteolysis of the labeled protein, the assignment of deuteriums to individual residues is typically not obtained, thereby limiting a detailed understanding of HX and the dynamics of protein structure. Here we use gas-phase fragmentation of peptic peptides by electron transfer dissociation (ETD) to measure...... the HX of individual amide linkages in the amyloidogenic protein beta2-microglobulin. A comparison of the deuterium levels of 60 individual backbone amides of beta2-microglobulin measured by HX-ETD-MS analysis to the corresponding values measured by NMR spectroscopy shows an excellent correlation...

  2. Estimation of extractable protein in botanical fractions of legume and grass species

    DEFF Research Database (Denmark)

    Solati, Zeinab; Jørgensen, Uffe; Eriksen, Jørgen

    2018-01-01

    With a globally strong interest in bio-based products such as fuels and chemicals, a feasible source of protein for the industry with positive economic impacts could be from leaves. However, more knowledge is needed on how to improve the content of extractable protein. Grasses and legumes have a ...

  3. Introduction to Biological Mass Spectroscopy: Determining Identity and Species of Origin of Two Proteins

    Science.gov (United States)

    Reimann, Curt T.; Mie, Axel; Nilsson, Carina; Cohen, Arieh

    2005-01-01

    An examination of the two proteins, namely, cytochrome c from horse and cow is conducted and it is indicated that cytochrome c is a mitochondrial protein. Mitochondria multiply by cell division and do not undergo sexual reproduction and mitochondria DNA is passed on via the mitochondria that are inherited from the female parent organism.

  4. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each...

  5. Species Differences in the Carbohydrate Binding Preferences of Surfactant Protein D

    DEFF Research Database (Denmark)

    Crouch, Erika C.; Smith, Kelly; McDonald, Barbara

    2006-01-01

    Interactions of surfactant protein D (SP-D) with micro-organisms and organic antigens involve binding to the trimeric neck plus carbohydrate recognition domain (neck+CRD). In these studies, we compared the ligand binding of homologous human, rat, and mouse trimeric neck+CRD fusion proteins, each ...

  6. An integrated protein localization and interaction map for Potato yellow dwarf virus, type species of the genus Nucleorhabdovirus

    International Nuclear Information System (INIS)

    Bandyopadhyay, Anindya; Kopperud, Kristin; Anderson, Gavin; Martin, Kathleen; Goodin, Michael

    2010-01-01

    The genome of Potato yellow dwarf virus (PYDV; Nucleorhabdovirus type species) was determined to be 12,875 nucleotides (nt). The antigenome is organized into seven open reading frames (ORFs) ordered 3'-N-X-P-Y-M-G-L-5', which likely encode the nucleocapsid, phospho, movement, matrix, glyco and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. The ORFs are flanked by a 3' leader RNA of 149 nt and a 5' trailer RNA of 97 nt, and are separated by conserved intergenic junctions. Phylogenetic analyses indicated that PYDV is closely related to other leafhopper-transmitted rhabdoviruses. Functional protein assays were used to determine the subcellular localization of PYDV proteins. Surprisingly, the M protein was able to induce the intranuclear accumulation of the inner nuclear membrane in the absence of any other viral protein. Finally, bimolecular fluorescence complementation was used to generate the most comprehensive protein interaction map for a plant-adapted rhabdovirus to date.

  7. A Single Protein S-acyl Transferase Acts through Diverse Substrates to Determine Cryptococcal Morphology, Stress Tolerance, and Pathogenic Outcome.

    Directory of Open Access Journals (Sweden)

    Felipe H Santiago-Tirado

    2015-05-01

    Full Text Available Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter's opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT, one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics.

  8. Single-stranded DNA-binding protein recruits DNA polymerase V to primer termini on RecA-coated DNA.

    Science.gov (United States)

    Arad, Gali; Hendel, Ayal; Urbanke, Claus; Curth, Ute; Livneh, Zvi

    2008-03-28

    Translesion DNA synthesis (TLS) by DNA polymerase V (polV) in Escherichia coli involves accessory proteins, including RecA and single-stranded DNA-binding protein (SSB). To elucidate the role of SSB in TLS we used an in vitro exonuclease protection assay and found that SSB increases the accessibility of 3' primer termini located at abasic sites in RecA-coated gapped DNA. The mutant SSB-113 protein, which is defective in protein-protein interactions, but not in DNA binding, was as effective as wild-type SSB in increasing primer termini accessibility, but deficient in supporting polV-catalyzed TLS. Consistently, the heterologous SSB proteins gp32, encoded by phage T4, and ICP8, encoded by herpes simplex virus 1, could replace E. coli SSB in the TLS reaction, albeit with lower efficiency. Immunoprecipitation experiments indicated that polV directly interacts with SSB and that this interaction is disrupted by the SSB-113 mutation. Taken together our results suggest that SSB functions to recruit polV to primer termini on RecA-coated DNA, operating by two mechanisms: 1) increasing the accessibility of 3' primer termini caused by binding of SSB to DNA and 2) a direct SSB-polV interaction mediated by the C terminus of SSB.

  9. Exploring transduction mechanisms of protein transduction domains (PTDs) in living cells utilizing single-quantum dot tracking (SQT) technology.

    Science.gov (United States)

    Suzuki, Yasuhiro

    2012-01-01

    Specific protein domains known as protein transduction domains (PTDs) can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs), we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP) in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT), to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.

  10. Exploring Transduction Mechanisms of Protein Transduction Domains (PTDs in Living Cells Utilizing Single-Quantum Dot Tracking (SQT Technology

    Directory of Open Access Journals (Sweden)

    Yasuhiro Suzuki

    2012-01-01

    Full Text Available Specific protein domains known as protein transduction domains (PTDs can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs, we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT, to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.

  11. Bovine spongiform encephalopathy induces misfolding of alleged prion-resistant species cellular prion protein without altering its pathobiological features.

    Science.gov (United States)

    Vidal, Enric; Fernández-Borges, Natalia; Pintado, Belén; Ordóñez, Montserrat; Márquez, Mercedes; Fondevila, Dolors; Torres, Juan María; Pumarola, Martí; Castilla, Joaquín

    2013-05-01

    Bovine spongiform encephalopathy (BSE) prions were responsible for an unforeseen epizootic in cattle which had a vast social, economic, and public health impact. This was primarily because BSE prions were found to be transmissible to humans. Other species were also susceptible to BSE either by natural infection (e.g., felids, caprids) or in experimental settings (e.g., sheep, mice). However, certain species closely related to humans, such as canids and leporids, were apparently resistant to BSE. In vitro prion amplification techniques (saPMCA) were used to successfully misfold the cellular prion protein (PrP(c)) of these allegedly resistant species into a BSE-type prion protein. The biochemical and biological properties of the new prions generated in vitro after seeding rabbit and dog brain homogenates with classical BSE were studied. Pathobiological features of the resultant prion strains were determined after their inoculation into transgenic mice expressing bovine and human PrP(C). Strain characteristics of the in vitro-adapted rabbit and dog BSE agent remained invariable with respect to the original cattle BSE prion, suggesting that the naturally low susceptibility of rabbits and dogs to prion infections should not alter their zoonotic potential if these animals became infected with BSE. This study provides a sound basis for risk assessment regarding prion diseases in purportedly resistant species.

  12. A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production

    DEFF Research Database (Denmark)

    de Vries, R.P.; Burgers, K.; van de Vondervoort, P.J.I

    2004-01-01

    A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produc....... vadensis were much higher than those of A. niger. These characteristics make A. vadensis a very promising candidate for homologous, and possibly heterologous, protein production.......A new species of the group of black aspergilli, Aspergillus vadensis, was analyzed for its potential as a host for homologous and heterologous protein production. Unlike the other black aspergilli, this strain does not acidify the culture medium when nitrate is the nitrogen source and only produces....... The production of FaeA in A. vadensis resulted in larger amounts of intact protein than production in A. tubingensis and was similar to production in an A. niger protease disruptant, confirming in vivo the low proteolytic activity of A. vadensis. The protoplast formation and transformation efficiencies of A...

  13. A network model to correlate conformational change and the impedance spectrum of single proteins

    Science.gov (United States)

    Alfinito, Eleonora; Pennetta, Cecilia; Reggiani, Lino

    2008-02-01

    Integrated nanodevices based on proteins or biomolecules are attracting increasing interest in today's research. In fact, it has been shown that proteins such as azurin and bacteriorhodopsin manifest some electrical properties that are promising for the development of active components of molecular electronic devices. Here we focus on two relevant kinds of protein: bovine rhodopsin, prototype of G-protein-coupled-receptor (GPCR) proteins, and the enzyme acetylcholinesterase (AChE), whose inhibition is one of the most qualified treatments of Alzheimer's disease. Both these proteins exert their function starting with a conformational change of their native structure. Our guess is that such a change should be accompanied with a detectable variation of their electrical properties. To investigate this conjecture, we present an impedance network model of proteins, able to estimate the different impedance spectra associated with the different configurations. The distinct types of conformational change of rhodopsin and AChE agree with their dissimilar electrical responses. In particular, for rhodopsin the model predicts variations of the impedance spectra up to about 30%, while for AChE the same variations are limited to about 10%, which supports the existence of a dynamical equilibrium between its native and complexed states.

  14. Single-cell genomics reveals features of a Colwellia species that was dominant during the Deepwater Horizon oil spill

    Directory of Open Access Journals (Sweden)

    Olivia eMason

    2014-07-01

    Full Text Available During the Deepwater Horizon (DWH oil spill in the Gulf of Mexico a deep-sea hydrocarbon plume developed resulting in a rapid succession of bacteria. Colwellia eventually supplanted Oceanospirillales, which dominated the plume early in the spill. These successional changes may have resulted, in part, from the changing composition and abundance of hydrocarbons over time. Colwellia abundance peaked when gaseous and simple aromatic hydrocarbons increased, yet the metabolic pathway used by Colwellia in hydrocarbon disposition is unknown. Here we used single-cell genomics to gain insights into the genome properties of a Colwellia enriched during the DWH deep-sea plume. A single amplified genome (SAG of a Colwellia cell isolated from a DWH plume, closely related (avg. 98% 16S rRNA gene similarity to other plume Colwellia, was sequenced and annotated. The SAG was similar to the sequenced isolate Colwellia psychrerythraea 34H (84% avg. nucleotide identity. Both had genes for denitrification, chemotaxis and motility, adaptations to cold environments, and a suite of nutrient acquisition genes. The Colwellia SAG may be capable of gaseous and aromatic hydrocarbon degradation, which contrasts with a DWH plume Oceanospirillales SAG genome which encoded non-gaseous n-alkane and cycloalkane degradation. The disparate hydrocarbon degradation pathways are consistent with hydrocarbons that were abundant at different times in the deep-sea plume; first, non-gaseous n-alkanes and cycloalkanes that could be degraded by Oceanospirillales, followed by gaseous, and simple aromatic hydrocarbons that may have been degraded by Colwellia. These insights into the genomic properties of a Colwellia species, which were supported by existing metagenomic sequence data from the plume and DWH contaminated sediments, help further our understanding of the successional changes in the dominant microbial players in the plume over the course of the DWH spill.

  15. New in protein structure and function annotation: hotspots, single nucleotide polymorphisms and the 'Deep Web'.

    Science.gov (United States)

    Bromberg, Yana; Yachdav, Guy; Ofran, Yanay; Schneider, Reinhard; Rost, Burkhard

    2009-05-01

    The rapidly increasing quantity of protein sequence data continues to widen the gap between available sequences and annotations. Comparative modeling suggests some aspects of the 3D structures of approximately half of all known proteins; homology- and network-based inferences annotate some aspect of function for a similar fraction of the proteome. For most known protein sequences, however, there is detailed knowledge about neither their function nor their structure. Comprehensive efforts towards the expert curation of sequence annotations have failed to meet the demand of the rapidly increasing number of available sequences. Only the automated prediction of protein function in the absence of homology can close the gap between available sequences and annotations in the foreseeable future. This review focuses on two novel methods for automated annotation, and briefly presents an outlook on how modern web software may revolutionize the field of protein sequence annotation. First, predictions of protein binding sites and functional hotspots, and the evolution of these into the most successful type of prediction of protein function from sequence will be discussed. Second, a new tool, comprehensive in silico mutagenesis, which contributes important novel predictions of function and at the same time prepares for the onset of the next sequencing revolution, will be described. While these two new sub-fields of protein prediction represent the breakthroughs that have been achieved methodologically, it will then be argued that a different development might further change the way biomedical researchers benefit from annotations: modern web software can connect the worldwide web in any browser with the 'Deep Web' (ie, proprietary data resources). The availability of this direct connection, and the resulting access to a wealth of data, may impact drug discovery and development more than any existing method that contributes to protein annotation.

  16. Computational approach to predict species-specific type III secretion system (T3SS) effectors using single and multiple genomes.

    Science.gov (United States)

    Hobbs, Christopher K; Porter, Vanessa L; Stow, Maxwell L S; Siame, Bupe A; Tsang, Herbert H; Leung, Ka Yin

    2016-12-19

    Many gram-negative bacteria use type III secretion systems (T3SSs) to translocate effector proteins into host cells. T3SS effectors can give some bacteria a competitive edge over others within the same environment and can help bacteria to invade the host cells and allow them to multiply rapidly within the host. Therefore, developing efficient methods to identify effectors scattered in bacterial genomes can lead to a better understanding of host-pathogen interactions and ultimately to important medical and biotechnological applications. We used 21 genomic and proteomic attributes to create a precise and reliable T3SS effector prediction method called Genome Search for Effectors Tool (GenSET). Five machine learning algorithms were trained on effectors selected from different organisms and a trained (voting) algorithm was then applied to identify other effectors present in the genome testing sets from the same (GenSET Phase 1) or different (GenSET Phase 2) organism. Although a select group of attributes that included the codon adaptation index, probability of expression in inclusion bodies, N-terminal disorder, and G + C content (filtered) were better at discriminating between positive and negative sets, algorithm performance was better when all 21 attributes (unfiltered) were used. Performance scores (sensitivity, specificity and area under the curve) from GenSET Phase 1 were better than those reported for six published methods. More importantly, GenSET Phase 1 ranked more known effectors (70.3%) in the top 40 ranked proteins and predicted 10-80% more effectors than three available programs in three of the four organisms tested. GenSET Phase 2 predicted 43.8% effectors in the top 40 ranked proteins when tested on four related or unrelated organisms. The lower prediction rates from GenSET Phase 2 may be due to the presence of different translocation signals in effectors from different T3SS families. The species-specific GenSET Phase 1 method offers an alternative

  17. Physical manipulation of single-molecule DNA using microbead and its application to analysis of DNA-protein interaction

    International Nuclear Information System (INIS)

    Kurita, Hirofumi; Yasuda, Hachiro; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

    2009-01-01

    We carried out an individual DNA manipulation using an optical trapping for a microbead. This manipulation system is based on a fluorescent microscopy equipped with an IR laser. Both ends of linear DNA molecule were labeled with a biotin and a thiol group, respectively. Then the biotinylated end was attached to a microbead, and the other was immobilized on a thiol-linkable glass surface. We controlled the form of an individual DNA molecule by moving the focal point of IR laser, which trapped the microbead. In addition, we applied single-molecule approach to analyze DNA hydrolysis. We also used microchannel for single-molecule observation of DNA hydrolysis. The shortening of DNA in length caused by enzymatic hydrolysis was observed in real-time. The single-molecule DNA manipulation should contribute to elucidate detailed mechanisms of DNA-protein interactions

  18. Is the C-terminal insertional signal in Gram-negative bacterial outer membrane proteins species-specific or not?

    Directory of Open Access Journals (Sweden)

    Paramasivam Nagarajan

    2012-09-01

    Full Text Available Abstract Background In Gram-negative bacteria, the outer membrane is composed of an asymmetric lipid bilayer of phopspholipids and lipopolysaccharides, and the transmembrane proteins that reside in this membrane are almost exclusively β-barrel proteins. These proteins are inserted into the membrane by a highly conserved and essential machinery, the BAM complex. It recognizes its substrates, unfolded outer membrane proteins (OMPs, through a C-terminal motif that has been speculated to be species-specific, based on theoretical and experimental results from only two species, Escherichia coli and Neisseria meningitidis, where it was shown on the basis of individual sequences and motifs that OMPs from the one cannot easily be over expressed in the other, unless the C-terminal motif was adapted. In order to determine whether this species specificity is a general phenomenon, we undertook a large-scale bioinformatics study on all predicted OMPs from 437 fully sequenced proteobacterial strains. Results We were able to verify the incompatibility reported between Escherichia coli and Neisseria meningitidis, using clustering techniques based on the pairwise Hellinger distance between sequence spaces for the C-terminal motifs of individual organisms. We noticed that the amino acid position reported to be responsible for this incompatibility between Escherichia coli and Neisseria meningitidis does not play a major role for determining species specificity of OMP recognition by the BAM complex. Instead, we found that the signal is more diffuse, and that for most organism pairs, the difference between the signals is hard to detect. Notable exceptions are the Neisseriales, and Helicobacter spp. For both of these organism groups, we describe the specific sequence requirements that are at the basis of the observed difference. Conclusions Based on the finding that the differences between the recognition motifs of almost all organisms are small, we assume that

  19. Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins.

    Science.gov (United States)

    Hill, R E; Mackessy, S P

    2000-12-01

    R.E. Hill and S.P. Mackessy. Characterization of venom (Duvernoy's secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon XX, xx-yy, 2000. - Venomous colubrids, which include more than 700 snake species worldwide, represent a vast potential source of novel biological compounds. The present study characterized venom (Duvernoy's gland secretion) collected from twelve species of opisthoglyphous (rear-fanged) colubrid snakes, an extremely diverse assemblage of non-venomous to highly venomous snakes. Most venoms displayed proteolytic activity (casein), though activity levels varied considerably. Low phosphodiesterase activity was detected in several venoms (Amphiesma stolata, Diadophis punctatus, Heterodon nasicus kennerlyi, H. n. nasicus and Thamnophis elegans vagrans), and acetylcholinesterase was found in Boiga irregularis saliva and venom, but no venoms displayed hyaluronidase, thrombin-like or kallikrein-like activities. High phospholipase A(2) (PLA(2)) activity was found in Trimorphodon biscutatus lambda venom, and moderate levels were detected in Boiga dendrophila and D. p. regalis venoms as well as B. dendrophila and H. n. nasicus salivas. Non-reducing SDS-PAGE revealed 7-20 protein bands (3.5 to over 200 kD, depending on species) for all venoms analyzed, and electrophoretic profiles of venoms were typically quite distinct from saliva profiles. Components from A. stolata, Hydrodynastes gigas, Tantilla nigriceps and T. e. vagrans venoms showed protease activity when run on gelatin zymogram gels. N-terminal protein sequences for three 26 kD venom components of three species (H. gigas, H. torquata, T. biscutatus) and one 3.5 kD component (T. nigriceps) were also obtained, and the 3.5 kD peptide showed apparent sequence homology with human vascular endothelial growth factor; these data represent the first sequences of colubrid venom components. Protease, phosphodiesterase and PLA(2) activities are also common to elapid

  20. Cross-species protein sequence and gene structure prediction with fine-tuned Webscipio 2.0 and Scipio.

    Science.gov (United States)

    Hatje, Klas; Keller, Oliver; Hammesfahr, Björn; Pillmann, Holger; Waack, Stephan; Kollmar, Martin

    2011-07-28

    Obtaining transcripts of homologs of closely related organisms and retrieving the reconstructed exon-intron patterns of the genes is a very important process during the analysis of the evolution of a protein family and the comparative analysis of the exon-intron structure of a certain gene from different species. Due to the ever-increasing speed of genome sequencing, the gap to genome annotation is growing. Thus, tools for the correct prediction and reconstruction of genes in related organisms become more and more important. The tool Scipio, which can also be used via the graphical interface WebScipio, performs significant hit processing of the output of the Blat program to account for sequencing errors, missing sequence, and fragmented genome assemblies. However, Scipio has so far been limited to high sequence similarity and unable to reconstruct short exons. Scipio and WebScipio have fundamentally been extended to better reconstruct very short exons and intron splice sites and to be better suited for cross-species gene structure predictions. The Needleman-Wunsch algorithm has been implemented for the search for short parts of the query sequence that were not recognized by Blat. Those regions might either be short exons, divergent sequence at intron splice sites, or very divergent exons. We have shown the benefit and use of new parameters with several protein examples from completely different protein families in searches against species from several kingdoms of the eukaryotes. The performance of the new Scipio version has been tested in comparison with several similar tools. With the new version of Scipio very short exons, terminal and internal, of even just one amino acid can correctly be reconstructed. Scipio is also able to correctly predict almost all genes in cross-species searches even if the ancestors of the species separated more than 100 Myr ago and if the protein sequence identity is below 80%. For our test cases Scipio outperforms all other software

  1. Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    Directory of Open Access Journals (Sweden)

    Huang Jian-dong

    2011-04-01

    Full Text Available Abstract Background SXT is an integrating conjugative element (ICE originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo and single strand annealing protein (S065, SXT-Bet encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively. Results SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn2+ or Mg2+ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb. When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo. Conclusions The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V

  2. An electrophoretic study of protein diversity in five species of land ...

    African Journals Online (AJOL)

    Swainson, 1821), Achatina achatina (Linnaeus, 1758), Achatina fulica (Bowdich, 1822), Archachatina papyracea (Pfeiffer, 1845) and an unidentified snail species) from some parts of southern and north-central states of Nigeria was used to ...

  3. Dynamics of a Birth-Pulse Single-Species Model with Restricted Toxin Input and Pulse Harvesting

    Directory of Open Access Journals (Sweden)

    Yi Ma

    2010-01-01

    Full Text Available We consider a birth-pulses single-species model with restricted toxin input and pulse harvesting in a polluted environment. Pollution accumulates as a slowly decaying stock and is assumed to affect the growth of the renewable resource population. Firstly, by using the discrete dynamical system determined by the stroboscopic map, we obtain an exact 1-period solution of system whose birth function is Ricker function or Beverton-Holt function and obtain the threshold conditions for their stability. Furthermore, we show that the timing of harvesting has a strong impact on the maximum annual sustainable yield. The best timing of harvesting is immediately after the birth pulses. Finally, we investigate the effect of the amount of toxin input on the stable resource population size. We find that when the birth rate is comparatively lower, the population size is decreasing with the increase of toxin input; that when the birth rate is high, the population size may begin to rise and then drop with the increase of toxin input.

  4. A measure of the denseness of a phylogenetic network. [by sequenced proteins from extant species

    Science.gov (United States)

    Holmquist, R.

    1978-01-01

    An objective measure of phylogenetic denseness is developed to examine various phylogenetic criteria: alpha- and beta-hemoglobin, myoglobin, cytochrome c, and the parvalbumin family. Attention is given to the number of nucleotide replacements separating homologous sequences, and to the topology of the network (in other words, to the qualitative nature of the network as defined by how closely the studied species are related). Applications include quantitative comparisons of species origin, relation, and rates of evolution.

  5. Annotating N termini for the human proteome project: N termini and Nα-acetylation status differentiate stable cleaved protein species from degradation remnants in the human erythrocyte proteome.

    Science.gov (United States)

    Lange, Philipp F; Huesgen, Pitter F; Nguyen, Karen; Overall, Christopher M

    2014-04-04

    A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as "missing", this is challenging. Moreover, proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier .

  6. Species-Specific Monoclonal Antibodies to Escherichia coli-Expressed p36 Cytosolic Protein of Mycoplasma hyopneumoniae

    Science.gov (United States)

    Caron, J.; Sawyer, N.; Moumen, B. Ben Abdel; Bouh, K. Cheikh Saad; Dea, S.

    2000-01-01

    The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an

  7. Red oak in southern New England and big-leaf mahogany in the Yucatan Peninsula: can mixed-species forests be sustainably managed for single-species production?

    Science.gov (United States)

    M. Kelty; L. Camara-Cabrales; J. Grogan

    2011-01-01

    Complex mixed-species forests are the focus of conservation efforts that seek to maintain native biodiversity. However, much of this forestland is privately owned and is managed for timber income as well as for conservation. Management of these high-diversity forests is particularly difficult when only one tree species produces the majority of high-value timber. We...

  8. Green fluorescent protein based indicators of dynamic redox changes and reactive oxygen species

    OpenAIRE

    Dooley, Colette

    2006-01-01

    Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expre...

  9. Comparative Proteomics of Rubber Latex Revealed Multiple Protein Species of REF/SRPP Family Respond Diversely to Ethylene Stimulation among Different Rubber Tree Clones.

    Science.gov (United States)

    Tong, Zheng; Wang, Dan; Sun, Yong; Yang, Qian; Meng, Xueru; Wang, Limin; Feng, Weiqiang; Li, Ling; Wurtele, Eve Syrkin; Wang, Xuchu

    2017-05-02

    Rubber elongation factor (REF) and small rubber particle protein (SRPP) are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF 138,175,258 and SRPP 117,204,243 , were characterized from Hevea brasiliensis Reyan (RY) 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF 258 has a β subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE), each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF 175 or REF 258 were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis.

  10. Comparative Proteomics of Rubber Latex Revealed Multiple Protein Species of REF/SRPP Family Respond Diversely to Ethylene Stimulation among Different Rubber Tree Clones

    Directory of Open Access Journals (Sweden)

    Zheng Tong

    2017-05-01

    Full Text Available Rubber elongation factor (REF and small rubber particle protein (SRPP are two key factors for natural rubber biosynthesis. To further understand the roles of these proteins in rubber formation, six different genes for latex abundant REF or SRPP proteins, including REF138,175,258 and SRPP117,204,243, were characterized from Hevea brasiliensis Reyan (RY 7-33-97. Sequence analysis showed that REFs have a variable and long N-terminal, whereas SRPPs have a variable and long C-terminal beyond the REF domain, and REF258 has a β subunit of ATPase in its N-terminal. Through two-dimensional electrophoresis (2-DE, each REF/SRPP protein was separated into multiple protein spots on 2-DE gels, indicating they have multiple protein species. The abundance of REF/SRPP proteins was compared between ethylene and control treatments or among rubber tree clones with different levels of latex productivity by analyzing 2-DE gels. The total abundance of each REF/SRPP protein decreased or changed a little upon ethylene stimulation, whereas the abundance of multiple protein species of the same REF/SRPP changed diversely. Among the three rubber tree clones, the abundance of the protein species also differed significantly. Especially, two protein species of REF175 or REF258 were ethylene-responsive only in the high latex productivity clone RY 8-79 instead of in RY 7-33-97 and PR 107. Some individual protein species were positively related to ethylene stimulation and latex productivity. These results suggested that the specific protein species could be more important than others for rubber production and post-translational modifications might play important roles in rubber biosynthesis.

  11. Reactive oxygen species modulator 1, a novel protein, combined with carcinoembryonic antigen in differentiating malignant from benign pleural effusion.

    Science.gov (United States)

    Chen, Xianmeng; Zhang, Na; Dong, Jiahui; Sun, Gengyun

    2017-05-01

    The differential diagnosis of malignant pleural effusion and benign pleural effusion remains a clinical problem. Reactive oxygen species modulator 1 is a novel protein overexpressed in various human tumors. The objective of this study was to evaluate the diagnostic value of joint detection of reactive oxygen species modulator 1 and carcinoembryonic antigen in the differential diagnosis of malignant pleural effusion and benign pleural effusion. One hundred two consecutive patients with pleural effusion (including 52 malignant pleural effusion and 50 benign pleural effusion) were registered in this study. Levels of reactive oxygen species modulator 1 and carcinoembryonic antigen were measured by enzyme-linked immunosorbent assay and radioimmunoassay, respectively. Results showed that the concentrations of reactive oxygen species modulator 1 both in pleural fluid and serum of patients with malignant pleural effusion were significantly higher than those of benign pleural effusion (both p carcinoembryonic antigen were 69.23% and 88.00%, respectively, at the cutoff value of 3.05 ng/mL, while serum carcinoembryonic antigen were 80.77% and 72.00% at the cutoff value of 2.60 ng/mL. The sensitivity could be raised to 88.17% in parallel detection of plural fluid reactive oxygen species modulator 1 and carcinoembryonic antigen concentration, and the specificity could be improved to 97.84% in serial detection.

  12. Combined Protein A and size exclusion high performance liquid chromatography for the single-step measurement of mAb, aggregates and host cell proteins.

    Science.gov (United States)

    Gjoka, Xhorxhi; Schofield, Mark; Cvetkovic, Aleksandar; Gantier, Rene

    2014-12-01

    Quantification of monoclonal antibody (mAb) monomer, mAb aggregates, and host cell proteins (HCPs) is critical for the optimization of the mAb production process. The present work describes a single high throughput analytical tool capable of tracking the concentration of mAb, mAb aggregate and HCPs in a growing cell culture batch. By combining two analytical HPLC methods, Protein A affinity and size-exclusion chromatography (SEC), it is possible to detect a relative increase or decrease in the concentration of all three entities simultaneously. A comparison of the combined Protein A-SEC assay to SEC alone was performed, demonstrating that it can be useful tool for the quantification of mAb monomer along with trending data for mAb aggregate and HCP. Furthermore, the study shows that the Protein A-SEC method is at least as accurate as other commonly used analytical methods such as ELISA and Bradford. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Perturbation of discrete sites on a single protein domain with RNA aptamers: targeting of different sides of the TATA-binding protein (TBP).

    Science.gov (United States)

    Hohmura, Ken I; Shi, Hua; Hirayoshi, Kazunori

    2013-01-01

    Control of interactions among proteins is critical in the treatment of diseases, but the specificity required is not easily incorporated into small molecules. Macromolecules could be more suitable as antagonists in this situation, and RNA aptamers have become particularly promising. Here we describe a novel selection procedure for RNA aptamers against a protein that constitutes a single structural domain, the Drosophila TATA-binding protein (TBP). In addition to the conventional filter partitioning method with free TBP as target, we performed another experiment, in which the TATA-bound form of TBP was targeted. Aptamers generated by both selections were able to bind specifically to TBP, but the two groups showed characteristics which were clearly different in terms of their capability to compete with TATA-DNA, their effects on the TATA-bound form of TBP, and their effects on in vitro transcription. The method used to generate these two groups of aptamers can be used with other targets to direct aptamer specificity to discrete sites on the surface of a protein.

  14. Fish allergy in patients with parvalbumin-specific immunoglobulin E depends on parvalbumin content rather than molecular differences in the protein among fish species.

    Science.gov (United States)

    Kobayashi, Ayako; Kobayashi, Yukihiro; Shiomi, Kazuo

    2016-10-01

    Allergenic characteristics of purified parvalbumins from different fish species have not been thoroughly investigated. We revealed that purified parvalbumins from nine different fish species have identical IgE-reactivities and high cross-reactivities. We also showed that fish allergenicity is associated with the parvalbumin content of the fish species, rather than species-specific differences in the molecular characteristics of the individual parvalbumin proteins.

  15. A synthetic cadmium metallothionein gene (PMCd1syn) of Paramecium species: expression, purification and characteristics of metallothionein protein.

    Science.gov (United States)

    Dar, Saira; Shuja, Rukhsana N; Shakoori, Abdul Rauf

    2013-02-01

    Metallothioneins (MTs) are metal binding proteins that are rich in cysteine residues constituting 10-30 % of the total protein, and in which the thiol groups bind to the metal ions. The increasing amount of metal ions in the medium have shown increased production of MTs by different organisms such as bacteria, protozoa and mammals like humans. PMCd1 is the first gene ever discovered in Paramecium, a ciliated protozoan, that could produce this MT in response to cadmium. In this study the PMCd1syn gene has been cloned in pET41a expression vector and expressed in an Escherichia coli BL21-codonplus strain for the first time. Since the gene PMCd1 amplified from Paramecium contained 10 codons, which could act as stop codons during expression in E. coli, this gene of 612 bps was synthesized to substitute these (stop) codons for the Paramecium sp. specific amino acids. For stability of the expressed protein, glutathione-S-transferase gene was fused with PMCd1syn gene and coexpressed. The cells expressing PMCd1syn demonstrated increased accumulation of cadmium. This is the first report of cadmium MT protein expressed from Paramecium species, particularly from synthetic MT gene (PMCd1syn). This fusion protein, the molecular weight of which has been confirmed to be 53.03 kDa with MALDI analysis, is rich in cysteine residues, and has been shown for the first time in this ciliate to bind to and sequester Cd(2+)-ions.

  16. Normal Growth in PKU Patients Under Low-Protein Diet in a Single-Center Cross-Sectional Study.

    Science.gov (United States)

    Matic, Jana; Zeltner, Nina A; Häberle, Johannes

    2018-02-25

    Dietary phenylalanine restriction in phenylketonuria (PKU) patients is usually mandatory in order to prevent cognitive impairment. The influence of a low-protein diet on growth has raised concerns in families and caregivers. This paper aims to investigate the growth in PKU patients treated with a low-protein diet including supplementation of amino acids and other nutrients according to standard protocols.We performed a single-center, cross-sectional study on growth in pediatric PKU patients (n = 51) treated with low-protein diet over a 20-month period. Height of healthy siblings (n = 44) and target height, calculated based on parents' height, served as controls.No statistically significant differences were found comparing mean height z-scores between patients and siblings (p = 0.261). Patients PKU patients treated with low-protein diet can achieve normal growth with patients making up the leeway after puberty. While prepubertal patients were shorter than expected based on their target height, older patients were within their expected target height. This study indicates that current practice of low-protein diet in PKU patients allows normal growth.

  17. Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

    International Nuclear Information System (INIS)

    Langowski, J.; Benight, A.S.; Fujimoto, B.S.; Schurr, J.M.; Schomburg, U.

    1985-01-01

    The influence of Escherichia coli single-strand binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNAs has been investigated by using dynamic light scattering at 632.8 and 351.1 nm and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNAs up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D 0 ) of both DNAs decrease by approximately 20%. Apparent diffusion coefficients (D/sub app/) obtained from dynamic light scattering display the well-known increase with K 2 (K = scattering vector), leveling off toward a plateau value (D/sub plat/) at high K 2 . For both DNAs, the difference D/sub plat/ - D 0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed

  18. Improved healing of transected rabbit Achilles tendon after a single injection of cartilage-derived morphogenetic protein-2.

    Science.gov (United States)

    Forslund, Carina; Aspenberg, Per

    2003-01-01

    Achilles tendon ruptures in humans might be treated more efficiently with the help of a growth factor. Cartilage-derived morphogenetic protein-2 has been shown to induce formation of tendon-like tissue. Cartilage-derived morphogenetic protein-2 has a positive effect on mechanical parameters for tendon healing in a rabbit model with Achilles tendon transection. Controlled laboratory study. The right Achilles tendon of 40 rabbits was transected without tendon suture. Cartilage-derived morphogenetic protein-2 (10 micro g) or vehicle control (acetate buffer) was injected locally 2 hours postoperatively. All tendons were tested biomechanically at 8 and 14 days, and treated tendons were histologically and radiographically evaluated at 56 days. At 14 days, both failure load and stiffness of treated tendons were increased by 35%. The treated tendons had significantly larger callus size at 8 and 14 days. Histologic and radiographic examination showed no signs of ossification in the treated tendons after 56 days. A single injection of cartilage-derived morphogenetic protein-2 led to a stronger and stiffer tendon callus than that in the controls without inducing bone formation. Similar results from a larger animal model would suggest a possible future use of cartilage-derived morphogenetic protein-2 in the treatment of human Achilles tendon ruptures.

  19. A novel multimodal chromatography based single step purification process for efficient manufacturing of an E. coli based biotherapeutic protein product.

    Science.gov (United States)

    Bhambure, Rahul; Gupta, Darpan; Rathore, Anurag S

    2013-11-01

    Methionine oxidized, reduced and fMet forms of a native recombinant protein product are often the critical product variants which are associated with proteins expressed as bacterial inclusion bodies in E. coli. Such product variants differ from native protein in their structural and functional aspects, and may lead to loss of biological activity and immunogenic response in patients. This investigation focuses on evaluation of multimodal chromatography for selective removal of these product variants using recombinant human granulocyte colony stimulating factor (GCSF) as the model protein. Unique selectivity in separation of closely related product variants was obtained using combined pH and salt based elution gradients in hydrophobic charge induction chromatography. Simultaneous removal of process related impurities was also achieved in flow-through leading to single step purification process for the GCSF. Results indicate that the product recovery of up to 90.0% can be obtained with purity levels of greater than 99.0%. Binding the target protein at pH

  20. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann

    2013-01-01

    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, whereas vertebrates contain two to four genes. In cnidarians, the gene appears to encode a secreted protein, but transmembrane isoforms of the protein have also evolved, and in many species, alternative splicing facilitates the expression of both transmembrane and secreted isoforms. In most species......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...

  1. Identification of proteins enriched in rice egg or sperm cells by single-cell proteomics.

    Directory of Open Access Journals (Sweden)

    Mafumi Abiko

    Full Text Available In angiosperms, female gamete differentiation, fertilization, and subsequent zygotic development occur in embryo sacs deeply embedded in the ovaries. Despite their importance in plant reproduction and development, how the egg cell is specialized, fuses with the sperm cell, and converts into an active zygote for early embryogenesis remains unclear. This lack of knowledge is partly attributable to the difficulty of direct analyses of gametes in angiosperms. In the present study, proteins from egg and sperm cells obtained from rice flowers were separated by one-dimensional polyacrylamide gel electrophoresis and globally identified by highly sensitive liquid chromatography coupled with tandem mass spectroscopy. Proteome analyses were also conducted for seedlings, callus, and pollen grains to compare their protein expression profiles to those of gametes. The proteomics data have been deposited to the ProteomeXchange with identifier PXD000265. A total of 2,138 and 2,179 expressed proteins were detected in egg and sperm cells, respectively, and 102 and 77 proteins were identified as preferentially expressed in egg and sperm cells, respectively. Moreover, several rice or Arabidopsis lines with mutations in genes encoding the putative gamete-enriched proteins showed clear phenotypic defects in seed set or seed development. These results suggested that the proteomic data presented in this study are foundational information toward understanding the mechanisms of reproduction and early development in angiosperms.

  2. A single amino acid mutation affects elicitor and expansins-like activities of cerato-platanin, a non-catalytic fungal protein.

    Directory of Open Access Journals (Sweden)

    Simone Luti

    Full Text Available Cerato-platanin (CP is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response. Indeed, CP is an elicitor of primary defences acting as a Pathogen-Associated Molecular Patterns (PAMP. So far, structure-function relationship study has been mainly performed on the bacterial BsEXLX1