WorldWideScience

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

    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...... produced a signalling-biased variant of Free Fatty Acid Receptor 2 in which Gi-mediated signalling by both short chain fatty acids and synthetic agonists was maintained whilst there was marked loss of agonist potency for signalling via Gq/11 and G12/13 G proteins. A single residue at the extracellular face...

  4. Prions: Beyond a Single Protein

    Science.gov (United States)

    Das, Alvin S.

    2016-01-01

    SUMMARY Since the term protein was first coined in 1838 and protein was discovered to be the essential component of fibrin and albumin, all cellular proteins were presumed to play beneficial roles in plants and mammals. However, in 1967, Griffith proposed that proteins could be infectious pathogens and postulated their involvement in scrapie, a universally fatal transmissible spongiform encephalopathy in goats and sheep. Nevertheless, this novel hypothesis had not been evidenced until 1982, when Prusiner and coworkers purified infectious particles from scrapie-infected hamster brains and demonstrated that they consisted of a specific protein that he called a “prion.” Unprecedentedly, the infectious prion pathogen is actually derived from its endogenous cellular form in the central nervous system. Unlike other infectious agents, such as bacteria, viruses, and fungi, prions do not contain genetic materials such as DNA or RNA. The unique traits and genetic information of prions are believed to be encoded within the conformational structure and posttranslational modifications of the proteins. Remarkably, prion-like behavior has been recently observed in other cellular proteins—not only in pathogenic roles but also serving physiological functions. The significance of these fascinating developments in prion biology is far beyond the scope of a single cellular protein and its related disease. PMID:27226089

  5. Protein determination in single corns

    International Nuclear Information System (INIS)

    Knorr, J.; Schiekel, M.; Franke, W.; Focke, F.

    1994-01-01

    Determination of protein content in food materials is usually done by analyzing the nitrogen amount by wet chemical Kjeldahl method. An improved accuracy accompanied by smaller analyzing intervals can be achieved using nondestructive neutron activation. Analyses have been performed using 14 MeV neutrons to determine the content of N and P in single wheat corns. Irradiation parameters have been optimized to prevent serious radiation damage in grains. About 200 single corns have been investigated with total net weights ranging from 30 to 70 mg. The tested arrangement allows determination of nitrogen amount in a single corn down to 0.3 mg with an accuracy of better than 4 %. Mean nitrogen concentrations in the range from 9 to 19% per corn have been detected. (author) 5 refs.; 6 figs

  6. A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner

    Science.gov (United States)

    Koehler, Alexander; Kolesnikova, Larissa; Welzel, Ulla; Schudt, Gordian; Herwig, Astrid

    2015-01-01

    ABSTRACT Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the viral matrix protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARVVP40(D184N)] grew to higher titers than wild-type recombinant MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other viral proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better viral assembly function of VP40D184N and its lower inhibitory effect on viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling. IMPORTANCE The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the viral genome. Analyzing how these mutations affect the functions of viral proteins and the ability of the virus to grow within new host cells helps in the understanding

  7. Diversity in protein glycosylation among insect species.

    Directory of Open Access Journals (Sweden)

    Gianni Vandenborre

    Full Text Available BACKGROUND: A very common protein modification in multicellular organisms is protein glycosylation or the addition of carbohydrate structures to the peptide backbone. Although the Class of the Insecta is the largest animal taxon on Earth, almost all information concerning glycosylation in insects is derived from studies with only one species, namely the fruit fly Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the differences in glycoproteomes between insects belonging to several economically important insect orders were studied. Using GNA (Galanthus nivalis agglutinin affinity chromatography, different sets of glycoproteins with mannosyl-containing glycan structures were purified from the flour beetle (Tribolium castaneum, the silkworm (Bombyx mori, the honeybee (Apis mellifera, the fruit fly (D. melanogaster and the pea aphid (Acyrthosiphon pisum. To identify and characterize the purified glycoproteins, LC-MS/MS analysis was performed. For all insect species, it was demonstrated that glycoproteins were related to a broad range of biological processes and molecular functions. Moreover, the majority of glycoproteins retained on the GNA column were unique to one particular insect species and only a few glycoproteins were present in the five different glycoprotein sets. Furthermore, these data support the hypothesis that insect glycoproteins can be decorated with mannosylated O-glycans. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that oligomannose N-glycosylation events are highly specific depending on the insect species. In addition, we also demonstrated that protein O-mannosylation in insect species may occur more frequently than currently believed.

  8. Dataset of protein species from human liver

    Directory of Open Access Journals (Sweden)

    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.

  9. Biological Evaluation of Single Cell Protein

    International Nuclear Information System (INIS)

    Hasan, I.A.; Mohamed, N.E.; El-Sayed, E.A.; Younis, N.A.

    2011-01-01

    In this study, the nutritional value of single cell protein (SCP) was evaluated as a non conventional protein source produced by fermenting fungal local strains of Trichoderma longibrachiatum, Aspergillus niger, Aspergillus terreus and Penicillium funiculosum with alkali treated sugar cane bagasse. Amino acid analysis revealed that the produced SCP contains essential and non essential amino acids. Male mice were fed on normal (basal) diet which contains 18% conventional protein and served as control group. In the second (T1) and the third (T2) group, the animals were fed on a diet in which 15% and 30% of conventional protein source were replaced by SCP, respectively. At intervals of 15, 30, 45 and 60 days, mice were sacrificed and the blood samples were collected for the biochemical evaluation. The daily averages of body weight were significantly higher with group T2 than group T1. Where as, the kidney weights in groups (T1) and (T2) were significantly increased as compared with control. A non significant difference between the tested groups in the enzyme activities of AST, ALT and GSH content of liver tissue were recorded. While, cholesterol and triglycerides contents showed a significant decrease in both (T1) and (T2) groups as compared with control. The recorded values of the serum hormone (T4), ALP activities, albumin and A/G ratio did not changed by the previous treatments. Serum levels of total protein, urea, creatinine and uric acid were higher for groups (T1) and (T2) than the control group. In conclusion, partial substitution of soy bean protein in mice diet with single cell protein (15%) improved the mice growth without any adverse effects on some of the physiological functions tested

  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 cell protein from mandarin orange peel

    Energy Technology Data Exchange (ETDEWEB)

    Mishio, M.; Magai, J.

    1981-01-01

    As the hydrolysis of mandarin orange peel with macerating enzyme (40 degrees C, 24 h) produced 0.59 g g-1 reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120 degrees C with 0.8 N H2S04), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel. When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (g g-1) were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30 degrees C using 100g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts. (Refs. 12).

  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. 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

  15. Can a Single Amphibian Species Be a Good Biodiversity Indicator?

    Directory of Open Access Journals (Sweden)

    David Sewell

    2009-11-01

    Full Text Available Although amphibians have been widely promoted as indicators of biodiversity and environmental change, rigorous tests are lacking. Here key indicator criteria are distilled from published papers, and a species that has been promoted as a bioindicator, the great crested newt, is tested against them. Although a link was established between the presence of great crested newts and aquatic plant diversity, this was not repeated with the diversity of macroinvertebrates. Equally, amphibians do not meet many of the published criteria of bioindicators. Our research suggests that a suite of indicators, rather than a single species, will usually be required.

  16. 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 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 ...

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

    Directory of Open Access Journals (Sweden)

    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

  18. 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

  19. 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+

  20. Protein quality of three different species of earthrvorms

    African Journals Online (AJOL)

    Keywords: Earthworm meal, nutritive value, protein source, poultry. 99 ... species on which the development of a high density production .... 116,28. Arginine. Broiler starter. 6,37. 87,61. 95,90. 116,78. 167,30. 113,50 ... Protein intake (ql7 days).

  1. Chemical score of different protein sources to four Macrobrachium species

    OpenAIRE

    Montoya-Martínez, Cynthia; Nolasco-Soria, Héctor; Carrillo-Farnés, Olimpia; Civera-Cerecedo, Roberto; Álvarez-González, Carlos; Vega-Villasante, Fernando

    2016-01-01

    Food production for aquaculture requires finding other protein sources or ingredients as potential alternatives in the formulation of aquaculture feeds, due to the shortage and high price of protein sources that are most commonly used. The aim of this analysis was to evaluate the relationship between the essential amino acids in 13 types of proteins available in the market with the essential amino acids found in the muscle of four of the most important farmed prawn species of the genus Macrob...

  2. 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.

  3. 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

    Fishery management advice has traditionally been given on a stock-by-stock basis. Recent problems in implementing this advice, particularly for the demersal fisheries of the North Sea, have highlighted the limitations of the approach. In the long term, it would be desirable to give advice...... 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...

  4. 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.

  5. A single ectomycorrhizal fungal species can enable a Pinus invasion.

    Science.gov (United States)

    Hayward, Jeremy; Horton, Thomas R; Pauchard, Aníbal; Nuñnez, Martin A

    2015-05-01

    Like all obligately ectomycorrhizal plants, pines require ectomycorrhizal fungal symbionts to complete their life cycle. Pines introduced into regions far from their native range are typically incompatible with local ectomycorrhizal fungi, and, when they invade, coinvade with fungi from their native range. While the identities and distributions of coinvasive fungal symbionts of pine invasions are poorly known, communities that have been studied are notably depauperate. However, it is not yet clear whether any number of fungal coinvaders is able to support a Pinaceae invasion, or whether very depauperate communities are unable to invade. Here, we ask whether there is evidence for a minimum species richness of fungal symbionts necessary to support a pine/ectomycorrhizal fungus coinvasion. We sampled a Pinus contorta invasion front near Coyhaique, Chile, using molecular barcoding to identify ectomycorrhizal fungi. We report that the site has a total richness of four species, and that many invasive trees appear to be supported by only a single ectomycorrhizal fungus, Suillus luteus. We conclude that a single ectomycorrhizal (ECM) fungus can suffice to enable a pine invasion.

  6. Customization of Protein Single Nanowires for Optical Biosensing.

    Science.gov (United States)

    Sun, Yun-Lu; Sun, Si-Ming; Wang, Pan; Dong, Wen-Fei; Zhang, Lei; Xu, Bin-Bin; Chen, Qi-Dai; Tong, Li-Min; Sun, Hong-Bo

    2015-06-24

    An all-protein single-nanowire optical biosensor is constructed by a facile and general femtosecond laser direct writing approach with nanoscale structural customization. As-formed protein single nanowires show excellent optical properties (fine waveguiding performance and bio-applicable transmission windows), and are utilized as evanescent optical nanobiosensors for label-free biotin detection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. 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.

  8. 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 ...

  9. 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 ...

  10. 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

  11. Optical probing of single fluorescent molecules and proteins

    NARCIS (Netherlands)

    Garcia Parajo, M.F.; Veerman, J.A.; Bouwhuis, R.; Bouwhuis, Rudo; van Hulst, N.F.; Vallée, R.A.L.

    2001-01-01

    Single-molecule detection and analysis of organic fluorescent molecules and proteins are presented, with emphasis o­n the underlying principles methodology and the application of single-molecule analysis at room temperature. This Minireview is mainly focused o­n the application of confocal and

  12. Statistical inference in single molecule measurements of protein adsorption

    Science.gov (United States)

    Armstrong, Megan J.; Tsitkov, Stanislav; Hess, Henry

    2018-02-01

    Significant effort has been invested into understanding the dynamics of protein adsorption on surfaces, in particular to predict protein behavior at the specialized surfaces of biomedical technologies like hydrogels, nanoparticles, and biosensors. Recently, the application of fluorescent single molecule imaging to this field has permitted the tracking of individual proteins and their stochastic contribution to the aggregate dynamics of adsorption. However, the interpretation of these results is complicated by (1) the finite time available to observe effectively infinite adsorption timescales and (2) the contribution of photobleaching kinetics to adsorption kinetics. Here, we perform a protein adsorption simulation to introduce specific survival analysis methods that overcome the first complication. Additionally, we collect single molecule residence time data from the adsorption of fibrinogen to glass and use survival analysis to distinguish photobleaching kinetics from protein adsorption kinetics.

  13. Multiplex single-molecule interaction profiling of DNA barcoded proteins

    Science.gov (United States)

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

  14. 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.

  15. 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.

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

    Science.gov (United States)

    Darmanis, Spyros; Gallant, Caroline Julie; Marinescu, Voichita Dana; Niklasson, Mia; Segerman, Anna; Flamourakis, Georgios; Fredriksson, Simon; Assarsson, Erika; Lundberg, Martin; Nelander, Sven; Westermark, Bengt; Landegren, Ulf

    2016-01-12

    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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  17. Semisynthetic protein nanoreactor for single-molecule chemistry

    OpenAIRE

    Lee, Joongoo; Bayley, Hagan

    2015-01-01

    The modulation of ionic current flowing through an individual protein pore provides information at the single-molecule level about chemical reactions occurring within the pore. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. H...

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

    Science.gov (United States)

    2011-01-01

    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 recognition proteins in C

  19. 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

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

    Science.gov (United States)

    Yao, Yao; Docter, Margreet; van Ginkel, Jetty; de Ridder, Dick; Joo, Chirlmin

    2015-10-01

    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 fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences.

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

    International Nuclear Information System (INIS)

    Yao, Yao; Docter, Margreet; Van Ginkel, Jetty; Joo, Chirlmin; De Ridder, Dick

    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 fundamental limitations in sensitivity. Here we propose a method for single-molecule (SM) protein sequencing. A major challenge lies in the fact that proteins are composed of 20 different amino acids, which demands 20 molecular reporters. We computationally demonstrate that it suffices to measure only two types of amino acids to identify proteins and suggest an experimental scheme using SM fluorescence. When achieved, this highly sensitive approach will result in a paradigm shift in proteomics, with major impact in the biological and medical sciences. (paper)

  2. MICROORGANISMS: A MARVELOUS SOURCE OF SINGLE CELL PROTEINS

    Directory of Open Access Journals (Sweden)

    Agam Nangul

    2013-08-01

    Full Text Available The increasing global population living below the poverty line is driving the scientific community to search for non-conventional protein sources that can replace conventional expensive ones. Microbial proteins, or single-cell protein (SCP, represent a potential future nutrient source for human food and animal feed. These microbial proteins can be grown rapidly on substrates with minimum dependence on soil, water and climate conditions. They can be produced from algae, fungi and bacteria the chief sources of SCP. It is convenient to use microorganisms for production of SCP as they grow rapidly and have high protein content. Industrially, they can be produced from algal biomass, yeast, fungi. There are several other ways of getting SCP as well. Despite numerous advantages of SCP, they have disadvantages and toxic effects too, especially related to mycotoxins and bacterial toxins.

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

    African Journals Online (AJOL)

    BSN

    origin is unicellular or simple multicellular organism such as bacteria, yeasts, fungi, ... Pilot plant produe1io11 of single cell proteins now take place in several centre.ii in ... animal feed but little or no information has been documented as per its ...

  4. Surface Passivation for Single-molecule Protein Studies

    Science.gov (United States)

    Chandradoss, Stanley D.; Haagsma, Anna C.; Lee, Young Kwang; Hwang, Jae-Ho; Nam, Jwa-Min; Joo, Chirlmin

    2014-01-01

    Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation. PMID:24797261

  5. 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.

  6. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    International Nuclear Information System (INIS)

    Timmins, P.A.; Pebay-Peyroula, E.

    1994-01-01

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H 2 O/D 2 O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished

  7. Protein-detergent interactions in single crystals of membrane proteins studied by neutron crystallography

    Energy Technology Data Exchange (ETDEWEB)

    Timmins, P.A. [ILL, Grenoble (France); Pebay-Peyroula, E. [IBS-UJF Grenoble (France)

    1994-12-31

    The detergent micelles surrounding membrane protein molecules in single crystals can be investigated using neutron crystallography combined with H{sub 2}O/D{sub 2}O contrast variation. If the protein structure is known then the contrast variation method allows phases to be determined at a contrast where the detergent dominates the scattering. The application of various constraints allows the resulting scattering length density map to be realistically modeled. The method has been applied to two different forms of the membrane protein porin. In one case both hydrogenated and partially deuterated protein were used, allowing the head group and tail to be distinguished.

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

    OpenAIRE

    Zhang, Yi; Zhang, Qiaoling; Li, Jinghao; Zhang, Qingling

    2014-01-01

    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 ind...

  9. Addressable droplet microarrays for single cell protein analysis.

    Science.gov (United States)

    Salehi-Reyhani, Ali; Burgin, Edward; Ces, Oscar; Willison, Keith R; Klug, David R

    2014-11-07

    Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

  10. 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

  11. Compressive Force Spectroscopy: From Living Cells to Single Proteins.

    Science.gov (United States)

    Wang, Jiabin; Liu, Meijun; Shen, Yi; Sun, Jielin; Shao, Zhifeng; Czajkowsky, Daniel Mark

    2018-03-23

    One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development.

  12. 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...

  13. Force spectroscopy studies on protein-ligand interactions: a single protein mechanics perspective.

    Science.gov (United States)

    Hu, Xiaotang; Li, Hongbin

    2014-10-01

    Protein-ligand interactions are ubiquitous and play important roles in almost every biological process. The direct elucidation of the thermodynamic, structural and functional consequences of protein-ligand interactions is thus of critical importance to decipher the mechanism underlying these biological processes. A toolbox containing a variety of powerful techniques has been developed to quantitatively study protein-ligand interactions in vitro as well as in living systems. The development of atomic force microscopy-based single molecule force spectroscopy techniques has expanded this toolbox and made it possible to directly probe the mechanical consequence of ligand binding on proteins. Many recent experiments have revealed how ligand binding affects the mechanical stability and mechanical unfolding dynamics of proteins, and provided mechanistic understanding on these effects. The enhancement effect of mechanical stability by ligand binding has been used to help tune the mechanical stability of proteins in a rational manner and develop novel functional binding assays for protein-ligand interactions. Single molecule force spectroscopy studies have started to shed new lights on the structural and functional consequence of ligand binding on proteins that bear force under their biological settings. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  14. Click strategies for single-molecule protein fluorescence.

    Science.gov (United States)

    Milles, Sigrid; Tyagi, Swati; Banterle, Niccolò; Koehler, Christine; VanDelinder, Virginia; Plass, Tilman; Neal, Adrian P; Lemke, Edward A

    2012-03-21

    Single-molecule methods have matured into central tools for studies in biology. Foerster resonance energy transfer (FRET) techniques, in particular, have been widely applied to study biomolecular structure and dynamics. The major bottleneck for a facile and general application of these studies arises from the need to label biological samples site-specifically with suitable fluorescent dyes. In this work, we present an optimized strategy combining click chemistry and the genetic encoding of unnatural amino acids (UAAs) to overcome this limitation for proteins. We performed a systematic study with a variety of clickable UAAs and explored their potential for high-resolution single-molecule FRET (smFRET). We determined all parameters that are essential for successful single-molecule studies, such as accessibility of the probes, expression yield of proteins, and quantitative labeling. Our multiparameter fluorescence analysis allowed us to gain new insights into the effects and photophysical properties of fluorescent dyes linked to various UAAs for smFRET measurements. This led us to determine that, from the extended tool set that we now present, genetically encoding propargyllysine has major advantages for state-of-the-art measurements compared to other UAAs. Using this optimized system, we present a biocompatible one-step dual-labeling strategy of the regulatory protein RanBP3 with full labeling position freedom. Our technique allowed us then to determine that the region encompassing two FxFG repeat sequences adopts a disordered but collapsed state. RanBP3 serves here as a prototypical protein that, due to its multiple cysteines, size, and partially disordered structure, is not readily accessible to any of the typical structure determination techniques such as smFRET, NMR, and X-ray crystallography.

  15. Phytoplankton IF-FISH: Species-specific labeling of cellular proteins by immunofluorescence (IF) with simultaneous species identification by fluorescence immunohybridization (FISH).

    Science.gov (United States)

    Meek, Megan E; Van Dolah, Frances M

    2016-05-01

    Phytoplankton rarely occur as unialgal populations. Therefore, to study species-specific protein expression, indicative of physiological status in natural populations, methods are needed that will both assay for a protein of interest and identify the species expressing it. Here we describe a protocol for IF-FISH, a dual labeling procedure using immunofluorescence (IF) labeling of a protein of interest followed by fluorescence in situ hybridization (FISH) to identify the species expressing that protein. The protocol was developed to monitor expression of the cell cycle marker proliferating cell nuclear antigen (PCNA) in the red tide dinoflagellate, Karenia brevis, using a large subunit (LSU) rRNA probe to identify K. brevis in a mixed population of morphologically similar Karenia species. We present this protocol as proof of concept that IF-FISH can be successfully applied to phytoplankton cells. This method is widely applicable for the analysis of single-cell protein expression of any protein of interest within phytoplankton communities. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. 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...

  17. Whey utilization for single-cell protein production

    Energy Technology Data Exchange (ETDEWEB)

    Barraquio, V; Silverio, L G; Revilleza, R P; Fernadez, W L

    1980-01-01

    The production of single-cell protein by yeast assimilation of lactose in soft cheese whey was studied using Candida pseudotropicalis as a test organism. Under shake-flask cultivation conditions with deproteinized whey as the medium, lactose (initially 4.20%) was completely assimilated in 48h; cell mass was 5.56 mg/mL after 72h; and average protein content of the dried mass was approximately 11.8%. Batch cultivation using undeproteinized whey resulted in a faster lactose utilization rate from an initial 3.93% to a residual 0.56% in 12 h; cell mass was 8.41 mg/mL in 10 h; and average protein was approximately 37.7%. In a semicontinuous culture with 10 to the power of 7 viable cells/mL as initial cell concentration, 15.69 mg/mL cell mass with a mean protein content of approximately 21.4% could be produced and lactose could be considerably consumed (from an initial 4.75% to a residual 0.42%) within 13-14 h. Supplementation with (NH/sub 4/)/sub 2/S0/sub 4/ and KH/sub 2/P0/sub 4/ did not increase cell mass (12.47 mg/mL in 12 h) and hasten lactose assimulation (from initial 4.49% to residual 0.3% in 12 h). Average protein content was approximately 31%. Cell mass yield was established as 0.29 mg yeast cell/mg lactose consumed. Factors that might have affected protein content are also discussed.

  18. Towards single-molecule observation of protein synthesis

    International Nuclear Information System (INIS)

    Dulin, David; Le Gall, Antoine; Bouyer, Philippe; Perronet, Karen; Westbrook, Nathalie; Soler, Nicolas; Fourmy, Dominique; Yoshizawa, Satoko

    2009-01-01

    The ribosome is the molecular motor responsible for the protein synthesis within all cells. Ribosome motions along the messenger RNA (mRNA) to read the genetic code are asynchronous and occur along multiple kinetic paths. Consequently, a study at the single macromolecule level is desirable to unravel the complex dynamics involved. In this communication, we present the development of an advanced surface chemistry to attach an active ribosome to the microscope coverslip and follow the amino-acid incorporation by fluorescence microscopy. The ribosome is labeled with a quantum dot (QD) in order to localize it on the surface while a specific amino acid (lysine) is marked with Bodipy-FL. This fluorescent dye is small enough to enter the ribosomal channel thus leaving intact ribosomal activity. It should then be possible to observe the protein synthesis in real time as the labeled amino acids are incorporated into the polypeptide chain. (Author)

  19. Single cell protein production from mandarin orange peel

    Energy Technology Data Exchange (ETDEWEB)

    Nishio, N.; Nagai, S.

    1981-01-01

    As the hydrolysis of mandarin orange peel with macerating enzyme (40/sup 0/C,24 h)produced 0.59 g g/sup -1/ reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120/sup 0/C with 0.8 N H/sub 2/SO/sub 4/), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel. When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (gg/sup -1/)were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30/sup 0/C using 100 g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts.

  20. 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.

  1. 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

  2. Antibiofilm Effect of DNase against Single and Mixed Species Biofilm

    Science.gov (United States)

    Sharma, Komal

    2018-01-01

    Biofilms are aggregates of microorganisms that coexist in socially coordinated micro-niche in a self-produced polymeric matrix on pre-conditioned surfaces. The biofilm matrix reduces the efficacy of antibiofilm strategies. DNase degrades the extracellular DNA (e-DNA) present in the matrix, rendering the matrix weak and susceptible to antimicrobials. In the current study, the effect of DNase I was evaluated during biofilm formation (pre-treatment), on preformed biofilms (post-treatment) and both (dual treatment). The DNase I pre-treatment was optimized for P. aeruginosa PAO1 (model biofilm organism) at 10 µg/mL and post-treatment at 10 µg/mL with 15 min of contact duration. Inclusion of Mg2+ alongside DNase I post-treatment resulted in 90% reduction in biofilm within only 5 min of contact time (irrespective of age of biofilm). On extension of these findings, DNase I was found to be less effective against mixed species biofilm than individual biofilms. DNase I can be used as potent antibiofilm agent and with further optimization can be effectively used for biofilm prevention and reduction in situ. PMID:29562719

  3. 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

  4. 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

  5. The action of chemical and mechanical stresses on single and dual species biofilm removal of drinking water bacteria.

    Science.gov (United States)

    Gomes, I B; Lemos, M; Mathieu, L; Simões, M; Simões, L C

    2018-08-01

    The presence of biofilms in drinking water distribution systems (DWDS) is a global public health concern as they can harbor pathogenic microorganisms. Sodium hypochlorite (NaOCl) is the most commonly used disinfectant for microbial growth control in DWDS. However, its effect on biofilm removal is still unclear. This work aims to evaluate the effects of the combination of chemical (NaOCl) and mechanical stresses on the removal of single and dual species biofilms of two bacteria isolated from DWDS and considered opportunistic, Acinectobacter calcoaceticus and Stenotrophomonas maltophilia. A rotating cylinder reactor was successfully used for the first time in drinking water biofilm studies with polyvinyl chloride as substratum. The single and dual species biofilms presented different characteristics in terms of metabolic activity, mass, density, thickness and content of proteins and polysaccharides. Their complete removal was not achieved even when a high NaOCl concentrations and an increasing series of shear stresses (from 2 to 23Pa) were applied. In general, NaOCl pre-treatment did not improve the impact of mechanical stress on biofilm removal. Dual species biofilms were colonized mostly by S. maltophilia and were more susceptible to chemical and mechanical stresses than these single species. The most efficient treatment (93% biofilm removal) was the combination of NaOCl at 175mg·l -1 with mechanical stress against dual species biofilms. Of concern was the high tolerance of S. maltophilia to chemical and mechanical stresses in both single and dual species biofilms. The overall results demonstrate the inefficacy of NaOCl on biofilm removal even when combined with high shear stresses. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    2016-07-01

    diseases are a regular occurrence globally (Figure 1). The Zika virus is the latest example gaining widespread attention. Many of the (re-)emerging...for establishing infection and/or modulating pathogenesis (Figures 2 and 3). 3 Figure 2. Schematic of several virus -host protein interactions within...8725 John J. Kingman Road, MS 6201 Fort Belvoir, VA 22060-6201 T E C H N IC A L R E P O R T DTRA-TR-16-79 Cross-species virus -host

  7. 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.

  8. 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.

  9. 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.

  10. 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. .

  11. 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.

  12. 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.

  13. Maintaining animal assemblages through single-species management: the case of threatened caribou in boreal forest.

    Science.gov (United States)

    Bichet, Orphé; Dupuch, Angélique; Hébert, Christian; Le Borgne, Hélène Le; Fortin, Daniel

    2016-03-01

    With the intensification of human activities, preserving animal populations is a contemporary challenge of critical importance. In this context, the umbrella species concept is appealing because preserving a single species should result in the protection of multiple co-occurring species. Practitioners, though, face the task of having to find suitable umbrellas to develop single-species management guidelines. In North America, boreal forests must be managed to facilitate the recovery of the threatened boreal caribou (Rangifer tarandus). Yet, the effect of caribou conservation on co-occurring animal species remains poorly documented. We tested if boreal caribou can constitute an effective umbrella for boreal fauna. Birds, small mammals, and insects were sampled along gradients of post-harvest and post-fire forest succession. Predictive models of occupancy were developed from the responses of 95 species to characteristics of forest stands and their surroundings. We then assessed the similarity of species occupancy expected between simulated harvested landscapes and a 90 000-km2 uncut landscape. Managed landscapes were simulated based on three levels of disturbance, two timber-harvest rotation cycles, and dispersed or aggregated cut-blocks. We found that management guidelines that were more likely to maintain caribou populations should also better preserve animal assemblages. Relative to fragmentation or harvest cycle, we detected a stronger effect of habitat loss on species assemblages. Disturbing 22%, 35%, and 45% of the landscape should result, respectively, in 80%, 60%, and 40% probability for caribou populations to be sustainable; in turn, this should result in regional species assemblages with Jaccard similarity indices of 0.86, 0.79, and 0.74, respectively, relative to the uncut landscape. Our study thus demonstrates the value of single-species management for animal conservation. Our quantitative approach allows for the evaluation of management guidelines prior

  14. 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.

  15. Differences in a ribosomal DNA sequence of Strongylus species allows identification of single eggs.

    Science.gov (United States)

    Campbell, A J; Gasser, R B; Chilton, N B

    1995-03-01

    In the current study, molecular techniques were evaluated for the species identification of individual strongyle eggs. Adult worms of Strongylus edentatus, S. equinus and S. vulgaris were collected at necropsy from horses from Australia and the U.S.A. Genomic DNA was isolated and a ribosomal transcribed spacer (ITS-2) amplified and sequenced using polymerase chain reaction (PCR) techniques. The length of the ITS-2 sequence of S. edentatus, S. equinus and S. vulgaris ranged between 217 and 235 nucleotides. Extensive sequence analysis demonstrated a low degree (0-0.9%) of intraspecific variation in the ITS-2 for the Strongylus species examined, whereas the levels of interspecific differences (13-29%) were significantly greater. Interspecific differences in the ITS-2 sequences allowed unequivocal species identification of single worms and eggs using PCR-linked restriction fragment length polymorphism. These results demonstrate the potential of the ribosomal spacers as genetic markers for species identification of single strongyle eggs from horse faeces.

  16. Trans-species Engineering of Glycosylated Therapeutic Proteins

    DEFF Research Database (Denmark)

    Yang, Zhang

    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......Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...

  17. DeepQA: Improving the estimation of single protein model quality with deep belief networks

    OpenAIRE

    Cao, Renzhi; Bhattacharya, Debswapna; Hou, Jie; Cheng, Jianlin

    2016-01-01

    Background Protein quality assessment (QA) useful for ranking and selecting protein models has long been viewed as one of the major challenges for protein tertiary structure prediction. Especially, estimating the quality of a single protein model, which is important for selecting a few good models out of a large model pool consisting of mostly low-quality models, is still a largely unsolved problem. Results We introduce a novel single-model quality assessment method DeepQA based on deep belie...

  18. Detection of four important Eimeria species by multiplex PCR in a single assay.

    Science.gov (United States)

    You, Myung-Jo

    2014-06-01

    The oocysts of some of the recognized species of chicken coccidiosis are difficult to distinguish morphologically. Diagnostic laboratories are increasingly utilizing DNA-based technologies for the specific identification of Eimeria species. This study reports a multiplex polymerase chain reaction (PCR) assay based on internal transcribed spacer-1 (ITS-1) for the simultaneous diagnosis of the Eimeria tenella, Eimeria acervulina, Eimeria maxima, and Eimeria necatrix species, which infect domestic fowl. Primer pairs specific to each species were designed in order to generate a ladder of amplification products ranging from 20 to 25 bp, and a common optimum annealing temperature for these species was determined to be 52.5 °C. Sensitivity tests were performed for each species, showing a detection threshold of 1-5 pg. All the species were amplified homogeneously, and a homogenous band ladder was observed, indicating that the assay permitted the simultaneous detection of all the species in a single-tube reaction. In the phylogenic study, there was a clear species clustering, which was irrespective of geographical location, for all the ITS-1 sequences used. This multiplex PCR assay represents a rapid and potential cost-effective diagnostic method for the detection of some key Eimeria species that infect domestic fowl. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. 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

  20. Reactive oxygen species formation during tetanic contractions in single isolated Xenopus myofibers

    OpenAIRE

    Zuo, Li; Nogueira, Leonardo; Hogan, Michael C.

    2011-01-01

    Contracting skeletal muscle produces reactive oxygen species (ROS) that have been shown to affect muscle function and adaptation. However, real-time measurement of ROS in contracting myofibers has proven to be difficult. We used amphibian (Xenopus laevis) muscle to test the hypothesis that ROS are formed during contractile activity in isolated single skeletal muscle fibers and that this contraction-induced ROS formation affects fatigue development. Single myofibers were loaded with 5 μM dihyd...

  1. 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...

  2. Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding

    Science.gov (United States)

    Shahi, Payam; Kim, Samuel C.; Haliburton, John R.; Gartner, Zev J.; Abate, Adam R.

    2017-03-01

    Proteins are the primary effectors of cellular function, including cellular metabolism, structural dynamics, and information processing. However, quantitative characterization of proteins at the single-cell level is challenging due to the tiny amount of protein available. Here, we present Abseq, a method to detect and quantitate proteins in single cells at ultrahigh throughput. Like flow and mass cytometry, Abseq uses specific antibodies to detect epitopes of interest; however, unlike these methods, antibodies are labeled with sequence tags that can be read out with microfluidic barcoding and DNA sequencing. We demonstrate this novel approach by characterizing surface proteins of different cell types at the single-cell level and distinguishing between the cells by their protein expression profiles. DNA-tagged antibodies provide multiple advantages for profiling proteins in single cells, including the ability to amplify low-abundance tags to make them detectable with sequencing, to use molecular indices for quantitative results, and essentially limitless multiplexing.

  3. 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

    environmental gradients and minimizing urbanization may have a greater benefit to ecosystem functioning then a single-species management focus.

  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. 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.

  6. 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

  7. Proteomic analysis of seed storage proteins in wild rice species of the Oryza genus.

    Science.gov (United States)

    Jiang, Chunmiao; Cheng, Zaiquan; Zhang, Cheng; Yu, Tengqiong; Zhong, Qiaofang; Shen, J Qingxi; Huang, Xingqi

    2014-01-01

    The total protein contents of rice seeds are significantly higher in the three wild rice species (Oryza rufipogon Grill., Oryza officinalis Wall. and Oryza meyeriana Baill.) than in the cultivated rice (Oryza sativa L.). However, there is still no report regarding a systematic proteomic analysis of seed proteins in the wild rice species. Also, the relationship between the contents of seed total proteins and rice nutritional quality has not been thoroughly investigated. The total seed protein contents, especially the glutelin contents, of the three wild rice species were higher than those of the two cultivated rice materials. Based on the protein banding patterns of SDS-PAGE, O. rufipogon was similar to the two cultivated rice materials, followed by O. officinalis, while O. meyeriana exhibited notable differences. Interestingly, O. meyeriana had high contents of glutelin and low contents of prolamine, and lacked 26 kDa globulin band and appeared a new 28 kDa protein band. However, for O. officinali a 16 kDa protein band was absent and a row of unique 32 kDa proteins appeared. In addition, we found that 13 kDa prolamine band disappeared while special 14 kDa and 12 kDa protein bands were present in O. officinalis. Two-dimensional gel electrophoresis (2-DE) analysis revealed remarkable differences in protein profiles of the wild rice species and the two cultivated rice materials. Also, the numbers of detected protein spots of the three wild rice species were significantly higher than those of two cultivated rice. A total of 35 differential protein spots were found for glutelin acidic subunits, glutelin precursors and glutelin basic subunits in wild rice species. Among those, 18 protein spots were specific and 17 major spots were elevated. Six differential protein spots for glutelin acidic subunits were identified, including a glutelin type-A 2 precursor and five hypothetical proteins. This was the first report on proteomic analysis of the three wild rice species

  8. Intramolecular three-colour single pair FRET of intrinsically disordered proteins with increased dynamic range.

    Science.gov (United States)

    Milles, Sigrid; Koehler, Christine; Gambin, Yann; Deniz, Ashok A; Lemke, Edward A

    2012-10-01

    Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.

  9. Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae.

    Science.gov (United States)

    Vernal, Javier; Serpa, Viviane I; Tavares, Carolina; Souza, Emanuel M; Pedrosa, Fábio O; Terenzi, Hernán

    2007-05-01

    An open reading frame encoding a protein similar in size and sequence to the Escherichia coli single-stranded DNA binding protein (SSB protein) was identified in the Herbaspirillum seropedicae genome. This open reading frame was cloned into the expression plasmid pET14b. The SSB protein from H. seropedicae, named Hs_SSB, was overexpressed in E. coli strain BL21(DE3) and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein. The apparent molecular mass of the native Hs_SSB was estimated by gel filtration, suggesting that the native protein is a tetramer made up of four similar subunits. The purified protein binds to single-stranded DNA (ssDNA) in a similar manner to other SSB proteins. The production of this recombinant protein in good yield opens up the possibility of obtaining its 3D-structure and will help further investigations into DNA metabolism.

  10. Cardiovascular Small Heat Shock Protein HSPB7 Is a Kinetically Privileged Reactive Electrophilic Species (RES) Sensor.

    Science.gov (United States)

    Surya, Sanjna L; Long, Marcus J C; Urul, Daniel A; Zhao, Yi; Mercer, Emily J; EIsaid, Islam M; Evans, Todd; Aye, Yimon

    2018-02-08

    Small heat shock protein (sHSP)-B7 (HSPB7) is a muscle-specific member of the non-ATP-dependent sHSPs. The precise role of HSPB7 is enigmatic. Here, we disclose that zebrafish Hspb7 is a kinetically privileged sensor that is able to react rapidly with native reactive electrophilic species (RES), when only substoichiometric amounts of RES are available in proximity to Hspb7 expressed in living cells. Among the two Hspb7-cysteines, this RES sensing is fulfilled by a single cysteine (C117). Purification and characterizations in vitro reveal that the rate for RES adduction is among the most efficient reported for protein-cysteines with native carbonyl-based RES. Covalent-ligand binding is accompanied by structural changes (increase in β-sheet-content), based on circular dichroism analysis. Among the two cysteines, only C117 is conserved across vertebrates; we show that the human ortholog is also capable of RES sensing in cells. Furthermore, a cancer-relevant missense mutation reduces this RES-sensing property. This evolutionarily conserved cysteine-biosensor may play a redox-regulatory role in cardioprotection.

  11. A REVIEW OF SINGLE SPECIES TOXICITY TESTS: ARE THE TESTS RELIABLE PREDICTORS OF AQUATIC ECOSYSTEM COMMUNITY RESPONSES?

    Science.gov (United States)

    This document provides a comprehensive review to evaluate the reliability of indicator species toxicity test results in predicting aquatic ecosystem impacts, also called the ecological relevance of laboratory single species toxicity tests.

  12. 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.

  13. Probing Enzyme-Surface Interactions via Protein Engineering and Single-Molecule Techniques

    Science.gov (United States)

    2017-06-26

    SECURITY CLASSIFICATION OF: The overall objective of this research was to exploit protein engineering and fluorescence single-molecule methods to...enhance our understanding of the interaction of proteins and surfaces. Given this objective, the specific aims of this research were to: 1) exploit the...incorporation of unnatural amino acids in proteins to introduce single-molecule probes (i.e., fluorophores for fluorescence resonance energy transfer

  14. YEAST A SINGLE CELL PROTEIN: CHARACTERISTICS and METABOLISM

    OpenAIRE

    AMATA, I.A

    2013-01-01

    Most of the developing countries of the world are facing a major problem of malnutrition. Due to rapid growth in the population, food and feed scarcity are prevalent leading to a deficiency of protein and essential nutrients amongst human beings and livestock. It is therefore important to take necessary measures to stem this trend by increasing protein production and making it available and more affordable to the population by utilizing methods available for the production of alternative sour...

  15. 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.

  16. Using Single-Protein Tracking to Study Cell Migration.

    Science.gov (United States)

    Orré, Thomas; Mehidi, Amine; Massou, Sophie; Rossier, Olivier; Giannone, Grégory

    2018-01-01

    To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.

  17. Reactive species formed on proteins exposed to singlet oxygen

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2004-01-01

    Singlet oxygen ((1)O(2)) is believed to be generated in biological systems by a range of endogenous processes (e.g. enzymatic and chemical reactions) and exogenous stimuli (e.g. UV or visible light in the presence of a sensitiser). Kinetic data is consistent with proteins being a major target...... 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...

  18. Improving N-terminal protein annotation of Plasmodium species based on signal peptide prediction of orthologous proteins

    Directory of Open Access Journals (Sweden)

    Neto Armando

    2012-11-01

    Full Text Available Abstract Background Signal peptide is one of the most important motifs involved in protein trafficking and it ultimately influences protein function. Considering the expected functional conservation among orthologs it was hypothesized that divergence in signal peptides within orthologous groups is mainly due to N-terminal protein sequence misannotation. Thus, discrepancies in signal peptide prediction of orthologous proteins were used to identify misannotated proteins in five Plasmodium species. Methods Signal peptide (SignalP and orthology (OrthoMCL were combined in an innovative strategy to identify orthologous groups showing discrepancies in signal peptide prediction among their protein members (Mixed groups. In a comparative analysis, multiple alignments for each of these groups and gene models were visually inspected in search of misannotated proteins and, whenever possible, alternative gene models were proposed. Thresholds for signal peptide prediction parameters were also modified to reduce their impact as a possible source of discrepancy among orthologs. Validation of new gene models was based on RT-PCR (few examples or on experimental evidence already published (ApiLoc. Results The rate of misannotated proteins was significantly higher in Mixed groups than in Positive or Negative groups, corroborating the proposed hypothesis. A total of 478 proteins were reannotated and change of signal peptide prediction from negative to positive was the most common. Reannotations triggered the conversion of almost 50% of all Mixed groups, which were further reduced by optimization of signal peptide prediction parameters. Conclusions The methodological novelty proposed here combining orthology and signal peptide prediction proved to be an effective strategy for the identification of proteins showing wrongly N-terminal annotated sequences, and it might have an important impact in the available data for genome-wide searching of potential vaccine and drug

  19. Limited tryptic proteolysis of the benzodiazepine binding proteins in different species reveals structural homologies.

    Science.gov (United States)

    Friedl, W; Lentes, K U; Schmitz, E; Propping, P; Hebebrand, J

    1988-12-01

    Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.

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

    African Journals Online (AJOL)

    use

    Key word: microbial protein, Oreochromis niloticus, feeding, cost benefit, aquaculture. ... most aqua feeds, is an important ingredient in aqua- culture diets. Though it has ... Such alternatives must satisfy the nutritional needs of the fish species ...

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

    Science.gov (United States)

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

    2015-09-01

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

  2. LC-MS/MS Identification of Species-Specific Muscle Peptides in Processed Animal Proteins.

    Science.gov (United States)

    Marchis, Daniela; Altomare, Alessandra; Gili, Marilena; Ostorero, Federica; Khadjavi, Amina; Corona, Cristiano; Ru, Giuseppe; Cappelletti, Benedetta; Gianelli, Silvia; Amadeo, Francesca; Rumio, Cristiano; Carini, Marina; Aldini, Giancarlo; Casalone, Cristina

    2017-12-06

    An innovative analytical strategy has been applied to identify signature peptides able to distinguish among processed animal proteins (PAPs) derived from bovine, pig, fish, and milk products. Proteomics was first used to elucidate the proteome of each source. Starting from the identified proteins and using a funnel based approach, a set of abundant and well characterized peptides with suitable physical-chemical properties (signature peptides) and specific for each source was selected. An on-target LC-ESI-MS/MS method (MRM mode) was set up using standard peptides and was then applied to selectively identify the PAP source and also to distinguish proteins from bovine carcass and milk proteins. We believe that the method described meets the request of the European Commission which has developed a strategy for gradually lifting the "total ban" toward "species to species ban", therefore requiring official methods for species-specific discrimination in feed.

  3. Protein folding and translocation : single-molecule investigations

    NARCIS (Netherlands)

    Leeuwen, Rudolphus Gerardus Henricus van

    2006-01-01

    This thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly

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

    African Journals Online (AJOL)

    ADOWIE PERE

    2018-03-13

    Mar 13, 2018 ... as orange, pineapple, banana, watermelon and cucumber waste as growth ... compared to plant and animal proteins with good ... not affected by weather condition, short generation .... found to be the least source of chemical composition ... Food waste. Proximate composition (%). Moisture. Ash. Crude fibre.

  5. Single-Molecule Studies of Bacterial Protein Translocation

    NARCIS (Netherlands)

    Kedrov, Alexej; Kusters, Ilja; Driessen, Arnold J. M.

    2013-01-01

    In prokaryotes, a large share of the proteins are secreted from the cell through a process that requires their translocation across the cytoplasmic membrane. This process is mediated by the universally conserved Sec system with homologues in the endoplasmic reticulum and thylakoid membranes of

  6. Genetic analysis of RPA single-stranded DNA binding protein in Haloferax volcanii

    OpenAIRE

    Stroud, A. L.

    2012-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein that is present in all three domains of life. The roles of RPA include stabilising and protecting single- stranded DNA from nuclease degradation during DNA replication and repair. To achieve this, RPA uses an oligosaccharide-binding fold (OB fold) to bind single- stranded DNA. Haloferax volcanii encodes three RPAs – RPA1, RPA2 and RPA3, of which rpa1 and rpa3 are in operons with genes encoding associated proteins (APs). ...

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

    Science.gov (United States)

    Henry, Kevin A.; Sulea, Traian; van Faassen, Henk; Hussack, Greg; Purisima, Enrico O.; MacKenzie, C. Roger; Arbabi-Ghahroudi, Mehdi

    2016-01-01

    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. PMID:27631624

  8. HitPredict version 4: comprehensive reliability scoring of physical protein-protein interactions from more than 100 species.

    Science.gov (United States)

    López, Yosvany; Nakai, Kenta; Patil, Ashwini

    2015-01-01

    HitPredict is a consolidated resource of experimentally identified, physical protein-protein interactions with confidence scores to indicate their reliability. The study of genes and their inter-relationships using methods such as network and pathway analysis requires high quality protein-protein interaction information. Extracting reliable interactions from most of the existing databases is challenging because they either contain only a subset of the available interactions, or a mixture of physical, genetic and predicted interactions. Automated integration of interactions is further complicated by varying levels of accuracy of database content and lack of adherence to standard formats. To address these issues, the latest version of HitPredict provides a manually curated dataset of 398 696 physical associations between 70 808 proteins from 105 species. Manual confirmation was used to resolve all issues encountered during data integration. For improved reliability assessment, this version combines a new score derived from the experimental information of the interactions with the original score based on the features of the interacting proteins. The combined interaction score performs better than either of the individual scores in HitPredict as well as the reliability score of another similar database. HitPredict provides a web interface to search proteins and visualize their interactions, and the data can be downloaded for offline analysis. Data usability has been enhanced by mapping protein identifiers across multiple reference databases. Thus, the latest version of HitPredict provides a significantly larger, more reliable and usable dataset of protein-protein interactions from several species for the study of gene groups. Database URL: http://hintdb.hgc.jp/htp. © The Author(s) 2015. Published by Oxford University Press.

  9. 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.

  10. Single oligomer spectra probe chromophore nanoenvironments of tetrameric fluorescent proteins

    NARCIS (Netherlands)

    Blum, Christian; Meixner, Alfred J; Subramaniam, Vinod

    2006-01-01

    When analyzing the emission of a large number of individual chromophores embedded in a matrix, the spread of the observed parameters is a characteristic property for the particular chromophore-matrix system. To quantitatively assess the influence of the matrix on the single molecule emission

  11. Single Oligomer Spectra Probe Chromophore Nanoenvironments of Tetrameric Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Meixner, Alfred J.; Subramaniam, Vinod

    2006-01-01

    When analyzing the emission of a large number of individual chromophores embedded in a matrix, the spread of the observed parameters is a characteristic property for the particular chromophore−matrix system. To quantitatively assess the influence of the matrix on the single molecule emission

  12. 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

  13. Drought Stress and Its Impact on Protein in Three Species of Vitex

    Directory of Open Access Journals (Sweden)

    John A. De Britto

    2011-09-01

    Full Text Available Drought is one of the most important natural phenomenon which affects on plant growth. When drought stress is imposed different molecular and biochemical responses took place in the plants. The protein profile of three species of Vitex (Vitex trifolia L., Vitex altissima L. and Vitex negundo L. under normally irrigated condition and severe drought plants was analyzed through SDS-PAGE. Drought stress significantly affects proteins in plants when compared the normal conditioned plants. Several new protein bands were identified in the stressed plants. It seems that Vitex species can be adapted to drought stress conditions. Hence it was concluded that number of new proteins were synthesized in stressed plants for their adaptation in the stressed conditions. These proteins could be used as markers in identifying the stressed plants.

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

    Science.gov (United States)

    2008-07-11

    Nucleotide Base pairs Hydrogen bonds FIG. 1: Ladder structure of DNA showing the Watson - Crick bonding of the bases A, T, G, and C which are suspended by a...protected against unwanted action of chemicals and proteins. The three-dimensional structure of DNA is the famed Watson - Crick double-helix, the equilibrium...quantitative analysis [88]. [1] A. Kornberg and T. A. Baker, DNA Replication (W. H. Freeman, New York, 1992). [2] J. D. Watson and F. H. C. Crick

  15. Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

    Science.gov (United States)

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

    2014-11-27

    In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

  16. A comparative approach using ecotoxicological methods from single-species bioassays to model ecosystems.

    Science.gov (United States)

    Haegerbaeumer, Arne; Höss, Sebastian; Ristau, Kai; Claus, Evelyn; Möhlenkamp, Christel; Heininger, Peter; Traunspurger, Walter

    2016-12-01

    Soft sediments are often hotspots of chemical contamination, and a thorough ecotoxicological assessment of this habitat can help to identify the causes of stress and to improve the health of the respective ecosystems. As an important component of the ecologically relevant meiobenthic fauna, nematodes can be used for sediment assessments, with various assay tools ranging from single-species toxicity tests to field studies. In the present study, microcosms containing sediment were used to investigate direct and indirect effects of zinc on natural nematode assemblages, and acute community toxicity tests considering only direct toxicity were conducted. The responses of the various freshwater nematode species in both approaches were compared with those of Caenorhabditis elegans, determined in standardized tests (ISO 10872). At a median lethal concentration (LC50) of 20 mg Zn/L, C. elegans represented the median susceptibility of 15 examined nematode species examined in the acute community toxicity tests. In the microcosms, Zn affected the nematodes dose-dependently, with changes in species composition first detected at 13 mg Zn/kg to 19 mg Zn/kg sediment dry weight. The observed species sensitivities in the microcosms corresponded better to field observations than to the results of the acute community toxicity tests. Environ Toxicol Chem 2016;35:2987-2997. © 2016 SETAC. © 2016 SETAC.

  17. The effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on protein-protein interactions.

    Science.gov (United States)

    Yates, Christopher M; Sternberg, Michael J E

    2013-11-01

    Non-synonymous single nucleotide polymorphisms (nsSNPs) are single base changes leading to a change to the amino acid sequence of the encoded protein. Many of these variants are associated with disease, so nsSNPs have been well studied, with studies looking at the effects of nsSNPs on individual proteins, for example, on stability and enzyme active sites. In recent years, the impact of nsSNPs upon protein-protein interactions has also been investigated, giving a greater insight into the mechanisms by which nsSNPs can lead to disease. In this review, we summarize these studies, looking at the various mechanisms by which nsSNPs can affect protein-protein interactions. We focus on structural changes that can impair interaction, changes to disorder, gain of interaction, and post-translational modifications before looking at some examples of nsSNPs at human-pathogen protein-protein interfaces and the analysis of nsSNPs from a network perspective. © 2013.

  18. 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.

  19. 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.

  20. 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.

  1. Physiological differentiation within a single-species biofilm fueled by serpentinization.

    Science.gov (United States)

    Brazelton, William J; Mehta, Mausmi P; Kelley, Deborah S; Baross, John A

    2011-01-01

    Carbonate chimneys at the Lost City hydrothermal field are coated in biofilms dominated by a single phylotype of archaea known as Lost City Methanosarcinales. In this study, we have detected surprising physiological complexity in single-species biofilms, which is typically indicative of multispecies biofilm communities. Multiple cell morphologies were visible within the biofilms by transmission electron microscopy, and some cells contained intracellular membranes that may facilitate methane oxidation. Both methane production and oxidation were detected at 70 to 80°C and pH 9 to 10 in samples containing the single-species biofilms. Both processes were stimulated by the presence of hydrogen (H(2)), indicating that methane production and oxidation are part of a syntrophic interaction. Metagenomic data included a sequence encoding AMP-forming acetyl coenzyme A synthetase, indicating that acetate may play a role in the methane-cycling syntrophy. A wide range of nitrogen fixation genes were also identified, many of which were likely acquired via lateral gene transfer (LGT). Our results indicate that cells within these single-species biofilms may have differentiated into multiple physiological roles to form multicellular communities linked by metabolic interactions and LGT. Communities similar to these Lost City biofilms are likely to have existed early in the evolution of life, and we discuss how the multicellular characteristics of ancient hydrogen-fueled biofilm communities could have stimulated ecological diversification, as well as unity of biochemistry, during the earliest stages of cellular evolution. Our previous work at the Lost City hydrothermal field has shown that its carbonate chimneys host microbial biofilms dominated by a single uncultivated "species" of archaea. In this paper, we integrate evidence from these previous studies with new data on the metabolic activity and cellular morphology of these archaeal biofilms. We conclude that the archaeal biofilm

  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

    , 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......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....../kg CP). Digestible rumen escape protein (DEP) varied significantly between species (P Laminaria, Mastocarpus and Palmaria can supply...

  3. On the involvement of single-bond rotation in the primary photochemistry of photoactive yellow protein

    NARCIS (Netherlands)

    Stahl, A.D.; Hospes, M.; Singhal, K.; van Stokkum, I.; van Grondelle, R.; Groot, M.L.; Hellingwerf, K.J.

    2011-01-01

    Prior experimental observations, as well as theoretical considerations, have led to the proposal that C4-C7 single-bond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid

  4. 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...

  5. 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

  6. Delay-induced wave instabilities in single-species reaction-diffusion systems

    Science.gov (United States)

    Otto, Andereas; Wang, Jian; Radons, Günter

    2017-11-01

    The Turing (wave) instability is only possible in reaction-diffusion systems with more than one (two) components. Motivated by the fact that a time delay increases the dimension of a system, we investigate the presence of diffusion-driven instabilities in single-species reaction-diffusion systems with delay. The stability of arbitrary one-component systems with a single discrete delay, with distributed delay, or with a variable delay is systematically analyzed. We show that a wave instability can appear from an equilibrium of single-species reaction-diffusion systems with fluctuating or distributed delay, which is not possible in similar systems with constant discrete delay or without delay. More precisely, we show by basic analytic arguments and by numerical simulations that fast asymmetric delay fluctuations or asymmetrically distributed delays can lead to wave instabilities in these systems. Examples, for the resulting traveling waves are shown for a Fisher-KPP equation with distributed delay in the reaction term. In addition, we have studied diffusion-induced instabilities from homogeneous periodic orbits in the same systems with variable delay, where the homogeneous periodic orbits are attracting resonant periodic solutions of the system without diffusion, i.e., periodic orbits of the Hutchinson equation with time-varying delay. If diffusion is introduced, standing waves can emerge whose temporal period is equal to the period of the variable delay.

  7. 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.

  8. 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.

  9. 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...

  10. Single-Molecule Force Spectroscopy Trajectories of a Single Protein and Its Polyproteins Are Equivalent: A Direct Experimental Validation Based on A Small Protein NuG2.

    Science.gov (United States)

    Lei, Hai; He, Chengzhi; Hu, Chunguang; Li, Jinliang; Hu, Xiaodong; Hu, Xiaotang; Li, Hongbin

    2017-05-22

    Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2) 4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2) 4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. 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

  12. 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

  13. 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.

  14. Trichoderma Reesei single cell protein production from rice straw pulp in solid state fermentation

    Science.gov (United States)

    Zaki, M.; Said, S. D.

    2018-04-01

    The dependency on fish meal as a major protein source for animal feed can lead toit priceinstability in line with the increasing in meat production and consumption in Indonesia. In order todeal with this problem, an effort to produce an alternative protein sources production is needed. This scenario is possible due to the abundantavailability of agricultural residues such as rice straw whichcould be utilized as substrate for production of single cell proteins as an alternative proteinsource. This work investigated the potential utilization of rice straw pulp and urea mixture as substrate for the production of local Trichoderma reesei single cell protein in solid state fermentation system. Some parameters have been analyzed to evaluate the effect of ratio of rice straw pulp to urea on mixed single cell protein biomass (mixed SCP biomass) composition, such as total crude protein (analyzed by kjedhal method) and lignin content (TAPPI method).The results showed that crude protein content in mixed SCP biomassincreases with the increasing in fermentation time, otherwise it decreases with the increasing insubstrate carbon to nitrogen (C/N) ratio. Residual lignin content in mixed SCP biomass decreases from 7% to 0.63% during fermentationproceeded of 21 days. The highest crude protein content in mixed SCP biomasswas obtained at substrate C/N ratio 20:1 of 25%.

  15. Protein single-model quality assessment by feature-based probability density functions.

    Science.gov (United States)

    Cao, Renzhi; Cheng, Jianlin

    2016-04-04

    Protein quality assessment (QA) has played an important role in protein structure prediction. We developed a novel single-model quality assessment method-Qprob. Qprob calculates the absolute error for each protein feature value against the true quality scores (i.e. GDT-TS scores) of protein structural models, and uses them to estimate its probability density distribution for quality assessment. Qprob has been blindly tested on the 11th Critical Assessment of Techniques for Protein Structure Prediction (CASP11) as MULTICOM-NOVEL server. The official CASP result shows that Qprob ranks as one of the top single-model QA methods. In addition, Qprob makes contributions to our protein tertiary structure predictor MULTICOM, which is officially ranked 3rd out of 143 predictors. The good performance shows that Qprob is good at assessing the quality of models of hard targets. These results demonstrate that this new probability density distribution based method is effective for protein single-model quality assessment and is useful for protein structure prediction. The webserver of Qprob is available at: http://calla.rnet.missouri.edu/qprob/. The software is now freely available in the web server of Qprob.

  16. Expression of membrane-associated proteins within single emulsion cell facsimiles.

    Science.gov (United States)

    Chanasakulniyom, Mayuree; Martino, Chiara; Paterson, David; Horsfall, Louise; Rosser, Susan; Cooper, Jonathan M

    2012-07-07

    MreB is a structural membrane-associated protein which is one of the key components of the bacterial cytoskeleton. Although it plays an important role in shape maintenance of rod-like bacteria, the understanding of its mechanism of action is still not fully understood. This study shows how segmented flow and microdroplet technology can be used as a new tool for biological in vitro investigation of this protein. In this paper, we demonstrate cell-free expression in a single emulsion system to express red fluorescence protein (RFP) and MreB linked RFP (MreB-RFP). We follow the aggregation and localisation of the fusion protein MreB-RFP in this artificial cell-like environment. The expression of MreB-RFP in single emulsion droplets leads to the formation of micrometer-scale protein patches distributed at the water/oil interface.

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

    Directory of Open Access Journals (Sweden)

    Jeong Y

    2015-11-01

    Full Text Available Yoon Jeong,1,2 Kwan Hong Lee,1,2 Hansoo Park,3 Jonghoon Choi1,2 1Department of Bionano Technology, Graduate School, Hanyang University, Seoul, 2Department of Bionano Engineering, Hanyang University ERICA, Ansan, 3School of Integrative Engineering, Chung-Ang University, Seoul, South Korea Abstract: 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. Keywords: microwell array, antibody’s orientation, single cell analysis, secreted cytokine, protein-G-terminated surface

  18. 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.

  19. 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.

  20. 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

  1. A species-specific nucleosomal signature defines a periodic distribution of amino acids in proteins.

    Science.gov (United States)

    Quintales, Luis; Soriano, Ignacio; Vázquez, Enrique; Segurado, Mónica; Antequera, Francisco

    2015-04-01

    Nucleosomes are the basic structural units of chromatin. Most of the yeast genome is organized in a pattern of positioned nucleosomes that is stably maintained under a wide range of physiological conditions. In this work, we have searched for sequence determinants associated with positioned nucleosomes in four species of fission and budding yeasts. We show that mononucleosomal DNA follows a highly structured base composition pattern, which differs among species despite the high degree of histone conservation. These nucleosomal signatures are present in transcribed and non-transcribed regions across the genome. In the case of open reading frames, they correctly predict the relative distribution of codons on mononucleosomal DNA, and they also determine a periodicity in the average distribution of amino acids along the proteins. These results establish a direct and species-specific connection between the position of each codon around the histone octamer and protein composition.

  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. 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.

  4. 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

    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...... 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...... 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...

  5. In silico characterization of boron transporter (BOR1 protein sequences in Poaceae species

    Directory of Open Access Journals (Sweden)

    Ertuğrul Filiz

    2013-01-01

    Full Text Available Boron (B is essential for the plant growth and development, and its primary function is connected with formation of the cell wall. Moreover, boron toxicity is a shared problem in semiarid and arid regions. In this study, boron transporter protein (BOR1 sequences from some Poaceae species (Hordeum vulgare subsp. vulgare, Zea mays, Brachypodium distachyon, Oryza sativa subsp. japonica, Oryza sativa subsp. indica, Sorghum bicolor, Triticum aestivum were evaluated by bioinformatics tools. Physicochemical analyses revealed that most of BOR1 proteins were basic character and had generally aliphatic amino acids. Analysis of the domains showed that transmembrane domains were identified constantly and three motifs were detected with 50 amino acids length. Also, the motif SPNPWEPGSYDHWTVAKDMFNVPPAYIFGAFIPATMVAGLYYFDHSVASQ was found most frequently with 25 repeats. The phylogenetic tree showed divergence into two main clusters. B. distachyon species were clustered separately. Finally, this study contributes to the new BOR1 protein characterization in grasses and create scientific base for in silico analysis in future.

  6. 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

  7. 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

  8. SNPchiMp v.3: integrating and standardizing single nucleotide polymorphism data for livestock species.

    Science.gov (United States)

    Nicolazzi, Ezequiel L; Caprera, Andrea; Nazzicari, Nelson; Cozzi, Paolo; Strozzi, Francesco; Lawley, Cindy; Pirani, Ali; Soans, Chandrasen; Brew, Fiona; Jorjani, Hossein; Evans, Gary; Simpson, Barry; Tosser-Klopp, Gwenola; Brauning, Rudiger; Williams, John L; Stella, Alessandra

    2015-04-10

    In recent years, the use of genomic information in livestock species for genetic improvement, association studies and many other fields has become routine. In order to accommodate different market requirements in terms of genotyping cost, manufacturers of single nucleotide polymorphism (SNP) arrays, private companies and international consortia have developed a large number of arrays with different content and different SNP density. The number of currently available SNP arrays differs among species: ranging from one for goats to more than ten for cattle, and the number of arrays available is increasing rapidly. However, there is limited or no effort to standardize and integrate array- specific (e.g. SNP IDs, allele coding) and species-specific (i.e. past and current assemblies) SNP information. Here we present SNPchiMp v.3, a solution to these issues for the six major livestock species (cow, pig, horse, sheep, goat and chicken). Original data was collected directly from SNP array producers and specific international genome consortia, and stored in a MySQL database. The database was then linked to an open-access web tool and to public databases. SNPchiMp v.3 ensures fast access to the database (retrieving within/across SNP array data) and the possibility of annotating SNP array data in a user-friendly fashion. This platform allows easy integration and standardization, and it is aimed at both industry and research. It also enables users to easily link the information available from the array producer with data in public databases, without the need of additional bioinformatics tools or pipelines. In recognition of the open-access use of Ensembl resources, SNPchiMp v.3 was officially credited as an Ensembl E!mpowered tool. Availability at http://bioinformatics.tecnoparco.org/SNPchimp.

  9. 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.

  10. Single-limb force data for two lemur species while vertically clinging.

    Science.gov (United States)

    Johnson, Laura E; Hanna, Jandy; Schmitt, Daniel

    2015-11-01

    Vertical clinging and climbing have been integral to hypotheses about primate origins, yet little is known about how an animal with nails instead of claws resists gravity while on large, vertical, and cylindrical substrates. Here we test models of how force is applied to maintain posture, predicting (1) the shear component force (Fs ) at the hands will be higher than the feet; (2) the normal component force (Fn ) at the feet will be relatively high compared to the hands; (3) the component force resisting gravity (Fg ) at the feet will be relatively high compared to the hands; (4) species with a high frequency of vertical clinging postures will have low Fg at the hands due to relatively short forelimbs. Using a novel instrumented support, single-limb force data were collected during clinging postures for the hands and feet and compared across limbs and species for Propithecus verreauxi (N = 2), a habitual vertical clinger and leaper, and Varecia variegata (N = 3), a habitual above-branch arboreal quadruped. For both species, hand Fs were significantly higher than at the feet and Fn and Fg at the feet were significantly higher than at the hands. Between species, P. verreauxi has relatively low Fg at the hands and Fn at the feet than V. vareigata. These results support previous models and show that hindlimb loading dominance, characteristic of primate locomotion, is found during clinging behaviors and may allow the forelimbs to be used for foraging while clinging. These findings provide insight into selective pressures on force distribution in primates and primate locomotor evolution. © 2015 Wiley Periodicals, Inc.

  11. 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...

  12. 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.

  13. Development of a Single Locus Sequence Typing (SLST) Scheme for Typing Bacterial Species Directly from Complex Communities.

    Science.gov (United States)

    Scholz, Christian F P; Jensen, Anders

    2017-01-01

    The protocol describes a computational method to develop a Single Locus Sequence Typing (SLST) scheme for typing bacterial species. The resulting scheme can be used to type bacterial isolates as well as bacterial species directly from complex communities using next-generation sequencing technologies.

  14. Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

    Science.gov (United States)

    Arcand, Mathieu; Roby, Philippe; Bossé, Roger; Lipari, Francesco; Padrós, Jaime; Beaudet, Lucille; Marcil, Alexandre; Dahan, Sophie

    2010-04-20

    We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

  15. Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

    Science.gov (United States)

    Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

    2018-06-01

    G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

  16. 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

  17. 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

  18. Photocleavable DNA Barcoding Antibodies for Multiplexed Protein Analysis in Single Cells.

    Science.gov (United States)

    Ullal, Adeeti V; Weissleder, Ralph

    2015-01-01

    We describe a DNA-barcoded antibody sensing technique for single cell protein analysis in which the barcodes are photocleaved and digitally detected without amplification steps (Ullal et al., Sci Transl Med 6:219, 2014). After photocleaving the unique ~70 mer DNA barcodes we use a fluorescent hybridization technology for detection, similar to what is commonly done for nucleic acid readouts. This protocol offers a simple method for multiplexed protein detection using 100+ antibodies and can be performed on clinical samples as well as single cells.

  19. Protein logic: a statistical mechanical study of signal integration at the single-molecule level.

    Science.gov (United States)

    de Ronde, Wiet; Rein ten Wolde, Pieter; Mugler, Andrew

    2012-09-05

    Information processing and decision-making is based upon logic operations, which in cellular networks has been well characterized at the level of transcription. In recent years, however, both experimentalists and theorists have begun to appreciate that cellular decision-making can also be performed at the level of a single protein, giving rise to the notion of protein logic. Here we systematically explore protein logic using a well-known statistical mechanical model. As an example system, we focus on receptors that bind either one or two ligands, and their associated dimers. Notably, we find that a single heterodimer can realize any of the 16 possible logic gates, including the XOR gate, by variation of biochemical parameters. We then introduce what to our knowledge is a novel idea: that a set of receptors with fixed parameters can encode functionally unique logic gates simply by forming different dimeric combinations. An exhaustive search reveals that the simplest set of receptors (two single-ligand receptors and one double-ligand receptor) can realize several different groups of three unique gates, a result for which the parametric analysis of single receptors and dimers provides a clear interpretation. Both results underscore the surprising functional freedom readily available to cells at the single-protein level. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  20. Ants farm subterranean aphids mostly in single clone groups - an example of prudent husbandry for carbohydrates and proteins?

    Directory of Open Access Journals (Sweden)

    Ivens Aniek BF

    2012-07-01

    Full Text Available Abstract Background Mutualistic interactions are wide-spread but the mechanisms underlying their evolutionary stability and ecological dynamics remain poorly understood. Cultivation mutualisms in which hosts consume symbionts occur in phylogenetically diverse groups, but often have symbiont monocultures for each host. This is consistent with the prediction that symbionts should avoid coexistence with other strains so that host services continue to benefit relatives, but it is less clear whether hosts should always favor monocultures and what mechanisms they might have to manipulate symbiont diversity. Few mutualisms have been studied in sufficient genetic detail to address these issues, so we decided to characterize symbiont diversity in the complex mutualism between multiple root aphid species and Lasius flavus ants. After showing elsewhere that three of these aphid species have low dispersal and mostly if not exclusively asexual reproduction, we here investigate aphid diversity within and between ant nest mounds. Results The three focal species (Geoica utricularia, Forda marginata and Tetraneura ulmi had considerable clonal diversity at the population level. Yet more than half of the ant mounds contained just a single aphid species, a significantly higher percentage than expected from a random distribution. Over 60% of these single-species mounds had a single aphid clone, and clones tended to persist across subsequent years. Whenever multiple species/clones co-occurred in the same mound, they were spatially separated with more than 95% of the aphid chambers containing individuals of a single clone. Conclusions L. flavus “husbandry” is characterized by low aphid “livestock” diversity per colony, especially at the nest-chamber level, but it lacks the exclusive monocultures known from other cultivation mutualisms. The ants appear to eat most of the early instar aphids, so that adult aphids are unlikely to face limited phloem resources and

  1. In silico study of protein to protein interaction analysis of AMP-activated protein kinase and mitochondrial activity in three different farm animal species

    Science.gov (United States)

    Prastowo, S.; Widyas, N.

    2018-03-01

    AMP-activated protein kinase (AMPK) is cellular energy censor which works based on ATP and AMP concentration. This protein interacts with mitochondria in determine its activity to generate energy for cell metabolism purposes. For that, this paper aims to compare the protein to protein interaction of AMPK and mitochondrial activity genes in the metabolism of known animal farm (domesticated) that are cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In silico study was done using STRING V.10 as prominent protein interaction database, followed with biological function comparison in KEGG PATHWAY database. Set of genes (12 in total) were used as input analysis that are PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PPARGC1, ACC, CPT1B, NRF2 and SOD. The first 7 genes belong to gene in AMPK family, while the last 5 belong to mitochondrial activity genes. The protein interaction result shows 11, 8 and 5 metabolism pathways in Bos taurus, Sus scrofa and Gallus gallus, respectively. The top pathway in Bos taurus is AMPK signaling pathway (10 genes), Sus scrofa is Adipocytokine signaling pathway (8 genes) and Gallus gallus is FoxO signaling pathway (5 genes). Moreover, the common pathways found in those 3 species are Adipocytokine signaling pathway, Insulin signaling pathway and FoxO signaling pathway. Genes clustered in Adipocytokine and Insulin signaling pathway are PRKAA2, PPARGC1A, PRKAB1 and PRKAG2. While, in FoxO signaling pathway are PRKAA2, PRKAB1, PRKAG2. According to that, we found PRKAA2, PRKAB1 and PRKAG2 are the common genes. Based on the bioinformatics analysis, we can demonstrate that protein to protein interaction shows distinct different of metabolism in different species. However, further validation is needed to give a clear explanation.

  2. Proteomics approach to identify unique xylem sap proteins in Pierce's disease-tolerant Vitis species.

    Science.gov (United States)

    Basha, Sheikh M; Mazhar, Hifza; Vasanthaiah, Hemanth K N

    2010-03-01

    Pierce's disease (PD) is a destructive bacterial disease of grapes caused by Xylella fastidiosa which is xylem-confined. The tolerance level to this disease varies among Vitis species. Our research was aimed at identifying unique xylem sap proteins present in PD-tolerant Vitis species. The results showed wide variation in the xylem sap protein composition, where a set of polypeptides with pI between 4.5 and 4.7 and M(r) of 31 kDa were present in abundant amount in muscadine (Vitis rotundifolia, PD-tolerant), in reduced levels in Florida hybrid bunch (Vitis spp., PD-tolerant) and absent in bunch grapes (Vitis vinifera, PD-susceptible). Liquid chromatography/mass spectrometry/mass spectrometry analysis of these proteins revealed their similarity to beta-1, 3-glucanase, peroxidase, and a subunit of oxygen-evolving enhancer protein 1, which are known to play role in defense and oxygen generation. In addition, the amount of free amino acids and soluble sugars was found to be significantly lower in xylem sap of muscadine genotypes compared to V. vinifera genotypes, indicating that the higher nutritional value of bunch grape sap may be more suitable for Xylella growth. These data suggest that the presence of these unique proteins in xylem sap is vital for PD tolerance in muscadine and Florida hybrid bunch grapes.

  3. Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

    Science.gov (United States)

    Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

    2012-11-14

    DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

  4. A comparison of fisheries biological reference points estimated from temperature-specific multi-species and single-species climate-enhanced stock assessment models

    Science.gov (United States)

    Holsman, Kirstin K.; Ianelli, James; Aydin, Kerim; Punt, André E.; Moffitt, Elizabeth A.

    2016-12-01

    Multi-species statistical catch at age models (MSCAA) can quantify interacting effects of climate and fisheries harvest on species populations, and evaluate management trade-offs for fisheries that target several species in a food web. We modified an existing MSCAA model to include temperature-specific growth and predation rates and applied the modified model to three fish species, walleye pollock (Gadus chalcogrammus), Pacific cod (Gadus macrocephalus) and arrowtooth flounder (Atheresthes stomias), from the eastern Bering Sea (USA). We fit the model to data from 1979 through 2012, with and without trophic interactions and temperature effects, and use projections to derive single- and multi-species biological reference points (BRP and MBRP, respectively) for fisheries management. The multi-species model achieved a higher over-all goodness of fit to the data (i.e. lower negative log-likelihood) for pollock and Pacific cod. Variability from water temperature typically resulted in 5-15% changes in spawning, survey, and total biomasses, but did not strongly impact recruitment estimates or mortality. Despite this, inclusion of temperature in projections did have a strong effect on BRPs, including recommended yield, which were higher in single-species models for Pacific cod and arrowtooth flounder that included temperature compared to the same models without temperature effects. While the temperature-driven multi-species model resulted in higher yield MBPRs for arrowtooth flounder than the same model without temperature, we did not observe the same patterns in multi-species models for pollock and Pacific cod, where variability between harvest scenarios and predation greatly exceeded temperature-driven variability in yield MBRPs. Annual predation on juvenile pollock (primarily cannibalism) in the multi-species model was 2-5 times the annual harvest of adult fish in the system, thus predation represents a strong control on population dynamics that exceeds temperature

  5. 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.

  6. Single vector system for efficient N-myristoylation of recombinant proteins in E. coli.

    Directory of Open Access Journals (Sweden)

    Julian M Glück

    Full Text Available BACKGROUND: N-myristoylation is a crucial covalent modification of numerous eukaryotic and viral proteins that is catalyzed by N-myristoyltransferase (NMT. Prokaryotes are lacking endogenous NMT activity. Recombinant production of N-myristoylated proteins in E. coli cells can be achieved by coexpression of heterologous NMT with the target protein. In the past, dual plasmid systems were used for this purpose. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a single vector system for efficient coexpression of substrate and enzyme suitable for production of co- or posttranslationally modified proteins. The approach was validated using the HIV-1 Nef protein as an example. A simple and efficient protocol for production of highly pure and completely N-myristoylated Nef is presented. The yield is about 20 mg myristoylated Nef per liter growth medium. CONCLUSIONS/SIGNIFICANCE: The single vector strategy allows diverse modifications of target proteins recombinantly coexpressed in E. coli with heterologous enzymes. The method is generally applicable and provides large amounts of quantitatively processed target protein that are sufficient for comprehensive biophysical and structural studies.

  7. Integrating single-cell transcriptomic data across different conditions, technologies, and species.

    Science.gov (United States)

    Butler, Andrew; Hoffman, Paul; Smibert, Peter; Papalexi, Efthymia; Satija, Rahul

    2018-06-01

    Computational single-cell RNA-seq (scRNA-seq) methods have been successfully applied to experiments representing a single condition, technology, or species to discover and define cellular phenotypes. However, identifying subpopulations of cells that are present across multiple data sets remains challenging. Here, we introduce an analytical strategy for integrating scRNA-seq data sets based on common sources of variation, enabling the identification of shared populations across data sets and downstream comparative analysis. We apply this approach, implemented in our R toolkit Seurat (http://satijalab.org/seurat/), to align scRNA-seq data sets of peripheral blood mononuclear cells under resting and stimulated conditions, hematopoietic progenitors sequenced using two profiling technologies, and pancreatic cell 'atlases' generated from human and mouse islets. In each case, we learn distinct or transitional cell states jointly across data sets, while boosting statistical power through integrated analysis. Our approach facilitates general comparisons of scRNA-seq data sets, potentially deepening our understanding of how distinct cell states respond to perturbation, disease, and evolution.

  8. Differential effects of methylmercury on the synthesis of protein species in dorsal root ganglia of the rat

    International Nuclear Information System (INIS)

    Kasama, Hidetaka; Itoh, Kazuo; Omata, Saburo; Sugano, Hiroshi

    1989-01-01

    Dorsal root ganglia from control and methylmercury(MeHg)-treated rats were incubated in vitro with 35 S-methionine and the proteins synthesized were analyzed by two-dimensional electrophoresis. The double labelling method, in which proteins of control dorsal root ganglia labelled in vitro with 3 H-leucine were added to each of the two samples as an internal standard, was used to minimize unavoidable errors arising from the resolving procedure itself. The results obtained showed that the effect of MeHg on the synthesis of proteins in dorsal root ganglia was not uniform for individual protein species in the latent period of MeHg intoxication. Among 200 protein species investigated, 157 showed inhibition of synthesis close to that of the total proteins in the tissue (68% of the control). Among the remaining protein species, 20 showed real stimulation of synthesis, whereas 7 were moderately inhibited and 16 were inhibited more strongly than the total proteins in the tissue. These results suggest that the effect of MeHg on the synthetic rates for protein species in dorsal root ganglia differs with the species, and that unusual elevation or reduction of the synthesis of some protein species caused by MeHg may lead to impairment of normal nerve functions. (orig.)

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

    Science.gov (United States)

    Apfelbach, Raimund; Parsons, Michael H; Soini, Helena A; Novotny, Milos V

    2015-01-01

    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 toward 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 understand all the

  10. 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.

  11. 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.

  12. 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.

  13. DeepQA: improving the estimation of single protein model quality with deep belief networks.

    Science.gov (United States)

    Cao, Renzhi; Bhattacharya, Debswapna; Hou, Jie; Cheng, Jianlin

    2016-12-05

    Protein quality assessment (QA) useful for ranking and selecting protein models has long been viewed as one of the major challenges for protein tertiary structure prediction. Especially, estimating the quality of a single protein model, which is important for selecting a few good models out of a large model pool consisting of mostly low-quality models, is still a largely unsolved problem. We introduce a novel single-model quality assessment method DeepQA based on deep belief network that utilizes a number of selected features describing the quality of a model from different perspectives, such as energy, physio-chemical characteristics, and structural information. The deep belief network is trained on several large datasets consisting of models from the Critical Assessment of Protein Structure Prediction (CASP) experiments, several publicly available datasets, and models generated by our in-house ab initio method. Our experiments demonstrate that deep belief network has better performance compared to Support Vector Machines and Neural Networks on the protein model quality assessment problem, and our method DeepQA achieves the state-of-the-art performance on CASP11 dataset. It also outperformed two well-established methods in selecting good outlier models from a large set of models of mostly low quality generated by ab initio modeling methods. DeepQA is a useful deep learning tool for protein single model quality assessment and protein structure prediction. The source code, executable, document and training/test datasets of DeepQA for Linux is freely available to non-commercial users at http://cactus.rnet.missouri.edu/DeepQA/ .

  14. Comparison of proteins involved in chondroitin sulfate utilization by three colonic Bacteroides species.

    OpenAIRE

    Lipeski, L; Guthrie, E P; O'Brien, M; Kotarski, S F; Salyers, A A

    1986-01-01

    Three species of colonic bacteria can ferment the mucopolysaccharide chondroitin sulfate: Bacteroides ovatus, Bacteroides sp. strain 3452A (an unnamed DNA homology group), and B. thetaiotaomicron. Proteins associated with the utilization of chondroitin sulfate by B. thetaiotaomicron have been characterized previously. In this report we compare chondroitin lyases and chondroitin sulfate-associated outer membrane polypeptides of B. ovatus and Bacteroides sp. strain 3452A with those of B. thetai...

  15. 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...

  16. Single Molecule Effects of Osteogenesis Imperfecta Mutations in Tropocollagen Protein Domains

    Science.gov (United States)

    2008-12-02

    Single molecule effects of osteogenesis imperfecta mutations in tropocollagen protein domains Alfonso Gautieri,1,2 Simone Vesentini,2 Alberto...2008 proteinscience.org Abstract: Osteogenesis imperfecta (OI) is a genetic disease characterized by fragile bones, skeletal deformities and, in severe...diagnosis and treatment, an effort referred to as materiomics. Keywords: steered molecular dynamics; osteogenesis imperfecta ; Young’s modulus; collagen

  17. 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.

  18. 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

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

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Hatzakis, Nikos

    2014-01-01

    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...

  20. 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

  1. Direct Correlation between Motile Behavior and Protein Abundance in Single Cells.

    Directory of Open Access Journals (Sweden)

    Yann S Dufour

    2016-09-01

    Full Text Available Understanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB at different levels, we quantitatively mapped motile phenotype (tumble bias to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.

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

    Science.gov (United States)

    Gribble, Kristin E; Mark Welch, David B

    2012-08-01

    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. 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. 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 rapidly, and novel alleles may be maintained and increase in

  3. 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

  4. Pathogenic Leptospira species express surface-exposed proteins belonging to the bacterial immunoglobulin superfamily

    Science.gov (United States)

    Matsunaga, James; Barocchi, Michele A.; Croda, Julio; Young, Tracy A.; Sanchez, Yolanda; Siqueira, Isadora; Bolin, Carole A.; Reis, Mitermayer G.; Riley, Lee W.; Haake, David A.; Ko, Albert I.

    2005-01-01

    Summary Proteins with bacterial immunoglobulin-like (Big) domains, such as the Yersinia pseudotuberculosis invasin and Escherichia coli intimin, are surface-expressed proteins that mediate host mammalian cell invasion or attachment. Here, we report the identification and characterization of a new family of Big domain proteins, referred to as Lig (leptospiral Ig-like) proteins, in pathogenic Leptospira. Screening of L. interrogans and L. kirschneri expression libraries with sera from leptospirosis patients identified 13 lambda phage clones that encode tandem repeats of the 90 amino acid Big domain. Two lig genes, designated ligA and ligB, and one pseudo-gene, ligC, were identified. The ligA and ligB genes encode amino-terminal lipoprotein signal peptides followed by 10 or 11 Big domain repeats and, in the case of ligB, a unique carboxy-terminal non-repeat domain. The organization of ligC is similar to that of ligB but contains mutations that disrupt the reading frame. The lig sequences are present in pathogenic but not saprophytic Leptospira species. LigA and LigB are expressed by a variety of virulent leptospiral strains. Loss of Lig protein and RNA transcript expression is correlated with the observed loss of virulence during culture attenuation of pathogenic strains. High-pressure freeze substitution followed by immunocytochemical electron microscopy confirmed that the Lig proteins were localized to the bacterial surface. Immunoblot studies with patient sera found that the Lig proteins are a major antigen recognized during the acute host infection. These observations demonstrate that the Lig proteins are a newly identified surface protein of pathogenic Leptospira, which by analogy to other bacterial immunoglobulin superfamily virulence factors, may play a role in host cell attachment and invasion during leptospiral pathogenesis. PMID:12890019

  5. Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.

    Science.gov (United States)

    Rasinger, J D; Marbaix, H; Dieu, M; Fumière, O; Mauro, S; Palmblad, M; Raes, M; Berntssen, M H G

    2016-09-16

    The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. 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.

  7. 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.

  8. Assessing transmissible spongiform encephalopathy species barriers with an in vitro prion protein conversion assay

    Science.gov (United States)

    Johnson, Christopher J.; Carlson, Christina M.; Morawski, Aaron R.; Manthei, Alyson; Cashman, Neil R.

    2015-01-01

    Studies to understanding interspecies transmission of transmissible spongiform encephalopathies (TSEs, prion diseases) are challenging in that they typically rely upon lengthy and costly in vivo animal challenge studies. A number of in vitro assays have been developed to aid in measuring prion species barriers, thereby reducing animal use and providing quicker results than animal bioassays. Here, we present the protocol for a rapid in vitroprion conversion assay called the conversion efficiency ratio (CER) assay. In this assay cellular prion protein (PrPC) from an uninfected host brain is denatured at both pH 7.4 and 3.5 to produce two substrates. When the pH 7.4 substrate is incubated with TSE agent, the amount of PrPC that converts to a proteinase K (PK)-resistant state is modulated by the original host’s species barrier to the TSE agent. In contrast, PrPC in the pH 3.5 substrate is misfolded by any TSE agent. By comparing the amount of PK-resistant prion protein in the two substrates, an assessment of the host’s species barrier can be made. We show that the CER assay correctly predicts known prion species barriers of laboratory mice and, as an example, show some preliminary results suggesting that bobcats (Lynx rufus) may be susceptible to white-tailed deer (Odocoileus virginianus) chronic wasting disease agent.

  9. Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

    Directory of Open Access Journals (Sweden)

    McClafferty Heather

    2005-01-01

    Full Text Available Abstract Background Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. Results C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. Conclusions C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP

  10. 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.

  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. 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

  13. A systematic identification of species-specific protein succinylation sites using joint element features information

    Directory of Open Access Journals (Sweden)

    Hasan MM

    2017-08-01

    Full Text Available Md Mehedi Hasan,1 Mst Shamima Khatun,2 Md Nurul Haque Mollah,2 Cao Yong,3 Dianjing Guo1 1School of Life Sciences and the State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong, People’s Republic of China; 2Laboratory of Bioinformatics, Department of Statistics, University of Rajshahi, Rajshahi, Bangladesh; 3Department of Mechanical Engineering and Automation, Harbin Institute of Technology, Shenzhen Graduate School, Shenzhen, People’s Republic of China Abstract: Lysine succinylation, an important type of protein posttranslational modification, plays significant roles in many cellular processes. Accurate identification of succinylation sites can facilitate our understanding about the molecular mechanism and potential roles of lysine succinylation. However, even in well-studied systems, a majority of the succinylation sites remain undetected because the traditional experimental approaches to succinylation site identification are often costly, time-consuming, and laborious. In silico approach, on the other hand, is potentially an alternative strategy to predict succinylation substrates. In this paper, a novel computational predictor SuccinSite2.0 was developed for predicting generic and species-specific protein succinylation sites. This predictor takes the composition of profile-based amino acid and orthogonal binary features, which were used to train a random forest classifier. We demonstrated that the proposed SuccinSite2.0 predictor outperformed other currently existing implementations on a complementarily independent dataset. Furthermore, the important features that make visible contributions to species-specific and cross-species-specific prediction of protein succinylation site were analyzed. The proposed predictor is anticipated to be a useful computational resource for lysine succinylation site prediction. The integrated species-specific online tool of SuccinSite2.0 is publicly

  14. Generic amyloidogenicity of mammalian prion proteins from species susceptible and resistant to prions.

    Science.gov (United States)

    Nyström, Sofie; Hammarström, Per

    2015-05-11

    Prion diseases are lethal, infectious diseases associated with prion protein (PrP) misfolding. A large number of mammals are susceptible to both sporadic and acquired prion diseases. Although PrP is highly conserved and ubiquitously expressed in all mammals, not all species exhibit prion disease. By employing full length recombinant PrP from five known prion susceptible species (human, cattle, cat, mouse and hamster) and two species considered to be prion resistant (pig and dog) the amyloidogenicity of these PrPs has been delineated. All the mammalian PrPs, even from resistant species, were swiftly converted from the native state to amyloid-like structure when subjected to a native condition conversion assay. The PrPs displayed amyloidotypic tinctorial and ultrastructural hallmarks. Self-seeded conversion of the PrPs displayed significantly decreased lag phases demonstrating that nucleation dependent polymerization is a dominating mechanism in the fibrillation process. Fibrils from Aβ1-40, Aβ1-42, Lysozyme, Insulin and Transthyretin did not accelerate conversion of HuPrP whereas fibrils from HuPrP90-231 and HuPrP121-231 as well as full length PrPs of all PrPs efficiently seeded conversion showing specificity of the assay requiring the C-terminal PrP sequence. Our findings have implications for PrP misfolding and could have ramifications in the context of prion resistant species and silent carriers.

  15. 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.

  16. 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 proteins have previously been found to be phosphorylated on tyrosine and arginine residues. While tyrosine phosphorylation was shown to enhance the DNA-binding properties of SsbA, arginine phosphorylation was not functionally characterized.Materials and methods: We used mass spectrometry analysis to detect...... phosphorylation of SsbA purified from B. subtilis cells. The detected phosphorylation site was assessed for its influence on DNA-binding in vitro, using electrophoretic mobility shift assays. The ability of B. subtilis serine/threonine kinases to phosphorylate SsbA was assessed using in vitro phosphorylation...

  17. Numerical investigation of three-dimensional single-species plasma equilibria on magnetic surfaces

    International Nuclear Information System (INIS)

    Lefrancois, Remi G.; Pedersen, Thomas Sunn; Boozer, Allen H.; Kremer, Jason P.

    2005-01-01

    Presented for the first time are numerical solutions to the three-dimensional nonlinear equilibrium equation for single-species plasmas confined on magnetic surfaces and surrounded by an equipotential boundary. The major-radial shift of such plasmas is found to be outward, qualitatively similar to the Shafranov shift of quasineutral plasmas confined on magnetic surfaces. However, this is the opposite of what occurs in the pure toroidal field equilibria of non-neutral plasmas (i.e., in the absence of magnetic surfaces). The effect of varying the number of Debye lengths in the plasma for the three-dimensional (3D) model is in agreement with previous 2D calculations: the potential varies significantly on magnetic surfaces for plasmas with few Debye lengths (a d ), and tends to be constant on surfaces when many Debye lengths are present (a > or approx. 10λ d ). For the case of a conducting boundary that does not conform to the outer magnetic surface, the plasma is shifted towards the conductor and the potential varies significantly on magnetic surfaces near the plasma edge. Debye shielding effects are clearly demonstrated when a nonuniform bias is applied to the boundary. Computed equilibrium profiles are presented for the Columbia Non-Neutral Torus [T. S. Pedersen, A. H. Boozer, J. P. Kermer, R. Lefrancois, F. Dahlgren, N. Pomphrey, W. Reiersen, and W. Dorland, Fusion Sci. Technol. 46, 200 (2004)], a stellarator designed to confine non-neutral plasmas

  18. Solvable single-species aggregation-annihilation model for chain-shaped cluster growth

    International Nuclear Information System (INIS)

    Ke Jianhong; Lin Zhenquan; Zheng Yizhuang; Chen Xiaoshuang; Lu Wei

    2007-01-01

    We propose a single-species aggregation-annihilation model, in which an aggregation reaction between two clusters produces an active cluster and an annihilation reaction produces an inert one. By means of the mean-field rate equation, we respectively investigate the kinetic scaling behaviours of three distinct systems. The results exhibit that: (i) for the general aggregation-annihilation system, the size distribution of active clusters consistently approaches the conventional scaling form; (ii) for the system with the self-degeneration of the cluster's activities, it takes the modified scaling form; and (iii) for the system with the self-closing of active clusters, it does not scale. Moreover, the size distribution of inert clusters with small size takes a power-law form, while that of large inert clusters obeys the scaling law. The results also show that all active clusters will eventually transform into inert ones and the inert clusters of any size can be produced by such an aggregation-annihilation process. This model can be used to mimic the chain-shaped cluster growth and can provide some useful predictions for the kinetic behaviour of the system

  19. 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.

  20. Persistence and extinction of a stochastic single-species model under regime switching in a polluted environment II.

    Science.gov (United States)

    Liu, Meng; Wang, Ke

    2010-12-07

    This is a continuation of our paper [Liu, M., Wang, K., 2010. Persistence and extinction of a stochastic single-species model under regime switching in a polluted environment, J. Theor. Biol. 264, 934-944]. Taking both white noise and colored noise into account, a stochastic single-species model under regime switching in a polluted environment is studied. Sufficient conditions for extinction, stochastic nonpersistence in the mean, stochastic weak persistence and stochastic permanence are established. The threshold between stochastic weak persistence and extinction is obtained. The results show that a different type of noise has a different effect on the survival results. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 μg LPS intraperitoneally and 7 hr later 250 μCi L-[ 35 S]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 μCi [ 35 S]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

  7. Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan.

    Science.gov (United States)

    Gamboa, Maribet; Tsuchiya, Maria Claret; Matsumoto, Suguru; Iwata, Hisato; Watanabe, Kozo

    2017-11-01

    Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization of time-of-flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co-occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen-related and energy-production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies. © 2017 Wiley Periodicals, Inc.

  8. Small scale endemism in Brazil's Atlantic Forest: 14 new species of Mesabolivar (Araneae, Pholcidae), each known from a single locality.

    Science.gov (United States)

    Huber, Bernhard A

    2015-04-07

    In an ongoing mega-transect project that aims at analyzing pholcid spider diversity and distribution in the Atlantic Forest of Brazil, many species appear restricted to small geographic ranges. Of the 84 species collected between 2003 and 2011 at 17 sites between Bahia and Santa Catarina, 51 species (61%) were found at only one locality. The present paper focuses on such species in the genus Mesabolivar, and compares diversity and distribution patterns of this genus within and outside the Atlantic Forest. The percentage of species known from single localities is higher in the Atlantic Forest (34 of 52 species; 65%) than outside the Atlantic Forest (10 of 25; 40%). Distribution rages of species in the Atlantic Forest are significantly smaller than of species outside the Atlantic Forest (mean maximum distances between localities: 184 versus 541 km; medians: 10 km versus 220 km). The following species are newly described (arranged from north to south), each currently known from the respective type locality only: M. caipora; M. kathrinae; M. bonita; M. pau (Bahia); M. monteverde; M. perezi (Espírito Santo); M. giupponii; M. goitaca; M. sai (Rio de Janeiro); M. tamoio; M. unicornis; M. gabettae; M. inornatus (São Paulo); M. itapoa (Santa Catarina).

  9. Observation of Single-Protein and DNA Macromolecule Collisions on Ultramicroelectrodes.

    Science.gov (United States)

    Dick, Jeffrey E; Renault, Christophe; Bard, Allen J

    2015-07-08

    Single-molecule detection is the ultimate sensitivity in analytical chemistry and has been largely unavailable in electrochemical analysis. Here, we demonstrate the feasibility of detecting electrochemically inactive single biomacromolecules, such as enzymes, antibodies, and DNA, by blocking a solution redox reaction when molecules adsorb and block electrode sites. By oxidizing a large concentration of potassium ferrocyanide on an ultramicroelectrode (UME, radius ≤150 nm), time-resolved, discrete adsorption events of antibodies, enzymes, DNA, and polystyrene nanospheres can be differentiated from the background by their "footprint". Further, by assuming that the mass transport of proteins to the electrode surface is controlled mainly by diffusion, a size estimate using the Stokes-Einstein relationship shows good agreement of electrochemical data with known protein sizes.

  10. 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.

  11. APOLLO: a quality assessment service for single and multiple protein models.

    Science.gov (United States)

    Wang, Zheng; Eickholt, Jesse; Cheng, Jianlin

    2011-06-15

    We built a web server named APOLLO, which can evaluate the absolute global and local qualities of a single protein model using machine learning methods or the global and local qualities of a pool of models using a pair-wise comparison approach. Based on our evaluations on 107 CASP9 (Critical Assessment of Techniques for Protein Structure Prediction) targets, the predicted quality scores generated from our machine learning and pair-wise methods have an average per-target correlation of 0.671 and 0.917, respectively, with the true model quality scores. Based on our test on 92 CASP9 targets, our predicted absolute local qualities have an average difference of 2.60 Å with the actual distances to native structure. http://sysbio.rnet.missouri.edu/apollo/. Single and pair-wise global quality assessment software is also available at the site.

  12. Species abundance distributions : moving beyond single prediction theories to integration within an ecological framework

    NARCIS (Netherlands)

    McGill, Brian J.; Etienne, Rampal S.; Gray, John S.; Alonso, David; Anderson, Marti J.; Benecha, Habtamu Kassa; Dornelas, Maria; Enquist, Brian J.; Green, Jessica L.; He, Fangliang; Hurlbert, Allen H.; Magurran, Anne E.; Marquet, Pablo A.; Maurer, Brian A.; Ostling, Annette; Soykan, Candan U.; Ugland, Karl I.; White, Ethan P.

    2007-01-01

    Species abundance distributions (SADs) follow one of ecology's oldest and most universal laws - every community shows a hollow curve or hyperbolic shape on a histogram with many rare species and just a few common species. Here, we review theoretical, empirical and statistical developments in the

  13. Species abundance distributions: moving beyond single prediction theories to integration within an ecological framework

    NARCIS (Netherlands)

    McGill, B.J.; Etienne, R.S.; Gray, J.S.; Alonso, D.; Anderson, M.J.; Benecha, H.K.

    2007-01-01

    Species abundance distributions (SADs) follow one of ecology's oldest and most universal laws ¿ every community shows a hollow curve or hyperbolic shape on a histogram with many rare species and just a few common species. Here, we review theoretical, empirical and statistical developments in the

  14. Determination of selenium in BCR single cell protein via destructive neutron activation analysis

    International Nuclear Information System (INIS)

    Goeij, J.J.M. de; Zegers, C.

    1978-10-01

    The amount of selenium in single cell protein (SCP), a product of BP Research Centre at Sunbury-at-Thames, England, was determined by neutron activation analysis. The SCP-samples were irradiated in the reactor of the Interuniversity Reactor Institute at Delft, in a neutron flux of 1.0 x 10 13 n/cm 2 s for 24 hours. After chemical destruction of the samples the amount of selenium was determined by measuring the γ-peaks of selenium-75

  15. Polymorphism of myofibrillar proteins of rabbit skeletal-muscle fibres. An electrophoretic study of single fibres.

    OpenAIRE

    Salviati, G; Betto, R; Danieli Betto, D

    1982-01-01

    Rabbit predominantly fast-twitch-fibre and predominantly slow-twitch-fibre skeletal muscles of the hind limbs, the psoas, the diaphragm and the masseter muscles were fibre-typed by one-dimensional polyacrylamide-gel electrophoresis of the myofibrillar proteins of chemically skinned single fibres. Investigation of the distribution of fast-twitch-fibre and slow-twitch-fibre isoforms of myosin light chains and the type of myosin heavy chains, based on peptide 'maps' published in Cleveland. Fisch...

  16. 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...

  17. 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.

  18. Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

    Science.gov (United States)

    Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

    2017-02-01

    Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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

  4. Bacterial single-stranded DNA-binding proteins are phosphorylated on tyrosine

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Petranovic, Dina; Macek, B

    2006-01-01

    for phosphotyrosine-containing proteins in Streptomyces griseus by immunoaffinity chromatography identified bacterial SSBs as a novel target of bacterial tyrosine kinases. Since genes encoding protein-tyrosine kinases (PTKs) have not been recognized in streptomycetes, and SSBs from Streptomyces coelicolor (Sc......SSB) and Bacillus subtilis (BsSSB) share 38.7% identity, we used a B.subtilis protein-tyrosine kinase YwqD to phosphorylate two cognate SSBs (BsSSB and YwpH) in vitro. We demonstrate that in vivo phosphorylation of B.subtilis SSB occurs on tyrosine residue 82, and this reaction is affected antagonistically...... by kinase YwqD and phosphatase YwqE. Phosphorylation of B.subtilis SSB increased binding almost 200-fold to single-stranded DNA in vitro. Tyrosine phosphorylation of B.subtilis, S.coelicolor and Escherichia coli SSBs occured while they were expressed in E.coli, indicating that tyrosine phosphorylation...

  5. 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.

  6. A single peroxisomal targeting signal mediates matrix protein import in diatoms.

    Directory of Open Access Journals (Sweden)

    Nicola H Gonzalez

    Full Text Available Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

  7. Single Event Resolution of Plant Plasma Membrane Protein Endocytosis by TIRF Microscopy.

    Science.gov (United States)

    Johnson, Alexander; Vert, Grégory

    2017-01-01

    Endocytosis is a key process in the internalization of extracellular materials and plasma membrane proteins, such as receptors and transporters, thereby controlling many aspects of cell signaling and cellular homeostasis. Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, nutrient delivery, toxin avoidance, and pathogen defense. The precise mechanisms of endocytosis in plants remain quite elusive. The lack of direct visualization and examination of single events of endocytosis has greatly hampered our ability to precisely monitor the cell surface lifetime and the recruitment profile of proteins driving endocytosis or endocytosed cargos in plants. Here, we discuss the necessity to systematically implement total internal reflection fluorescence microcopy (TIRF) in the Plant Cell Biology community and present reliable protocols for high spatial and temporal imaging of endocytosis in plants using clathrin-mediated endocytosis as a test case, since it represents the major route for internalization of cell-surface proteins in plants. We developed a robust method to directly visualize cell surface proteins using TIRF microscopy combined to a high throughput, automated and unbiased analysis pipeline to determine the temporal recruitment profile of proteins to single sites of endocytosis, using the departure of clathrin as a physiological reference for scission. Using this 'departure assay', we assessed the recruitment of two different AP-2 subunits, alpha and mu, to the sites of endocytosis and found that AP2A1 was recruited in concert with clathrin, while AP2M was not. This validated approach therefore offers a powerful solution to better characterize the plant endocytic machinery and the dynamics of one's favorite cargo protein.

  8. Motion of single MreB bacterial actin proteins in Caulobacter show treadmilling in vivo

    Science.gov (United States)

    Moerner, W. E.; Kim, Soyeon; Gitai, Zemer; Kinkhabwala, Anika; McAdams, Harley; Shapiro, Lucy

    2006-03-01

    Ensemble imaging of a bacterial actin homologue, the MreB protein, suggests that the MreB proteins form a dynamic filamentous spiral along the long axis of the cell in Caulobacter crescentus. MreB contracts and expands along the cell axis and plays an important role in cell shape and polarity maintenance, as well as chromosome segregation and translocation of the origin of replication during cell division. In this study we investigated the real-time polymerization of MreB in Caulobacter crescentus using single-molecule fluorescence imaging. With time-lapse imaging, polymerized MreB could be distinguished from cytoplasmic MreB monomers, because single monomeric MreB showed fast motion characteristic of Brownian diffusion, while single polymerized MreB displayed slow, directed motion. This directional movement of labeled MreB in the growing polymer implies that treadmilling is the predominant mechanism in MreB filament formation. These single-molecule imaging experiments provide the first available information on the velocity of bacterial actin polymerization in a living cell.

  9. Substoichiometric hydroxynonenylation of a single protein recapitulates whole-cell-stimulated antioxidant response.

    Science.gov (United States)

    Parvez, Saba; Fu, Yuan; Li, Jiayang; Long, Marcus J C; Lin, Hong-Yu; Lee, Dustin K; Hu, Gene S; Aye, Yimon

    2015-01-14

    Lipid-derived electrophiles (LDEs) that can directly modify proteins have emerged as important small-molecule cues in cellular decision-making. However, because these diffusible LDEs can modify many targets [e.g., >700 cysteines are modified by the well-known LDE 4-hydroxynonenal (HNE)], establishing the functional consequences of LDE modification on individual targets remains devilishly difficult. Whether LDE modifications on a single protein are biologically sufficient to activate discrete redox signaling response downstream also remains untested. Herein, using T-REX (targetable reactive electrophiles and oxidants), an approach aimed at selectively flipping a single redox switch in cells at a precise time, we show that a modest level (∼34%) of HNEylation on a single target is sufficient to elicit the pharmaceutically important antioxidant response element (ARE) activation, and the resultant strength of ARE induction recapitulates that observed from whole-cell electrophilic perturbation. These data provide the first evidence that single-target LDE modifications are important individual events in mammalian physiology.

  10. 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.

  11. Species identification of processed animal proteins (PAPs) in animal feed containing feed materials from animal origin.

    Science.gov (United States)

    Axmann, Sonja; Adler, Andreas; Brandstettner, Agnes Josephine; Spadinger, Gabriela; Weiss, Roland; Strnad, Irmengard

    2015-01-01

    Since June 2013 the total feed ban of processed animal proteins (PAPs) was partially lifted. Now it is possible to mix fish feed with PAPs from non-ruminants (pig and poultry). To guarantee that fish feed, which contains non-ruminant PAPs, is free of ruminant PAPs, it has to be analysed with a ruminant PCR assay to comply with the total ban of feeding PAPs from ruminants. However, PCR analysis cannot distinguish between ruminant DNA, which originates from proteins such as muscle and bones, and ruminant DNA, which comes from feed materials of animal origin such as milk products or fat. Thus, there is the risk of obtaining positive ruminant PCR signals based on these materials. The paper describes the development of the combination of two analysis methods, micro-dissection and PCR, to eliminate the problem of 'false-positive' PCR signals. With micro-dissection, single particles can be isolated and subsequently analysed with PCR.

  12. [FUNCTION OF INTERCELLULAR ADHESION A, FIBRINOGEN BINDING PROTEIN, AND ACCUMULATION-ASSOCIATED PROTEIN GENES IN FORMATION OF STAPHYLOCOCCUS EPIDERMIDIS-CANDIDA ALBICANS MIXED SPECIES BIOFILMS].

    Science.gov (United States)

    Wang, Xiaoyan; Chen, Ying; Huang, Yunchao; Zhou, Youquan; Zhao, Guangqiang; Ye, Lianhua; Lei, Yujie; Tang, Qi

    2015-01-01

    To explore the function of intercellular adhesion A (icaA), fibrinogen binding protein (fbe), and accumulation-associated protein (aap) genes in formation of Staphylococcus epidermidis-Candida albicans mixed species biofilms. The experiment was divided into 3 groups: single culture of Staphylococcus epidermidis ATCC35984 (S. epidermidis group) or Candida albicans ATCC10231 (C. albicans group), and co-culture of two strains (mixed group) to build in vitro biofilm model. Biofilm mass was detected by crystal violet semi-quantitative adherence assay at 2, 4, 6, 8, 12, 24, 48, and 72 hours after incubation. XTT assay was performed to determine the growth kinetics in the same time. Scanning electron microscopy (SEM) was used to observe the ultrastructure of the biofilms after 24 and 72 hours of incubation. The expressions of icaA, fbe, and aap genes were analyzed by real-time fluorescent quantitative PCR. Crystal violet semi-quantitative adherence assay showed that the biofilms thickened at 12 hours in the S. epidermidis and mixed groups; after co-cultured for 72 hours the thickness of biofilm in mixed group was more than that in the S. epidermidis group, and there was significant difference between 2 groups at the other time (P 0.05). In C. albicans group, the biofilm started to grow at 12 hours of cultivation, but the thickness of the biofilm was significantly lower than that in the mixed group in all the time points (P 0.05) except at 12 hours (P 0.05); the A value of mixed group was significantly higher than that of the C. albicans group after 6 hours (P biofilms with complex structure formed in all groups. The real-time fluorescent quantitative PCR showed the expressions of fbe, icaA, and aap genes in mixed group increased 1.93, 1.52, and 1.46 times respectively at 72 hours compared with the S. epidermidis group (P biofilms have more complex structure and are thicker than single species biofilms of Staphylococcus epidermidis or Candida albicans, which is related to

  13. Mobilization and Role of Starch, Protein, and Fat Reserves during Seed Germination of Six Wild Grassland Species.

    Science.gov (United States)

    Zhao, Ming; Zhang, Hongxiang; Yan, Hong; Qiu, Lu; Baskin, Carol C

    2018-01-01

    Since seed reserves can influence seed germination, the quantitative and qualitative differences in seed reserves may relate to the germination characteristics of species. The purpose of our study was to evaluate the correlation between germination and seed reserves, as well as their mobilization during germination of six grassland species ( Chloris virgata , Kochia scoparia , Lespedeza hedysaroides , Astragalus adsurgens , Leonurus artemisia , and Dracocephalum moldavica ) and compare the results with domesticated species. We measured starch, protein, and fat content in dry seeds and the initial absorption of water during imbibition. Starch, soluble protein, fat, and soluble sugar content also were determined at five stages during germination. Starch, protein, and fat reserves in dry seeds were not significantly correlated with germination percentage and rate (speed), but soluble sugar and soluble protein contents at different germination stages were positively significantly correlated with germination rate for the six species. Starch was mainly used during seed imbibition, and soluble protein was used from the imbibition stage to the highest germination stage. Fat content for all species remained relatively constant throughout germination for six species, regardless of the proportion of other seed reserves in the seeds. Our results for fat utilization differ from those obtained for cultivated grasses and legumes. These results provide new insight on the role of seed reserves as energy resources in germination for wild species.

  14. Mobilization and Role of Starch, Protein, and Fat Reserves during Seed Germination of Six Wild Grassland Species

    Directory of Open Access Journals (Sweden)

    Ming Zhao

    2018-02-01

    Full Text Available Since seed reserves can influence seed germination, the quantitative and qualitative differences in seed reserves may relate to the germination characteristics of species. The purpose of our study was to evaluate the correlation between germination and seed reserves, as well as their mobilization during germination of six grassland species (Chloris virgata, Kochia scoparia, Lespedeza hedysaroides, Astragalus adsurgens, Leonurus artemisia, and Dracocephalum moldavica and compare the results with domesticated species. We measured starch, protein, and fat content in dry seeds and the initial absorption of water during imbibition. Starch, soluble protein, fat, and soluble sugar content also were determined at five stages during germination. Starch, protein, and fat reserves in dry seeds were not significantly correlated with germination percentage and rate (speed, but soluble sugar and soluble protein contents at different germination stages were positively significantly correlated with germination rate for the six species. Starch was mainly used during seed imbibition, and soluble protein was used from the imbibition stage to the highest germination stage. Fat content for all species remained relatively constant throughout germination for six species, regardless of the proportion of other seed reserves in the seeds. Our results for fat utilization differ from those obtained for cultivated grasses and legumes. These results provide new insight on the role of seed reserves as energy resources in germination for wild species.

  15. 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...

  16. Fragment-based modelling of single stranded RNA bound to RNA recognition motif containing proteins

    Science.gov (United States)

    de Beauchene, Isaure Chauvot; de Vries, Sjoerd J.; Zacharias, Martin

    2016-01-01

    Abstract Protein-RNA complexes are important for many biological processes. However, structural modeling of such complexes is hampered by the high flexibility of RNA. Particularly challenging is the docking of single-stranded RNA (ssRNA). We have developed a fragment-based approach to model the structure of ssRNA bound to a protein, based on only the protein structure, the RNA sequence and conserved contacts. The conformational diversity of each RNA fragment is sampled by an exhaustive library of trinucleotides extracted from all known experimental protein–RNA complexes. The method was applied to ssRNA with up to 12 nucleotides which bind to dimers of the RNA recognition motifs (RRMs), a highly abundant eukaryotic RNA-binding domain. The fragment based docking allows a precise de novo atomic modeling of protein-bound ssRNA chains. On a benchmark of seven experimental ssRNA–RRM complexes, near-native models (with a mean heavy-atom deviation of <3 Å from experiment) were generated for six out of seven bound RNA chains, and even more precise models (deviation < 2 Å) were obtained for five out of seven cases, a significant improvement compared to the state of the art. The method is not restricted to RRMs but was also successfully applied to Pumilio RNA binding proteins. PMID:27131381

  17. 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.

  18. 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.

  19. 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.

  20. Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species.

    Science.gov (United States)

    Saito, Takahiro; Kikuchi, Aoi; Kaneko, Akira; Isozumi, Rie; Teramoto, Isao; Kimura, Masatsugu; Hirasawa, Noriyasu; Hiratsuka, Masahiro

    2018-06-01

    Malaria is caused by five species of Plasmodium in humans. Microscopy is currently used for pathogen detection, requiring considerable training and technical expertise as the parasites are often difficult to differentiate morphologically. Rapid diagnostic tests are as reliable as microscopy and offer faster diagnoses but possess lower detection limits and are incapable of distinguishing among the parasitic species. To improve global health efforts towards malaria control, a rapid, sensitive, species-specific, and economically viable diagnostic method is needed. In this study, we designed a malaria diagnostic method involving a multiplex single-tube nested PCR targeting Plasmodium mitochondrial cytochrome c oxidase III and single-stranded tag hybridization chromatographic printed-array strip. The detection sensitivity was found to be at least 40 times higher than that of agarose gel electrophoresis with ethidium bromide. This system also enables the identification of both single- and mixed-species malaria infections. The assay was validated with 152 Kenyan samples; using nested PCR as the standard, the assay's sensitivity and specificity were 88.7% and 100.0%, respectively. The turnaround time required, from PCR preparation to signal detection, is 90min. Our method should improve the diagnostic speed, treatment efficacy, and control of malaria, in addition to facilitating surveillance within global malaria eradication programs. Copyright © 2018 Elsevier B.V. All rights reserved.

  1. Evaluation of four single-locus markers for leishmania species discrimination by sequencing

    NARCIS (Netherlands)

    van der Auwera, Gert; Ravel, Christophe; Verweij, Jaco J.; Bart, Aldert; Schönian, Gabriele; Felger, Ingrid

    2014-01-01

    Several genetic markers have been described for discriminating Leishmania species. In most reported cases, one or a few polymorphisms are the basis of species identification, and the methods were validated on a limited number of strains from a particular geographical region. Therefore, most

  2. 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...

  3. Nuclear magnetic resonance detection and spectroscopy of single proteins using quantum logic.

    Science.gov (United States)

    Lovchinsky, I; Sushkov, A O; Urbach, E; de Leon, N P; Choi, S; De Greve, K; Evans, R; Gertner, R; Bersin, E; Müller, C; McGuinness, L; Jelezko, F; Walsworth, R L; Park, H; Lukin, M D

    2016-02-19

    Nuclear magnetic resonance spectroscopy is a powerful tool for the structural analysis of organic compounds and biomolecules but typically requires macroscopic sample quantities. We use a sensor, which consists of two quantum bits corresponding to an electronic spin and an ancillary nuclear spin, to demonstrate room temperature magnetic resonance detection and spectroscopy of multiple nuclear species within individual ubiquitin proteins attached to the diamond surface. Using quantum logic to improve readout fidelity and a surface-treatment technique to extend the spin coherence time of shallow nitrogen-vacancy centers, we demonstrate magnetic field sensitivity sufficient to detect individual proton spins within 1 second of integration. This gain in sensitivity enables high-confidence detection of individual proteins and allows us to observe spectral features that reveal information about their chemical composition. Copyright © 2016, American Association for the Advancement of Science.

  4. Decision Tree Algorithm-Generated Single-Nucleotide Polymorphism Barcodes of rbcL Genes for 38 Brassicaceae Species Tagging.

    Science.gov (United States)

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

    2018-01-01

    DNA barcode sequences are accumulating in large data sets. A barcode is generally a sequence larger than 1000 base pairs and generates a computational burden. Although the DNA barcode was originally envisioned as straightforward species tags, the identification usage of barcode sequences is rarely emphasized currently. Single-nucleotide polymorphism (SNP) association studies provide us an idea that the SNPs may be the ideal target of feature selection to discriminate between different species. We hypothesize that SNP-based barcodes may be more effective than the full length of DNA barcode sequences for species discrimination. To address this issue, we tested a r ibulose diphosphate carboxylase ( rbcL ) S NP b arcoding (RSB) strategy using a decision tree algorithm. After alignment and trimming, 31 SNPs were discovered in the rbcL sequences from 38 Brassicaceae plant species. In the decision tree construction, these SNPs were computed to set up the decision rule to assign the sequences into 2 groups level by level. After algorithm processing, 37 nodes and 31 loci were required for discriminating 38 species. Finally, the sequence tags consisting of 31 rbcL SNP barcodes were identified for discriminating 38 Brassicaceae species based on the decision tree-selected SNP pattern using RSB method. Taken together, this study provides the rational that the SNP aspect of DNA barcode for rbcL gene is a useful and effective sequence for tagging 38 Brassicaceae species.

  5. Differentiation of Populus species using chloroplast single nucleotide polymorphism (SNP) markers--essential for comprehensible and reliable poplar breeding.

    Science.gov (United States)

    Schroeder, H; Hoeltken, A M; Fladung, M

    2012-03-01

    Within the genus Populus several species belonging to different sections are cross-compatible. Hence, high numbers of interspecies hybrids occur naturally and, additionally, have been artificially produced in huge breeding programmes during the last 100 years. Therefore, determination of a single poplar species, used for the production of 'multi-species hybrids' is often difficult, and represents a great challenge for the use of molecular markers in species identification. Within this study, over 20 chloroplast regions, both intergenic spacers and coding regions, have been tested for their ability to differentiate different poplar species using 23 already published barcoding primer combinations and 17 newly designed primer combinations. About half of the published barcoding primers yielded amplification products, whereas the new primers designed on the basis of the total sequenced cpDNA genome of Populus trichocarpa Torr. & Gray yielded much higher amplification success. Intergenic spacers were found to be more variable than coding regions within the genus Populus. The highest discrimination power of Populus species was found in the combination of two intergenic spacers (trnG-psbK, psbK-psbl) and the coding region rpoC. In barcoding projects, the coding regions matK and rbcL are often recommended, but within the genus Populus they only show moderate variability and are not efficient in species discrimination. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

  6. 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

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

    Science.gov (United States)

    MacDowell, Alastair A; Celestre, Rich 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, Tom; Berger, James M; Agard, David A; Padmore, Howard A

    2004-11-01

    At the Advanced Light Source, three protein crystallography beamlines have been built that use as a source one of the three 6 T 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, owing 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.

  8. 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.

  9. 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.

  10. 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.

  11. Mathematical modeling and comparison of protein size distribution in different plant, animal, fungal and microbial species reveals a negative correlation between protein size and protein number, thus providing insight into the evolution of proteomes

    Directory of Open Access Journals (Sweden)

    Tiessen Axel

    2012-02-01

    Full Text Available Abstract Background The sizes of proteins are relevant to their biochemical structure and for their biological function. The statistical distribution of protein lengths across a diverse set of taxa can provide hints about the evolution of proteomes. Results Using the full genomic sequences of over 1,302 prokaryotic and 140 eukaryotic species two datasets containing 1.2 and 6.1 million proteins were generated and analyzed statistically. The lengthwise distribution of proteins can be roughly described with a gamma type or log-normal model, depending on the species. However the shape parameter of the gamma model has not a fixed value of 2, as previously suggested, but varies between 1.5 and 3 in different species. A gamma model with unrestricted shape parameter described best the distributions in ~48% of the species, whereas the log-normal distribution described better the observed protein sizes in 42% of the species. The gamma restricted function and the sum of exponentials distribution had a better fitting in only ~5% of the species. Eukaryotic proteins have an average size of 472 aa, whereas bacterial (320 aa and archaeal (283 aa proteins are significantly smaller (33-40% on average. Average protein sizes in different phylogenetic groups were: Alveolata (628 aa, Amoebozoa (533 aa, Fornicata (543 aa, Placozoa (453 aa, Eumetazoa (486 aa, Fungi (487 aa, Stramenopila (486 aa, Viridiplantae (392 aa. Amino acid composition is biased according to protein size. Protein length correlated negatively with %C, %M, %K, %F, %R, %W, %Y and positively with %D, %E, %Q, %S and %T. Prokaryotic proteins had a different protein size bias for %E, %G, %K and %M as compared to eukaryotes. Conclusions Mathematical modeling of protein length empirical distributions can be used to asses the quality of small ORFs annotation in genomic releases (detection of too many false positive small ORFs. There is a negative correlation between average protein size and total number of

  12. 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

  13. Persistence and extinction of a stochastic single-specie model under regime switching in a polluted environment.

    Science.gov (United States)

    Liu, Meng; Wang, Ke

    2010-06-07

    A new single-species model disturbed by both white noise and colored noise in a polluted environment is developed and analyzed. Sufficient criteria for extinction, stochastic nonpersistence in the mean, stochastic weak persistence in the mean, stochastic strong persistence in the mean and stochastic permanence of the species are established. The threshold between stochastic weak persistence in the mean and extinction is obtained. The results show that both white and colored environmental noises have sufficient effect to the survival results. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  14. Ultrastructural Comparison of Processing of Protein and Pigment in the Ink Gland of Four Species of Sea Hares

    Directory of Open Access Journals (Sweden)

    Jeffrey S. Prince

    2015-01-01

    Full Text Available The ink glands of four sea hare species (Aplysia californica, A. parvula, A. juliana, and Dolabrifera dolabrifera were compared to determine where ink protein is synthesized, how it is incorporated into protein storage vesicles, and the degree of variation in the structure of the ink gland. Ink protein was synthesized in RER cells and stored in amber and white vesicles. Lack of competent RER cells in the ink gland of D. dolabrifera was correlated with the absence of ink protein. Ink protein had similar characteristics in all three Aplysia species but, again, it was absent in D. dolabrifera. Its uptake involved pinocytosis by protein vesicle cell membranes. Granulate cells showed little variation in structure among the four species, the opposite was the case for RER cells. The conversion of the red algal pigment, phycoerythrin, to phycoerythrobilin (PEB occurs in the digestive gland but the change of PEB to aplysioviolin (APV, the form of pigment released by the ink gland, occurs in the ink gland itself by both granulate cells and pigment vesicles. The literature describes five types of vesicles based upon color and contents in the ink gland of these four species. We report only three types of vesicle: colored (purple, protein (white and amber, and transparent (includes clear vesicles.

  15. 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

  16. Packaging of single DNA molecules by the yeast mitochondrial protein Abf2p.

    Science.gov (United States)

    Brewer, Laurence R; Friddle, Raymond; Noy, Aleksandr; Baldwin, Enoch; Martin, Shelley S; Corzett, Michele; Balhorn, Rod; Baskin, Ronald J

    2003-10-01

    Mitochondrial and nuclear DNA are packaged by proteins in a very different manner. Although protein-DNA complexes called "nucleoids" have been identified as the genetic units of mitochondrial inheritance in yeast and man, little is known about their physical structure. The yeast mitochondrial protein Abf2p was shown to be sufficient to compact linear dsDNA, without the benefit of supercoiling, using optical and atomic force microscopy single molecule techniques. The packaging of DNA by Abf2p was observed to be very weak as evidenced by a fast Abf2p off-rate (k(off) = 0.014 +/- 0.001 s(-1)) and the extremely small forces (<0.6 pN) stabilizing the condensed protein-DNA complex. Atomic force microscopy images of individual complexes showed the 190-nm structures are loosely packaged relative to nuclear chromatin. This organization may leave mtDNA accessible for transcription and replication, while making it more vulnerable to damage.

  17. 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.

  18. 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.

  19. 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

  20. 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...

  1. 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.

  2. 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

    (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...... 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...... and methionine sulfone, respectively, was assessed. The established IDA method was validated for the absolute quantification of commercially available lysozyme and ceruloplasmin standards including the calculation of a total combined uncertainty budget....

  3. Conjugated action of two species-specific invasion proteins for fetoplacental listeriosis.

    Science.gov (United States)

    Disson, Olivier; Grayo, Solène; Huillet, Eugénie; Nikitas, Georgios; Langa-Vives, Francina; Dussurget, Olivier; Ragon, Marie; Le Monnier, Alban; Babinet, Charles; Cossart, Pascale; Lecuit, Marc

    2008-10-23

    The ability to cross host barriers is an essential virulence determinant of invasive microbial pathogens. Listeria monocytogenes is a model microorganism that crosses human intestinal and placental barriers, and causes severe maternofetal infections by an unknown mechanism. Several studies have helped to characterize the bacterial invasion proteins InlA and InlB. However, their respective species specificity has complicated investigations on their in vivo role. Here we describe two novel and complementary animal models for human listeriosis: the gerbil, a natural host for L. monocytogenes, and a knock-in mouse line ubiquitously expressing humanized E-cadherin. Using these two models, we uncover the essential and interdependent roles of InlA and InlB in fetoplacental listeriosis, and thereby decipher the molecular mechanism underlying the ability of a microbe to target and cross the placental barrier.

  4. 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.

  5. Persistence and extinction of a stochastic single-species population model in a polluted environment with impulsive toxicant input

    Directory of Open Access Journals (Sweden)

    Meng Liu

    2013-10-01

    Full Text Available A stochastic single-species population system in a polluted environment with impulsive toxicant input is proposed and studied. Sufficient conditions for extinction, non-persistence in the mean, strong persistence in the mean and stochastic permanence of the population are established. The threshold between strong persistence in the mean and extinction is obtained. Some simulation figures are introduced to illustrate the main results.

  6. 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.

  7. 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.

  8. 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.

  9. 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 PEimeria infection and in identifying molecular targets for diagnostic screening and development of alternative preventative and therapeutic methods. PMID:21297942

  10. 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.

  11. Protein dynamics revealed in the excitonic spectra of single LH2 complexes

    International Nuclear Information System (INIS)

    Valkunas, Leonas; Janusonis, Julius; Rutkauskas, Danielis; Grondelle, Rienk van

    2007-01-01

    The fluorescence emission spectrum of single peripheral light-harvesting (LH2) complexes of the photosynthetic purple bacterium Rhodopseudomonas acidophila exhibits remarkable dynamics on a time scale of several minutes. Often the spectral properties are quasi-stable; sometimes large spectral jumps to the blue or to the red are observed. To explain the dynamics, every pigment is proposed to be in two conformational substates with different excitation energies, which originate from the conformational state of the protein as a result of pigment-protein interaction. Due to the excitonic coupling in the ring of 18 pigments, the two-state assumption generates a substantial amount of distinct spectroscopic states, which reflect part of the inhomogeneous distributed spectral properties of LH2. To describe the observed dynamics, spontaneous and light-induced transitions are introduced between the two states. For each 'realization of the disorder', the spectral properties are calculated using a disordered exciton model combined with the modified Redfield theory to obtain realistic spectral line shapes. The single-molecule fluorescence peak (FLP) distribution, the distribution dependence on the excitation intensity, and the FLP time traces are well described within the framework of this model

  12. Helical filaments of human Dmc1 protein on single-stranded DNA: a cautionary tale

    Science.gov (United States)

    Yu, Xiong; Egelman, Edward H.

    2010-01-01

    Proteins in the RecA/Rad51/RadA family form nucleoprotein filaments on DNA that catalyze a strand exchange reaction as part of homologous genetic recombination. Because of the centrality of this system to many aspects of DNA repair, the generation of genetic diversity, and cancer when this system fails or is not properly regulated, these filaments have been the object of many biochemical and biophysical studies. A recent paper has argued that the human Dmc1 protein, a meiotic homolog of bacterial RecA and human Rad51, forms filaments on single stranded DNA with ∼ 9 subunits per turn in contrast to the filaments formed on double stranded DNA with ∼ 6.4 subunits per turn, and that the stoichiometry of DNA binding is different between these two filaments. We show using scanning transmission electron microscopy (STEM) that the Dmc1 filament formed on single stranded DNA has a mass per unit length expected from ∼ 6.5 subunits per turn. More generally, we show how ambiguities in helical symmetry determination can generate incorrect solutions, and why one sometimes must use other techniques, such as biochemistry, metal shadowing, or STEM to resolve these ambiguities. While three-dimensional reconstruction of helical filaments from EM images is a powerful tool, the intrinsic ambiguities that may be present with limited resolution are not sufficiently appreciated. PMID:20600108

  13. A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species

    Science.gov (United States)

    Amornchai, Premjit; Wuthiekanun, Vanaporn; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Chantratita, Narisara; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.

    2013-01-01

    Background The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis. PMID:23359622

  14. HitPredict version 4: comprehensive reliability scoring of physical protein?protein interactions from more than 100 species

    OpenAIRE

    L?pez, Yosvany; Nakai, Kenta; Patil, Ashwini

    2015-01-01

    HitPredict is a consolidated resource of experimentally identified, physical protein?protein interactions with confidence scores to indicate their reliability. The study of genes and their inter-relationships using methods such as network and pathway analysis requires high quality protein?protein interaction information. Extracting reliable interactions from most of the existing databases is challenging because they either contain only a subset of the available interactions, or a mixture of p...

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

    Science.gov (United States)

    2013-01-01

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

  16. 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

  17. 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.

  18. 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.

  19. Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

    Science.gov (United States)

    Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

    2013-10-01

    Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

  20. A single mutation in the envelope protein modulates flavivirus antigenicity, stability, and pathogenesis.

    Directory of Open Access Journals (Sweden)

    Leslie Goo

    2017-02-01

    Full Text Available The structural flexibility or 'breathing' of the envelope (E protein of flaviviruses allows virions to sample an ensemble of conformations at equilibrium. The molecular basis and functional consequences of virus conformational dynamics are poorly understood. Here, we identified a single mutation at residue 198 (T198F of the West Nile virus (WNV E protein domain I-II hinge that regulates virus breathing. The T198F mutation resulted in a ~70-fold increase in sensitivity to neutralization by a monoclonal antibody targeting a cryptic epitope in the fusion loop. Increased exposure of this otherwise poorly accessible fusion loop epitope was accompanied by reduced virus stability in solution at physiological temperatures. Introduction of a mutation at the analogous residue of dengue virus (DENV, but not Zika virus (ZIKV, E protein also increased accessibility of the cryptic fusion loop epitope and decreased virus stability in solution, suggesting that this residue modulates the structural ensembles sampled by distinct flaviviruses at equilibrium in a context dependent manner. Although the T198F mutation did not substantially impair WNV growth kinetics in vitro, studies in mice revealed attenuation of WNV T198F infection. Overall, our study provides insight into the molecular basis and the in vitro and in vivo consequences of flavivirus breathing.

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

    Science.gov (United States)

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

    2008-09-01

    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.

  2. 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

  3. 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.

  4. 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.

  5. 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 ...

  6. A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species

    NARCIS (Netherlands)

    Boonsilp, Siriphan; Thaipadungpanit, Janjira; Amornchai, Premjit; Wuthiekanun, Vanaporn; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Chantratita, Narisara; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.

    2013-01-01

    The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. We modified the existing scheme by replacing one of the

  7. 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.

  8. PhosphOrtholog: a web-based tool for cross-species mapping of orthologous protein post-translational modifications.

    Science.gov (United States)

    Chaudhuri, Rima; Sadrieh, Arash; Hoffman, Nolan J; Parker, Benjamin L; Humphrey, Sean J; Stöckli, Jacqueline; Hill, Adam P; James, David E; Yang, Jean Yee Hwa

    2015-08-19

    Most biological processes are influenced by protein post-translational modifications (PTMs). Identifying novel PTM sites in different organisms, including humans and model organisms, has expedited our understanding of key signal transduction mechanisms. However, with increasing availability of deep, quantitative datasets in diverse species, there is a growing need for tools to facilitate cross-species comparison of PTM data. This is particularly important because functionally important modification sites are more likely to be evolutionarily conserved; yet cross-species comparison of PTMs is difficult since they often lie in structurally disordered protein domains. Current tools that address this can only map known PTMs between species based on known orthologous phosphosites, and do not enable the cross-species mapping of newly identified modification sites. Here, we addressed this by developing a web-based software tool, PhosphOrtholog ( www.phosphortholog.com ) that accurately maps protein modification sites between different species. This facilitates the comparison of datasets derived from multiple species, and should be a valuable tool for the proteomics community. Here we describe PhosphOrtholog, a web-based application for mapping known and novel orthologous PTM sites from experimental data obtained from different species. PhosphOrtholog is the only generic and automated tool that enables cross-species comparison of large-scale PTM datasets without relying on existing PTM databases. This is achieved through pairwise sequence alignment of orthologous protein residues. To demonstrate its utility we apply it to two sets of human and rat muscle phosphoproteomes generated following insulin and exercise stimulation, respectively, and one publicly available mouse phosphoproteome following cellular stress revealing high mapping and coverage efficiency. Although coverage statistics are dataset dependent, PhosphOrtholog increased the number of cross-species mapped sites

  9. Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions.

    Science.gov (United States)

    Götz, Markus; Wortmann, Philipp; Schmid, Sonja; Hugel, Thorsten

    2018-01-30

    Single-molecule Förster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail. Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates. By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

  10. Lutzomyia longipalpis in Brazil: a complex or a single species? A mini-review

    Directory of Open Access Journals (Sweden)

    Luiz GSR Bauzer

    2007-02-01

    Full Text Available Lutzomyia longipalpis is the main vector of Leishmania infantum chagasi, the causative agent of American visceral leishmaniasis (AVL. Although there is strong evidence that Lu. longipalpis is a species complex, not all data concerning populations from Brazil support this hypothesis. The issue is still somewhat controversial for this large part of Lu. longipalpis distribution range even though that it is the Latin American region contributing to most of the cases of AVL. In this mini-review we consider in detail the current data for the Brazilian populations and conclude that Lu. longipalpis is a complex of incipient vector species with a complexity similar to Anopheles gambiae s.s. in Africa.

  11. Single Molecule Science for Personalized Nanomedicine: Atomic Force Microscopy of Biopolymer-Protein Interactions

    Science.gov (United States)

    Hsueh, Carlin

    Nanotechnology has a unique and relatively untapped utility in the fields of medicine and dentistry at the level of single-biopolymer and -molecule diagnostics. In recent years atomic force microscopy (AFM) has garnered much interest due to its ability to obtain atomic-resolution of molecular structures and probe biophysical behaviors of biopolymers and proteins in a variety of biologically significant environments. The work presented in this thesis focuses on the nanoscale manipulation and observation of biopolymers to develop an innovative technology for personalized medicine while understanding complex biological systems. These studies described here primarily use AFM to observe biopolymer interactions with proteins and its surroundings with unprecedented resolution, providing a better understanding of these systems and interactions at the nanoscale. Transcriptional profiling, the measure of messenger RNA (mRNA) abundance in a single cell, is a powerful technique that detects "behavior" or "symptoms" at the tissue and cellular level. We have sought to develop an alternative approach, using our expertise in AFM and single molecule nanotechnology, to achieve a cost-effective high throughput method for sensitive detection and profiling of subtle changes in transcript abundance. The technique does not require amplification of the mRNA sample because the AFM provides three-dimensional views of molecules with unprecedented resolution, requires minimal sample preparation, and utilizes a simple tagging chemistry on cDNA molecules. AFM images showed collagen polymers in teeth and of Drebrin-A remodeling of filamentous actin structure and mechanics. AFM was used to image collagen on exposed dentine tubules and confirmed tubule occlusion with a desensitizing prophylaxis paste by Colgate-Palmolive. The AFM also superseded other microscopy tools in resolving F-actin helix remodeling and possible cooperative binding by a neuronal actin binding protein---Drebrin-A, an

  12. Transparent Nanopore Cavity Arrays Enable Highly Parallelized Optical Studies of Single Membrane Proteins on Chip.

    Science.gov (United States)

    Diederichs, Tim; Nguyen, Quoc Hung; Urban, Michael; Tampé, Robert; Tornow, Marc

    2018-06-13

    Membrane proteins involved in transport processes are key targets for pharmaceutical research and industry. Despite continuous improvements and new developments in the field of electrical readouts for the analysis of transport kinetics, a well-suited methodology for high-throughput characterization of single transporters with nonionic substrates and slow turnover rates is still lacking. Here, we report on a novel architecture of silicon chips with embedded nanopore microcavities, based on a silicon-on-insulator technology for high-throughput optical readouts. Arrays containing more than 14 000 inverted-pyramidal cavities of 50 femtoliter volumes and 80 nm circular pore openings were constructed via high-resolution electron-beam lithography in combination with reactive ion etching and anisotropic wet etching. These cavities feature both, an optically transparent bottom and top cap. Atomic force microscopy analysis reveals an overall extremely smooth chip surface, particularly in the vicinity of the nanopores, which exhibits well-defined edges. Our unprecedented transparent chip design provides parallel and independent fluorescent readout of both cavities and buffer reservoir for unbiased single-transporter recordings. Spreading of large unilamellar vesicles with efficiencies up to 96% created nanopore-supported lipid bilayers, which are stable for more than 1 day. A high lipid mobility in the supported membrane was determined by fluorescent recovery after photobleaching. Flux kinetics of α-hemolysin were characterized at single-pore resolution with a rate constant of 0.96 ± 0.06 × 10 -3 s -1 . Here, we deliver an ideal chip platform for pharmaceutical research, which features high parallelism and throughput, synergistically combined with single-transporter resolution.

  13. Species-specific sensitivity of coagulase-negative Staphylococci to single antibiotics and their combinations.

    Science.gov (United States)

    Szymańska, Grazyna; Szemraj, Magdalena; Szewczyk, Eligia M

    2011-01-01

    The activity of beta-lactam antibiotics (oxacillin, cloxacillin, cephalotin), vancomycin, gentamicin and rifampicin applied in vitro individually and in combination against 37 nosocomial methicillin-resistant strains of coagulase-negative staphylococci (CNS) was assessed to demonstrate the heterogeneity of this group of bacteria and estimate the chance of the efficacy of such therapy. The strains belonged to four species: Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus cohnii, Staphylococcus hominis. They originated from a hospital environment and from the skin of medical staff of the intensive care unit of a paediatric ward at a university hospital. All strains were methicillin-resistant, according to CLSI standards, but individual strains differed in MIC(ox) values. Susceptibility to other tested antibiotics was also characteristic for the species. The increased susceptibility to antibiotics in combinations, tested by calculating the fractional inhibitory concentration (FIC) index, concerned 26 out of 37 investigated strains and it was a feature of a particular species. Combinations of vancomycin and cephalotin against S. epidermidis and oxacillin with vancomycin were significant, as well as cephalotin and rifampicin in growth inhibition of multiresistant S. haemolyticus strains.

  14. The presence of INA proteins on the surface of single cells of Pseudomonas syringae R10.79 isolated from rain

    Science.gov (United States)

    Šantl-Temkiv, Tina; Ling, Meilee; Holm, Stine; Finster, Kai; Boesen, Thomas

    2016-04-01

    One of the important open questions in atmospheric ice nucleation is the impact of bioaerosols on the ice content of mix phase clouds (DeMott and Prenni 2010). Biogenic ice nuclei have a unique capacity of facilitating ice formation at temperatures between -1 and -10 °C. The model biogenic ice nuclei are produced by a few species of plant-surface bacteria, such as Pseudomonas syringae, that are commonly transported through the atmosphere. These bacterial species have highly specialized proteins, the so-called ice nucleation active (INA) proteins, which are exposed at the outer membrane surface of the cell where they promote ice particle formation. The mechanisms behind the onset of INA protein synthesis in single bacterial cells are not well understood. We performed a laboratory study in order to (i) investigate the presence of INA proteins on single bacterial cells and (ii) understand the conditions that induce INA protein production. We previously isolated an INA-positive strain of Pseudomonas syringae from rain samples collected in Denmark. Bacterial cells initiated ice nucleation activity at temperatures ≤-2°C and the cell fragments at temperatures ≤-8°C (Šantl-Temkiv et al 2015). We determined the amino-acid sequence of the INA protein and used the sequence to produce custom-made antibodies (GenScript, Germany). These antibodies were used to specifically stain and visualize the INA protein on the surfaces of single cells, which can then be quantified by a technique called flow cytometry. The synthesis of INA proteins by individual cells was followed during a batch growth experiment. An unusually high proportion of cells that were adapting to the new conditions prior to growth produced INA proteins (~4.4% of all cells). A smaller fraction of actively growing cells was carrying INA proteins (~1.2 % of all cells). The cells that stopped growing due to unfavorable conditions had the lowest fraction of cells carrying INA proteins (~0.5 % of all cells). To

  15. Analyzing the structural aspects of Isoprenoid biosynthesis pathway proteins in Ocimum species

    Directory of Open Access Journals (Sweden)

    Muktesh Chandra

    2017-10-01

    Full Text Available Generally thought that the extremely diverse array of secondary metabolites observed within Ocimum species defends against a comparable diverse array of biotic pests, pathogens and herbivores encountered around its natural range. Along with defense the diverse array of secondary metabolite also leads to the therapeutic and remedial property which justifies Ocimum as natural medicinal and aromatic casket. Many of the defense compounds, aroma compounds and medicinal derivatives are secondary metabolites isolated from trichome glands, mainly consist of terpenoids as well as phenylpropanoids. Various pathways fabricating these compounds are known viz. mevalonate pathway (MVA, phenylpropanoid pathway and MEP pathways. The enzyme cascade responsible for various secondary metabolites, need to be explored in various aspects. Here we had studied the MVA pathway enzymes in O. basilicum and O. gratissimum to figure out variations in enzyme structures due to speciation. Hence, in depth analysis of the transcriptome of O. basilicum and O. gratissimum, varrying in qualitative and quantitative aspects of essential oil were carried out. The transcriptome data from NCBI server was assembled using bioinformatic approaches. nr database at NCBI repository used for annotation, which assigned 60% contigs to known functions. Contigs corresponding to Mevalonate pathway enzymes are isolated using perl pipelines developed in our lab, which were further assembled using CLC workbench to remove redundancy and make larger stretch of sequence. Blastx of these larger sequences assigned them function and they are mapped to validated sequences to make full length. Data from both species led us to overall seven enzymes (total 14 of MVA pathway. These enzymes are studied in detail for various physio-chemical properties, steriochemical properties and motif/domain for protein-protein interaction (PPI study. Homolog models of all enzymes were predicted, against templates from RCSB

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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

  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. Immunological and cytotoxicological characterization of tuberculin purified protein derivative (PPD) conjugated to single-walled carbon nanotubes.

    Science.gov (United States)

    Zeinali, Majid; Jammalan, Mostafa; Ardestani, Sussan K; Mosaveri, Nader

    2009-09-22

    Tuberculosis (TB) represents one of the leading killers among all infectious disease. Protection against TB depends on the activation of T-helper type I (Th1) immune response. Carbon nanotubes (CNTs) have attracted considerable attention because of their potential applications as new nanovehicle. In the current study, tuberculin purified protein derivative (PPD) was conjugated to carboxylated single-walled carbon nanotubes (SWCNTs). Cytotoxicity of the carboxylated SWCNT and SWCNT-PPD conjugate was analyzed with MTT assay and by reactive oxygen species (ROS) and nitric oxide (NO) generation. Male BALB/c mice were immunized with BCG, PPD, SWCNT-PPD conjugate and PPD in complete Freund's adjuvant (CFA). Induction of cellular immune response was analyzed by measuring the levels of Th1 cytokines (IFN-gamma and IL-12) and Th2 cytokines (IL-10 and IL-5). Immunization with non-conjugated PPD or PPD in Freund's adjuvant induced a Th2 cytokine response while immunization with BCG resulted to a mixed Th1/Th2 cytokine response. In contrast, PPD in conjugation with SWCNT generated preferentially a Th1-type cytokine response in the absence of potential cytotoxic effects.

  3. 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).

  4. Determination of trace elements in BCR single cell protein via destructive neutron activation analyses

    International Nuclear Information System (INIS)

    Tjioe, P.S.; Goeij, J.J.M. de; Nooijen, J.L.; Kroon, J.J.

    1978-10-01

    The amount of some trace elements in single cell protein (SCP), a product of BP Research Centre at Sunbury-at-Thames, England, was determined by neutron activation analysis. The SCP-samples were irradiated in the reactor of the Interuniversity Reactor Institute at Delft in a neutron flux of 1.0x10 13 n/cm 2 s for 12 hours. Samples of Bowen's Kale were used as reference material. After a decay of two or three days the samples were chemically destroyed, and the trace elements were separated. The quantity of the following elements was determined by measuring the γ-activity by means of a scintillation counter: antimony, cadmium, mercury, arsenic and selenium. The amounts of these elements in the SCP and in the reference material were tabled

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

    International Nuclear Information System (INIS)

    Hu Pingan; Zhang Jia; Wen Zhenzhong; Zhang Can

    2011-01-01

    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 x 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.

  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. 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.

  8. Chemo-mechanical pushing of proteins along single-stranded DNA.

    Science.gov (United States)

    Sokoloski, Joshua E; Kozlov, Alexander G; Galletto, Roberto; Lohman, Timothy M

    2016-05-31

    Single-stranded (ss)DNA binding (SSB) proteins bind with high affinity to ssDNA generated during DNA replication, recombination, and repair; however, these SSBs must eventually be displaced from or reorganized along the ssDNA. One potential mechanism for reorganization is for an ssDNA translocase (ATP-dependent motor) to push the SSB along ssDNA. Here we use single molecule total internal reflection fluorescence microscopy to detect such pushing events. When Cy5-labeled Escherichia coli (Ec) SSB is bound to surface-immobilized 3'-Cy3-labeled ssDNA, a fluctuating FRET signal is observed, consistent with random diffusion of SSB along the ssDNA. Addition of Saccharomyces cerevisiae Pif1, a 5' to 3' ssDNA translocase, results in the appearance of isolated, irregularly spaced saw-tooth FRET spikes only in the presence of ATP. These FRET spikes result from translocase-induced directional (5' to 3') pushing of the SSB toward the 3' ssDNA end, followed by displacement of the SSB from the DNA end. Similar ATP-dependent pushing events, but in the opposite (3' to 5') direction, are observed with EcRep and EcUvrD (both 3' to 5' ssDNA translocases). Simulations indicate that these events reflect active pushing by the translocase. The ability of translocases to chemo-mechanically push heterologous SSB proteins along ssDNA provides a potential mechanism for reorganization and clearance of tightly bound SSBs from ssDNA.

  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-stranded DNA-binding protein * photoconvertible fluorescent protein Eos Subject RIV: EA - Cell Biology Impact factor: 1.559, year: 2015

  10. Mercuric ions inhibit mitogen-activated protein kinase dephosphorylation by inducing reactive oxygen species

    International Nuclear Information System (INIS)

    Haase, Hajo; Engelhardt, Gabriela; Hebel, Silke; Rink, Lothar

    2011-01-01

    Mercury intoxication profoundly affects the immune system, in particular, signal transduction of immune cells. However, the mechanism of the interaction of mercury with cellular signaling pathways, such as mitogen activated protein kinases (MAPK), remains elusive. Therefore, the objective of this study is to investigate three potential ways in which Hg 2+ ions could inhibit MAPK dephosphorylation in the human T-cell line Jurkat: (1) by direct binding to phosphatases; (2) by releasing cellular zinc (Zn 2+ ); and (3) by inducing reactive oxygen species (ROS). Hg 2+ causes production of ROS, measured by dihydrorhodamine 123, and triggers ROS-mediated Zn 2+ release, detected with FluoZin-3. Yet, phosphatase-inhibition is not mediated by binding of Zn 2+ or Hg 2+ . Rather, phosphatases are inactivated by at least two forms of thiol oxidation; initial inhibition is reversible with reducing agents such as Tris(2-carboxyethyl)phosphine. Prolonged inhibition leads to non-reversible phosphatase oxidation, presumably oxidizing the cysteine thiol to sulfinic- or sulfonic acid. Notably, phosphatases are a particularly sensitive target for Hg 2+ -induced oxidation, because phosphatase activity is inhibited at concentrations of Hg 2+ that have only minor impact on over all thiol oxidation. This phosphatase inhibition results in augmented, ROS-dependent MAPK phosphorylation. MAPK are important regulators of T-cell function, and MAPK-activation by inhibition of phosphatases seems to be one of the molecular mechanisms by which mercury affects the immune system.

  11. 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)

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

    Science.gov (United States)

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

    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.

  13. SAAS: Short Amino Acid Sequence - A Promising Protein Secondary Structure Prediction Method of Single Sequence

    Directory of Open Access Journals (Sweden)

    Zhou Yuan Wu

    2013-07-01

    Full Text Available In statistical methods of predicting protein secondary structure, many researchers focus on single amino acid frequencies in α-helices, β-sheets, and so on, or the impact near amino acids on an amino acid forming a secondary structure. But the paper considers a short sequence of amino acids (3, 4, 5 or 6 amino acids as integer, and statistics short sequence's probability forming secondary structure. Also, many researchers select low homologous sequences as statistical database. But this paper select whole PDB database. In this paper we propose a strategy to predict protein secondary structure using simple statistical method. Numerical computation shows that, short amino acids sequence as integer to statistics, which can easy see trend of short sequence forming secondary structure, and it will work well to select large statistical database (whole PDB database without considering homologous, and Q3 accuracy is ca. 74% using this paper proposed simple statistical method, but accuracy of others statistical methods is less than 70%.

  14. High performance dendrimer functionalized single-walled carbon nanotubes field effect transistor biosensor for protein detection

    Science.gov (United States)

    Rajesh, Sharma, Vikash; Puri, Nitin K.; Mulchandani, Ashok; Kotnala, Ravinder K.

    2016-12-01

    We report a single-walled carbon nanotube (SWNT) field-effect transistor (FET) functionalized with Polyamidoamine (PAMAM) dendrimer with 128 carboxyl groups as anchors for site specific biomolecular immobilization of protein antibody for C-reactive protein (CRP) detection. The FET device was characterized by scanning electron microscopy and current-gate voltage (I-Vg) characteristic studies. A concentration-dependent decrease in the source-drain current was observed in the regime of clinical significance, with a detection limit of ˜85 pM and a high sensitivity of 20% change in current (ΔI/I) per decade CRP concentration, showing SWNT being locally gated by the binding of CRP to antibody (anti-CRP) on the FET device. The low value of the dissociation constant (Kd = 0.31 ± 0.13 μg ml-1) indicated a high affinity of the device towards CRP analyte arising due to high anti-CRP loading with a better probe orientation on the 3-dimensional PAMAM structure.

  15. 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.

  16. 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

  17. The effect of seasonal harvesting on a single-species discrete population model with stage structure and birth pulses

    International Nuclear Information System (INIS)

    Gao Shujing; Chen Lansun

    2005-01-01

    In this paper, we propose an exploited single-species discrete model with stage structure for the dynamics in a fish population for which births occur in a single pulse once per time period. Using the stroboscopic map, we obtain an exact cycle of the system, and obtain the threshold conditions for its stability. Bifurcation diagrams are constructed with the birth rate as the bifurcation parameter, and these are observed to display complex dynamic behaviors, including chaotic bands with period windows, pitch-fork and tangent bifurcation. This suggests that birth pulse provides a natural period or cyclicity that makes the dynamical behavior more complex. Moreover, we show that the timing of harvesting has a strong impact on the persistence of the fish population, on the volume of mature fish stock and on the maximum annual-sustainable yield. An interesting result is obtained that, after the birth pulse, the earlier culling the mature fish, the larger harvest can tolerate

  18. 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.

  19. 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

  20. 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

  1. Ileal and faecal protein digestibility measurement in humans and other non-ruminants - a comparative species view

    NARCIS (Netherlands)

    Hendriks, W.H.; Baal, van J.; Bosch, G.

    2012-01-01

    A comparative non-ruminant species view of the contribution of the large intestinal metabolism to inaccuracies in nitrogen and amino acid absorption measurements is provided to assess potential implications for the determination of crude protein/amino acid digestibility in adult humans consuming

  2. Fluctuations in protein synthesis from a single RNA template: stochastic kinetics of ribosomes.

    Science.gov (United States)

    Garai, Ashok; Chowdhury, Debashish; Ramakrishnan, T V

    2009-01-01

    Proteins are polymerized by cyclic machines called ribosomes, which use their messenger RNA (mRNA) track also as the corresponding template, and the process is called translation. We explore, in depth and detail, the stochastic nature of the translation. We compute various distributions associated with the translation process; one of them--namely, the dwell time distribution--has been measured in recent single-ribosome experiments. The form of the distribution, which fits best with our simulation data, is consistent with that extracted from the experimental data. For our computations, we use a model that captures both the mechanochemistry of each individual ribosome and their steric interactions. We also demonstrate the effects of the sequence inhomogeneities of real genes on the fluctuations and noise in translation. Finally, inspired by recent advances in the experimental techniques of manipulating single ribosomes, we make theoretical predictions on the force-velocity relation for individual ribosomes. In principle, all our predictions can be tested by carrying out in vitro experiments.

  3. 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.

  4. 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.

  5. Proteins Related to the Type I Secretion System Are Associated with Secondary SecA_DEAD Domain Proteins in Some Species of Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi.

    Directory of Open Access Journals (Sweden)

    Olga K Kamneva

    Full Text Available A number of bacteria belonging to the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae super-phylum contain unusual ribosome-bearing intracellular membranes. The evolutionary origins and functions of these membranes are unknown. Some proteins putatively associated with the presence of intracellular membranes in PVC bacteria contain signal peptides. Signal peptides mark proteins for translocation across the cytoplasmic membrane in prokaryotes, and the membrane of the endoplasmic reticulum in eukaryotes, by highly conserved Sec machinery. This suggests that proteins might be targeted to intracellular membranes in PVC bacteria via the Sec pathway. Here, we show that canonical signal peptides are significantly over-represented in proteins preferentially present in PVC bacteria possessing intracellular membranes, indicating involvement of Sec translocase in their cellular targeting. We also characterized Sec proteins using comparative genomics approaches, focusing on the PVC super-phylum. While we were unable to detect unique changes in Sec proteins conserved among membrane-bearing PVC species, we identified (1 SecA ATPase domain re-arrangements in some Planctomycetes, and (2 secondary SecA_DEAD domain proteins in the genomes of some Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi. This is the first report of potentially duplicated SecA in Gram-negative bacteria. The phylogenetic distribution of secondary SecA_DEAD domain proteins suggests that the presence of these proteins is not related to the occurrence of PVC endomembranes. Further genomic analysis showed that secondary SecA_DEAD domain proteins are located within genomic neighborhoods that also encode three proteins possessing domains specific for the Type I secretion system.

  6. Species B adenovirus serotypes 3, 7, 11 and 35 share similar binding sites on the membrane cofactor protein CD46 receptor.

    Science.gov (United States)

    Fleischli, Christoph; Sirena, Dominique; Lesage, Guillaume; Havenga, Menzo J E; Cattaneo, Roberto; Greber, Urs F; Hemmi, Silvio

    2007-11-01

    We recently characterized the domains of the human cofactor protein CD46 involved in binding species B2 adenovirus (Ad) serotype 35. Here, the CD46 binding determinants are mapped for the species B1 Ad serotypes 3 and 7 and for the species B2 Ad11. Ad3, 7 and 11 bound and transduced CD46-positive rodent BHK cells at levels similar to Ad35. By using antibody-blocking experiments, hybrid CD46-CD4 receptor constructs and CD46 single point mutants, it is shown that Ad3, 7 and 11 share many of the Ad35-binding features on CD46. Both CD46 short consensus repeat domains SCR I and SCR II were necessary and sufficient for optimal binding and transgene expression, provided that they were positioned at an appropriate distance from the cell membrane. Similar to Ad35, most of the putative binding residues of Ad3, 7 and 11 were located on the same glycan-free, solvent-exposed face of the SCR I or SCR II domains, largely overlapping with the binding surface of the recently solved fiber knob Ad11-SCR I-II three-dimensional structure. Differences between species B1 and B2 Ads were documented with competition experiments based on anti-CD46 antibodies directed against epitopes flanking the putative Ad-binding sites, and with competition experiments based on soluble CD46 protein. It is concluded that the B1 and B2 species of Ad engage CD46 through similar binding surfaces.

  7. 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.

  8. Species trees from consensus single nucleotide polymorphism (SNP) data: Testing phylogenetic approaches with simulated and empirical data.

    Science.gov (United States)

    Schmidt-Lebuhn, Alexander N; Aitken, Nicola C; Chuah, Aaron

    2017-11-01

    Datasets of hundreds or thousands of SNPs (Single Nucleotide Polymorphisms) from multiple individuals per species are increasingly used to study population structure, species delimitation and shallow phylogenetics. The principal software tool to infer species or population trees from SNP data is currently the BEAST template SNAPP which uses a Bayesian coalescent analysis. However, it is computationally extremely demanding and tolerates only small amounts of missing data. We used simulated and empirical SNPs from plants (Australian Craspedia, Asteraceae, and Pelargonium, Geraniaceae) to compare species trees produced (1) by SNAPP, (2) using SVD quartets, and (3) using Bayesian and parsimony analysis with several different approaches to summarising data from multiple samples into one set of traits per species. Our aims were to explore the impact of tree topology and missing data on the results, and to test which data summarising and analyses approaches would best approximate the results obtained from SNAPP for empirical data. SVD quartets retrieved the correct topology from simulated data, as did SNAPP except in the case of a very unbalanced phylogeny. Both methods failed to retrieve the correct topology when large amounts of data were missing. Bayesian analysis of species level summary data scoring the two alleles of each SNP as independent characters and parsimony analysis of data scoring each SNP as one character produced trees with branch length distributions closest to the true trees on which SNPs were simulated. For empirical data, Bayesian inference and Dollo parsimony analysis of data scored allele-wise produced phylogenies most congruent with the results of SNAPP. In the case of study groups divergent enough for missing data to be phylogenetically informative (because of additional mutations preventing amplification of genomic fragments or bioinformatic establishment of homology), scoring of SNP data as a presence/absence matrix irrespective of allele

  9. Degenerate mixing of plasma waves on cold, magnetized single-species plasmas

    Science.gov (United States)

    Anderson, M. W.; O'Neil, T. M.; Dubin, D. H. E.; Gould, R. W.

    2011-10-01

    In the cold-fluid dispersion relation ω =ωp/[1+(k⊥/kz)2]1/2 for Trivelpiece-Gould waves on an infinitely long magnetized plasma cylinder, the transverse and axial wavenumbers appear only in the combination k⊥/kz. As a result, for any frequency ω plasma column, these degenerate waves reflect into one another at the ends; thus, each standing-wave normal mode of the bounded plasma is a mixture of many degenerate waves, not a single standing wave as is often assumed. A striking feature of the many-wave modes is that the short-wavelength waves often add constructively along resonance cones given by dz /dr=±(ωp2/ω2-1)1/2. Also, the presence of short wavelengths in the admixture for a predominantly long-wavelength mode enhances the viscous damping beyond what the single-wave approximation would predict. Here, numerical solutions are obtained for modes of a cylindrical plasma column with rounded ends. Exploiting the fact that the modes of a spheroidal plasma are known analytically (the Dubin modes), a perturbation analysis is used to investigate the mixing of low-order, nearly degenerate Dubin modes caused by small deformations of a plasma spheroid.

  10. Degenerate mixing of plasma waves on cold, magnetized single-species plasmas

    International Nuclear Information System (INIS)

    Anderson, M. W.; O'Neil, T. M.; Dubin, D. H. E.; Gould, R. W.

    2011-01-01

    In the cold-fluid dispersion relation ω=ω p /[1+(k perpendicular /k z ) 2 ] 1/2 for Trivelpiece-Gould waves on an infinitely long magnetized plasma cylinder, the transverse and axial wavenumbers appear only in the combination k perpendicular /k z . As a result, for any frequency ω p , there are infinitely many degenerate waves, all having the same value of k perpendicular /k z . On a cold finite-length plasma column, these degenerate waves reflect into one another at the ends; thus, each standing-wave normal mode of the bounded plasma is a mixture of many degenerate waves, not a single standing wave as is often assumed. A striking feature of the many-wave modes is that the short-wavelength waves often add constructively along resonance cones given by dz/dr=±(ω p 2 /ω 2 -1) 1/2 . Also, the presence of short wavelengths in the admixture for a predominantly long-wavelength mode enhances the viscous damping beyond what the single-wave approximation would predict. Here, numerical solutions are obtained for modes of a cylindrical plasma column with rounded ends. Exploiting the fact that the modes of a spheroidal plasma are known analytically (the Dubin modes), a perturbation analysis is used to investigate the mixing of low-order, nearly degenerate Dubin modes caused by small deformations of a plasma spheroid.

  11. Molecular analysis of an odorant-binding protein gene in two sympatric species of Lutzomyia longipalpis s.l.

    Directory of Open Access Journals (Sweden)

    Ana Karina Kerche Dias

    2013-01-01

    Full Text Available Lutzomyia longipalpis s.l. is the main vector of American visceral leishmaniasis (AVL and occurs as a species complex. DNA samples from two Brazilian sympatric species that differ in pheromone and courtship song production were used to analyse molecular polymorphisms in an odorant-binding protein ( obp29 gene. OBPs are proteins related to olfaction and are involved in activities fundamental to survival, such as foraging, mating and choice of oviposition site. In this study, the marker obp29 was found to be highly polymorphic in Lu. longipalpis s.l. , with no fixed differences observed between the two species. A pairwise fixation index test indicated a moderate level of genetic differentiation between the samples analysed.

  12. Functional groups in a single pteridosperm species: Variability and circumscription (Pennsylvanian, Nova Scotia, Canada)

    Science.gov (United States)

    Zodrow, E.L.; Mastalerz, Maria

    2007-01-01

    Multiple foliar specimens of the Late Pennsylvanian fossil pteridosperm [gymnosperm] Alethopteris zeilleri (Ragot) Wagner were collected from one restricted stratigraphical horizon in the Canadian Sydney Coalfield. Variability of functional-group distribution using FTIR technique was studied in compressions, adaxial versus abaxial cuticles, and in unseparated cuticles as a function of maceration time from 48 to 168??h. The results obtained document spectral variability that could be expected within specimens of one species. For example, CH2/CH3 and Al/ox ratios can differ by as much as 20% of the values. Moreover, the experiments performed confirm that by using a previously established maceration protocol, long maceration periods do not bias FTIR spectra in terms of oxygenation overprinting. The inference that this cuticle is robust, under the given diagenetic level, probably reflects a reassuring degree of chemical fidelity of the Pennsylvanian plant to support Carboniferous chemotaxonomic observations. ?? 2006 Elsevier B.V. All rights reserved.

  13. Kinetic effects in Alfven wave heating Part 2 propagation and absorption with a single minority species

    International Nuclear Information System (INIS)

    Li, Wann-Quan; Ross, D.W.; Mahajan, Swadesh M.

    1989-06-01

    Kinetic effects of Alfven wave spatial resonances near the plasma edge are investigated numerically and analytically in a cylindrical tokamak model. In Part 1, cold plasma surface Alfven eigenmodes (SAE's) in a pure plasma are examined. Numerical calculations of antenna-driven waves exhibiting absorption resonances at certain discrete frequencies are first reviewed. From a simplified kinetic equation, an analytical dispersion relation is then obtained with the antenna current set equal to zero. The real and imaginary parts of its roots, which are the complex eigenfrequencies, agree with the central frequencies and widths, respectively, of the numerical antenna-driven resonances. These results serve as an introduction to the companion paper, in which it is shown that, in the presence of a minority species, certain SAE's, instead of heating the plasma exterior, can dissipate substantial energy in the two-ion hybrid layer near the plasma center. 11 refs., 8 figs., 1 tab

  14. 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...

  15. Quantification of protein based on single-molecule counting by total internal reflection fluorescence microscopy with adsorption equilibrium

    International Nuclear Information System (INIS)

    Wang Lei; Xu Guang; Shi Zhikun; Jiang Wei; Jin Wenrui

    2007-01-01

    We developed a sensitive single-molecule imaging method for quantification of protein by total internal reflection fluorescence microscopy with adsorption equilibrium. In this method, the adsorption equilibrium of protein was achieved between solution and glass substrate. Then, fluorescence images of protein molecules in a evanescent wave field were taken by a highly sensitive electron multiplying charge coupled device. Finally, the number of fluorescent spots corresponding to the protein molecules in the images was counted. Alexa Fluor 488-labeled goat anti-rat IgG(H + L) was chosen as the model protein. The spot number showed an excellent linear relationship with protein concentration. The concentration linear range was 5.4 x 10 -11 to 8.1 x 10 -10 mol L -1

  16. 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...

  17. Discrete and structurally unique proteins (tāpirins) mediate attachment of extremely thermophilic Caldicellulosiruptor species to cellulose.

    Science.gov (United States)

    Blumer-Schuette, Sara E; Alahuhta, Markus; Conway, Jonathan M; Lee, Laura L; Zurawski, Jeffrey V; Giannone, Richard J; Hettich, Robert L; Lunin, Vladimir V; Himmel, Michael E; Kelly, Robert M

    2015-04-24

    A variety of catalytic and noncatalytic protein domains are deployed by select microorganisms to deconstruct lignocellulose. These extracellular proteins are used to attach to, modify, and hydrolyze the complex polysaccharides present in plant cell walls. Cellulolytic enzymes, often containing carbohydrate-binding modules, are key to this process; however, these enzymes are not solely responsible for attachment. Few mechanisms of attachment have been discovered among bacteria that do not form large polypeptide structures, called cellulosomes, to deconstruct biomass. In this study, bioinformatics and proteomics analyses identified unique, discrete, hypothetical proteins ("tāpirins," origin from Māori: to join), not directly associated with cellulases, that mediate attachment to cellulose by species in the noncellulosomal, extremely thermophilic bacterial genus Caldicellulosiruptor. Two tāpirin genes are located directly downstream of a type IV pilus operon in strongly cellulolytic members of the genus, whereas homologs are absent from the weakly cellulolytic Caldicellulosiruptor species. Based on their amino acid sequence, tāpirins are specific to these extreme thermophiles. Tāpirins are also unusual in that they share no detectable protein domain signatures with known polysaccharide-binding proteins. Adsorption isotherm and trans vivo analyses demonstrated the carbohydrate-binding module-like affinity of the tāpirins for cellulose. Crystallization of a cellulose-binding truncation from one tāpirin indicated that these proteins form a long β-helix core with a shielded hydrophobic face. Furthermore, they are structurally unique and define a new class of polysaccharide adhesins. Strongly cellulolytic Caldicellulosiruptor species employ tāpirins to complement substrate-binding proteins from the ATP-binding cassette transporters and multidomain extracellular and S-layer-associated glycoside hydrolases to process the carbohydrate content of lignocellulose.

  18. 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 the vegetative growth stage had little effect on the parameters investigated. For the first time, H-1 HR-MAS NMR spectra of grains taken during grain-filling were analysed by an advanced multiway model. In addition to the results from the chemical protein analysis and the H-1 HR-MAS NMR spectra of single kernels...... was to examine the implications of different drought treatments on the protein fractions in grains of winter wheat using H-1 nuclear magnetic resonance spectroscopy followed by chemometric analysis. Triticum aestivum L. cv. Vinjett was studied in a semi-field experiment and subjected to drought episodes either...... 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...

  19. The dynamics of single protein molecules is non-equilibrium and self-similar over thirteen decades in time

    Science.gov (United States)

    Hu, Xiaohu; Hong, Liang; Dean Smith, Micholas; Neusius, Thomas; Cheng, Xiaolin; Smith, Jeremy C.

    2016-02-01

    Internal motions of proteins are essential to their function. The time dependence of protein structural fluctuations is highly complex, manifesting subdiffusive, non-exponential behaviour with effective relaxation times existing over many decades in time, from ps up to ~102 s (refs ,,,). Here, using molecular dynamics simulations, we show that, on timescales from 10-12 to 10-5 s, motions in single proteins are self-similar, non-equilibrium and exhibit ageing. The characteristic relaxation time for a distance fluctuation, such as inter-domain motion, is observation-time-dependent, increasing in a simple, power-law fashion, arising from the fractal nature of the topology and geometry of the energy landscape explored. Diffusion over the energy landscape follows a non-ergodic continuous time random walk. Comparison with single-molecule experiments suggests that the non-equilibrium self-similar dynamical behaviour persists up to timescales approaching the in vivo lifespan of individual protein molecules.

  20. 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.

  1. A Single Sphingomyelin Species Promotes Exosomal Release of Endoglin into the Maternal Circulation in Preeclampsia.

    Science.gov (United States)

    Ermini, Leonardo; Ausman, Jonathan; Melland-Smith, Megan; Yeganeh, Behzad; Rolfo, Alessandro; Litvack, Michael L; Todros, Tullia; Letarte, Michelle; Post, Martin; Caniggia, Isabella

    2017-09-22

    Preeclampsia (PE), an hypertensive disorder of pregnancy, exhibits increased circulating levels of a short form of the auxillary TGF-beta (TGFB) receptor endoglin (sENG). Until now, its release and functionality in PE remains poorly understood. Here we show that ENG selectively interacts with sphingomyelin(SM)-18:0 which promotes its clustering with metalloproteinase 14 (MMP14) in SM-18:0 enriched lipid rafts of the apical syncytial membranes from PE placenta where ENG is cleaved by MMP14 into sENG. The SM-18:0 enriched lipid rafts also contain type 1 and 2 TGFB receptors (TGFBR1 and TGFBR2), but not soluble fms-like tyrosine kinase 1 (sFLT1), another protein secreted in excess in the circulation of women with PE. The truncated ENG is then released into the maternal circulation via SM-18:0 enriched exosomes together with TGFBR1 and 2. Such an exosomal TGFB receptor complex could be functionally active and block the vascular effects of TGFB in the circulation of PE women.

  2. 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

  3. 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.

  4. Assigning Significance in Label-Free Quantitative Proteomics to Include Single-Peptide-Hit Proteins with Low Replicates

    OpenAIRE

    Li, Qingbo

    2010-01-01

    When sample replicates are limited in a label-free proteomics experiment, selecting differentially regulated proteins with an assignment of statistical significance remains difficult for proteins with a single-peptide hit or a small fold-change. This paper aims to address this issue. An important component of the approach employed here is to utilize the rule of Minimum number of Permuted Significant Pairings (MPSP) to reduce false positives. The MPSP rule generates permuted sample pairings fr...

  5. 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

    -6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine...... the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism....

  6. Lipopeptide biosurfactants from Paenibacillus polymyxa inhibit single and mixed species biofilms.

    Science.gov (United States)

    Quinn, Gerry A; Maloy, Aaron P; McClean, Stephen; Carney, Brian; Slater, John W

    2012-01-01

    Although biofilms are recognised as important in microbial colonisation, solutions to their inhibition are predominantly based on planktonic assays. These solutions have limited efficacy against biofilms. Here, a series of biofilm-orientated tests were used to identify anti-biofilm compounds from marine micro-flora. This led to the isolation of a complex of anti-biofilm compounds from an extract of Paenibacillus polymyxa (PPE). A combination of rpHPLC and mass spectrometry identified the principle components of PPE as fusaricidin B (LI-FO4b) and polymyxin D1, with minor contributions from surfactins. This complex (PPE) reduced the biofilm biomass of Bacillus subtilis, Micrococcus luteus, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus bovis. In contrast, ampicillin was only effective against S. aureus. PPE also inhibited a self-assembling marine biofilm (SAMB) in co-incubation assays by 99.3% ± 1.9 and disrupted established SAMB by 72.4% ± 4.4, while ampicillin showed no significant reduction. The effectiveness of this complex of lipopeptides against single and multispecies biofilms suggests a future role in biofilm prevention strategies.

  7. The different types of sperm morphology and behavior within a single species

    Science.gov (United States)

    Hirohashi, Noritaka; Iwata, Yoko

    2013-01-01

    Some coastal squids exhibit male dimorphism (large and small body size) that is linked to mating behaviors. Large “consort” males compete with other, rival males to copulate with a female, and thereby transfer their spermatophores to her internal site around the oviduct. Small “sneaker” males rush to a single female or copulating pair and transfer spermatophores to her external body surface around the seminal receptacle near the mouth. We previously found that in Loligo bleekeri, sneaker sperm are ~50% longer than consort sperm, and only the sneaker sperm, once ejaculated from the spermatophore (sperm mass), form a cluster because of chemoattraction toward their own respiratory CO2. Here, we report that sperm clusters are able to move en masse. Because a fraction of ejaculated sperm from a sneaker’s spermatophore are eventually located in the female’s seminal receptacle, we hypothesize that sperm clustering facilitates collective migration to the seminal receptacle or an egg micropyle. Sperm clustering is regarded as a cooperative behavior that may have evolved by sperm competition and/or physical and physiological constraints imposed by male mating tactics. PMID:24567779

  8. Assessing nitrogen fixation in mixed- and single-species plantations of Eucalyptus globulus and Acacia mearnsii.

    Science.gov (United States)

    Forrester, David I; Schortemeyer, Marcus; Stock, William D; Bauhus, Jürgen; Khanna, Partap K; Cowie, Annette L

    2007-09-01

    Mixtures of Eucalyptus globulus Labill. and Acacia mearnsii de Wildeman are twice as productive as E. globulus monocultures growing on the same site in East Gippsland, Victoria, Australia, possibly because of increased nitrogen (N) availability owing to N(2) fixation by A. mearnsii. To investigate whether N(2) fixation by A. mearnsii could account for the mixed-species growth responses, we assessed N(2) fixation by the accretion method and the (15)N natural abundance method. Nitrogen gained by E. globulus and A. mearnsii mixtures and monocultures was calculated by the accretion method with plant and soil samples collected 10 years after plantation establishment. Nitrogen in biomass and soil confirmed that A. mearnsii influenced N dynamics. Assuming that the differences in soil, forest floor litter and biomass N of plots containing A. mearnsii compared with E. globulus monocultures were due to N(2) fixation, the 10-year annual mean rates of N(2) fixation were 38 and 86 kg ha(-1) year(-1) in 1:1 mixtures and A. mearnsii monocultures, respectively. Nitrogen fixation by A. mearnsii could not be quantified on the basis of the natural abundance of (15)N because such factors as mycorrhization type and fractionation of N isotopes during N cycling within the plant confounded the effect of the N source on the N isotopic signature of plants. This study shows that A. mearnsii fixed significant quantities of N(2) when mixed with E. globulus. A decline in delta(15)N values of E. globulus and A. mearnsii with time, from 2 to 10 years, is further evidence that N(2) was fixed and cycled through the stands. The increased aboveground biomass production of E. globulus trees in mixtures when compared with monocultures can be attributed to increases in N availability.

  9. Deproteinization assessment using isotopically enriched compounds to trace the coprecipitation of low-molecular-weight selenium species with proteins.

    Science.gov (United States)

    Godin, Simon; Bouzas-Ramos, Diego; Fontagné-Dicharry, Stéphanie; Bouyssière, Brice; Bueno, Maïté

    2017-08-01

    Studies have shown that information related to the presence of low-molecular-weight metabolites is frequently lost after deproteinization of complex matrices, such as blood and plasma, during sample preparation. Therefore, the effect of several deproteinization reagents on low-molecular-weight selenium species has been compared by species-specific isotope labeling. Two isotopically enriched selenium tracers were used to mimic models of small inorganic anionic ( 77 Se-selenite) and organic zwitterionic ( 76 Se-selenomethionine) species. The results presented here show that the use of a methanol-acetonitrile-acetone (1:1:1 v/v/v) mixture provided approximately two times less tracer loss from plasma samples in comparison with the classic procedure using acetonitrile, which may not be optimal as it leads to important losses of low-molecular-weight selenium species. In addition, the possible interactions between selenium tracers and proteins were investigated, revealing that both coprecipitation phenomena and association with proteins were potentially responsible for selenite tracer losses during protein precipitation in blood samples. However, coprecipitation phenomena were found to be fully responsible for losses of both tracers observed in plasma samples and of the selenomethionine tracer in blood samples. This successfully applied strategy is anticipated to be useful for more extensive future studies in selenometabolomics. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Similar rates of protein adaptation in Drosophila miranda and D. melanogaster, two species with different current effective population sizes

    Directory of Open Access Journals (Sweden)

    Bachtrog Doris

    2008-12-01

    Full Text Available Abstract Background Adaptive protein evolution is common in several Drosophila species investigated. Some studies point to very weak selection operating on amino-acid mutations, with average selection intensities on the order of Nes ~ 5 in D. melanogaster and D. simulans. Species with lower effective population sizes should undergo less adaptation since they generate fewer mutations and selection is ineffective on a greater proportion of beneficial mutations. Results Here I study patterns of polymorphism and divergence at 91 X-linked loci in D. miranda, a species with a roughly 5-fold smaller effective population size than D. melanogaster. Surprisingly, I find a similar fraction of amino-acid mutations being driven to fixation by positive selection in D. miranda and D. melanogaster. Genes with higher rates of amino-acid evolution show lower levels of neutral diversity, a pattern predicted by recurrent adaptive protein evolution. I fit a hitchhiking model to patterns of polymorphism in D. miranda and D. melanogaster and estimate an order of magnitude higher selection coefficients for beneficial mutations in D. miranda. Conclusion This analysis suggests that effective population size may not be a major determinant in rates of protein adaptation. Instead, adaptation may not be mutation-limited, or the distribution of fitness effects for beneficial mutations might differ vastly between different species or populations. Alternative explanation such as biases in estimating the fraction of beneficial mutations or slightly deleterious mutation models are also discussed.

  11. 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.

  12. A Single Amino Acid Substitution in an ORANGE Protein Promotes Carotenoid Overaccumulation in Arabidopsis1[OPEN

    Science.gov (United States)

    Yuan, Hui; Owsiany, Katherine; Sheeja, T.E.; Zhou, Xiangjun; Rodriguez, Caroline; Li, Yongxi; Welsch, Ralf; Chayut, Noam; Yang, Yong; Thannhauser, Theodore W.; Parthasarathy, Mandayam V.; Xu, Qiang; Deng, Xiuxin; Fei, Zhangjun; Schaffer, Ari; Katzir, Nurit; Burger, Joseph; Tadmor, Yaakov; Li, Li

    2015-01-01

    Carotenoids are crucial for plant growth and human health. The finding of ORANGE (OR) protein as a pivotal regulator of carotenogenesis offers a unique opportunity to comprehensively understand the regulatory mechanisms of carotenoid accumulation and develop crops with enhanced nutritional quality. Here, we demonstrated that alteration of a single amino acid in a wild-type OR greatly enhanced its ability to promote carotenoid accumulation. Whereas overexpression of OR from Arabidopsis (Arabidopsis thaliana; AtOR) or from the agronomically important crop sorghum (Sorghum bicolor; SbOR) increased carotenoid levels up to 2-fold, expression of AtORHis (R90H) or SbORHis (R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found that AtORAla (R90A) functioned similarly to AtORHis to promote carotenoid overproduction. Neither AtOR nor AtORHis greatly affected carotenogenic gene expression. AtORHis exhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtORHis triggered biogenesis of membranous chromoplasts in the Arabidopsis calli, which shared structures similar to chromoplasts found in the curd of the orange cauliflower (Brassica oleracea) mutant. By contrast, AtOR did not cause plastid-type changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability of AtORHis in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstrates ORHis/Ala as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlying ORHis-regulated carotenoid accumulation. PMID:26224804

  13. 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.

  14. Developmental changes in translatable RNA species and protein synthesis during sporulation in the aquatic fungus Blastocladiella emersonii

    International Nuclear Information System (INIS)

    Silva, A.M. da; Costa Maia, J.C. da; Juliani, M.H.

    1986-01-01

    Protein synthesis during sporulation in Blastocladiella emersonii is developmentally regulated as revealed using ( 35 S)methionine pulse labeling and two-dimensional gel electrophoresis. A large increase in the synthesis of several proteins is associated with particular stages. A large number of basic proteins are synthesized exclusively during late sporulation. Changes in translatable mRNA species were also detected by two-dimensional gel electrophoresis of the polypeptides produced in a cell-free rabbit reticulocyte lysate primed with RNA prepared at different stages of sporulation. The synthesis of several proteins during sporulation seems to be transcriptionally controlled. Most of the sporulation-specific messages are not present in the mature zoospores. (Author)

  15. Transient intermediates are populated in the folding pathways of single-domain two-state folding protein L

    Science.gov (United States)

    Maity, Hiranmay; Reddy, Govardhan

    2018-04-01

    Small single-domain globular proteins, which are believed to be dominantly two-state folders, played an important role in elucidating various aspects of the protein folding mechanism. However, recent single molecule fluorescence resonance energy transfer experiments [H. Y. Aviram et al. J. Chem. Phys. 148, 123303 (2018)] on a single-domain two-state folding protein L showed evidence for the population of an intermediate state and it was suggested that in this state, a β-hairpin present near the C-terminal of the native protein state is unfolded. We performed molecular dynamics simulations using a coarse-grained self-organized-polymer model with side chains to study the folding pathways of protein L. In agreement with the experiments, an intermediate is populated in the simulation folding pathways where the C-terminal β-hairpin detaches from the rest of the protein structure. The lifetime of this intermediate structure increased with the decrease in temperature. In low temperature conditions, we also observed a second intermediate state, which is globular with a significant fraction of the native-like tertiary contacts satisfying the features of a dry molten globule.

  16. 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.

  17. In vitro evaluation of single- and multi-strain probiotics: Inter-species inhibition between probiotic strains, and inhibition of pathogens.

    Science.gov (United States)

    Chapman, C M C; Gibson, G R; Rowland, I

    2012-08-01

    Many studies comparing the effects of single- and multi-strain probiotics on pathogen inhibition compare treatments with different concentrations. They also do not examine the possibility of inhibition between probiotic strains with a mixture. We tested the ability of 14 single-species probiotics to inhibit each other using a cross-streak assay, and agar spot test. We then tested the ability of 15 single-species probiotics and 5 probiotic mixtures to inhibit Clostridium difficile, Escherichia coli and S. typhimurium, using the agar spot test. Testing was done with mixtures created in two ways: one group contained component species incubated together, the other group of mixtures was made using component species which had been incubated separately, equalised to equal optical density, and then mixed in equal volumes. Inhibition was observed for all combinations of probiotics, suggesting that when used as such there may be inhibition between probiotics, potentially reducing efficacy of the mixture. Significant inter-species variation was seen against each pathogen. When single species were tested against mixtures, the multi-species preparations displayed significantly (p probiotic species will inhibit each other when incubated together in vitro, in many cases a probiotic mixture was more effective at inhibiting pathogens than its component species when tested at approximately equal concentrations of biomass. This suggests that using a probiotic mixture might be more effective at reducing gastrointestinal infections, and that creating a mixture using species with different effects against different pathogens may have a broader spectrum of action that a single provided by a single strain. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species.

    Science.gov (United States)

    Yu, Meng; Stevens, Vicky; Berry, Jody D; Crameri, Gary; McEachern, Jennifer; Tu, Changchun; Shi, Zhengli; Liang, Guodong; Weingartl, Hana; Cardosa, Jane; Eaton, Bryan T; Wang, Lin-Fa

    2008-02-29

    Knowledge of immunodominant regions in major viral antigens is important for rational design of effective vaccines and diagnostic tests. Although there have been many reports of such work done for SARS-CoV, these were mainly focused on the immune responses of humans and mice. In this study, we aim to search for and compare immunodominant regions of the spike (S) and nucleocapsid (N) proteins which are recognized by sera from different animal species, including mouse, rat, rabbit, civet, pig and horse. Twelve overlapping recombinant protein fragments were produced in Escherichia coli, six each for the S and N proteins, which covered the entire coding region of the two proteins. Using a membrane-strip based Western blot approach, the reactivity of each antigen fragment against a panel of animal sera was determined. Immunodominant regions containing linear epitopes, which reacted with sera from all the species tested, were identified for both proteins. The S3 fragment (aa 402-622) and the N4 fragment (aa 220-336) were the most immunodominant among the six S and N fragments, respectively. Antibodies raised against the S3 fragment were able to block the binding of a panel of S-specific monoclonal antibodies (mAb) to SARS-CoV in ELISA, further demonstrating the immunodominance of this region. Based on these findings, one-step competition ELISAs were established which were able to detect SARS-CoV antibodies from human and at least seven different animal species. Considering that a large number of animal species are known to be susceptible to SARS-CoV, these assays will be a useful tool to trace the origin and transmission of SARS-CoV and to minimise the risk of animal-to-human transmission.

  19. Protein expression parallels thermal tolerance and ecologic changes in the diversification of a diving beetle species complex.

    Science.gov (United States)

    Hidalgo-Galiana, A; Monge, M; Biron, D G; Canals, F; Ribera, I; Cieslak, A

    2016-01-01

    Physiological changes associated with evolutionary and ecological processes such as diversification, range expansion or speciation are still incompletely understood, especially for non-model species. Here we study differences in protein expression in response to temperature in a western Mediterranean diving beetle species complex, using two-dimensional differential gel electrophoresis with one Moroccan and one Iberian population each of Agabus ramblae and Agabus brunneus. We identified proteins with significant expression differences after thermal treatments comparing them with a reference EST library generated from one of the species of the complex (A. ramblae). The colonisation during the Middle Pleistocene of the Iberian peninsula by A. ramblae, where maximum temperatures and seasonality are lower than in the ancestral north African range, was associated with changes in the response to 27 °C in proteins related to energy metabolism. The subsequent speciation of A. brunneus from within populations of Iberian A. ramblae was associated with changes in the expression of several stress-related proteins (mostly chaperons) when exposed to 4 °C. These changes are in agreement with the known tolerance to lower temperatures of A. brunneus, which occupies a larger geographical area with a wider range of climatic conditions. In both cases, protein expression changes paralleled the evolution of thermal tolerance and the climatic conditions experienced by the species. However, although the colonisation of the Iberian peninsula did not result in morphological change, the speciation process of A. brunneus within Iberia involved genetic isolation and substantial differences in male genitalia and body size and shape.

  20. Ralstonia syzygii, the Blood Disease Bacterium and Some Asian R. solanacearum Strains Form a Single Genomic Species Despite Divergent Lifestyles

    Science.gov (United States)

    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

  1. The Synergistic Effect of Proteins and Reactive Oxygen Species on Electrochemical Behaviour of 316L Stainless Steel for Biomedical Applications

    Science.gov (United States)

    Simionescu, N.; Benea, L.; Dumitrascu, V. M.

    2018-06-01

    The stainless steels, especially 316L type is the most used metallic biomaterials for biomedical applications due to their good biocompatibility, low price, excellent corrosion resistance, availability, easy processing and high strength. Due to these favorable properties 316L stainless steel has become the most attractive biomaterial for dental implants, stents and orthopedic implants. However an implant material in the human body is exposed to an action effect of other molecules, including proteins (such as albumin) and reactive oxygen species (such as hydrogen peroxide - H2O2 ) produced by bacteria and immune cells. In the literature there are few studies to follow the effect of proteins and reactive oxygen species on 316L stainless steel used as implant material and are still unclear. The degree of corrosion resistance is the first criterion in the use of a metallic biomaterial in the oral or body environment. The aim of this research work is to investigate the influence of proteins (albumin) and reactive oxygen species (H2O2 ) in combination, taking into account the synergistic effect of these two factors on 316L stainless steel. Albumin is present in the body near implants and reactive oxygen species could appear in inflammatory processes as well. The study shows that the presence of albumin and reactive species influences the corrosion resistance of 316L stainless steel in biological solutions. In this research work the corrosion behavior of 316L stainless steel is analyzed by electrochemical methods such as: open circuit potential (OCP), Electrochemical Impedance Spectroscopy (EIS). It was found that, the electrochemical results are in a good agreement with micro photographs taken before and after corrosion assays. The albumin and reactive oxygen species have influence on 316L stainless steel behavior.

  2. Nutrient recovery from industrial wastewater as single cell protein by a co-culture of green microalgae and methanotrophs

    DEFF Research Database (Denmark)

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

    2018-01-01

    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 (~95% removal for monocultures and 91% for the algal-bacterial consortium). However, their growth stopped before......, for all cultures the protein content (45% of dry weight, DW, for methanotrophs; 52.5% of DW for algae; and 27.6% of DW for consortium) and amino acid profile was suitable for substitution of conventional protein sources. Further research should focus on increasing productivity of biomass grown...

  3. 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...istory of This Database Site Policy | Contact Us Cluster based on sequence compariso

  4. 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.

  5. 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.

  6. GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM.

    Science.gov (United States)

    Hauer, Florian; Gerle, Christoph; Fischer, Niels; Oshima, Atsunori; Shinzawa-Itoh, Kyoko; Shimada, Satoru; Yokoyama, Ken; Fujiyoshi, Yoshinori; Stark, Holger

    2015-09-01

    We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. 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.

  8. Production of single-cell protein from enzymatic hydrolyzate of rice straw

    Energy Technology Data Exchange (ETDEWEB)

    Taniguchi, M.; Kometani, Y.; Tanaka, M.; Matsuno, R.; Kamikubo, T.

    1982-01-01

    The components of rice straw, pretreated with sodium chlorite, cellulose and hemicellulose were solubilized with culture filtrate of Pellicularia filamentosa or Trichoderma reesei. The ratio of glucose to total sugar in the solution obtained from the cellulose component with the culture filtrate of Pellicularia filamentosa was approximately twice that of Trichoderma reesei. Ten yeast strains (Candida utilis, C. tropicalis, C. guilliermondii, C. parapsilosis, Torulopsis xylinus, Trichosporon cutaneum, Debaryomyces hansenii, Rhodotorula glutinis, Saccharomyces fragilis and Saccharomyces cerevisiae) were cultivated as test organisms for single-cell protein (SCP) production on sugar solutions obtained from the straw, cellulose and hemicellulose components, pretreated with the culture filtrate of Pellicularia filamentosa. Sugar consumption, in terms of total sugar and cell yield, of the culture with the sugar solution obtained from pretreated straw were; 70% and 6.8 g/l for Candida tropicalis, 56% and 6.4 g/l for Torulopsis xylinus, 76% and 10.1 g/l for Trichosporon cutaneum, and 74% and 7.6 g/l for Candida guilliermondii. In addition, the highest consumption with respect to total sugar (87%) and the best dry cell yield (15.6 g/l) were observed with the culture of Trichosporon cutaneum using the sugar solution obtained from the hemicellulose component. (Refs. 17).

  9. 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

  10. 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

  11. Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

    Science.gov (United States)

    Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

    2015-08-20

    For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. 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

  13. 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 orga...nism species. DOI 10.18908/lsdba.nbdc00464-001 Description of data contents Amino acid sequences of predicted proteins...Database Description Download License Update History of This Database Site Policy | Contact Us Amino acid sequences of predicted prot...eins and their annotation for 95 organism species. - Gclust Server | LSDB Archive ...

  14. Cultivation and utilization of Jerusalem artichoke for ethanol, single cell protein, and high-fructose syrup production

    Energy Technology Data Exchange (ETDEWEB)

    Bajpai, P.K.; Bajpai, Pratima (Thapar Corporate Research and Development Center, Patiala (IN). Div. of Chemical and Biochemical Engineering)

    1991-04-01

    Jerusalem artichoke has one of the highest carbohydrate yields of the known agricultural crops and has many distinct advantages over traditional crops. This brief review presents data on the yield and composition of Jerusalem artichoke, techniques of carbohydrate extraction and its utilization for the production of ethanol, single cell protein (SCP), and high-fructose syrup, along with economic considerations. (author).

  15. Selection of single grain seeds by 14N(n,γ)15N nuclear reaction for protein improvement

    International Nuclear Information System (INIS)

    Andras, L.; Csoke, A.; Nagy, A.Z.; Balint, A.

    1979-01-01

    A new non-destructive screening technique was developed for determining the protein (total nitrogen) content of single grain seeds. Here, our first experiment is described where in the case of maize samples, 300 s was used to perform one measurement on a seed with a semiautomatic device. (author)

  16. Studying repair of a single protein-bound nick in vivo using the Flp-nick system

    DEFF Research Database (Denmark)

    Nielsen, Ida; Andersen, Anni Hangaard; Bjergbæk, Lotte

    2012-01-01

    The Flp-nick system is a simple in vivo system developed for studying the cellular responses to a protein-bound nick at a single genomic site in the budding yeast Saccharomyces cerevisiae. The Flp-nick system takes advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp ...

  17. Note: Pulsed single longitudinal mode optical parametric oscillator for sub-Doppler spectroscopy of jet cooled transient species

    Science.gov (United States)

    Zhang, Qiang; Zhu, Boxing; Zhang, Deping; Gu, Jingwang; Zhao, Dongfeng; Chen, Yang

    2017-12-01

    We present a pulsed single longitudinal mode optical parametric oscillator that was recently constructed for sub-Doppler spectroscopic studies of transient species in a supersonic slit jet expansion environment. The system consists of a Littman-type grazing-incidence-grating resonator and a KTP crystal and is pumped at 532 nm. By spatially filtering the pump laser beam and employing an active cavity-length-stabilization scheme, a frequency down-conversion efficiency up to 18% and generation of Fourier-transform limited pulses with a typical pulse duration of ˜5.5 ns and a bandwidth less than 120 MHz have been achieved. In combination with a slit jet expansion, a sub-Doppler spectrum of SiC2 has been recorded at ˜498 nm, showing a spectral resolution of Δν/ν ≈ 6.2 × 10-7.

  18. Alterations in the nuclear matrix protein mass correlate with heat-induced inhibition of DNA single-strand-break repair

    International Nuclear Information System (INIS)

    Warters, R.L.; Brizgys, L.M.; Lyons, B.W.

    1987-01-01

    The total protein mass co-isolating with the nuclear matrix or nucleoid from Chinese hamster ovary (CHO) cells was observed to increase in heated cells as a function of increasing exposure temperature between 43 0 C and 45 0 C or of exposure time at any temperature. The sedimentation distance of the CHO cell nucleoid in sucrose gradients increased with increasing exposure time at 45 0 C. Both these nuclear alterations correlated in a log-linear manner with heat-induced inhibition of DNA strand break repair. A two-fold threshold increase in nuclear matrix protein mass preceded any substantial inhibition of repair of DNA single-strand breaks. When preheated cells were incubated at 37 0 C the nuclear matrix protein mass and nucleoid sedimentation recovered with a half-time of about 5 h, while DNA single-strand-break repair recovered with a half-time of about 2 h. When preheated cells were placed at 41 0 C a further increase was observed in the nuclear matrix protein mass and the half-time of DNA strand break repair, while nucleoid sedimentation recovered toward control values. These results implicate alterations in the protein mass of the nuclear matrix in heat-induced inhibition of repair of DNA single-strand breaks. (author)

  19. 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.

  20. Comparative analysis of the repertoire of G protein-coupled receptors of three species of the fungal genus Trichoderma

    Science.gov (United States)

    2013-01-01

    Background Eukaryotic organisms employ cell surface receptors such as the seven-transmembrane G protein-coupled receptors (GPCRs) as sensors to connect to the environment. GPCRs react to a variety of extracellular cues and are considered to play central roles in the signal transduction in fungi. Several species of the filamentous ascomycete Trichoderma are potent mycoparasites, i.e. can attack and parasitize other fungi, which turns them into successful bio-fungicides for the protection of plants against fungal phytopathogens. The identification and characterization of GPCRs will provide insights into how Trichoderma communicates with its environment and senses the presence of host fungi. Results We mined the recently published genomes of the two mycoparasitic biocontrol agents Trichoderma atroviride and Trichoderma virens and compared the identified GPCR-like proteins to those of the saprophyte Trichoderma reesei. Phylogenetic analyses resulted in 14 classes and revealed differences not only among the three Trichoderma species but also between Trichoderma and other fungi. The class comprising proteins of the PAQR family was significantly expanded both in Trichoderma compared to other fungi as well as in the two mycoparasites compared to T. reesei. Expression analysis of the PAQR-encoding genes of the three Trichoderma species revealed that all except one were actually transcribed. Furthermore, the class of receptors with a DUF300 domain was expanded in T. atroviride, and T. virens showed an expansion of PTH11-like receptors compared to T. atroviride and T. reesei. Conclusions Comparative genome analyses of three Trichoderma species revealed a great diversity of putative GPCRs with genus- and species- specific differences. The expansion of certain classes in the mycoparasites T. atroviride and T. virens is likely to reflect the capability of these fungi to establish various ecological niches and interactions with other organisms such as fungi and plants. These

  1. Comparative analysis of the repertoire of G protein-coupled receptors of three species of the fungal genus Trichoderma.

    Science.gov (United States)

    Gruber, Sabine; Omann, Markus; Zeilinger, Susanne

    2013-05-16

    Eukaryotic organisms employ cell surface receptors such as the seven-transmembrane G protein-coupled receptors (GPCRs) as sensors to connect to the environment. GPCRs react to a variety of extracellular cues and are considered to play central roles in the signal transduction in fungi. Several species of the filamentous ascomycete Trichoderma are potent mycoparasites, i.e. can attack and parasitize other fungi, which turns them into successful bio-fungicides for the protection of plants against fungal phytopathogens. The identification and characterization of GPCRs will provide insights into how Trichoderma communicates with its environment and senses the presence of host fungi. We mined the recently published genomes of the two mycoparasitic biocontrol agents Trichoderma atroviride and Trichoderma virens and compared the identified GPCR-like proteins to those of the saprophyte Trichoderma reesei. Phylogenetic analyses resulted in 14 classes and revealed differences not only among the three Trichoderma species but also between Trichoderma and other fungi. The class comprising proteins of the PAQR family was significantly expanded both in Trichoderma compared to other fungi as well as in the two mycoparasites compared to T. reesei. Expression analysis of the PAQR-encoding genes of the three Trichoderma species revealed that all except one were actually transcribed. Furthermore, the class of receptors with a DUF300 domain was expanded in T. atroviride, and T. virens showed an expansion of PTH11-like receptors compared to T. atroviride and T. reesei. Comparative genome analyses of three Trichoderma species revealed a great diversity of putative GPCRs with genus- and species- specific differences. The expansion of certain classes in the mycoparasites T. atroviride and T. virens is likely to reflect the capability of these fungi to establish various ecological niches and interactions with other organisms such as fungi and plants. These GPCRs consequently represent

  2. Adsorption behaviour of SF6 decomposed species onto Pd4-decorated single-walled CNT: a DFT study

    Science.gov (United States)

    Cui, Hao; Zhang, Xiaoxing; Zhang, Jun; Tang, Ju

    2018-07-01

    Metal nanocluster decorated single-walled carbon nanotubes (SWCNT) with improved adsorption behaviour towards gaseous molecules compared with intrinsic ones, have been widely accepted as a workable media for gas interaction due to their strong catalysis. In this work, Pd4 cluster is determined as a catalytic centre to theoretically study the adsorption property of Pd4-decorated SWCNT upon SF6 decomposed species. Results indicate that Pd4-SWCNT possessing good responses and sensitivities towards three composed species of SF6 could realise selective detection for them according to the different conductivity changes resulting from the varying adsorption ability. The response of Pd4-SWCNT upon three molecules in order is SOF2 > H2S > SO2, and the conductivity of the proposed material is about to increase in SOF2 and H2S systems, while declining in SO2 system. Such conclusions would be helpful for experimentalists to explore novel SWCNT-based sensors in evaluating the operating state of SF6 insulation devices.

  3. 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.

  4. 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.

  5. Fast electron transfer through a single molecule natively structured redox protein

    DEFF Research Database (Denmark)

    Della Pia, Eduardo Antonio; Chi, Qijin; Macdonald, J. Emyr

    2012-01-01

    The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conduc...

  6. 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...

  7. Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

    Science.gov (United States)

    Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

    2008-08-25

    Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

  8. 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

  9. 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.

  10. 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.

  11. 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 ...

  12. 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 ...

  13. Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

    Directory of Open Access Journals (Sweden)

    Berkhout Ben

    2006-12-01

    Full Text Available Abstract 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 split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU in Salisbury, UK. Results All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%. Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates carry a single ORF, 660 nt in size, encoding a single protein of 219 amino acids, which is a homologue of the ORF3 proteins encoded by HCoV-NL63 and PEDV. Conclusion Thus, the genome organization of the group 1b coronaviruses HCoV-NL63, PEDV and HCoV-229E is identical. It is possible that extensive culturing of the HCoV-229E laboratory strain resulted in truncation of ORF4. This may indicate that the protein is not essential in cell culture, but the highly conserved amino acid sequence of the ORF4 protein among clinical isolates suggests that the protein plays an important role in vivo.

  14. 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.

  15. 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

  16. 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 pleural fluid reactive oxygen species modulator 1 were 61.54% and 82.00%, respectively, with the optimized cutoff value of 589.70 pg/mL. However, the diagnostic sensitivity and specificity of serum reactive oxygen species modulator 1 were only 41.38% and 86.21%, respectively, with the cutoff value of 27.22 ng/mL, indicating that serum reactive oxygen species modulator 1 may not be a good option in the differential diagnosis of malignant pleural effusion and benign pleural effusion. The sensitivity and specificity of pleural fluid 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.

  17. Single-molecule resolution of protein dynamics on polymeric membrane surfaces: the roles of spatial and population heterogeneity.

    Science.gov (United States)

    Langdon, Blake B; Mirhossaini, Roya B; Mabry, Joshua N; Sriram, Indira; Lajmi, Ajay; Zhang, Yanxia; Rojas, Orlando J; Schwartz, Daniel K

    2015-02-18

    Although polymeric membranes are widely used in the purification of protein pharmaceuticals, interactions between biomolecules and membrane surfaces can lead to reduced membrane performance and damage to the product. In this study, single-molecule fluorescence microscopy provided direct observation of bovine serum albumin (BSA) and human monoclonal antibody (IgG) dynamics at the interface between aqueous buffer and polymeric membrane materials including regenerated cellulose and unmodified poly(ether sulfone) (PES) blended with either polyvinylpyrrolidone (PVP), polyvinyl acetate-co-polyvinylpyrrolidone (PVAc-PVP), or polyethylene glycol methacrylate (PEGM) before casting. These polymer surfaces were compared with model surfaces composed of hydrophilic bare fused silica and hydrophobic trimethylsilane-coated fused silica. At extremely dilute protein concentrations (10(-3)-10(-7) mg/mL), protein surface exchange was highly dynamic with protein monomers desorbing from the surface within ∼1 s after adsorption. Protein oligomers (e.g., nonspecific dimers, trimers, or larger aggregates), although less common, remained on the surface for 5 times longer than monomers. Using newly developed super-resolution methods, we could localize adsorption sites with ∼50 nm resolution and quantify the spatial heterogeneity of the various surfaces. On a small anomalous subset of the adsorption sites, proteins adsorbed preferentially and tended to reside for significantly longer times (i.e., on "strong" sites). Proteins resided for shorter times overall on surfaces that were more homogeneous and exhibited fewer strong sites (e.g., PVAc-PVP/PES). We propose that strong surface sites may nucleate protein aggregation, initiated preferentially by protein oligomers, and accelerate ultrafiltration membrane fouling. At high protein concentrations (0.3-1.0 mg/mL), fewer strong adsorption sites were observed, and surface residence times were reduced. This suggests that at high concentrations

  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. 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.

  20. Novel Phosphorylation and Ubiquitination Sites Regulate Reactive Oxygen Species-dependent Degradation of Anti-apoptotic c-FLIP Protein*

    Science.gov (United States)

    Wilkie-Grantham, Rachel P.; Matsuzawa, Shu-Ichi; Reed, John C.

    2013-01-01

    The cytosolic protein c-FLIP (cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein) is an inhibitor of death receptor-mediated apoptosis that is up-regulated in a variety of cancers, contributing to apoptosis resistance. Several compounds found to restore sensitivity of cancer cells to TRAIL, a TNF family death ligand with promising therapeutic potential, act by targeting c-FLIP ubiquitination and degradation by the proteasome. The generation of reactive oxygen species (ROS) has been implicated in c-FLIP protein degradation. However, the mechanism by which ROS post-transcriptionally regulate c-FLIP protein levels is not well understood. We show here that treatment of prostate cancer PPC-1 cells with the superoxide generators menadione, paraquat, or buthionine sulfoximine down-regulates c-FLIP long (c-FLIPL) protein levels, which is prevented by the proteasome inhibitor MG132. Furthermore, pretreatment of PPC-1 cells with a ROS scavenger prevented ubiquitination and loss of c-FLIPL protein induced by menadione or paraquat. We identified lysine 167 as a novel ubiquitination site of c-FLIPL important for ROS-dependent degradation. We also identified threonine 166 as a novel phosphorylation site and demonstrate that Thr-166 phosphorylation is required for ROS-induced Lys-167 ubiquitination. The mutation of either Thr-166 or Lys-167 was sufficient to stabilize c-FLIP protein levels in PPC-1, HEK293T, and HeLa cancer cells treated with menadione or paraquat. Accordingly, expression of c-FLIP T166A or K167R mutants protected cells from ROS-mediated sensitization to TRAIL-induced cell death. Our findings reveal novel ROS-dependent post-translational modifications of the c-FLIP protein that regulate its stability, thus impacting sensitivity of cancer cells to TRAIL. PMID:23519470

  1. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.

    Science.gov (United States)

    Justiz-Vaillant, A A; Akpaka, P E; McFarlane-Anderson, N; Smikle, M F

    2013-01-01

    The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera). These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.

  2. 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)

  3. 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

  4. 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 .

  5. 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.

  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. 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.

  8. Interaction of single and multi-layer graphene oxide with fetal bovine serum: assessing the protein corona formation

    Energy Technology Data Exchange (ETDEWEB)

    Franqui, Lidiane Silva; Farias, Marcelo Alexandre de; Portugal, Rodrigo Villares; Costa, Carlos Alberto; Leme, Adriana Franco Paes; Martinez, Diego Stefani Teodoro, E-mail: lidiane.franqui@pos.ft.unicamp.br [Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP (Brazil); Coluci, Vitor Rafael [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

    2016-07-01

    Full text: When in contact with biological systems, nanomaterials surface adsorbs biomolecules present in the biological medium, mainly proteins, yielding a molecular coating 'protein corona' which affects the biological response and toxicity of the nanomaterials. Several factors can influence the protein corona formation, such as nanomaterial physicochemical properties and the nature of biological medium. In this work, we have performed a comparative study between the single and multi-layer graphene oxide nanomaterials (SL-GO and ML-GO, respectively) after their interaction with DMEM cell culture medium containing fetal bovine serum (FBS). Bare GOs and FBS protein corona-coated GOs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), cryogenic transmission electron microscopy (Cryo-TEM) and X-ray photoelectron spectroscopy (XPS). The protein corona composition was characterized by gel electrophoresis (SDS-PAGE) and mass spectrometry (LC-MS/MS). Our results showed that, after interaction with FBS, GO particle size increased due to the protein corona formation. Besides, the presence of proteins also has significantly increased the dispersion stability of SLGO and ML-GO over time. Whereas the main proteins have been identified in both SL-GO and ML-GO, SL-GO has adsorbed larger amounts of proteins than ML-GO. Finally, the number of GO layers influences its interactions with FBS proteins and dispersion stability in DMEM medium. These results point out implications for in vitro cytotoxicity assessment and biomedical applications of these promising carbon nanomaterials. (author)

  9. Interaction of single and multi-layer graphene oxide with fetal bovine serum: assessing the protein corona formation

    International Nuclear Information System (INIS)

    Franqui, Lidiane Silva; Farias, Marcelo Alexandre de; Portugal, Rodrigo Villares; Costa, Carlos Alberto; Leme, Adriana Franco Paes; Martinez, Diego Stefani Teodoro; Coluci, Vitor Rafael

    2016-01-01

    Full text: When in contact with biological systems, nanomaterials surface adsorbs biomolecules present in the biological medium, mainly proteins, yielding a molecular coating 'protein corona' which affects the biological response and toxicity of the nanomaterials. Several factors can influence the protein corona formation, such as nanomaterial physicochemical properties and the nature of biological medium. In this work, we have performed a comparative study between the single and multi-layer graphene oxide nanomaterials (SL-GO and ML-GO, respectively) after their interaction with DMEM cell culture medium containing fetal bovine serum (FBS). Bare GOs and FBS protein corona-coated GOs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), cryogenic transmission electron microscopy (Cryo-TEM) and X-ray photoelectron spectroscopy (XPS). The protein corona composition was characterized by gel electrophoresis (SDS-PAGE) and mass spectrometry (LC-MS/MS). Our results showed that, after interaction with FBS, GO particle size increased due to the protein corona formation. Besides, the presence of proteins also has significantly increased the dispersion stability of SLGO and ML-GO over time. Whereas the main proteins have been identified in both SL-GO and ML-GO, SL-GO has adsorbed larger amounts of proteins than ML-GO. Finally, the number of GO layers influences its interactions with FBS proteins and dispersion stability in DMEM medium. These results point out implications for in vitro cytotoxicity assessment and biomedical applications of these promising carbon nanomaterials. (author)

  10. Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species

    DEFF Research Database (Denmark)

    Li, Hui; Jubelirer, Sara; Garcia Costas, Amaya M

    2009-01-01

    include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were...

  11. 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.

  12. 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.

  13. 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

  14. 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.

  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. A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Junghwa [Chemical Genomics National Research Lab., Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Jung, Hye Jin [Department of Pharmaceutical Engineering, Sun Moon University, Asansi, Chungnam 330-150 (Korea, Republic of); Jeong, Seung Hun; Kim, Hyoung Kyu; Han, Jin [National Research Laboratory for Mitochondrial Signaling, Department of Physiology, College of Medicine, Department of Health Sciences and Technology, Cardiovascular and Metabolic Disease Center, Inje University, Busan (Korea, Republic of); Kwon, Ho Jeong, E-mail: kwonhj@yonsei.ac.kr [Chemical Genomics National Research Lab., Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752 (Korea, Republic of)

    2014-12-12

    Highlights: • We constructed mitochondrial protein UQCRB mutant stable cell lines on the basis of a human case report. • These mutant cell lines exhibit pro-angiogenic activity with enhanced VEGF expression. • Proliferation of mutant cell lines was regulated by UQCRB inhibitors. • UQCRB may have a functional role in angiogenesis. - Abstract: Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identification and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders.

  17. A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species

    International Nuclear Information System (INIS)

    Chang, Junghwa; Jung, Hye Jin; Jeong, Seung Hun; Kim, Hyoung Kyu; Han, Jin; Kwon, Ho Jeong

    2014-01-01

    Highlights: • We constructed mitochondrial protein UQCRB mutant stable cell lines on the basis of a human case report. • These mutant cell lines exhibit pro-angiogenic activity with enhanced VEGF expression. • Proliferation of mutant cell lines was regulated by UQCRB inhibitors. • UQCRB may have a functional role in angiogenesis. - Abstract: Ubiquinol-cytochrome c reductase binding protein (UQCRB) is one of the subunits of mitochondrial complex III and is a target protein of the natural anti-angiogenic small molecule terpestacin. Previously, the biological role of UQCRB was thought to be limited to the maintenance of complex III. However, the identification and validation of UQCRB as a target protein of terpestacin enabled the role of UQCRB in oxygen sensing and angiogenesis to be elucidated. To explore the biological role of this protein further, UQCRB mutant stable cell lines were generated on the basis of a human case report. We demonstrated that these cell lines exhibited glycolytic and pro-angiogenic activities via mitochondrial reactive oxygen species (mROS)-mediated HIF1 signal transduction. Furthermore, a morphological abnormality in mitochondria was detected in UQCRB mutant stable cell lines. In addition, the proliferative effect of the UQCRB mutants was significantly regulated by the UQCRB inhibitors terpestacin and A1938. Collectively, these results provide a molecular basis for UQCRB-related biological processes and reveal potential key roles of UQCRB in angiogenesis and mitochondria-mediated metabolic disorders

  18. Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization.

    Science.gov (United States)

    Kwiatkowski, M; Wurlitzer, M; Krutilin, A; Kiani, P; Nimer, R; Omidi, M; Mannaa, A; Bussmann, T; Bartkowiak, K; Kruber, S; Uschold, S; Steffen, P; Lübberstedt, J; Küpker, N; Petersen, H; Knecht, R; Hansen, N O; Zarrine-Afsar, A; Robertson, W D; Miller, R J D; Schlüter, H

    2016-02-16

    Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation. Enzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  19. 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.

  20. Combined effect of protein and oxygen on reactive oxygen and nitrogen species in the plasma treatment of tissue

    Science.gov (United States)

    Gaur, Nishtha; Szili, Endre J.; Oh, Jun-Seok; Hong, Sung-Ha; Michelmore, Andrew; Graves, David B.; Hatta, Akimitsu; Short, Robert D.

    2015-09-01

    The influence of protein and molecular, ground state oxygen (O2) on the plasma generation, and transport of reactive oxygen and nitrogen species (RONS) in tissue are investigated. A tissue target, comprising a 1 mm thick gelatin film (a surrogate for real tissue), is placed on top of a 96-well plate; each well is filled with phosphate buffered saline (PBS, pH 7.4) containing one fluorescent or colorimetric reporter that is specific for one of three RONS (i.e., H2O2, NO2-, or OH•) or a broad spectrum reactive oxygen species reporter (2,7-dichlorodihydrofluorescein). A helium cold atmospheric plasma (CAP) jet contacts the top of the gelatin surface, and the concentrations of RONS generated in PBS are measured on a microplate reader. The data show that H2O2, NO2-, or OH• are generated in PBS underneath the target. Independently, measurements are made of the O2 concentration in the PBS with and without the gelatin target. Adding bovine serum albumin protein to the PBS or gelatin shows that protein either raises or inhibits RONS depending upon the O2 concentration. Our results are discussed in the context of plasma-soft tissue interactions that are important in the development of CAP technology for medicine, biology, and food manufacturing.

  1. 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.

  2. 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

  3. 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%.

  4. 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...

  5. Altered Gene Expression in Three Plant Species in Response to Treatment with Nep1, a Fungal Protein That Causes Necrosis

    Science.gov (United States)

    Keates, Sarah E.; Kostman, Todd A.; Anderson, James D.; Bailey, Bryan A.

    2003-01-01

    Nep1 is an extracellular fungal protein that causes necrosis when applied to many dicotyledonous plants, including invasive weed species. Using transmission electron microscopy, it was determined that application of Nep1 (1.0 μg mL–1, 0.1% [v/v] Silwet-L77) to Arabidopsis and two invasive weed species, spotted knapweed (Centaurea maculosa) and dandelion (Taraxacum officinale), caused a reduction in the thickness of the cuticle and a breakdown of chloroplasts 1 to 4 h after treatment. Membrane breakdown was most severe in cells closest to the surface of application. Differential display was used to isolate cDNA clones from the three species showing differential expression in response to Nep1 treatment. Differential gene expression was observed for a putative serpin (CmSER-1) and a calmodulin-like (CmCAL-1) protein from spotted knapweed, and a putative protein phosphatase 2C (ToPP2C-1) and cytochrome P-450 (ToCYP-1) protein from dandelion. In addition, differential expression was observed for genes coding for a putative protein kinase (AtPK-1), a homolog (AtWI-12) of wound-induced WI12, a homolog (AtLEA-1) of late embryogenesis abundant LEA-5, a WRKY-18 DNA-binding protein (AtWRKY-18), and a phospholipase D (AtPLD-1) from Arabidopsis. Genes showing elevated mRNA levels in Nep1-treated (5 μg mL–1, 0.1% [v/v] Silwet-L77) leaves 15 min after Nep1 treatment included CmSER-1 and CmCAL-1 for spotted knapweed, ToCYP-1 and CmCAL-1 for dandelion, and AtPK-1, AtWRKY-18, AtWI-12, and AtLEA-1 for Arabidopsis. Levels of mRNA for AtPLD-1 (Arabidopsis) and ToPP2C-1 (dandelion) decreased rapidly in Silwet-l77-treated plants between 15 min and 4 h of treatment, but were maintained or decreased more slowly over time in Nep1-treated (5 μg mL–1, 0.1% [v/v] Silwet-L77) leaves. In general, increases in mRNA band intensities were in the range of two to five times, with only ToCYP-1 in dandelion exceeding an increase of 10 times. The identified genes have been shown to be involved

  6. 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

  7. Single-step azide introduction in proteins via an aqueous diazo transfer

    NARCIS (Netherlands)

    van Dongen, S.F.M.; Teeuwen, R.L.M.; Nallani, M.; van Berkel, S.S.; Cornelissen, J.J.L.M.; Nolte, R.J.M.; van Hest, J.C.M.

    The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at

  8. Single-Step Azide Introduction in Proteins via an Aqueous Diazo Transfer

    NARCIS (Netherlands)

    van Dongen, Stijn; Teeuwen, R.L.M.; Nallani, Madhavan; van Berkel, S.S; Cornelissen, Jeroen Johannes Lambertus Maria; Nolte, Roeland; van Hest, Jan

    2009-01-01

    The controlled introduction of azides in proteins provides targetable handles for selective protein manipulation. We present here an efficient diazo transfer protocol that can be applied in an aqueous solution, leading to the facile introduction of azides in the side chains of lysine residues and at

  9. Mechanism of SOS PR-domain autoinhibition revealed by single-molecule assays on native protein from lysate.

    Science.gov (United States)

    Lee, Young Kwang; Low-Nam, Shalini T; Chung, Jean K; Hansen, Scott D; Lam, Hiu Yue Monatrice; Alvarez, Steven; Groves, Jay T

    2017-04-28

    The guanine nucleotide exchange factor (GEF) Son of Sevenless (SOS) plays a critical role in signal transduction by activating Ras. Here we introduce a single-molecule assay in which individual SOS molecules are captured from raw cell lysate using Ras-functionalized supported membrane microarrays. This enables characterization of the full-length SOS protein, which has not previously been studied in reconstitution due to difficulties in purification. Our measurements on the full-length protein reveal a distinct role of the C-terminal proline-rich (PR) domain to obstruct the engagement of allosteric Ras independently of the well-known N-terminal domain autoinhibition. This inhibitory role of the PR domain limits Grb2-independent recruitment of SOS to the membrane through binding of Ras·GTP in the SOS allosteric binding site. More generally, this assay strategy enables characterization of the functional behaviour of GEFs with single-molecule precision but without the need for purification.

  10. Single molecule force spectroscopy data and BD- and MD simulations on the blood protein von Willebrand factor

    Directory of Open Access Journals (Sweden)

    Sandra Posch

    2016-09-01

    Full Text Available We here give information for a deeper understanding of single molecule force spectroscopy (SMFS data through the example of the blood protein von Willebrand factor (VWF. It is also shown, how fitting of rupture forces versus loading rate profiles in the molecular dynamics (MD loading-rate range can be used to demonstrate the qualitative agreement between SMFS and MD simulations. The recently developed model by Bullerjahn, Sturm, and Kroy (BSK was used for this demonstration. Further, Brownian dynamics (BD simulations, which can be utilized to estimate the lifetimes of intramolecular VWF interactions under physiological shear, are described. For interpretation and discussion of the methods and data presented here, we would like to directly point the reader to the related research paper, “Mutual A domain interactions in the force sensing protein von Willebrand Factor” (Posch et al., 2016 [1]. Keywords: Atomic force microscopy, Single molecule force spectroscopy, Molecular dynamics simulation, Brownian dynamics simulation, von Willebrand factor

  11. Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

    Science.gov (United States)

    De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

    2011-10-01

    Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

  12. Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli

    International Nuclear Information System (INIS)

    Moreau, P.L.

    1988-01-01

    Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA

  13. Preparation of genomic DNA from a single species of uncultured magnetotactic bacterium by multiple-displacement amplification.

    Science.gov (United States)

    Arakaki, Atsushi; Shibusawa, Mie; Hosokawa, Masahito; Matsunaga, Tadashi

    2010-03-01

    Magnetotactic bacteria comprise a phylogenetically diverse group that is capable of synthesizing intracellular magnetic particles. Although various morphotypes of magnetotactic bacteria have been observed in the environment, bacterial strains available in pure culture are currently limited to a few genera due to difficulties in their enrichment and cultivation. In order to obtain genetic information from uncultured magnetotactic bacteria, a genome preparation method that involves magnetic separation of cells, flow cytometry, and multiple displacement amplification (MDA) using phi29 polymerase was used in this study. The conditions for the MDA reaction using samples containing 1 to 100 cells were evaluated using a pure-culture magnetotactic bacterium, "Magnetospirillum magneticum AMB-1," whose complete genome sequence is available. Uniform gene amplification was confirmed by quantitative PCR (Q-PCR) when 100 cells were used as a template. This method was then applied for genome preparation of uncultured magnetotactic bacteria from complex bacterial communities in an aquatic environment. A sample containing 100 cells of the uncultured magnetotactic coccus was prepared by magnetic cell separation and flow cytometry and used as an MDA template. 16S rRNA sequence analysis of the MDA product from these 100 cells revealed that the amplified genomic DNA was from a single species of magnetotactic bacterium that was phylogenetically affiliated with magnetotactic cocci in the Alphaproteobacteria. The combined use of magnetic separation, flow cytometry, and MDA provides a new strategy to access individual genetic information from magnetotactic bacteria in environmental samples.

  14. A single European aspen (Populus tremula) tree individual may potentially harbour dozens of Cenococcum geophilum ITS genotypes and hundreds of species of ectomycorrhizal fungi.

    Science.gov (United States)

    Bahram, Mohammad; Põlme, Sergei; Kõljalg, Urmas; Tedersoo, Leho

    2011-02-01

    Ectomycorrhizal fungi (EcMF) form diverse communities and link different host plants into mycorrhizal networks, yet little is known about the magnitude of mycobiont diversity of a single tree individual. This study addresses species richness and spatial structure of EcMF in the root system of a single European aspen (Populus tremula) individual in an old-growth boreal mixed forest ecosystem in Estonia. Combining morphological and molecular identification methods for both plant and fungi, 122 species of EcMF were recovered from 103 root samples of the single tree. Richness estimators predicted the total EcMF richness to range from 182 to 207 species, reflecting the observation of 62.3% singletons and doubletons within the community. Fine-scale genetic diversity in Cenococcum geophilum indicates the presence of 23 internal transcribed spacer genotypes. EcMF community was significantly spatially autocorrelated only at the lineage level up to 3 m distance, but not at the species level. Proximity of other hosts had a significant effect on the spatial distribution of EcMF lineages. This study demonstrates that a single tree may host as many EcMF species and individuals as recovered on multiple hosts in diverse communities over larger areas. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  15. 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.

  16. A network model to correlate conformational change and the impedance spectrum of single proteins

    Energy Technology Data Exchange (ETDEWEB)

    Alfinito, Eleonora; Pennetta, Cecilia; Reggiani, Lino [Dipartimento di Ingegneria dell' Innovazione, Universita del Salento, Via Arnesano, Lecce (Italy); Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM) (Italy)

    2008-02-13

    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.

  17. Discrimination between ovine Babesia and Theileria species in China based on the ribosomal protein S8 (RPS8) gene.

    Science.gov (United States)

    Tian, Zhancheng; Liu, Guangyuan; Yin, Hong; Luo, Jianxun; Guan, Guiquan; Luo, Jin; Xie, Junren; Zheng, Jinfeng; Yuan, Xiaosong; Wang, Fangfang; Shen, Hui; Tian, Meiyuan

    2013-10-18

    Ovine babesiosis and theileriosis are important hemoprotozoal diseases of sheep and goats in tropical and subtropical regions that lead to economic losses in these animals. PCR-restriction fragment length polymorphism (PCR-RFLP) is a reliable molecular diagnostic tool for discriminating Theileria or Babesia species in the same host. In this study, the DNA sequences of a ribosomal protein S8 (RPS8) gene from four species of piroplasms in China were used to develop a species-specific PCR-RFLP diagnostic tool. The sensitivity of the PCR assays was 0.1 pg DNA for B. motasi and 1 pg DNA for T. uilenbergi and 10 pg DNA for Babesia sp. Xinjiang-2005 and T. luwenshuni. The clear size difference of the PCR products allowed for a direct discrimination for B. motasi, Babesia sp. Xinjiang-2005 and ovine Theileria species (T. uilenbergi and T. luwenshuni), except that the mixed infection between T. uilenbergi and T. luwenshuni may be difficult to distinguish, simply after the electrophoretic separation of the amplification products. Further T. uilenbergi and T. luwenshuni diagnoses were made by digesting the PCR product with SacI. The established method could be applicable for the survey of parasite dynamics, and epidemiological studies as well as prevention and control of the disease. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. 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 produces...... very low levels of extracellular proteases, mainly serine metalloproteases. The stability of A. tubingensis feruloyl esterase A (FaeA) was compared upon production in wild-type A. vadensis, A. tubingensis, and an A. niger strain in which the three main protease-encoding genes were disrupted....... 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...

  19. Localizing internal friction along the reaction coordinate of protein folding by combining ensemble and single-molecule fluorescence spectroscopy

    Science.gov (United States)

    Borgia, Alessandro; Wensley, Beth G.; Soranno, Andrea; Nettels, Daniel; Borgia, Madeleine B.; Hoffmann, Armin; Pfeil, Shawn H.; Lipman, Everett A.; Clarke, Jane; Schuler, Benjamin

    2012-01-01

    Theory, simulations and experimental results have suggested an important role of internal friction in the kinetics of protein folding. Recent experiments on spectrin domains provided the first evidence for a pronounced contribution of internal friction in proteins that fold on the millisecond timescale. However, it has remained unclear how this contribution is distributed along the reaction and what influence it has on the folding dynamics. Here we use a combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, microfluidic mixing and denaturant- and viscosity-dependent protein-folding kinetics to probe internal friction in the unfolded state and at the early and late transition states of slow- and fast-folding spectrin domains. We find that the internal friction affecting the folding rates of spectrin domains is highly localized to the early transition state, suggesting an important role of rather specific interactions in the rate-limiting conformational changes. PMID:23149740

  20. 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.

  1. 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

    affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation...... by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor...

  2. Involvement of Reactive Oxygen Species and Mitochondrial Proteins in Biophoton Emission in Roots of Soybean Plants under Flooding Stress.

    Science.gov (United States)

    Kamal, Abu Hena Mostafa; Komatsu, Setsuko

    2015-05-01

    To understand the mechanism of biophoton emission, ROS and mitochondrial proteins were analyzed in soybean plants under flooding stress. Enzyme activity and biophoton emission were increased in the flooding stress samples when assayed in reaction mixes specific for antioxidant enzymes and reactive oxygen species; although the level of the hydroxyl radicals was increased at day 4 (2 days of flooding) compared to nonflooding at day 4, the emission of biophotons did not change. Mitochondria were isolated and purified from the roots of soybean plants grown under flooding stress by using a Percoll gradient, and proteins were analyzed by a gel-free proteomic technique. Out of the 98 mitochondrial proteins that significantly changed abundance under flooding stress, 47 increased and 51 decreased at day 4. The mitochondrial enzymes fumarase, glutathione-S-transferase, and aldehyde dehydrogenase increased at day 4 in protein abundance and enzyme activity. Enzyme activity and biophoton emission decreased at day 4 by the assay of lipoxygenase under stress. Aconitase, acyl CoA oxidase, succinate dehydrogenase, and NADH ubiquinone dehydrogenase were up-regulated at the transcription level. These results indicate that oxidation and peroxide scavenging might lead to biophoton emission and oxidative damage in the roots of soybean plants under flooding stress.

  3. 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

  4. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    Energy Technology Data Exchange (ETDEWEB)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Pack, Chan-Gi [Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198 (Japan); Terajima, Hideki [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan); Yajima, Junichiro; Nishizaka, Takayuki [Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588 (Japan); Kinjo, Masataka [Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021 (Japan); Taguchi, Hideki, E-mail: taguchi@bio.titech.ac.jp [Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)

    2011-02-25

    Research highlights: {yields} We develop a method to track a quantum dot-conjugated protein in yeast cells. {yields} We incorporate the conjugated quantum dot proteins into yeast spheroplasts. {yields} We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  5. Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells

    International Nuclear Information System (INIS)

    Tsuji, Toshikazu; Kawai-Noma, Shigeko; Pack, Chan-Gi; Terajima, Hideki; Yajima, Junichiro; Nishizaka, Takayuki; Kinjo, Masataka; Taguchi, Hideki

    2011-01-01

    Research highlights: → We develop a method to track a quantum dot-conjugated protein in yeast cells. → We incorporate the conjugated quantum dot proteins into yeast spheroplasts. → We track the motions by conventional or 3D tracking microscopy. -- Abstract: Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically conjugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells.

  6. 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

  7. Rapid concentration and dialysis of proteins with single hollow fibers: possible applications in analysis of protein secretion by isolated cells and steroid radioimmunoassays

    International Nuclear Information System (INIS)

    Rommerts, F.F.G.; Clotscher, W.F.; Van der Molen, H.J.

    1977-01-01

    Single hollow fibers were used in specially made cells for fast concentration and dialysis of solutions containing macromolecules. Volumes on the order of 5 ml of diluted protein solutions could be concentrated to 50--100 μl or less within 7 min with a protein recovery of 60--80%. More than 99% of the molecules with a molecular weight less than 500 could be removed in less than 1 hr. A possible application of the rapid dialysis method for the mechanization of radioimmunoassays is indicated. It was shown that in the radioimmunoassay of steriods the unbound steroids could be removed after incubation with antiserum, within 10 min and without a change in volume

  8. The Cell Wall-Associated Proteins in the Dimorphic Pathogenic Species of Paracoccidioides.

    Science.gov (United States)

    Puccia, Rosana; Vallejo, Milene C; Longo, Larissa V G

    2017-01-01

    Paracoccidioides brasiliensis and P. lutzii cause human paracoccidioidomycosis (PCM). They are dimorphic ascomycetes that grow as filaments at mild temperatures up to 28oC and as multibudding pathogenic yeast cells at 37oC. Components of the fungal cell wall have an important role in the interaction with the host because they compose the cell outermost layer. The Paracoccidioides cell wall is composed mainly of polysaccharides, but it also contains proportionally smaller rates of proteins, lipids, and melanin. The polysaccharide cell wall composition and structure of Paracoccidioides yeast cells, filamentous and transition phases were studied in detail in the past. Other cell wall components have been better analyzed in the last decades. The present work gives to the readers a detailed updated view of cell wall-associated proteins. Proteins that have been localized at the cell wall compartment using antibodies are individually addressed. We also make an overview about PCM, the Paracoccidioides cell wall structure, secretion mechanisms, and fungal extracellular vesicles. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2

    Science.gov (United States)

    Breitkopf, Susanne B.; Yang, Xuemei; Begley, Michael J.; Kulkarni, Meghana; Chiu, Yu-Hsin; Turke, Alexa B.; Lauriol, Jessica; Yuan, Min; Qi, Jie; Engelman, Jeffrey A.; Hong, Pengyu; Kontaridis, Maria I.; Cantley, Lewis C.; Perrimon, Norbert; Asara, John M.

    2016-02-01

    Using a series of immunoprecipitation (IP) - tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.

  10. 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.

  11. 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

  12. 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 pHproduct variants using the combined pH and salt based elution gradient and removal of the host cell impurities in flow-through are the key novel features of the developed multimodal chromatographic purification step. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Ubiquitous distribution of fluorescent protein in muscles of four species and two subspecies of eel (genus Anguilla).

    Science.gov (United States)

    Funahashi, Aki; Itakura, Takao; Hassanin Abeer, A I; Komatsu, Masaharu; Hayashi, Seiichi; Kaminishi, Yoshio

    2017-03-01

    In this study, the localization of fluorescent protein (FP) was characterized in the muscles of four species and two subspecies of eels Anguilla anguilla, A. australis, A. bicolor bicolor (b.), A. bicolor pacifica (p.) and A. mossambica in addition to the previously reported A. japonica. The open reading frame of each eel FP was 417 bp encoding 139 amino acid residues. The deduced amino acid sequences among the four species and two subspecies exhibited 91.4-100% identity, and belonged to the fatty-acid-binding protein (FABP) family. The gene structure of eel FPs in A. japonica, A. anguilla, A. australis, A. bicolor b., A. bicolor p. and A. mossambica have four exons and three introns, and were common to that of FABP family. The apo eel FPs expressed by Escherichia coli with recombinant eel FP genes were analysed for the fluorescent properties in the presence of bilirubin. The excitation and emission spectra of holo eel FPs had the maximum wavelengths of 490-496 and 527-530 nm, respectively. The holo eel FPs indicated that the fluorescent intensities were stronger in A. japonica and A. bicolor than in A. mossambica, A. australis and A. anguilla. The comparison of amino acid sequences revealed two common substitutions in A. mossambica, A. australis and A. anguilla with weak fluorescent intensity.

  14. Real-Time Discrimination and Versatile Profiling of Spontaneous Reactive Oxygen Species in Living Organisms with a Single Fluorescent Probe.

    Science.gov (United States)

    Zhang, Ruilong; Zhao, Jun; Han, Guangmei; Liu, Zhengjie; Liu, Cui; Zhang, Cheng; Liu, Bianhua; Jiang, Changlong; Liu, Renyong; Zhao, Tingting; Han, Ming-Yong; Zhang, Zhongping

    2016-03-23

    Fluorescent probes are powerful tools for the investigations of reactive oxygen species (ROS) in living organisms by visualization and imaging. However, the multiparallel assays of several ROS with multiple probes are often limited by the available number of spectrally nonoverlapping chromophores together with large invasive effects and discrepant biological locations. Meanwhile, the spontaneous ROS profilings in various living organs/tissues are also limited by the penetration capability of probes across different biological barriers and the stability in reactive in vivo environments. Here, we report a single fluorescent probe to achieve the effective discrimination and profiling of hydroxyl radicals (•OH) and hypochlorous acid (HClO) in living organisms. The probe is constructed by chemically grafting an additional five-membered heterocyclic ring and a lateral triethylene glycol chain to a fluorescein mother, which does not only turn off the fluorescence of fluorescein, but also create the dual reactive sites to ROS and the penetration capability in passing through various biological barriers. The reactions of probe with •OH and HClO simultaneously result in cyan and green emissions, respectively, providing the real-time discrimination and quantitative analysis of the two ROS in cellular mitochondria. Surprisingly, the accumulation of probes in the intestine and liver of a normal-state zebrafish and the transfer pathway from intestine-to-blood-to-organ/tissue-to-kidney-to-excretion clearly present the profiling of spontaneous •OH and HClO in these metabolic organs. In particular, the stress generation of •OH at the fresh wound of zebrafish is successfully visualized for the first time, in spite of its extremely short lifetime.

  15. Demonstration of Fast and Accurate Discrimination and Quantification of Chemically Similar Species Utilizing a Single Cross-Selective Chemiresistor

    Science.gov (United States)

    2015-01-01

    Performance characteristics of gas-phase microsensors will determine the ultimate utility of these devices for a wide range of chemical monitoring applications. Commonly employed chemiresistor elements are quite sensitive to selected analytes, and relatively new methods have increased the selectivity to specific compounds, even in the presence of interfering species. Here, we have focused on determining whether purposefully driven temperature modulation can produce faster sensor-response characteristics, which could enable measurements for a broader range of applications involving dynamic compositional analysis. We investigated the response speed of a single chemiresitive In2O3 microhotplate sensor to four analytes (methanol, ethanol, acetone, 2-butanone) by systematically varying the oscillating frequency (semicycle periods of 20–120 ms) of a bilevel temperature cycle applied to the sensing element. It was determined that the fastest response (≈ 9 s), as indicated by a 98% signal-change metric, occurred for a period of 30 ms and that responses under such modulation were dramatically faster than for isothermal operation of the same device (>300 s). Rapid modulation between 150 and 450 °C exerts kinetic control over transient processes, including adsorption, desorption, diffusion, and reaction phenomena, which are important for charge transfer occurring in transduction processes and the observed response times. We also demonstrate that the fastest operation is accompanied by excellent discrimination within a challenging 16-category recognition problem (consisting of the four analytes at four separate concentrations). This critical finding demonstrates that both speed and high discriminatory capabilities can be realized through temperature modulation. PMID:24931319

  16. Highly multiplexed simultaneous detection of RNAs and proteins in single cells.

    Science.gov (United States)

    Frei, Andreas P; Bava, Felice-Alessio; Zunder, Eli R; Hsieh, Elena W Y; Chen, Shih-Yu; Nolan, Garry P; Gherardini, Pier Federico

    2016-03-01

    To enable the detection of expression signatures specific to individual cells, we developed PLAYR (proximity ligation assay for RNA), a method for highly multiplexed transcript quantification by flow and mass cytometry that is compatible with standard antibody staining. When used with mass cytometry, PLAYR allowed for the simultaneous quantification of more than 40 different mRNAs and proteins. In primary cells, we quantified multiple transcripts, with the identity and functional state of each analyzed cell defined on the basis of the expression of a separate set of transcripts or proteins. By expanding high-throughput deep phenotyping of cells beyond protein epitopes to include RNA expression, PLAYR opens a new avenue for the characterization of cellular metabolism.

  17. 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

    and does not represent a recent acquirement of the phage. The P1 and E. coli SSB proteins are fully functionally interchangeable. SSB-P1 is nonessential for phage growth in an exponentially growing E. coli host, and it is sufficient to promote bacterial growth in the absence of the E. coli SSB protein....... Expression studies showed that the P1 ssb gene is transcribed only, in an rpoS-independent fashion, during stationary-phase growth in E. coli. Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an ssb-null mutant when exposed to a bacterial host in the stationary...

  18. 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....... The deuterium labeling pattern of beta2-microglobulin is retained in the gaseous fragment ions by employing mild declustering conditions for electrospray ionization. A recently developed model peptide is used to arrive at such ion source declustering conditions that prevent the occurrence of intramolecular gas...

  19. A single WAP domain (SWD)-containing protein with antiviral activity from Pacific white shrimp Litopenaeus vannamei.

    Science.gov (United States)

    Yang, Linwei; Niu, Shengwen; Gao, Jiefeng; Zuo, Hongliang; Yuan, Jia; Weng, Shaoping; He, Jianguo; Xu, Xiaopeng

    2018-02-01

    The single whey acidic protein (WAP) domain (SWD)-containing proteins, also called type III crustins, are a group of antimicrobial peptides (AMPs) in crustaceans. At present, a number of SWDs have been identified in shrimp, which showed essential antibacterial activities. However, the roles of SWDs in antiviral immune responses have not been reported up to now. In this study, a novel SWD (LvSWD3) was identified from Pacific white shrimp, Litopenaeus vannamei, which contained a typical single WAP domain homologous to those of other crustacean SWDs. Although lacking the pro and arg-rich region between the signal peptide and the WAP domain, LvSWD3 was closely clustered with other shrimp SWDs in the phylogenetic tree. Similar to many shrimp SWDs, the highest expression of LvSWD3 was detected in hemocytes. The LvSWD3 expression exhibited only limited changes after challenges with Vibrio parahaemolyticus, Poly (I:C) and lipopolysaccharide, but was significantly up-regulated after white spot syndrome virus (WSSV) infection. Silencing of LvSWDs significantly accelerated the death of the WSSV-infected but not the V. parahaemolyticus-infected shrimp. The recombinant LvSWD3 protein did not show proteinase inhibitory and antibacterial activities but could significantly postpone the death of WSSV-infected shrimp and reduce the viral load in tissues. These suggested that LvSWD3 was a novel SWD with antiviral activity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype.

    Science.gov (United States)

    Folle, Ana Maite; Kitano, Eduardo S; Lima, Analía; Gil, Magdalena; Cucher, Marcela; Mourglia-Ettlin, Gustavo; Iwai, Leo K; Rosenzvit, Mara; Batthyány, Carlos; Ferreira, Ana María

    2017-01-01

    The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in

  1. Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype

    Science.gov (United States)

    Folle, Ana Maite; Kitano, Eduardo S.; Lima, Analía; Gil, Magdalena; Cucher, Marcela; Mourglia-Ettlin, Gustavo; Iwai, Leo K.; Rosenzvit, Mara; Batthyány, Carlos

    2017-01-01

    The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in

  2. EXTRACELLULAR PROTEINS PRODUCED BY DIFFERENT SPECIES OF THE FUNGUS TRICHODERMA ON SECONDARY PAPER MILL SLUDGE SUBSTRATE

    Directory of Open Access Journals (Sweden)

    Ida Vaskova,

    2012-01-01

    Full Text Available Kraft pulping is the most commonly used pulping process in the pulp and paper industry. In this process wood chips are chemically delignified using sodium sulfide and sodium hydroxide. Delignification is usually followed by mechanical fiberization and a bleaching process of the resulting wood pulp. In addition to lignin-free wood pulp, this process also produces waste that contains residues of used chemicals, lignin, cellulose, hemicelluloses, and small amounts of other wood components. Because of the worldwide large-scale production of paper, the sludge from paper mills contributes significantly to environmental pollution. Although there have been great efforts being made to utilize this lignin-rich material, sludge is mostly disposed in landfills or incinerated in a boiler. This research project used secondary sludge as a substrate for 7 wood-decay fungi taxonomically belonging to the genus Trichoderma. The examined fungi expressed the capability of consuming sludge components as a carbon source to produce extracellular proteins. The proteins were separated by gel electrophoresis. Before and after fungi cultivation, the sludge was analyzed by Fourier transform infrared spectroscopy (FTIR.

  3. Visualization of amino acid composition differences between processed protein from different animal species by self-organizing feature maps

    Directory of Open Access Journals (Sweden)

    Xingfan ZHOU,Zengling YANG,Longjian CHEN,Lujia HAN

    2016-06-01

    Full Text Available Amino acids are the dominant organic components of processed animal proteins, however there has been limited investigation of differences in their composition between various protein sources. Information on these differences will not only be helpful for their further utilization but also provide fundamental information for developing species-specific identification methods. In this study, self-organizing feature maps (SOFM were used to visualize amino acid composition of fish meal, and meat and bone meal (MBM produced from poultry, ruminants and swine. SOFM display the similarities and differences in amino acid composition between protein sources and effectively improve data transparency. Amino acid composition was shown to be useful for distinguishing fish meal from MBM due to their large concentration differences between glycine, lysine and proline. However, the amino acid composition of the three MBMs was quite similar. The SOFM results were consistent with those obtained by analysis of variance and principal component analysis but more straightforward. SOFM was shown to have a robust sample linkage capacity and to be able to act as a powerful means to link different sample for further data mining.

  4. High-quality Thermodynamic Data on the Stability Changes of Proteins Upon Single-site Mutations

    Energy Technology Data Exchange (ETDEWEB)

    Pucci, Fabrizio, E-mail: fapucci@ulb.ac.be; Bourgeas, Raphaël, E-mail: rbourgeas@ulb.ac.be; Rooman, Marianne, E-mail: mrooman@ulb.ac.be [Department of BioModeling, BioInformatics and BioProcesses, Université Libre de Bruxelles, CP 165/61, Roosevelt Avenue 50, 1050 Brussels, Belgium and Interuniversity Institute of Bioinformatics in Brussels, CP 263, Triumph Bld, 1050 Brussels (Belgium)

    2016-06-15

    We have set up and manually curated a dataset containing experimental information on the impact of amino acid substitutions in a protein on its thermal stability. It consists of a repository of experimentally measured melting temperatures (T{sub m}) and their changes upon point mutations (ΔT{sub m}) for proteins having a well-resolved x-ray structure. This high-quality dataset is designed for being used for the training or benchmarking of in silico thermal stability prediction methods. It also reports other experimentally measured thermodynamic quantities when available, i.e., the folding enthalpy (ΔH) and heat capacity (ΔC{sub P}) of the wild type proteins and their changes upon mutations (ΔΔH and ΔΔC{sub P}), as well as the change in folding free energy (ΔΔG) at a reference temperature. These data are analyzed in view of improving our insights into the correlation between thermal and thermodynamic stabilities, the asymmetry between the number of stabilizing and destabilizing mutations, and the difference in stabilization potential of thermostable versus mesostable proteins.

  5. 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

    Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane...

  6. Variations in protein and fat contents and their fractions in milk from two species fed different forages.

    Science.gov (United States)

    Kholif, S M; El-Shewy, A A; Morsy, T A; Abd El-Rahman, H H

    2015-02-01

    This study aimed at determining the variations in milk constituents which could be varied by feed and animal species. To achieve this goal, two groups of homoparity Baladi cows and Egyptian buffaloes (n = 20 per species) were used. Each group was divided into two subgroups (n = 10): subgroup I received legume forage (Egyptian clover) and subgroup II received grass forage (sorghum forage). All experimental animals were fed the diet consisting of concentrate, forage and rice straw as 50, 25 and 25% of dry matter intake respectively. Milk samples were taken for analysis. The trial lasted until the 3rd month of parturition. The main results indicated that lactating cattle fed legume forage significantly (p ≤ 0.01) had more content of casein nitrogen (513 mg/100 ml milk), lower content of glutamic acid (23.56 g/100 g milk protein) and more content of cis-9, trans-11 18:2 conjugated linoleic acid (CLA) (0.77 g/100 g milk fat) compared with 433, 26.67 and 0.53, respectively, for cattle fed grass forage. With regard to the species effect, results showed that buffalo milk appeared to contain significantly higher (p ≤ 0.01) contents of casein nitrogen, phenylalanine, glutamic and arachidonic acid compared with cow's milk. However, the latter was significantly (p ≤ 0.01) more in the cis-9, trans-11CLA (0.59 g/100 g milk fat) than that in buffalo milk (0.47 g/100 g milk fat). The results revealed that not only forage type played a critical role in determining the variations of milk nitrogen distribution, milk amino acids and fatty acids but also animal species had a significant effect on these parameters. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

  7. Species contributions to single biodiversity values under-estimate whole community contribution to a wider range of values to society.

    Science.gov (United States)

    Hiron, Matthew; Pärt, Tomas; Siriwardena, Gavin M; Whittingham, Mark J

    2018-05-03

    A major task for decision makers is deciding how to consider monetary, cultural and conservation values of biodiversity explicitly when planning sustainable land use. Thus, there is a great need to understand just what "valuing" biodiversity or species really means, e.g. regarding how many and which species are important in providing ecosystem services or other values. Constructing ecosystem-level indices, however, requires weighting the relative contribution of species to the different values. Using farmland birds, we illustrate how species contribute to different biodiversity values, namely utilitarian (pest seed predation potential), cultural (species occurrence in poetry), conservational (declines and rarity) and inherent (all species equal) value. Major contributions to each value are often made by a subset of the community and different species are important for different values, leading to no correlations or, in some cases, negative correlations between species' relative contributions to different values. Our results and methods using relative contributions of species to biodiversity values can aid decisions when weighing different values in policies and strategies for natural resource management. We conclude that acknowledging the importance of the range of biodiversity values that are apparent from different perspectives is critical if the full value of biodiversity to society is to be realised.

  8. Anti-Human Endoglin (hCD105 Immunotoxin—Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1

    Directory of Open Access Journals (Sweden)

    Begoña Barriuso

    2016-06-01

    Full Text Available Endoglin (CD105 is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT—containing recombinant musarmin 1 (single chain ribosome-inactivating proteins linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio propionate (SPDP. The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10−10 to 10−9 M.

  9. 15N incorporation into organ proteins of newborn rats following single pulse-labelling with different tracers

    International Nuclear Information System (INIS)

    Wutzke, K.D.; Plath, C.; Richter, I.; Heine, W.; Zhukova, T.P.; Sorokina, E.G.; Friedrich, M.

    1987-01-01

    A short-chain 15 N-peptide mixture characterized by an average chain length of 2.3 was obtained when 15 N-labelled yeast protein was hydrolyzed enzymatically by thermitase from Thermoactinomyces vulgaris. Fifteen newborn Wistar rats were given a single pulse of [ 15 N]glycine. [ 15 N]H 4 Cl and [ 15 N]yeast protein thermitasehydrolysate (YPTH) in a dosage of 50 mg 15 N excess kg -1 by gastric tube. In comparison with [ 15 N]glycine the 15 N incorporation rates of brain, muscle and liver were approximately 150% higher after [ 15 N]YPTH application. Uniform labelling, high 15 N enrichment, almost complete absorption, avoidance of imbalances and the low price make this tracer substance superior to other tracers conventionally used for organ labelling. (author)

  10. Anti-Human Endoglin (hCD105) Immunotoxin-Containing Recombinant Single Chain Ribosome-Inactivating Protein Musarmin 1.

    Science.gov (United States)

    Barriuso, Begoña; Antolín, Pilar; Arias, F Javier; Girotti, Alessandra; Jiménez, Pilar; Cordoba-Diaz, Manuel; Cordoba-Diaz, Damián; Girbés, Tomás

    2016-06-10

    Endoglin (CD105) is an accessory component of the TGF-β receptor complex, which is expressed in a number of tissues and over-expressed in the endothelial cells of tumor neovasculature. Targeting endoglin with immunotoxins containing type 2 ribosome-inactivating proteins has proved an effective tool to reduce blood supply to B16 mice tumor xenografts. We prepared anti-endoglin immunotoxin (IT)-containing recombinant musarmin 1 (single chain ribosome-inactivating proteins) linked to the mouse anti-human CD105 44G4 mouse monoclonal antibody via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). The immunotoxin specifically killed L929 fibroblast mouse cells transfected with the short form of human endoglin with IC50 values in the range of 5 × 10(-10) to 10(-9) M.

  11. Nitric oxide-related species-induced protein oxidation: reversible, irreversible, and protective effects on enzyme function of papain.

    Science.gov (United States)

    Väänänen, Antti J; Kankuri, Esko; Rauhala, Pekka

    2005-04-15

    Protein oxidation, irreversible modification, and inactivation may play key roles in various neurodegenerative disorders. Therefore, we studied the effects of the potentially in vivo occurring nitric oxide-related species on two different markers of protein oxidation: protein carbonyl generation on bovine serum albumine (BSA) and loss of activity of a cysteine-dependent protease, papain, in vitro by using Angeli's salt, papanonoate, SIN-1, and S-nitrosoglutathione (GSNO) as donors of nitroxyl, nitric oxide, peroxynitrite, and nitrosonium ions, respectively. Angeli's salt, SIN-1, and papanonoate (0-1000 microM) all generated a concentration-dependent increase in carbonyl formation on BSA (107, 60, and 45%, respectively). GSNO did not affect carbonyl formation. Papain was inhibited by Angeli's salt, SIN-1, papanonoate, and GSNO with IC50 values of 0.62, 2.3, 54, and 80 microM, respectively. Angeli's salt (3.16 microM)-induced papain inactivation was only partially reversible, while the effects of GSNO (316 microM) and papanonoate (316 microM) were reversible upon addition of excess DTT. The Angeli's salt-mediated DTT-irreversible inhibition of papain was prevented by GSNO or papanonoate pretreatment, hypothetically through mixed disulfide formation or S-nitrosylation of the catalytically critical thiol group of papain. These results, for the first time, compare the generation of carbonyls in proteins by Angeli's salt, papanonoate, and SIN-1. Furthermore, these results suggest that S-nitrosothiols may have a novel function in protecting critical thiols from irreversible oxidative damage.

  12. A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

    Directory of Open Access Journals (Sweden)

    Miguel A Martín-Acebes

    Full Text Available West Nile virus (WNV is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu on the highly basic internal capsid or core (C protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

  13. A single amino acid substitution in the core protein of West Nile virus increases resistance to acidotropic compounds.

    Science.gov (United States)

    Martín-Acebes, Miguel A; Blázquez, Ana-Belén; de Oya, Nereida Jiménez; Escribano-Romero, Estela; Shi, Pei-Yong; Saiz, Juan-Carlos

    2013-01-01

    West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH4Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.

  14. Tritium toxicity in postnatally developing brain: Effects of single administration on nucleic acids and protein

    International Nuclear Information System (INIS)

    Bhatia, A.L.; Saraswat, A.

    1988-01-01

    The brains of postnatally developing mice were studied at one, two, three, four, five and six weeks of age after injecting one day old neonates (1.95 ± 0.35 g) with 11.1 kBq and 111 kBq/ml of bondy water. The HTO-exposed developing animals though do not show any significant decline in their brain and body weight, their DNA concentration was found significantly depleted at one week by 19% after the treatment with 111 kBq dose and subsequently recovered by six week reaching 93% of the control. Protein concentration showed significant deficit in both the dose groups at all the postnatal invervals. Protein/DNA ratio increased in one and two weeks old mice and reduced from weeks onward. RNA/DNA ratio has also been found consistently low in irradiated groups. (orig.) [de

  15. A study of biodiversity using DSS method and seed storage protein comparison of populations in two species of Achillea L. in the west of Iran

    Directory of Open Access Journals (Sweden)

    Hajar Salehi

    2013-11-01

    Full Text Available Intarspecific and interspecific variations are the main reserves of biodiversity and both are important sources of speciation. On this basis, identifing and recognizing the intra and interspecific variations is important in order to recognition of biodiversity. This research was done to study biodiversity and electrophoresis comparison of seed storage proteins in the populations of the two species of the genus Achillea in Hamadan and Kurdistan provinces using of the method of determination of special station (DSS. For this purpose, 12 and 9 special stations were selected for the species A. tenuifolia and A. biebresteinii using the data published in the related flora. Seed storage proteins were extracted and then studied using electrophoresis techniques (SDS-PAGE. In survey of all special stations, 120 plant species were distinguished as associated species. The results of the floristic data for the both species determined six distinctive groups that indicated the existence of intraspecific diversity in this species. The result of analysis of ecological data and seed storage proteins for the two species was in accordance with the floristic data and showed six distinctive groups. The existence of the bands of no. 4, 5, 8, 12 and 13 in the special stations of A. tenuifolia and the bands of 14, 15 and 16 in the special stations of A. biebresteinii o separated the populations of the species in two quite different and distinctive groups.

  16. Growth response of drought-stressed Pinus sylvestris seedlings to single- and multi-species inoculation with ectomycorrhizal fungi.

    Directory of Open Access Journals (Sweden)

    Tabea Kipfer

    Full Text Available Many trees species form symbiotic associations with ectomycorrhizal (ECM fungi, which improve nutrient and water acquisition of their host. Until now it is unclear whether the species richness of ECM fungi is beneficial for tree seedling performance, be it during moist conditions or drought. We performed a pot experiment using Pinus sylvestris seedlings inoculated with four selected ECM fungi (Cenococcum geophilum, Paxillus involutus, Rhizopogon roseolus and Suillus granulatus to investigate (i whether these four ECM fungi, in monoculture or in species mixtures, affect growth of P. sylvestris seedlings, and (ii whether this effect can be attributed to species number per se or to species identity. Two different watering regimes (moist vs. dry were applied to examine the context-dependency of the results. Additionally, we assessed the activity of eight extracellular enzymes in the root tips. Shoot growth was enhanced in the presence of S. granulatus, but not by any other ECM fungal species. The positive effect of S. granulatus on shoot growth was more pronounced under moist (threefold increase than under dry conditions (twofold increase, indicating that the investigated ECM fungi did not provide additional support during drought stress. The activity of secreted extracellular enzymes was higher in S. granulatus than in any other species. In conclusion, our findings suggest that ECM fungal species composition may affect seedling performance in terms of aboveground biomass.

  17. Growth response of drought-stressed Pinus sylvestris seedlings to single- and multi-species inoculation with ectomycorrhizal fungi.

    Science.gov (United States)

    Kipfer, Tabea; Wohlgemuth, Thomas; van der Heijden, Marcel G A; Ghazoul, Jaboury; Egli, Simon

    2012-01-01

    Many trees species form symbiotic associations with ectomycorrhizal (ECM) fungi, which improve nutrient and water acquisition of their host. Until now it is unclear whether the species richness of ECM fungi is beneficial for tree seedling performance, be it during moist conditions or drought. We performed a pot experiment using Pinus sylvestris seedlings inoculated with four selected ECM fungi (Cenococcum geophilum, Paxillus involutus, Rhizopogon roseolus and Suillus granulatus) to investigate (i) whether these four ECM fungi, in monoculture or in species mixtures, affect growth of P. sylvestris seedlings, and (ii) whether this effect can be attributed to species number per se or to species identity. Two different watering regimes (moist vs. dry) were applied to examine the context-dependency of the results. Additionally, we assessed the activity of eight extracellular enzymes in the root tips. Shoot growth was enhanced in the presence of S. granulatus, but not by any other ECM fungal species. The positive effect of S. granulatus on shoot growth was more pronounced under moist (threefold increase) than under dry conditions (twofold increase), indicating that the investigated ECM fungi did not provide additional support during drought stress. The activity of secreted extracellular enzymes was higher in S. granulatus than in any other species. In conclusion, our findings suggest that ECM fungal species composition may affect seedling performance in terms of aboveground biomass.

  18. Using single strand conformational polymorphisms (SSCP) to identify Phytophthora species in Oregon forests affected by sudden oak death

    Science.gov (United States)

    E. Hansen; C. Hesse; P. Reeser; W. Sutton; L. Winton

    2006-01-01

    Phytophthora species are abundant in streams, widespread in soils and occasionally found in diseased plants in the tanoak forests of southwestern Oregon. It is time-consuming and expensive to identify hundreds of isolates to species using morphology or internal transribed spacer (ITS) sequencing. We modified a published Phytophthora...

  19. Specific leaf mass, fresh: dry weight ratio, sugar and protein contents in species of Lamiaceae from different light environments

    Directory of Open Access Journals (Sweden)

    M Castrillo

    2005-06-01

    Full Text Available 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 basilicum (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 (SLMand fresh: dry weight (FW/DW ratios were calculated. The higher values for soluble sugars contents were present in sun species: Lj, Pa, Ocb, Occ, Or. m, 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 be shade-tolerant and able to survive in sunny environments. Perhaps these species originated in shaded environments and have been adapting to sunny habitats.Rev.Biol.Trop.53(1-2:23-28.Epub 2005 Jun 24En once especies de la familia Lamiaceae: Plecthranthus

  20. Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species.

    Science.gov (United States)

    Morphew, Russell M; Wilkinson, Toby J; Mackintosh, Neil; Jahndel, Veronika; Paterson, Steve; McVeigh, Paul; Abbas Abidi, Syed M; Saifullah, Khalid; Raman, Muthusamy; Ravikumar, Gopalakrishnan; LaCourse, James; Maule, Aaron; Brophy, Peter M

    2016-09-02

    The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a foodborne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on triclabendazole (TCBZ), and overuse has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty-acid-binding protein (FABP) superfamily has proposed multifunctional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterized FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome, and EST data mining with proteomics and phylogenetics to rev