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Sample records for complex peptide mixtures

  1. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures.

    Science.gov (United States)

    Semashko, Tatiana A; Vorotnikova, Elena A; Sharikova, Valeriya F; Vinokurov, Konstantin S; Smirnova, Yulia A; Dunaevsky, Yakov E; Belozersky, Mikhail A; Oppert, Brenda; Elpidina, Elena N; Filippova, Irina Y

    2014-03-15

    This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes. Published by Elsevier Inc.

  2. Sample displacement chromatography as a method for purification of proteins and peptides from complex mixtures

    Science.gov (United States)

    Gajdosik, Martina Srajer; Clifton, James; Josic, Djuro

    2012-01-01

    Sample displacement chromatography (SDC) in reversed-phase and ion-exchange modes was introduced approximately twenty years ago. This method takes advantage of relative binding affinities of components in a sample mixture. During loading, there is a competition among different sample components for the sorption on the surface of the stationary phase. SDC was first used for the preparative purification of proteins. Later, it was demonstrated that this kind of chromatography can also be performed in ion-exchange, affinity and hydrophobic-interaction mode. It has also been shown that SDC can be performed on monoliths and membrane-based supports in both analytical and preparative scale. Recently, SDC in ion-exchange and hydrophobic interaction mode was also employed successfully for the removal of trace proteins from monoclonal antibody preparations and for the enrichment of low abundance proteins from human plasma. In this review, the principals of SDC are introduced, and the potential for separation of proteins and peptides in micro-analytical, analytical and preparative scale is discussed. PMID:22520159

  3. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

    Czech Academy of Sciences Publication Activity Database

    Semashko, T. A.; Vorotnikova, E. A.; Sharikova, V. F.; Vinokurov, Konstantin; Smirnova, Y. A.; Dunaevsky, Y. E.; Belozersky, M. A.; Oppert, B.; Elpidina, E. N.; Filippova, I. Y.

    2014-01-01

    Roč. 449, č. 1 (2014), s. 179-187 ISSN 0003-2697 Grant - others:Russian Foundation for Basic Research(RU) 12-04-01562-a; Russian Foundation for Basic Research(RU) 12-03-01057-a; ISTC (RU) 3455 Institutional support: RVO:60077344 Keywords : cysteine peptidases * substrates of peptidases * selective peptide substrates Subject RIV: CE - Biochemistry Impact factor: 2.219, year: 2014 http://www.sciencedirect.com/science/article/pii/S0003269713006180#

  4. Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures.

    Science.gov (United States)

    Li, Guo-Zhong; Vissers, Johannes P C; Silva, Jeffrey C; Golick, Dan; Gorenstein, Marc V; Geromanos, Scott J

    2009-03-01

    A novel database search algorithm is presented for the qualitative identification of proteins over a wide dynamic range, both in simple and complex biological samples. The algorithm has been designed for the analysis of data originating from data independent acquisitions, whereby multiple precursor ions are fragmented simultaneously. Measurements used by the algorithm include retention time, ion intensities, charge state, and accurate masses on both precursor and product ions from LC-MS data. The search algorithm uses an iterative process whereby each iteration incrementally increases the selectivity, specificity, and sensitivity of the overall strategy. Increased specificity is obtained by utilizing a subset database search approach, whereby for each subsequent stage of the search, only those peptides from securely identified proteins are queried. Tentative peptide and protein identifications are ranked and scored by their relative correlation to a number of models of known and empirically derived physicochemical attributes of proteins and peptides. In addition, the algorithm utilizes decoy database techniques for automatically determining the false positive identification rates. The search algorithm has been tested by comparing the search results from a four-protein mixture, the same four-protein mixture spiked into a complex biological background, and a variety of other "system" type protein digest mixtures. The method was validated independently by data dependent methods, while concurrently relying on replication and selectivity. Comparisons were also performed with other commercially and publicly available peptide fragmentation search algorithms. The presented results demonstrate the ability to correctly identify peptides and proteins from data independent acquisition strategies with high sensitivity and specificity. They also illustrate a more comprehensive analysis of the samples studied; providing approximately 20% more protein identifications, compared to

  5. Complex mixture analysis of peptides using LC/LC-MS/MS and data-dependent protein identification

    International Nuclear Information System (INIS)

    Wasinger, V.; Corthals, G.

    2001-01-01

    The comprehensive identification of proteins within complex solutions by mass-spectrometry largely depends on the sensitivity, resolving power and sampling efficiency of the technology. An integrated orthogonal approach using Strong Cation Exchange-Reverse Phase-MS/MS (SCX-RP-MS/MS) was used to evaluate the data-dependent Collision Induced Dissociation (CID) of yeast peptides. Reverse phase gradient times of 4, 10. 30, 90, and 180 minutes allowed the identification of hundreds of proteins in a nearly automated fashion from nuclear, membrane, and cytosolic distributions. Many proteins from typically difficult to resolve regions of two-dimensional gels, such as >100kDa, > pI 9.0 and Codon Adaptation Index < 0.2, were also identified using this multi-dimensional separation technology. Few low mass proteins (<10kDa) were identified. The impact of scan-range and duty-cycle on CID of peptides will be discussed

  6. Mutagenicity of complex mixtures

    International Nuclear Information System (INIS)

    Pelroy, R.A.

    1985-01-01

    The effect of coal-derived complex chemical mixtures on the mutagenicity of 6-aminochrysene (6-AC) was determined with Salmonella typhimurium TA98. Previous results suggested that the mutagenic potency of 6-AC for TA98 in the standard microsomal activation (Ames) assay increased if it was presented to the cells mixed with high-boiling coal liquids (CL) from the solvent refined coal (SRC) process. In this year's work, the apparent mutational synergism of CL and 6-AC was independently verified in a fluctuation bioassay which allowed quantitation of mutational frequencies and cell viability. The results of this assay system were similar to those in the Ames assay. Moreover, the fluctation assay revealed that mutagenesis and cellular toxicity induced by 6-AC were both strongly enhanced if 6-AC was presented to the cells mixed in a high-boiling CL. 4 figures

  7. Complex mixtures biostudies

    International Nuclear Information System (INIS)

    Springer, D.L.

    1987-01-01

    The objective of the project is to identify potential adverse biological activities associated with human exposures to complex organic mixtures (COM) from energy-related industries. Studies to identify the influence of chemical class fractions from a COM on the initiating activity of a known carcinogen, benzo(a)pyrene (BaP), demonstrated that the polycyclic aromatic hydrocarbons (PAH) and nitrogen-containing polycyclic aromatic compound (NPAC) fractions were the most effective inhibitors of initiation. In an effort to determine the contribution of BaP to the initiating activity of the COM, binding of radiolabeled BaP to mouse skin DNA was measured. Results indicated that binding of BaP to DNA decreased in the presence of the COM so that at initiating COM doses, BaP binding was near the limit detection. Addition of unlabeled BaP to the COM at an amount similar to that originally present in the COM did not significantly increase the binding. Studies to determine the rates of disappearance of carcinogenic PAH from the site of application on the skin indicated that half-lives for PAH differed by a factor of about 2. Analytical methods developed to identify PAH from COM which covalently bind to DNA demonstrated that the lower level of detection is approximately 200 picograms. Developmental studies demonstrated that both pregnant rats and mice treated dermally with a high-boiling COM developed fetuses with major malformations including cleft palate, small lungs, edema, and sagittal suture hemorrhages. 3 figures, 5 tables

  8. A microfluidic dialysis device for complex biological mixture SERS analysis

    KAUST Repository

    Perozziello, Gerardo; Candeloro, Patrizio; Gentile, Francesco T.; Coluccio, Maria Laura; Tallerico, Marco; De Grazia, Antonio; Nicastri, Annalisa; Perri, Angela Mena; Parrotta, Elvira; Pardeo, Francesca; Catalano, Rossella; Cuda, Giovanni; Di Fabrizio, Enzo M.

    2015-01-01

    In this paper, we present a microfluidic device fabricated with a simple and inexpensive process allowing rapid filtering of peptides from a complex mixture. The polymer microfluidic device can be used for sample preparation in biological

  9. Method of producing a peptide mixture

    DEFF Research Database (Denmark)

    2000-01-01

    The present invention relates to a method for industrial production of a peptide preparation having specific specifications by hydrolysis of a protein material, preferably based on whey. The method comprises several steps, which makes it easy to control the method so as to obtain a product which,.......g. because of low mineral content, is well suited for peritoneal dialysis and parenteral feeding. The method gives a high yield....

  10. Protein-peptide interactions in mixtures of whey peptides and whey proteins

    NARCIS (Netherlands)

    Creusot, N.; Gruppen, H.

    2007-01-01

    The effects of several conditions on the amounts and compositions of aggregates formed in mixtures of whey protein hydrolysate, made with Bacillus licheniformis protease, and whey protein isolate were investigated using response surface methodology. Next, the peptides present in the aggregates were

  11. Predicting skin permeability from complex chemical mixtures

    International Nuclear Information System (INIS)

    Riviere, Jim E.; Brooks, James D.

    2005-01-01

    Occupational and environmental exposure to topical chemicals is usually in the form of complex chemical mixtures, yet risk assessment is based on experimentally derived data from individual chemical exposures from a single, usually aqueous vehicle, or from computed physiochemical properties. We present an approach using hybrid quantitative structure permeation relationships (QSPeR) models where absorption through porcine skin flow-through diffusion cells is well predicted using a QSPeR model describing the individual penetrants, coupled with a mixture factor (MF) that accounts for physicochemical properties of the vehicle/mixture components. The baseline equation is log k p = c + mMF + aΣα 2 H + bΣβ 2 H + sπ 2 H + rR 2 + vV x where Σα 2 H is the hydrogen-bond donor acidity, Σβ 2 H is the hydrogen-bond acceptor basicity, π 2 H is the dipolarity/polarizability, R 2 represents the excess molar refractivity, and V x is the McGowan volume of the penetrants of interest; c, m, a, b, s, r, and v are strength coefficients coupling these descriptors to skin permeability (k p ) of 12 penetrants (atrazine, chlorpyrifos, ethylparathion, fenthion, methylparathion, nonylphenol, ρ-nitrophenol, pentachlorophenol, phenol, propazine, simazine, and triazine) in 24 mixtures. Mixtures consisted of full factorial combinations of vehicles (water, ethanol, propylene glycol) and additives (sodium lauryl sulfate, methyl nicotinate). An additional set of 4 penetrants (DEET, SDS, permethrin, ricinoleic acid) in different mixtures were included to assess applicability of this approach. This resulted in a dataset of 16 compounds administered in 344 treatment combinations. Across all exposures with no MF, R 2 for absorption was 0.62. With the MF, correlations increased up to 0.78. Parameters correlated to the MF include refractive index, polarizability and log (1/Henry's Law Constant) of the mixture components. These factors should not be considered final as the focus of these studies

  12. Tandem mass spectrometry: analysis of complex mixtures

    International Nuclear Information System (INIS)

    Singleton, K.E.

    1985-01-01

    Applications of tandem mass spectrometry (MS/MS) for the analysis of complex mixtures results in increased specificity and selectivity by using a variety of reagent gases in both negative and positive ion modes. Natural isotopic abundance ratios were examined in both simple and complex mixtures using parent, daughter and neutral loss scans. MS/MS was also used to discover new compounds. Daughter scans were used to identify seven new alkaloids in a cactus species. Three of these alkaloids were novel compounds, and included the first simple, fully aromatic isoquinoline alkaloids reported in Cactaceae. MS/MS was used to characterize the chemical reaction products of coal in studies designed to probe its macromolecular structure. Negative ion chemical ionization was utilized to study reaction products resulting from the oxidation of coal. Possible structural units in the precursor coal were predicted based on the reaction products identified, aliphatic and aromatic acids and their anhydrides. The MS/MS method was also used to characterize reaction products resulting from coal liquefaction and/or extraction. These studies illustrate the types of problems for which MS/MS is useful. Emphasis has been placed on characterization of complex mixtures by selecting experimental parameters which enhance the information obtained. The value of using MS/MS in conjunction with other analytical techniques as well as the chemical pretreatment is demonstrated

  13. A microfluidic dialysis device for complex biological mixture SERS analysis

    KAUST Repository

    Perozziello, Gerardo

    2015-08-01

    In this paper, we present a microfluidic device fabricated with a simple and inexpensive process allowing rapid filtering of peptides from a complex mixture. The polymer microfluidic device can be used for sample preparation in biological applications. The device is fabricated by micromilling and solvent assisted bonding, in which a microdialysis membrane (cut-off of 12-14 kDa) is sandwiched in between an upper and a bottom microfluidic chamber. An external frame connects the microfluidic device to external tubes, microvalves and syringe pumps. Bonding strength and interface sealing are pneumatically tested. Microfluidic protocols are also described by using the presented device to filter a sample composed of specific peptides (MW 1553.73 Da, at a concentration of 1.0 ng/μl) derived from the BRCA1 protein, a tumor-suppressor molecule which plays a pivotal role in the development of breast cancer, and albumin (MW 66.5 kDa, at a concentration of 35 μg/μl), the most represented protein in human plasma. The filtered samples coming out from the microfluidic device were subsequently deposited on a SERS (surface enhanced Raman scattering) substrate for further analysis by Raman spectroscopy. By using this approach, we were able to sort the small peptides from the bigger and highly concentrated protein albumin and to detect them by using a label-free technique at a resolution down to 1.0 ng/μl.

  14. Complex mixtures, complex responses: Assessing pharmaceutical mixtures using field and laboratory approaches

    Science.gov (United States)

    Schoenfuss, Heiko L.; Furlong, Edward T.; Phillips, Patrick J.; Scott, Tia-Marie; Kolpin, Dana W.; Cetkovic-Cvrlje, Marina; Lesteberg, Kelsey E.; Rearick, Daniel C.

    2016-01-01

    Pharmaceuticals are present in low concentrations (pharmaceutical formulation facilities. Using existing concentration data, the authors assessed pharmaceuticals in laboratory exposures of fathead minnows (Pimephales promelas) and added environmental complexity through effluent exposures. In the laboratory, larval and mature minnows were exposed to a simple opioid mixture (hydrocodone, methadone, and oxycodone), an opioid agonist (tramadol), a muscle relaxant (methocarbamol), a simple antidepressant mixture (fluoxetine, paroxetine, venlafaxine), a sleep aid (temazepam), or a complex mixture of all compounds. Larval minnow response to effluent exposure was not consistent. The 2010 exposures resulted in shorter exposed minnow larvae, whereas the larvae exposed in 2012 exhibited altered escape behavior. Mature minnows exhibited altered hepatosomatic indices, with the strongest effects in females and in mixture exposures. In addition, laboratory-exposed, mature male minnows exposed to all pharmaceuticals (except the selective serotonin reuptake inhibitor mixture) defended nest sites less rigorously than fish in the control group. Tramadol or antidepressant mixture exposure resulted in increased splenic T lymphocytes. Only male minnows exposed to whole effluent responded with increased plasma vitellogenin concentrations. Female minnows exposed to pharmaceuticals (except the opioid mixture) had larger livers, likely as a compensatory result of greater prominence of vacuoles in liver hepatocytes. The observed alteration of apical endpoints central to sustaining fish populations confirms that effluents containing waste streams from pharmaceutical formulation facilities can adversely impact fish populations but that the effects may not be temporally consistent. The present study highlights the importance of including diverse biological endpoints spanning levels of biological organization and life stages when assessing contaminant interactions.

  15. Health and environmental effects of complex chemical mixtures: proceedings

    Energy Technology Data Exchange (ETDEWEB)

    1985-01-01

    The Office of Health and Environmental Research (OHER) of the Department of Energy supports a broad long-term research program on human health and environmental effects from potential exposure to energy-related complex chemical mixtures. The program seeks basic mechanistic data on the effects of complex mixtures at the cellular, molecular, and whole animal levels to aid in predicting human health effects and seeks ecological data on biological and physical transformations in the mixtures, concentrations of the mixtures in various compartments of the environment, and potential routes for human exposure to these mixtures (e.g., food chain). On June 17-18, 1985, OHER held its First Annual Technical Meeting on the Complex Chemical Mixtures Program in Chicago, IL. The primary purpose of the meeting was to enable principal investigators to report the research status and accomplishments of ongoing complex chemical mixture studies supported by OHER. To help focus future research directions round table discussions were conducted.

  16. Complex contaminant mixtures in multistressor Appalachian riverscapes.

    Science.gov (United States)

    Merriam, Eric R; Petty, J Todd; Strager, Michael P; Maxwell, Aaron E; Ziemkiewicz, Paul F

    2015-11-01

    Runoff from watersheds altered by mountaintop mining in the Appalachian region (USA) is known to pollute headwater streams, yet regional-scale assessments of water quality have focused on salinization and selenium. The authors conducted a comprehensive survey of inorganic contaminants found in 170 stream segments distributed across a spectrum of historic and contemporary human land use. Principal component analysis identified 3 important dimensions of variation in water chemistry that were significantly correlated with contemporary surface mining (principal component 1: elevated dominant ions, sulfate, alkalinity, and selenium), coal geology and legacy mines (principal component 2: elevated trace metals), and residential development (principal component 3: elevated sodium and chloride). The combination of these 3 dominant sources of pollutants produced a complex stream-to-stream patchwork of contaminant mixtures. Seventy-five percent of headwater streams (catchments  5 km(2) ) were classified as having reference chemistries, and chemistries indicative of combined mining and development contaminants accounted for 47% of larger streams (compared with 26% of headwater streams). Extreme degradation of larger streams can be attributed to accumulation of contaminants from multiple human land use activities that include contemporary mountaintop mining, underground mining, abandoned mines, and untreated domestic wastewater. Consequently, water quality improvements in this region will require a multicontaminant remediation approach. © 2015 SETAC.

  17. Peptide de novo sequencing of mixture tandem mass spectra

    DEFF Research Database (Denmark)

    Gorshkov, Vladimir; Hotta, Stéphanie Yuki Kolbeck; Braga, Thiago Verano

    2016-01-01

    they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched...... complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced...... peptides. The improvement was in the range of 20–35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight...

  18. New dendrimer - Peptide host - Guest complexes: Towards dendrimers as peptide carriers

    DEFF Research Database (Denmark)

    Boas, Ulrik; Sontjens, S.H.M.; Jensen, Knud Jørgen

    2002-01-01

    Adamantyl urea and adamantyl thiourea modified poly(propylene imine) dendrimers act as hosts for N-terminal tert-butoxycarbonyl (Boc)-protected peptides and form chloroform-soluble complexes. investigations with NMR spectroscopy show that the peptide is bound to the dendrimer by ionic interactions...... between the dendrimer outer shell tertiary amines and the C-terminal carboxylic acid of the peptide, and also through host-urea to peptide-amide hydrogen bonding. The hydrogen-bonding nature of the peptide dendrimer interactions was further confirmed by using Fourier transform IR spectroscopy, for which...... the NH- and CO-stretch signals of the peptide amide moieties shift towards lower wave-numbers upon complexation with the dendrimer. Spatial analysis of the complexes with NOESY spectroscopy generally shows close proximity of the N-terminal Boc group of the peptide to the peripheral adamantyl groups...

  19. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Thingholm, Tine E; Jensen, Ole N

    2005-01-01

    based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased...... the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS...... was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented....

  20. New dendrimer - peptide host - guest complexes : towards dendrimers as peptide carriers

    NARCIS (Netherlands)

    Boas, U.; Sontjens, S.H.M.; Jensen, K.J.; Christensen, J.B.; Meijer, E.W.

    2002-01-01

    Adamantyl urea and adamantyl thiourea modified poly(propylene imine) dendrimers act as hosts for N-terminal tert-butoxycarbonyl (Boc)-protected peptides and form chloroform-soluble complexes. investigations with NMR spectroscopy show that the peptide is bound to the dendrimer by ionic interactions

  1. Aqueous Extract of Chrysanthemum morifolium Enhances the Antimelanogenic and Antioxidative Activities of the Mixture of Soy Peptide and Collagen Peptide

    Directory of Open Access Journals (Sweden)

    Min Gui

    2014-07-01

    Full Text Available The possible synergistic effect between the aqueous extract of Chrysanthemum morifolium (菊花 Jú Huā (AECM and the peptide mixture (PM containing soy peptide and collagen peptide was investigated in an ultraviolet (UV irradiation–induced skin damage mouse model. The irradiated mice were treated with the PM or PM+AECM (containing PM and AECM, respectively. Both PM and PM+AECM groups displayed an apparent photoprotective effect on the UV-irradiated skin damage of mice. Histological evaluation demonstrated that the epidermal hyperplasia and melanocytes in the basal epidermal layer of the UV-irradiated skin in mice decreased when treated with either PM or PM+AECM. Further study showed that soy peptide, collagen peptide, and AECM also inhibited the activities of mushroom tyrosinase with IC50 values of 82.3, 28.2, and 1.6 μg/ml, respectively. Additionally, PM+AECM reduced melanogenesis by 46.2% at the concentration of 10 mg/ml in B16 mouse melanoma cells. Meanwhile, the UV-induced increase of antioxidative indicators, including glutathione peroxidase (GSH-Px, superoxide dismutase (SOD, and malondialdehyde (MDA, was reduced significantly after treatment with 1.83 g/kg/dbw of PM+AECM. This evidence supported the synergistic antioxidative effect of AECM with PM. These results demonstrated that oral intake of PM and AECM had synergistic antimelanogenic and antioxidative effects in UV-irradiated mice.

  2. Different target surfaces for the analysis of peptides, peptide mixtures and peptide mass fingerprints by AP-MALDI ion trap-mass spectrometry.

    Science.gov (United States)

    Pittenauer, Ernst; Kassler, Alexander; Haubner, Roland; Allmaier, Günter

    2011-06-10

    The desorption/ionization behavior of individual peptides, an equimolare peptide mixture and a tryptic digest was investigated by AP-MALDI-IT-MS using four different target materials (gold-covered stainless steel (SS), titanium nitride-covered SS, hand-polished SS, and microdiamond-covered hardmetal) under identical conditions. Gold-covered as well as polished SS targets yielded comparable mass spectra for peptides and peptide mixture in the low pMol-range. The first target exhibited superior data down to the 10fMol-range. In contrast, titanium nitride-covered SS and microdiamond-covered hardmetal AP-MALDI-targets yielded poor sensitivity. These observations could be correlated with the surface roughness of the targets determined by 3D-confocal-white-light-microscopy. The roughest surfaces were found for titanium nitride-covered SS and microdiamond-covered hardmetal material showing both poor MS sensitivity. A less rough surface could be determined for the hand-polished SS target and the smoothest surface was found for the gold-covered target yielding the best sensitivity of all surfaces. These differences in the roughness having a strong impact on the ultimate sensitivity obtainable for peptide samples could be corroborated by electron microscopy. A peptide mixture covering a wide range of molecular weights and a tryptic protein digest (from 2-DE) exhibit the same behavior. This clearly indicates that the smooth gold-covered SS target is the surface of choice in AP-MALDI MS proteomics. Copyright © 2010. Published by Elsevier B.V.

  3. Characterisation of unresolved complex mixtures of hydrocarbons

    OpenAIRE

    Gough, Mark Adrian

    1989-01-01

    Metadata merged with duplicate record (http://hdl.handle.net/10026.1/666) on 20.12.2016 by CS (TIS). This is a digitised version of a thesis that was deposited in the University Library. If you are the author please contact PEARL Admin () to discuss options. The hydrocarbons of Recent Polluted.., sediments, in-reservoir and laboratory biodegraded crude oils, and certain petroleum products (e. g. lubricating oils) often display "humps" or Unresolved Complex...

  4. Chemometrics as a tool to analyse complex chemical mixtures

    DEFF Research Database (Denmark)

    Christensen, J. H.

    Chemical characterisation of contaminant mixtures is important for environmental forensics and risk assessment. The great challenge in future research lies in develop- ing suitable, rapid, reliable and objective methods for analysis of the composition of complex chemical mixtures. This thesis...... describes the development of such methods for assessing the identity (chemical fingerprinting) and fate (e.g. biodegradation) of petroleum hydrocarbon mixtures. The methods comply with the general concept that suitable methods must be rapid and inexpensive, objective with limited human in- tervention...... and at the same time must consider a substantial fraction of compounds in the complex mixture. A combination of a) limited sample preparation, b) rapid chemical screening analysis, c) fast and semi-automatic pre-processing, d) compre- hensive multivariate statistical data analysis and e) objective data evaluation...

  5. Structural organization and spectroscopy of peptide-actinide(IV) complexes

    International Nuclear Information System (INIS)

    Dahou, S.

    2010-01-01

    The contamination of living organisms by actinide elements is at the origin of both radiological and chemical toxicity that may lead to severe dysfunction. Most of the data available on the actinide interaction with biological systems are macroscopic physiological measurements and are lacking a molecular description of the systems. Because of the intricacy of these systems, classical biochemical methods are difficult to implement. Our strategy consisted in designing simplified biomimetic peptides, and describing the corresponding intramolecular interactions with actinides. A carboxylic pentapeptide of the form DDPDD has been at the starting point of this work in order to further assess the influence of the peptide sequence on the topology of the complexes.To do so, various linear (Asp/Ala permutations, peptoids) and cyclic analogues have been synthesized. Furthermore, in order to include the hydroxamic function (with a high affinity for Fe(III)) in the peptide, both desferrioxamine and acetohydroxamic acid have been investigated. However because of difficulties in synthesis, we have not been able to test these peptides. Three actinide cations have been considered at oxidation state +IV (Th, Np, Pu) and compared to Fe(III), often considered as a biological surrogate of Pu(IV). The spatial arrangement of the peptide around the cation has been probed by spectrophotometry and X-ray Absorption Spectroscopy. The spectroscopic data and EXAFS data adjustment lead us to rationalize the topology of the complexes as a function of the peptide sequence: mix hydroxy polynuclear species for linear and cyclic peptides, mononuclear for the desferrioxamine complexes. Furthermore, significant differences have appeared between Fe(III) and actinide(IV), related to differences of reactivity in aqueous medium. (author)

  6. Overtone Mobility Spectrometry: Part 4. OMS-OMS Analyses of Complex Mixtures

    Science.gov (United States)

    Kurulugama, Ruwan T.; Nachtigall, Fabiane M.; Valentine, Stephen J.; Clemmer, David E.

    2011-11-01

    A new, two-dimensional overtone mobility spectrometry (OMS-OMS) instrument is described for the analysis of complex peptide mixtures. OMS separations are based on the differences in mobilities of ions in the gas phase. The method utilizes multiple drift regions with modulated drift fields such that only ions with appropriate mobilities are transmitted to the detector. Here we describe a hybrid OMS-OMS combination that utilizes two independently operated OMS regions that are separated by an ion activation region. Mobility-selected ions from the first OMS region are exposed to energizing collisions and may undergo structural transitions before entering the second OMS region. This method generates additional peak capacity and allows for higher selectivity compared with the one-dimensional OMS method. We demonstrate the approach using a three-protein tryptic digest spiked with the peptide Substance P. The [M + 3H]3+ ion from Substance P can be completely isolated from other components in this complex mixture prior to introduction into the mass spectrometer.

  7. Evaluation of acute tryptophan depletion and sham depletion with a gelatin-based collagen peptide protein mixture

    DEFF Research Database (Denmark)

    Stenbæk, Dea Siggaard; Einarsdottir, H S; Goregliad-Fjaellingsdal, T

    2016-01-01

    Acute Tryptophan Depletion (ATD) is a dietary method used to modulate central 5-HT to study the effects of temporarily reduced 5-HT synthesis. The aim of this study is to evaluate a novel method of ATD using a gelatin-based collagen peptide (CP) mixture. We administered CP-Trp or CP+Trp mixtures...

  8. Bioassay-based risk assessment of complex mixtures

    International Nuclear Information System (INIS)

    Donnelly, K.C.; Huebner, H.J.

    1996-01-01

    The baseline risk assessment often plays an integral role in various decision-making processes at Superfund sites. The present study reports on risk characterizations prepared for seven complex mixtures using biological and chemical analysis. Three of the samples (A, B, and C) were complex mixtures of polycyclic aromatic hydrocarbons (PAHs) extracted from coal tar; while four samples extracted from munitions-contaminated soil contained primarily nitroaromatic hydrocarbons. The chemical-based risk assessment ranked sample C as least toxic, while the risk associated with samples A and B was approximately equal. The microbial bioassay was in general agreement for the coal tar samples. The weighted activity of the coal tar extracts in Salmonella was 4,960 for sample C, and 162,000 and 206,000 for samples A and B, respectively. The bacterial mutagenicity of 2,4,6-trinitrotoluene contaminated soils exhibited an indirect correlation with chemical-based risk assessment. The aqueous extract of sample 004 induced 1,292 net revertants in Salmonella, while the estimated risk to ingestion and dermal adsorption was 2E-9. The data indicate that the chemical-based risk assessment accurately predicted the genotoxicity of the PAHs, while the accuracy of the risk assessment for munitions contaminated soils was limited due to the presence of metabolites of TNT degradation. The biological tests used in this research provide a valuable compliment to chemical analysis for characterizing the genotoxic risk of complex mixtures

  9. Generation of two-dimensional binary mixtures in complex plasmas

    Science.gov (United States)

    Wieben, Frank; Block, Dietmar

    2016-10-01

    Complex plasmas are an excellent model system for strong coupling phenomena. Under certain conditions the dust particles immersed into the plasma form crystals which can be analyzed in terms of structure and dynamics. Previous experiments focussed mostly on monodisperse particle systems whereas dusty plasmas in nature and technology are polydisperse. Thus, a first and important step towards experiments in polydisperse systems are binary mixtures. Recent experiments on binary mixtures under microgravity conditions observed a phase separation of particle species with different radii even for small size disparities. This contradicts several numerical studies of 2D binary mixtures. Therefore, dedicated experiments are required to gain more insight into the physics of polydisperse systems. In this contribution first ground based experiments on two-dimensional binary mixtures are presented. Particular attention is paid to the requirements for the generation of such systems which involve the consideration of the temporal evolution of the particle properties. Furthermore, the structure of these two-component crystals is analyzed and compared to simulations. This work was supported by the Deutsche Forschungsgemeinschaft DFG in the framework of the SFB TR24 Greifswald Kiel, Project A3b.

  10. Bioassay-based risk assessment of complex mixtures

    Energy Technology Data Exchange (ETDEWEB)

    Donnelly, K.C.; Safe, S.H. [Texas A& M Univ., Houston, TX (United States); Randerath, K.; Randerath, E. [College Station and Baylor College of Medicine, Houston, TX (United States)

    1994-12-31

    To compare the standard chemical-based risk assessment with in vitro genotoxicity assays, two complex environmental mixtures from a wood preserving site were analyzed in the Salmonella/microsome and E. coli prophage induction assays. Using GC/MS, sample 003 was found to contain relatively low levels of polycyclic aromatic hydrocarbons (PNAs) and elevated levels of polychlorinated dibenzo-p-dioxins (PCDDs), while sample 005 had higher levels of PNAs and relatively low levels of PCDDs. The complex mixtures were sequentially extracted with methylene chloride and methanol for analysis in Salmonella, or extracted with 1:1 hexane: acetone mixture for analysis in the prophage induction assay. At a dose of 1.0 mg/plate in Salmonella strain TA98 with metabolic activation, the methanol extract of sample 003 induced 197 net revertants, while sample 005 induced 436 net revertants. In the prophage induction assay, with activation, the hexane:acetone extract of sample 003 induced a fold increase that was slightly lower than that observed with sample 005. The estimated incremental carcinogenic risk for dermal adsorption and ingestion was 1.5E-3 for sample 003, while for sample 005 the estimated risk was 1.5E-2. Thus, the sample which induced the maximum response in both bioassays also had the highest estimated cancer risk. However, the frequency of PNA-DNA adducts in both skin and liver tissues was appreciably higher with sample 005 than with sample 003.

  11. Application of Biologically-Based Lumping To Investigate the Toxicological Interactions of a Complex Gasoline Mixture

    Science.gov (United States)

    People are often exposed to complex mixtures of environmental chemicals such as gasoline, tobacco smoke, water contaminants, or food additives. However, investigators have often considered complex mixtures as one lumped entity. Valuable information can be obtained from these exp...

  12. Complex formation and vectorization of a phosphorothioate oligonucleotide with an amphipathic leucine- and lysine-rich peptide: study at molecular and cellular levels.

    Science.gov (United States)

    Boukhalfa-Heniche, Fatima-Zohra; Hernández, Belén; Gaillard, Stéphane; Coïc, Yves-Marie; Huynh-Dinh, Tam; Lecouvey, Marc; Seksek, Olivier; Ghomi, Mahmoud

    2004-04-15

    Optical spectroscopic techniques such as CD, Raman scattering, and fluorescence imaging allowed us to analyze the complex formation and vectorization of a single-stranded 20-mer phosphorothioate oligodeoxynucleotide with a 15-mer amphipathic peptide at molecular and cellular levels. Different solvent mixtures (methanol and water) and molecular ratios of peptide/oligodeoxynucleotide complexes were tested in order to overcome the problems related to solubility. Optimal conditions for both spectroscopic and cellular experiments were obtained with the molecular ratio peptide/oligodeoxynucleotide equal to 21:4, corresponding to a 7:5 ratio for their respective +/- charge ratio. At the molecular level, CD and Raman spectra were consistent with a alpha-helix conformation of the peptide in water or in a methanol-water mixture. The presence of methanol increased considerably the solubility of the peptide without altering its alpha-helix conformation, as evidenced by CD and Raman spectroscopies. UV absorption melting profile of the oligodeoxynucleotide gave rise to a flat melting profile, corresponding to its random structure in solution. Raman spectra of oligodeoxynucleotide/peptide complexes could only be studied in methanol/water mixture solutions. Drastic changes observed in Raman spectra have undoubtedly shown: (a) the perturbation occurred in the peptide secondary structure, and (b) possible interaction between the lysine residues of the peptide and the oligodeoxynucleotide. At the cellular level, the complex was prepared in a mixture of 10% methanol and 90% cell medium. Cellular uptake in optimal conditions for the oligodeoxynucleotide delivery with low cytotoxicity was controlled by fluorescence imaging allowing to specifically locate the compacted oligonucleotide labeled with fluorescein at its 5'-terminus with the peptide into human glioma cells after 1 h of incubation at 37 degrees C. Copyright 2004 Wiley Periodicals, Inc.

  13. The optimization of peptide cargo bound to MHC class I molecules by the peptide-loading complex.

    Science.gov (United States)

    Elliott, Tim; Williams, Anthony

    2005-10-01

    Major histocompatibility complex (MHC) class I complexes present peptides from both self and foreign intracellular proteins on the surface of most nucleated cells. The assembled heterotrimeric complexes consist of a polymorphic glycosylated heavy chain, non-polymorphic beta(2) microglobulin, and a peptide of typically nine amino acids in length. Assembly of the class I complexes occurs in the endoplasmic reticulum and is assisted by a number of chaperone molecules. A multimolecular unit termed the peptide-loading complex (PLC) is integral to this process. The PLC contains a peptide transporter (transporter associated with antigen processing), a thiooxido-reductase (ERp57), a glycoprotein chaperone (calreticulin), and tapasin, a class I-specific chaperone. We suggest that class I assembly involves a process of optimization where the peptide cargo of the complex is edited by the PLC. Furthermore, this selective peptide loading is biased toward peptides that have a longer off-rate from the assembled complex. We suggest that tapasin is the key chaperone that directs this action of the PLC with secondary contributions from calreticulin and possibly ERp57. We provide a framework model for how this may operate at the molecular level and draw parallels with the proposed mechanism of action of human leukocyte antigen-DM for MHC class II complex optimization.

  14. Molecular characterization of covalent complexes between tissue transglutaminase and gliadin peptides

    DEFF Research Database (Denmark)

    Fleckenstein, Burkhard; Qiao, Shuo-Wang; Larsen, Martin Røssel

    2004-01-01

    recognized by intestinal T cells from patients. Incubation of TG2 with gliadin peptides also results in the formation of covalent TG2-peptide complexes. Here we report the characterization of complexes between TG2 and two immunodominant gliadin peptides. Two types of covalent complexes were found......; the peptides are either linked via a thioester bond to the active site cysteine of TG2 or via isopeptide bonds to particular lysine residues of the enzyme. We quantified the number of gliadin peptides bound to TG2 under different conditions. After 30 min of incubation of TG2 at 1 microm with an equimolar ratio...... of peptides to TG2, approximately equal amounts of peptides were bound by thioester and isopeptide linkage. At higher peptide to TG2 ratios, more than one peptide was linked to TG2, and isopeptide bond formation dominated. The lysine residues in TG2 that act as acyl acceptors were identified by matrix...

  15. Dermal tumorigen PAH and complex mixtures for biological research

    International Nuclear Information System (INIS)

    Griest, W.H.; Guerin, M.R.; Ho, C.

    1985-01-01

    Thirteen commercially available, commonly reported four-five ring dermal tumorigen PAHs, were determined in a set of complex mixtures consisting of crude and upgraded coal liquids, and petroleum crude oils and their distillate fractions. Semi-preparative scale, normal phase high performance liquid chromatographic fractionation followed by capillary column gas chromatography or gas chromatography-mass spectroscopy were used for the measurements. Deuterated or carbon-14 labeled PAH served as internal standards or allowed recovery corrections. Approaches for the preparation and measurement of radiolabeled PAH were examined to provide chemical probes for biological study. Synthetic routes for production of 14 C labeled dihydrobenzo[a]pyrene and 14 C- or 3 H 10-azabenzo[a]pyrene are being studied to provide tracers for fundamental studies in tracheal transplant and skin penetration systems. (DT)

  16. Dynamics of major histocompatibility complex class I association with the human peptide-loading complex.

    Science.gov (United States)

    Panter, Michaela S; Jain, Ankur; Leonhardt, Ralf M; Ha, Taekjip; Cresswell, Peter

    2012-09-07

    Although the human peptide-loading complex (PLC) is required for optimal major histocompatibility complex class I (MHC I) antigen presentation, its composition is still incompletely understood. The ratio of the transporter associated with antigen processing (TAP) and MHC I to tapasin, which is responsible for MHC I recruitment and peptide binding optimization, is particularly critical for modeling of the PLC. Here, we characterized the stoichiometry of the human PLC using both biophysical and biochemical approaches. By means of single-molecule pulldown (SiMPull), we determined a TAP/tapasin ratio of 1:2, consistent with previous studies of insect-cell microsomes, rat-human chimeric cells, and HeLa cells expressing truncated TAP subunits. We also report that the tapasin/MHC I ratio varies, with the PLC population comprising both 2:1 and 2:2 complexes, based on mutational and co-precipitation studies. The MHC I-saturated PLC may be particularly prevalent among peptide-selective alleles, such as HLA-C4. Additionally, MHC I association with the PLC increases when its peptide supply is reduced by inhibiting the proteasome or by blocking TAP-mediated peptide transport using viral inhibitors. Taken together, our results indicate that the composition of the human PLC varies under normal conditions and dynamically adapts to alterations in peptide supply that may arise during viral infection. These findings improve our understanding of the quality control of MHC I peptide loading and may aid the structural and functional modeling of the human PLC.

  17. Bioavailability of tryptophan from a single oral dose of a trytophan-enriched peptide mixture in healthy men

    NARCIS (Netherlands)

    Brink, E.J.; Boelsma, E.; Steijns, J.; Hendriks, H.F.J.

    2004-01-01

    The aim of the study was to investigate the bioavailability of tryptophan (Trp) from a Trp-enriched peptide mixture in healthy men. A second objective was to investigate the effect of this Trp-enriched protein hydrolysate on potential parameters of serotonergic activity. serum serotonim melatonin

  18. Measuring pair-wise molecular interactions in a complex mixture

    Science.gov (United States)

    Chakraborty, Krishnendu; Varma, Manoj M.; Venkatapathi, Murugesan

    2016-03-01

    Complex biological samples such as serum contain thousands of proteins and other molecules spanning up to 13 orders of magnitude in concentration. Present measurement techniques do not permit the analysis of all pair-wise interactions between the components of such a complex mixture to a given target molecule. In this work we explore the use of nanoparticle tags which encode the identity of the molecule to obtain the statistical distribution of pair-wise interactions using their Localized Surface Plasmon Resonance (LSPR) signals. The nanoparticle tags are chosen such that the binding between two molecules conjugated to the respective nanoparticle tags can be recognized by the coupling of their LSPR signals. This numerical simulation is done by DDA to investigate this approach using a reduced system consisting of three nanoparticles (a gold ellipsoid with aspect ratio 2.5 and short axis 16 nm, and two silver ellipsoids with aspect ratios 3 and 2 and short axes 8 nm and 10 nm respectively) and the set of all possible dimers formed between them. Incident light was circularly polarized and all possible particle and dimer orientations were considered. We observed that minimum peak separation between two spectra is 5 nm while maximum is 184nm.

  19. Toxicological risk assessment of complex mixtures through the Wtox model

    Directory of Open Access Journals (Sweden)

    William Gerson Matias

    2015-01-01

    Full Text Available Mathematical models are important tools for environmental management and risk assessment. Predictions about the toxicity of chemical mixtures must be enhanced due to the complexity of eects that can be caused to the living species. In this work, the environmental risk was accessed addressing the need to study the relationship between the organism and xenobiotics. Therefore, ve toxicological endpoints were applied through the WTox Model, and with this methodology we obtained the risk classication of potentially toxic substances. Acute and chronic toxicity, citotoxicity and genotoxicity were observed in the organisms Daphnia magna, Vibrio scheri and Oreochromis niloticus. A case study was conducted with solid wastes from textile, metal-mechanic and pulp and paper industries. The results have shown that several industrial wastes induced mortality, reproductive eects, micronucleus formation and increases in the rate of lipid peroxidation and DNA methylation of the organisms tested. These results, analyzed together through the WTox Model, allowed the classication of the environmental risk of industrial wastes. The evaluation showed that the toxicological environmental risk of the samples analyzed can be classied as signicant or critical.

  20. Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase

    Science.gov (United States)

    Nguyen, Huong T. H.; Andrikopoulos, Prokopis C.; Rulíšek, Lubomír; Shaffer, Christopher J.; Tureček, František

    2018-05-01

    We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355 nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100 ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. [Figure not available: see fulltext.

  1. Mixture-based combinatorial libraries from small individual peptide libraries: a case study on α1-antitrypsin deficiency.

    Science.gov (United States)

    Chang, Yi-Pin; Chu, Yen-Ho

    2014-05-16

    The design, synthesis and screening of diversity-oriented peptide libraries using a "libraries from libraries" strategy for the development of inhibitors of α1-antitrypsin deficiency are described. The major buttress of the biochemical approach presented here is the use of well-established solid-phase split-and-mix method for the generation of mixture-based libraries. The combinatorial technique iterative deconvolution was employed for library screening. While molecular diversity is the general consideration of combinatorial libraries, exquisite design through systematic screening of small individual libraries is a prerequisite for effective library screening and can avoid potential problems in some cases. This review will also illustrate how large peptide libraries were designed, as well as how a conformation-sensitive assay was developed based on the mechanism of the conformational disease. Finally, the combinatorially selected peptide inhibitor capable of blocking abnormal protein aggregation will be characterized by biophysical, cellular and computational methods.

  2. Mixture

    Directory of Open Access Journals (Sweden)

    Silva-Aguilar Martín

    2011-01-01

    Full Text Available Metals are ubiquitous pollutants present as mixtures. In particular, mixture of arsenic-cadmium-lead is among the leading toxic agents detected in the environment. These metals have carcinogenic and cell-transforming potential. In this study, we used a two step cell transformation model, to determine the role of oxidative stress in transformation induced by a mixture of arsenic-cadmium-lead. Oxidative damage and antioxidant response were determined. Metal mixture treatment induces the increase of damage markers and the antioxidant response. Loss of cell viability and increased transforming potential were observed during the promotion phase. This finding correlated significantly with generation of reactive oxygen species. Cotreatment with N-acetyl-cysteine induces effect on the transforming capacity; while a diminution was found in initiation, in promotion phase a total block of the transforming capacity was observed. Our results suggest that oxidative stress generated by metal mixture plays an important role only in promotion phase promoting transforming capacity.

  3. PeptideNavigator: An interactive tool for exploring large and complex data sets generated during peptide-based drug design projects.

    Science.gov (United States)

    Diller, Kyle I; Bayden, Alexander S; Audie, Joseph; Diller, David J

    2018-01-01

    There is growing interest in peptide-based drug design and discovery. Due to their relatively large size, polymeric nature, and chemical complexity, the design of peptide-based drugs presents an interesting "big data" challenge. Here, we describe an interactive computational environment, PeptideNavigator, for naturally exploring the tremendous amount of information generated during a peptide drug design project. The purpose of PeptideNavigator is the presentation of large and complex experimental and computational data sets, particularly 3D data, so as to enable multidisciplinary scientists to make optimal decisions during a peptide drug discovery project. PeptideNavigator provides users with numerous viewing options, such as scatter plots, sequence views, and sequence frequency diagrams. These views allow for the collective visualization and exploration of many peptides and their properties, ultimately enabling the user to focus on a small number of peptides of interest. To drill down into the details of individual peptides, PeptideNavigator provides users with a Ramachandran plot viewer and a fully featured 3D visualization tool. Each view is linked, allowing the user to seamlessly navigate from collective views of large peptide data sets to the details of individual peptides with promising property profiles. Two case studies, based on MHC-1A activating peptides and MDM2 scaffold design, are presented to demonstrate the utility of PeptideNavigator in the context of disparate peptide-design projects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Heat shock protein-peptide complex-96 (Vitespen for the treatment of cancer

    Directory of Open Access Journals (Sweden)

    Robert J. Amato

    2011-12-01

    Full Text Available Heat shock proteins (HSPs are the most abundant and ubiquitous soluble intracellular proteins. Members of the HSP family bind peptides, they include antigenic peptides generated within cells. HSPs also interact with antigen-presenting cells (APCs through CD91 and other receptors, eliciting a cascade of events that includes re-presentation of HSP-chaperoned peptides by major histocompatability complex (MHC, translocation of nuclear factorkappaB (NFkB into the nuclei, and maturation of dendritic cells (DCs. These consequences point to a key role of heat shock proteins in fundamental immunological phenomena such as activation of APCs, indirect presentation (or crosspriming of antigenic peptides, and chaperoning of peptides during antigen presentation. The properties of HSPs also allow them to be used for immunotherapy of cancers and infections in novel ways. This paper reviews the development and clinical trial progress of vitespen, an HSP peptide complex vaccine based on tumor-derived glycoprotein 96.

  5. Ga+ TOF-SIMS lineshape analysis for resolution enhancement of MALDI MS spectra of a peptide mixture

    International Nuclear Information System (INIS)

    Malyarenko, D.I.; Chen, H.; Wilkerson, A.L.; Tracy, E.R.; Cooke, W.E.; Manos, D.M.; Sasinowski, M.; Semmes, O.J.

    2004-01-01

    The use of mass spectrometry to obtain molecular profiles indicative of alteration of concentrations of peptides in body fluids is currently the subject of intense investigation. For surface-based time-of-flight mass spectrometry the reliability and specificity of such profiling methods depend both on the resolution of the measuring instrument and on the preparation of samples. The present work is a part of a program to use Ga + beam TOF-SIMS alone, and as an adjunct to MALDI, in the development of reliable protein and peptide markers for diseases. Here, we describe techniques to prepare samples of relatively high-mass peptides, which serve as calibration standards and proxies for biomarkers. These are: Arg8-vasopressin, human angiotensin II, and somatostatin. Their TOF-SIMS spectra show repeatable characteristic features, with mass resolution exceeding 2000, including parent peaks and chemical adducts. The lineshape analysis for high-resolution parent peaks is shown to be useful for filter construction and deconvolution of inferior resolution SELDI-TOF spectra of calibration peptide mixture

  6. Structure of HLA-A*1101 in complex with a hepatitis B peptide homologue

    DEFF Research Database (Denmark)

    Blicher, Thomas; Kastrup, Jette Sandholm; Pedersen, Lars Østergaard

    2006-01-01

    A high-resolution structure of the human MHC-I molecule HLA-A*1101 is presented in which it forms a complex with a sequence homologue of a peptide that occurs naturally in hepatitis B virus DNA polymerase. The sequence of the bound peptide is AIMPARFYPK, while that of the corresponding natural...

  7. Laser absorption spectroscopy - Method for monitoring complex trace gas mixtures

    Science.gov (United States)

    Green, B. D.; Steinfeld, J. I.

    1976-01-01

    A frequency stabilized CO2 laser was used for accurate determinations of the absorption coefficients of various gases in the wavelength region from 9 to 11 microns. The gases investigated were representative of the types of contaminants expected to build up in recycled atmospheres. These absorption coefficients were then used in determining the presence and amount of the gases in prepared mixtures. The effect of interferences on the minimum detectable concentration of the gases was measured. The accuracies of various methods of solution were also evaluated.

  8. Gel and gel-free approaches for the quantitative characterisation of complex protein mixtures

    CSIR Research Space (South Africa)

    Buthelezi, S

    2012-10-01

    Full Text Available reliable set of methods for profiling proteins in a complex mixture in order to allow for the mining of low abundant species. To achieve this, several fractionation techniques were applied to samples of bovine hepatic tissue. These included two... further separated via low pH reverse phase (RP) chromatography before being introduced for mass spectrometric analysis. MATERIALS AND METHODS Figure 1: Study design to analyse a complex mixture of proteins extracted from hepatic tissue. To determine...

  9. Differential effects of a complex organochlorine mixture on the proliferation of breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Aube, Michel, E-mail: 4aubem@videotron.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Larochelle, Christian, E-mail: christian.larochelle@inspq.qc.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Ayotte, Pierre, E-mail: pierre.ayotte@inspq.qc.ca [Axe de recherche en sante des populations et environnementale, Centre de recherche du Centre hospitalier universitaire de Quebec and Universite Laval, 2875 Boulevard Laurier, Edifice Delta 2, bureau 600, Quebec, QC, Canada G1V 2M2 (Canada); Laboratoire de Toxicologie, Institut national de sante publique du Quebec, 945 avenue Wolfe, Quebec, QC, Canada G1V 5B3 (Canada)

    2011-04-15

    Organochlorine compounds (OCs) are a group of persistent chemicals that accumulate in fatty tissues with age. Although OCs has been tested individually for their capacity to induce breast cancer cell proliferation, few studies examined the effect of complex mixtures that comprise compounds frequently detected in the serum of women. We constituted such an OC mixture containing 15 different components in environmentally relevant proportions and assessed its proliferative effects in four breast cancer cell lines (MCF-7, T47D, CAMA-1, MDAMB231) and in non-cancerous CV-1 cells. We also determined the capacity of the mixture to modulate cell cycle stage of breast cancer cells and to induce estrogenic and antiandrogenic effects using gene reporter assays. We observed that low concentrations of the mixture (100x10{sup 3} and 50x10{sup 3} dilutions) stimulated the proliferation of MCF-7 cells while higher concentrations (10x10{sup 3} and 5x10{sup 3} dilutions) had the opposite effect. In contrast, the mixture inhibited the proliferation of non-hormone-dependent cell lines. The mixture significantly increased the number of MCF-7 cells entering the S phase, an effect that was blocked by the antiestrogen ICI 182,780. Low concentrations of the mixture also caused an increase in CAMA-1 cell proliferation but only in the presence estradiol and dihydrotestosterone (p<0.05 at the 50x10{sup 3} dilution). DDT analogs and polychlorinated biphenyls all had the capacity to stimulate the proliferation of CAMA-1 cells in the presence of sex steroids. Reporter gene assays further revealed that the mixture and several of its constituents (DDT analogs, aldrin, dieldrin, {beta}-hexachlorocyclohexane, toxaphene) induced estrogenic effects, whereas the mixture and several components (DDT analogs, aldrin, dieldrin and PCBs) inhibited the androgen signaling pathway. Our results indicate that the complex OC mixture increases the proliferation of MCF-7 cells due to its estrogenic potential. The

  10. Status Report on the High-Throughput Characterization of Complex Intact O-Glycopeptide Mixtures

    Science.gov (United States)

    Pap, Adam; Klement, Eva; Hunyadi-Gulyas, Eva; Darula, Zsuzsanna; Medzihradszky, Katalin F.

    2018-05-01

    A very complex mixture of intact, human N- and O-glycopeptides, enriched from the tryptic digest of urinary proteins of three healthy donors using a two-step lectin affinity enrichment, was analyzed by LC-MS/MS, leading to approximately 45,000 glycopeptide EThcD spectra. Two search engines, Byonic and Protein Prospector, were used for the interpretation of the data, and N- and O-linked glycopeptides were assigned from separate searches. The identification rate was very low in all searches, even when results were combined. Thus, we investigated the reasons why was it so, to help to improve the identification success rate. Focusing on O-linked glycopeptides, we noticed that in EThcD, larger glycan oxonium ions better survive the activation than those in HCD. These fragments, combined with reducing terminal Y ions, provide important information about the glycan(s) present, so we investigated whether filtering the peaklists for glycan oxonium ions indicating the presence of a tetra- or hexasaccharide structure would help to reveal all molecules containing such glycans. Our study showed that intact glycans frequently do not survive even mild supplemental activation, meaning one cannot rely on these oxonium ions exclusively. We found that ETD efficiency is still a limiting factor, and for highly glycosylated peptides, the only information revealed in EThcD was related to the glycan structures. The limited overlap of results delivered by the two search engines draws attention to the fact that automated data interpretation of O-linked glycopeptides is not even close to being solved. [Figure not available: see fulltext.

  11. Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes

    DEFF Research Database (Denmark)

    Schmidt, Esben G W; Buus, Soren; Thorn, Mette

    2009-01-01

    to in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood mononuclear...

  12. Structural requirements and biological significance of interactions between peptides and the major histocompatibility complex

    DEFF Research Database (Denmark)

    Grey, H M; Buus, S; Colon, S

    1989-01-01

    Previous studies indicate that T cells recognize a complex between the major histocompatibility complex (MHC) restriction-element and peptide-antigen fragments. Two aspects of this complex formation are considered in this paper: (1) what is the nature of the specificity of the interactions that a...... of binding to Ia (i.e. determinant selection was operative), we found that about 40% of Ia-binding peptides were not immunogenic (i.e. there were also 'holes in the T-cell repertoire')....... responsiveness, we present data that suggest both mechanisms operate in concert with one another. Thus only about 30% of a collection of peptides that in sum represent the sequence of a protein molecule were found to bind to Ia. Although immunogenicity was restricted to those peptides that were capable...

  13. The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes.

    Directory of Open Access Journals (Sweden)

    Siri Dørum

    Full Text Available BACKGROUND: Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2. In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. METHODS: A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. RESULTS: We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. CONCLUSION: TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease.

  14. The Preferred Substrates for Transglutaminase 2 in a Complex Wheat Gluten Digest Are Peptide Fragments Harboring Celiac Disease T-Cell Epitopes

    Science.gov (United States)

    Dørum, Siri; Arntzen, Magnus Ø.; Qiao, Shuo-Wang; Holm, Anders; Koehler, Christian J.; Thiede, Bernd; Sollid, Ludvig M.; Fleckenstein, Burkhard

    2010-01-01

    Background Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2). In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. Methods A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. Results We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. Conclusion TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease. PMID:21124911

  15. Towards a realistic risk characterization of complex mixtures using in vitro bioassays

    NARCIS (Netherlands)

    Montano Garces, M.

    2013-01-01

    This thesis aims to better understand and further improve the relevance and reliabilityof in vitro bioassaysfor a biobased risk characterisation of complex mixtures, with special focus on persistent organic pollutants (POPs) in sediments.

    In Chapter 1 the importance of complex

  16. Complex resistivity signatures of ethanol in sand-clay mixtures

    Science.gov (United States)

    Personna, Yves Robert; Slater, Lee; Ntarlagiannis, Dimitrios; Werkema, Dale D.; Szabo, Zoltan

    2013-01-01

    We performed complex resistivity (CR) measurements on laboratory columns to investigate changes in electrical properties as a result of varying ethanol (EtOH) concentration (0% to 30% v/v) in a sand–clay (bentonite) matrix. We applied Debye decomposition, a phenomenological model commonly used to fit CR data, to determine model parameters (time constant: τ, chargeability: m, and normalized chargeability: mn). The CR data showed a significant (P ≤ 0.001) time-dependent variation in the clay driven polarization response (~ 12 mrad) for 0% EtOH concentration. This temporal variation probably results from the clay–water reaction kinetics trending towards equilibrium in the sand–clay–water system. The clay polarization is significantly suppressed (P ≤ 0.001) for both measured phase (ϕ) and imaginary conductivity (σ″) with increasing EtOH concentration. Normalized chargeability consistently decreases (by up to a factor of ~ 2) as EtOH concentration increases from 0% to 10% and 10 to 20%, respectively. We propose that such suppression effects are associated with alterations in the electrical double layer (EDL) at the clay–fluid interface due to (a) strong EtOH adsorption on clay, and (b) complex intermolecular EtOH–water interactions and subsequent changes in ionic mobility on the surface in the EDL. Changes in the CR data following a change of the saturating fluid from EtOH 20% to plain water indicate strong hysteresis effects in the electrical response, which we attribute to persistent EtOH adsorption on clay. Our results demonstrate high sensitivity of CR measurements to clay–EtOH interactions in porous media, indicating the potential application of this technique for characterization and monitoring of ethanol contamination in sediments containing clays.

  17. Deciphering complex patterns of class-I HLA-peptide cross-reactivity via hierarchical grouping.

    Science.gov (United States)

    Mukherjee, Sumanta; Warwicker, Jim; Chandra, Nagasuma

    2015-07-01

    T-cell responses in humans are initiated by the binding of a peptide antigen to a human leukocyte antigen (HLA) molecule. The peptide-HLA complex then recruits an appropriate T cell, leading to cell-mediated immunity. More than 2000 HLA class-I alleles are known in humans, and they vary only in their peptide-binding grooves. The polymorphism they exhibit enables them to bind a wide range of peptide antigens from diverse sources. HLA molecules and peptides present a complex molecular recognition pattern, as many peptides bind to a given allele and a given peptide can be recognized by many alleles. A powerful grouping scheme that not only provides an insightful classification, but is also capable of dissecting the physicochemical basis of recognition specificity is necessary to address this complexity. We present a hierarchical classification of 2010 class-I alleles by using a systematic divisive clustering method. All-pair distances of alleles were obtained by comparing binding pockets in the structural models. By varying the similarity thresholds, a multilevel classification was obtained, with 7 supergroups, each further subclassifying to yield 72 groups. An independent clustering performed based only on similarities in their epitope pools correlated highly with pocket-based clustering. Physicochemical feature combinations that best explain the basis of clustering are identified. Mutual information calculated for the set of peptide ligands enables identification of binding site residues contributing to peptide specificity. The grouping of HLA molecules achieved here will be useful for rational vaccine design, understanding disease susceptibilities and predicting risk of organ transplants.

  18. Combining measurements to estimate properties and characterization extent of complex biochemical mixtures; applications to Heparan Sulfate

    Science.gov (United States)

    Pradines, Joël R.; Beccati, Daniela; Lech, Miroslaw; Ozug, Jennifer; Farutin, Victor; Huang, Yongqing; Gunay, Nur Sibel; Capila, Ishan

    2016-04-01

    Complex mixtures of molecular species, such as glycoproteins and glycosaminoglycans, have important biological and therapeutic functions. Characterization of these mixtures with analytical chemistry measurements is an important step when developing generic drugs such as biosimilars. Recent developments have focused on analytical methods and statistical approaches to test similarity between mixtures. The question of how much uncertainty on mixture composition is reduced by combining several measurements still remains mostly unexplored. Mathematical frameworks to combine measurements, estimate mixture properties, and quantify remaining uncertainty, i.e. a characterization extent, are introduced here. Constrained optimization and mathematical modeling are applied to a set of twenty-three experimental measurements on heparan sulfate, a mixture of linear chains of disaccharides having different levels of sulfation. While this mixture has potentially over two million molecular species, mathematical modeling and the small set of measurements establish the existence of nonhomogeneity of sulfate level along chains and the presence of abundant sulfate repeats. Constrained optimization yields not only estimations of sulfate repeats and sulfate level at each position in the chains but also bounds on these levels, thereby estimating the extent of characterization of the sulfation pattern which is achieved by the set of measurements.

  19. Conformational determinants of phosphotyrosine peptides complexed with the Src SH2 domain.

    Directory of Open Access Journals (Sweden)

    Joseph Nachman

    2010-06-01

    Full Text Available The inhibition of specific SH2 domain mediated protein-protein interactions as an effective chemotherapeutic approach in the treatment of diseases remains a challenge. That different conformations of peptide-ligands are preferred by different SH2 domains is an underappreciated observation from the structural analysis of phosphotyrosine peptide binding to SH2 domains that may aid in future drug design. To explore the nature of ligand binding, we use simulated annealing (SA to sample the conformational space of phosphotyrosine-containing peptides complexed with the Src SH2 domain. While in good agreement with the crystallographic and NMR studies of high-affinity phosphopeptide-SH2 domain complexes, the results suggest that the structural basis for phopsphopeptide- Src SH2 interactions is more complex than the "two-pronged plug two-hole socket" model. A systematic study of peptides of type pYEEX, where pY is phosphotyrosine and X is a hydrophobic residue, indicates that these peptides can assume two conformations, one extended and one helical, representing the balance between the interaction of residue X with the hydrophobic hole on the surface of the Src SH2 domain, and its contribution to the inherent tendency of the two glutamic acids to form an alpha-helix. In contrast, a beta-turn conformation, almost identical to that observed in the crystal structure of pYVNV bound to the Grb2 SH2 domain, predominates for pYXNX peptides, even in the presence of isoleucine at the third position. While peptide binding affinities, as measured by fluorescence polarization, correlate with the relative proportion of extended peptide conformation, these results suggest a model where all three residues C-terminal to the phosphotyrosine determine the conformation of the bound phosphopeptide. The information obtained in this work can be used in the design of specific SH2 domain inhibitors.

  20. A functional mimic of natural peroxidases : synthesis and catalytic activity of a non-heme iron peptide hydroperoxide complex

    NARCIS (Netherlands)

    Choma, CT; Schudde, EP; Kellogg, RM; Robillard, GT; Feringa, BL

    1998-01-01

    Site-selective attachment of unprotected peptides to a non-heme iron complex is achieved by displacing two halides on the catalyst by peptide caesium thiolates, This coupling approach should be compatible with any peptide sequence provided there is only a single reduced cysteine. The oxidation

  1. Mutagenic hazards of complex polycyclic aromatic hydrocarbon mixtures in contaminated soil

    Energy Technology Data Exchange (ETDEWEB)

    Lemieux, C.L.; Lambert, A.B.; Lundstedt, S.; Tysklind, M.; White, P.A. [Health Canada, Ottawa, ON (Canada). Safe Environment Program

    2008-04-15

    The objective of the present study was to evaluate hazard/risk assessment methods for complex environmental mixtures that involve a targeted, priority chemical approach based on the cumulative hazard/risk of known mixture components or analyses of sufficiently similar mixtures. Ten polycyclic aromatic hydrocarbon (PAH)-contaminated soils were separated into nonpolar and semipolar fractions, and both fractions elicited positive responses on the Salmonella reverse mutation assay. Targeted and nontargeted methods of hazard prediction routinely overestimated mutagenic activities for the nonpolar soil fractions, suggesting nonadditive interactions of PAHs in complex mixtures. This suggests that current risk assessment methods for complex mixtures may provide conservative estimates regarding soils contaminated with priority PAHs alone. Significant underestimations of total risk, however, will be obtained if the soils also contain unidentified PAHs as well as polycyclic aromatic compounds and related compounds that contribute to the total mutagenic activity. Furthermore, estimates of excess lifetime cancer risk associated with the nondietary ingestion of the PAH-contaminated soils studied here indicate that a traditional risk assessment model based on identified priority PAHs and an assumption of additivity generally underestimates the risk associated with the nonpolar soil fractions (in comparison to bioassay-derived risk estimates). Additional cancer risk may be associated with the more polar compounds that also are found at these contaminated sites and that rarely are included in the standard risk assessment methodology.

  2. Localization of an O-glycosylated site in the recombinant barley alpha-amylase 1 produced in yeast and correction of the amino acid sequence using matrix-assisted laser desorption/ionization mass spectrometry of peptide mixtures

    DEFF Research Database (Denmark)

    Andersen, Jens S.; Søgaard, M; Svensson, B

    1994-01-01

    Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of peptide mixtures was used to characterize recombinant barley alpha-amylase 1, produced in yeast. Three peptide mixtures were generated by cleavage with CNBr, digestion with endoproteinase Lys-C and Asp-N, respectively...

  3. Symposium on Short-Term Genetic Bioassays in the Evaluation of Complex Environmental Mixtures

    CERN Document Server

    Sandhu, Shahbeg; Lewtas, Joellen; Claxton, Larry; Strauss, Gary; Nesnow, Stephen

    1985-01-01

    With this proceedings of the fourth symposium on complex mixtures, we continue to revise and extend our knowledge of genetic methods for the evaluation of chemical mixtures in the environment. The early chapters of this volume are devoted to new bioassay techniques that are directly applicable to the monitoring of environments contaminated with genotoxic chemicals. Microbiological methods have been further refined to meet the special needs of atmospheric monitoring so that very small samples may now be efficiently tested. New in situ methods utilizing green plants actually avoid many of the usual difficulties of sample collection and preparation and offer special advantages in monitoring wastewater, sludges, and hazardous wastes. Insects also are being employed very effectively in the evaluation of gaseous air pollutants in controlled laboratory investigations. Increased emphasis has been placed on a comprehensive assessment of the potential of complex mixtures t9 cause various kinds of genetic damage. New as...

  4. Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

    DEFF Research Database (Denmark)

    Pedersen, Lasse Eggers; Harndahl, Mikkel; Rasmussen, Michael

    2011-01-01

    a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules....... A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data......In all vertebrate animals, CD8+ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC...

  5. A new high-throughput LC-MS method for the analysis of complex fructan mixtures

    DEFF Research Database (Denmark)

    Verspreet, Joran; Hansen, Anders Holmgaard; Dornez, Emmie

    2014-01-01

    In this paper, a new liquid chromatography-mass spectrometry (LC-MS) method for the analysis of complex fructan mixtures is presented. In this method, columns with a trifunctional C18 alkyl stationary phase (T3) were used and their performance compared with that of a porous graphitized carbon (PGC...

  6. Model of the complex of Parathyroid hormone-2 receptor and Tuberoinfundibular peptide of 39 residues

    Directory of Open Access Journals (Sweden)

    Persson Bengt

    2010-10-01

    Full Text Available Abstract Background We aim to propose interactions between the parathyroid hormone-2 receptor (PTH2R and its ligand the tuberoinfundibular peptide of 39 residues (TIP39 by constructing a homology model of their complex. The two related peptides parathyroid hormone (PTH and parathyroid hormone related protein (PTHrP are compared with the complex to examine their interactions. Findings In the model, the hydrophobic N-terminus of TIP39 is buried in a hydrophobic part of the central cavity between helices 3 and 7. Comparison of the peptide sequences indicates that the main discriminator between the agonistic peptides TIP39 and PTH and the inactive PTHrP is a tryptophan-phenylalanine replacement. The model indicates that the smaller phenylalanine in PTHrP does not completely occupy the binding site of the larger tryptophan residue in the other peptides. As only TIP39 causes internalisation of the receptor and the primary difference being an aspartic acid in position 7 of TIP39 that interacts with histidine 396 in the receptor, versus isoleucine/histidine residues in the related hormones, this might be a trigger interaction for the events that cause internalisation. Conclusions A model is constructed for the complex and a trigger interaction for full agonistic activation between aspartic acid 7 of TIP39 and histidine 396 in the receptor is proposed.

  7. Lagged kernel machine regression for identifying time windows of susceptibility to exposures of complex mixtures.

    Science.gov (United States)

    Liu, Shelley H; Bobb, Jennifer F; Lee, Kyu Ha; Gennings, Chris; Claus Henn, Birgit; Bellinger, David; Austin, Christine; Schnaas, Lourdes; Tellez-Rojo, Martha M; Hu, Howard; Wright, Robert O; Arora, Manish; Coull, Brent A

    2018-07-01

    The impact of neurotoxic chemical mixtures on children's health is a critical public health concern. It is well known that during early life, toxic exposures may impact cognitive function during critical time intervals of increased vulnerability, known as windows of susceptibility. Knowledge on time windows of susceptibility can help inform treatment and prevention strategies, as chemical mixtures may affect a developmental process that is operating at a specific life phase. There are several statistical challenges in estimating the health effects of time-varying exposures to multi-pollutant mixtures, such as: multi-collinearity among the exposures both within time points and across time points, and complex exposure-response relationships. To address these concerns, we develop a flexible statistical method, called lagged kernel machine regression (LKMR). LKMR identifies critical exposure windows of chemical mixtures, and accounts for complex non-linear and non-additive effects of the mixture at any given exposure window. Specifically, LKMR estimates how the effects of a mixture of exposures change with the exposure time window using a Bayesian formulation of a grouped, fused lasso penalty within a kernel machine regression (KMR) framework. A simulation study demonstrates the performance of LKMR under realistic exposure-response scenarios, and demonstrates large gains over approaches that consider each time window separately, particularly when serial correlation among the time-varying exposures is high. Furthermore, LKMR demonstrates gains over another approach that inputs all time-specific chemical concentrations together into a single KMR. We apply LKMR to estimate associations between neurodevelopment and metal mixtures in Early Life Exposures in Mexico and Neurotoxicology, a prospective cohort study of child health in Mexico City.

  8. Propensity of a single-walled carbon nanotube-peptide to mimic a KK10 peptide in an HLA-TCR complex

    Science.gov (United States)

    Feng, Mei; Bell, David R.; Zhou, Ruhong

    2017-12-01

    The application of nanotechnology to improve disease diagnosis, treatment, monitoring, and prevention is the goal of nanomedicine. We report here a theoretical study of a functionalized single-walled carbon nanotube (CNT) mimic binding to a human leukocyte antigen-T cell receptor (HLA-TCR) immune complex as a first attempt of a potential nanomedicine for human immunodeficiency virus (HIV) vaccine development. The carbon nanotube was coated with three arginine residues to imitate the HIV type 1 immunodominant viral peptide KK10 (gag 263-272: KRWIILGLNK), named CNT-peptide hereafter. Through molecular dynamics simulations, we explore the CNT-peptide and KK10 binding to an important HLA-TCR complex. Our results suggest that the CNT-peptide and KK10 bind comparably to the HLA-TCR complex, but the CNT-peptide forms stronger interactions with the TCR. Desorption simulations highlight the innate flexibility of KK10 over the CNT-peptide, resulting in a slightly higher desorption energy required for KK10 over the CNT-peptide. Our findings indicate that the designed CNT-peptide mimic has favorable propensity to activate TCR pathways and should be further explored to understand therapeutic potential.

  9. Directing the phase behavior of polyelectrolyte complexes using chiral patterned peptides

    Energy Technology Data Exchange (ETDEWEB)

    Pacalin, Naomi M.; Leon, Lorraine; Tirrell, Matthew

    2016-10-01

    Polyelectrolyte complexes (PECs) have a broad range of promising applications as soft materials due to their self-assembly and diversity of structure and chemical composition. Peptide polymer PECs are highly biocompatible and biodegradable, making them particularly useful for encapsulation of food additives and flavors, micellar drug delivery, medical and underwater adhesives, fetal membrane patches, and scaffolds for cell growth in tissue engineering. While parameters affecting PEC formation and stability in regards to charge effects are well researched, little is known about the effects of van der Waals interactions, hydrogen bonding, and secondary structure in these materials. Peptide chirality provides a unique opportunity to manipulate PEC phase to modulate the amount of solid-like (precipitate) or liquid-like (coacervate) character by influencing hydrogen bonding interactions among peptide chains. In previous work, we showed that chiral peptides form solid complexes, while complexes with even one racemic peptide were fluid. This raised the interesting question of how long a homochiral sequence must be to result in solid phase formation. In this work, we designed chiral patterned peptides of polyglutamic acid and polylysine ranging from 50 to 90% L-chiral residues with increasing numbers of sequential L-chiral residues before a chirality change. These polymers were mixed together to form PECs. We observed that 8 or more sequential L-chiral residues are necessary to achieve both the appearance of a precipitate phase and sustained beta-sheets in the complex, as determined by optical imaging and FTIR Spectroscopy. Less homochiral content results in formation of a coacervate phase. Thus, we show that chiral sequence can be used to control the phase transition of PECs. Understanding how to manipulate PEC phase using chiral sequence as presented here may enable tuning of the material properties to achieve the desired mechanical strength for coatings and polymer

  10. Directing the phase behavior of polyelectrolyte complexes using chiral patterned peptides

    Science.gov (United States)

    Pacalin, Naomi M.; Leon, Lorraine; Tirrell, Matthew

    2016-10-01

    Polyelectrolyte complexes (PECs) have a broad range of promising applications as soft materials due to their self-assembly and diversity of structure and chemical composition. Peptide polymer PECs are highly biocompatible and biodegradable, making them particularly useful for encapsulation of food additives and flavors, micellar drug delivery, medical and underwater adhesives, fetal membrane patches, and scaffolds for cell growth in tissue engineering. While parameters affecting PEC formation and stability in regards to charge effects are well researched, little is known about the effects of van der Waals interactions, hydrogen bonding, and secondary structure in these materials. Peptide chirality provides a unique opportunity to manipulate PEC phase to modulate the amount of solid-like (precipitate) or liquid-like (coacervate) character by influencing hydrogen bonding interactions among peptide chains. In previous work, we showed that chiral peptides form solid complexes, while complexes with even one racemic peptide were fluid. This raised the interesting question of how long a homochiral sequence must be to result in solid phase formation. In this work, we designed chiral patterned peptides of polyglutamic acid and polylysine ranging from 50 to 90% L-chiral residues with increasing numbers of sequential L-chiral residues before a chirality change. These polymers were mixed together to form PECs. We observed that 8 or more sequential L-chiral residues are necessary to achieve both the appearance of a precipitate phase and sustained β-sheets in the complex, as determined by optical imaging and FTIR Spectroscopy. Less homochiral content results in formation of a coacervate phase. Thus, we show that chiral sequence can be used to control the phase transition of PECs. Understanding how to manipulate PEC phase using chiral sequence as presented here may enable tuning of the material properties to achieve the desired mechanical strength for coatings and polymer

  11. Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy.

    Science.gov (United States)

    Vasconcelos, Luís; Lehto, Tõnis; Madani, Fatemeh; Radoi, Vlad; Hällbrink, Mattias; Vukojević, Vladana; Langel, Ülo

    2018-02-01

    Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Recoil implantation reactions in binary mixtures of catcher complexes and in mixed ligand catchers

    International Nuclear Information System (INIS)

    Sekine, Tsutomu; Sano, Masaaki; Yoshihara, Kenji

    1989-01-01

    Recoil implantation reactions were studied in binary mixtures of catcher complexes of tris(β-diketonato)metal(III) and in single-component catcher complexes of Cr(acac) n (dbm) 3-n where n=1 and 2. For the mixtures of M(acac) 3 and M(dbm) 3 , the products of 51 Cr(acac) 3 and 51 Cr(dbm) 3 were obtained as major components while 51 Cr(acac) 2 (dbm) and 51 Cr(acac)(dbm) 2 were seen as minor components. For the single component catcher complexes, predominant chemical species were parent retention type compounds. In addition to retentions there were product distributions which indicated a strong preference for acac pickup. The results were interpreted by a model which involves displacement reaction as a main process and ligand pickup reactions as side processes. (orig.)

  13. Crystal structure of a TAPBPR–MHC I complex reveals the mechanism of peptide editing in antigen presentation

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Jiansheng; Natarajan, Kannan; Boyd, Lisa F.; Morozov, Giora I.; Mage, Michael G.; Margulies, David H. (NIH); (Hebrew)

    2017-10-12

    Central to CD8+ T cell–mediated immunity is the recognition of peptide–major histocompatibility complex class I (p–MHC I) proteins displayed by antigen-presenting cells. Chaperone-mediated loading of high-affinity peptides onto MHC I is a key step in the MHC I antigen presentation pathway. However, the structure of MHC I with a chaperone that facilitates peptide loading has not been determined. We report the crystal structure of MHC I in complex with the peptide editor TAPBPR (TAP-binding protein–related), a tapasin homolog. TAPBPR remodels the peptide-binding groove of MHC I, resulting in the release of low-affinity peptide. Changes include groove relaxation, modifications of key binding pockets, and domain adjustments. This structure captures a peptide-receptive state of MHC I and provides insights into the mechanism of peptide editing by TAPBPR and, by analogy, tapasin.

  14. Application of Biologically Based Lumping To Investigate the Toxicokinetic Interactions of a Complex Gasoline Mixture.

    Science.gov (United States)

    Jasper, Micah N; Martin, Sheppard A; Oshiro, Wendy M; Ford, Jermaine; Bushnell, Philip J; El-Masri, Hisham

    2016-03-15

    People are often exposed to complex mixtures of environmental chemicals such as gasoline, tobacco smoke, water contaminants, or food additives. We developed an approach that applies chemical lumping methods to complex mixtures, in this case gasoline, based on biologically relevant parameters used in physiologically based pharmacokinetic (PBPK) modeling. Inhalation exposures were performed with rats to evaluate the performance of our PBPK model and chemical lumping method. There were 109 chemicals identified and quantified in the vapor in the chamber. The time-course toxicokinetic profiles of 10 target chemicals were also determined from blood samples collected during and following the in vivo experiments. A general PBPK model was used to compare the experimental data to the simulated values of blood concentration for 10 target chemicals with various numbers of lumps, iteratively increasing from 0 to 99. Large reductions in simulation error were gained by incorporating enzymatic chemical interactions, in comparison to simulating the individual chemicals separately. The error was further reduced by lumping the 99 nontarget chemicals. The same biologically based lumping approach can be used to simplify any complex mixture with tens, hundreds, or thousands of constituents.

  15. Nitriles form mixed-coligand complexes with 99mTc-HYNIC-Peptide

    International Nuclear Information System (INIS)

    Liu Guozheng; Wescott, Charles; Sato, Aaron; Wang Yi; Liu Ning; Zhang Yumin; Rusckowski, Mary; Hnatowich, Donald J.

    2002-01-01

    Using a 12-amino acid peptide conjugated with HYNIC as a model, we investigated nitriles as possible coligands for labeling with 99m Tc. After the preparation of the 99m Tc labeled HYNIC-peptide using tricine as coligand, the addition of acetonitile was found by reverse phase HPLC to block further coligand exchange with ethylenediamine diacetic acid (EDDA) at room temperature. The addition of this nitrile changed the pharmacokinetics of the 99m Tc labeled peptide in normal mice towards faster clearance and significant differences in accumulation in most tissues sampled. By replacing acetonitrile with cyanoacetate, a nitrile not present in the HPLC eluant, it was possible to show the existence of a new, more hydrophilic, species by reverse phase HPLC. We conclude that nitriles can act as coligands for HYNIC-conjugated peptides labeled with 99m Tc and tricine. Furthermore, the presence of acetonitrile during Sep-Pak or HPLC purification may inadvertently generate a mixed tricine/acetonitile coligand 99m Tc-HYNIC-peptide complex

  16. Intestinal Permeability of β-Lapachone and Its Cyclodextrin Complexes and Physical Mixtures.

    Science.gov (United States)

    Mangas-Sanjuan, Victor; Gutiérrez-Nieto, Jorge; Echezarreta-López, Magdalena; González-Álvarez, Isabel; González-Álvarez, Marta; Casabó, Vicente-Germán; Bermejo, Marival; Landin, Mariana

    2016-12-01

    β-Lapachone (βLAP) is a promising, poorly soluble, antitumoral drug. βLAP combination with cyclodextrins (CDs) improves its solubility and dissolution but there is not enough information about the impact of cyclodextrins on βLAP intestinal permeability. The objectives of this work were to characterize βLAP intestinal permeability and to elucidate cyclodextrins effect on the dissolution properties and on the intestinal permeability. The final goal was to evaluate CDs influence on the oral absorption of βLAP. Binary systems (physical mixtures and inclusion complexes) including βLAP and CDs (β-cyclodextrin: βCD, random-methyl-β-cyclodextrin: RMβCD and sulfobutylether-β-cyclodextrin: SBEβCD) have been prepared and analysed by differential scanning calorimetry. βLAP (and its combinations with CDs) absorption rate coefficients and effective permeability values have been determined in vitro in MDCK or MDCK-Mdr1 monolayers and in situ in rat by a closed loop perfusion technique. DSC results confirmed the formation of the inclusion complexes. βLAP-CDs inclusion complexes improve drug solubility and dissolution rate in comparison with physical mixtures. βLAP presented a high permeability value which can provide complete oral absorption. Its oral absorption is limited by its low solubility and dissolution rate. Cyclodextrin (both as physical mixtures and inclusion complexes) showed a positive effect on the intestinal permeability of βLAP. Complexation with CDs does not reduce βLAP intestinal permeability in spite of the potential negative effect of the reduction in free fraction of the drug. The use of RMβCD or SBEβCD inclusion complexes could benefit βLAP oral absorption by enhancing its solubility, dissolution rate and permeability.

  17. An assessment of the information content of likelihood ratios derived from complex mixtures.

    Science.gov (United States)

    Marsden, Clare D; Rudin, Norah; Inman, Keith; Lohmueller, Kirk E

    2016-05-01

    With the increasing sensitivity of DNA typing methodologies, as well as increasing awareness by law enforcement of the perceived capabilities of DNA typing, complex mixtures consisting of DNA from two or more contributors are increasingly being encountered. However, insufficient research has been conducted to characterize the ability to distinguish a true contributor (TC) from a known non-contributor (KNC) in these complex samples, and under what specific conditions. In order to investigate this question, sets of six 15-locus Caucasian genotype profiles were simulated and used to create mixtures containing 2-5 contributors. Likelihood ratios were computed for various situations, including varying numbers of contributors and unknowns in the evidence profile, as well as comparisons of the evidence profile to TCs and KNCs. This work was intended to illustrate the best-case scenario, in which all alleles from the TC were detected in the simulated evidence samples. Therefore the possibility of drop-out was not modeled in this study. The computer program DNAMIX was then used to compute LRs comparing the evidence profile to TCs and KNCs. This resulted in 140,000 LRs for each of the two scenarios. These complex mixture simulations show that, even when all alleles are detected (i.e. no drop-out), TCs can generate LRs less than 1 across a 15-locus profile. However, this outcome was rare, 7 of 140,000 replicates (0.005%), and associated only with mixtures comprising 5 contributors in which the numerator hypothesis includes one or more unknown contributors. For KNCs, LRs were found to be greater than 1 in a small number of replicates (75 of 140,000 replicates, or 0.05%). These replicates were limited to 4 and 5 person mixtures with 1 or more unknowns in the numerator. Only 5 of these 75 replicates (0.004%) yielded an LR greater than 1,000. Thus, overall, these results imply that the weight of evidence that can be derived from complex mixtures containing up to 5 contributors

  18. Investigating the inclusion properties of aromatic amino acids complexing beta-cyclodextrins in model peptides.

    Science.gov (United States)

    Caso, Jolanda Valentina; Russo, Luigi; Palmieri, Maddalena; Malgieri, Gaetano; Galdiero, Stefania; Falanga, Annarita; Isernia, Carla; Iacovino, Rosa

    2015-10-01

    Cyclodextrins are commonly used as complexing agents in biological, pharmaceutical, and industrial applications since they have an effect on protein thermal and proteolytic stability, refolding yields, solubility, and taste masking. β-cyclodextrins (β-CD), because of their cavity size are a perfectly suited complexing agent for many common guest moieties. In the case of peptide-cyclodextrin and protein-cyclodextrin host-guest complexes the aromatic amino acids are reported to be the principal responsible of the interaction. For these reasons, we have investigated the inclusion properties of nine designed tripeptides, obtained permuting the position of two L-alanines (Ala, A) with that of one L-tryptophan (Trp, W), L-phenylalanine (Phe, F), or L-tyrosine (Tyr, Y), respectively. Interestingly, the position of the aromatic side-chain in the sequence appears to modulate the β-CD:peptide binding constants, determined via UV-Vis and NMR spectroscopy, which in turn assumes values higher than those reported for the single amino acid. The tripeptides containing a tyrosine showed the highest binding constants, with the central position in the Ac-AYA-NH2 peptide becoming the most favorite for the interaction. A combined NMR and Molecular Docking approach permitted to build detailed complex models, highlighting the stabilizing interactions of the neighboring amino acids backbone atoms with the upper rim of the β-CD.

  19. Combining Passive Sampling with Toxicological Characterization of Complex Mixtures of Pollutants from the Aquatic Environment.

    Science.gov (United States)

    Jahnke, Annika; Witt, Gesine; Schäfer, Sabine; Haase, Nora; Escher, Beate I

    The combination of polymer-based passive sampling to collect complex environmental mixtures of pollutants, the transfer of these mixtures into bioassays, and their related toxicological characterization is still in its infancy. However, this approach has considerable potential to improve environmental hazard and risk assessment for two reasons. First, the passive sampler collects a broad range of chemicals representing the fraction of compounds available for diffusion and (bio)uptake, excluding a large part of the matrix; thus, extensive sample cleanup which could discriminate certain compounds can be avoided. Second, the toxicological characterization of samples using bioassays is complementary to chemical (target) analysis within environmental monitoring because it captures all chemicals exerting the same mode of toxic action and acting jointly in mixtures, thus providing a comprehensive picture of their overall combined effects. The scientific literature describes a range of examples from the water phase where passive sampling is usually carried out in the kinetic uptake regime for most chemicals although some may already have reached equilibrium. The composition of the chemical mixture changes from the water phase to the passive sampling material because of kinetic effects and polymer/water partition coefficients which depend on the chemicals' hydrophobicity. In contrast, only a few applications in sediment and biota have been described, but amongst these some pioneering studies have demonstrated the feasibility and potential of this combined approach. This chapter gives an overview of what has been carried out in this research area, focusing on opportunities and challenges, and points out desirable future developments with a focus on the importance of choosing a suitable combination of sampling and dosing to transfer (or re-establish) the environmental mixture into the bioassay.

  20. End-labeling of peptide nucleic acid with osmium complex. Voltammetry at carbon and mercury electrodes

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Trefulka, Mojmír; Fojta, Miroslav

    2009-01-01

    Roč. 11, č. 2 (2009), s. 359-362 ISSN 1388-2481 R&D Projects: GA AV ČR(CZ) KAN400310651; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : peptide nucleic acid end-labeling * osmium tetroxide complexes * electroactive labels Subject RIV: BO - Biophysics Impact factor: 4.243, year: 2009

  1. Efficient preparation of {sup 99m}Tc(III) '4+1' mixed-ligand complexes for peptide labeling with high specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Kunstler, Jens-Uwe [Biotectid GmbH, Deutscher Platz 5c, 04103 Leipzig (Germany); Seidel, Gesine [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden (Germany); Pietzsch, Hans-Jurgen, E-mail: h.j.pietzsch@fzd.d [Institute of Radiopharmacy, Forschungszentrum Dresden-Rossendorf, P.O. Box 510 119, 01314 Dresden (Germany)

    2010-09-15

    An improved labeling procedure for peptides attached to organometallic {sup 99m}Tc(III) '4+1' mixed-ligand complexes in which the radiometal is coordinated by a tripodal tetradentate chelator 2,2',2''-nitrilotriethanethiol (NS{sub 3}) and a monodentate isocyanide ligand is presented. The labeling procedure was evaluated by the synthesis of [{sup 99m}Tc(NS{sub 3})(L2-RGD)]. The containing radiopharmaceutically interesting RGD-peptide cyclo[Arg-Gly-Asp-D-Tyr-Lys] was modified with 4-isocyanobutanoic acid (L2) as linker conjugated to N{sup 6}-Lys to get the monodentate ligand L2-RGD. The structural identity of the {sup 99m}Tc-conjugate was confirmed by comparison to a Re reference compound. The Tc- and Re-conjugates had matching retention times under identical HPLC conditions. The {sup 99m}Tc-labeling was performed in a novel one-step procedure using the eluate of a {sup 99}Mo/{sup 99m}Tc generator, NS{sub 3}, the isocyanide modified peptide, SnCl{sub 2}, Na{sub 2}EDTA, mannitol and ascorbic acid in the reaction mixture. Using optimized reagents it is possible to label 50 nmol peptide with {sup 99m}Tc within 60 min at room temperature with a radiochemical yield higher than 95% and a specific activity of {approx}20 GBq/{mu}mol.

  2. Labeling of Complex III peptides in beef heart mitochondria and submitochondrial particles by diazonium bensene [35S]sulfonate

    International Nuclear Information System (INIS)

    Mendel-Hartvig, I.; Nelson, B.D.

    1978-01-01

    The labeling of Complex III peptides in DABS-treated mitochondria and submitochondrial particles from beef heart is reported. The results are consistent with the labeling of the two core proteins on the matrix surface of the inner membrane, and of a 29 000 molecular weight peptide on the cytoplasmic surface. (Auth.)

  3. Using metal complex-labeled peptides for charge transfer-based biosensing with semiconductor quantum dots

    Science.gov (United States)

    Medintz, Igor L.; Pons, Thomas; Trammell, Scott A.; Blanco-Canosa, Juan B.; Dawson, Philip E.; Mattoussi, Hedi

    2009-02-01

    Luminescent colloidal semiconductor quantum dots (QDs) have unique optical and photonic properties and are highly sensitive to charge transfer in their surrounding environment. In this study we used synthetic peptides as physical bridges between CdSe-ZnS core-shell QDs and some of the most common redox-active metal complexes to understand the charge transfer interactions between the metal complexes and QDs. We found that QD emission underwent quenching that was highly dependent on the choice of metal complex used. We also found that quenching traces the valence or number of metal complexes brought into close proximity of the nanocrystal surface. Monitoring of the QD absorption bleaching in the presence of the metal complex provided insight into the charge transfer mechanism. The data suggest that two distinct charge transfer mechanisms can take place. One directly to the QD core states for neutral capping ligands and a second to surface states for negatively charged capping ligands. A basic understanding of the proximity driven charge-transfer and quenching interactions allowed us to construct proteolytic enzyme sensing assemblies with the QD-peptide-metal complex conjugates.

  4. The Urtica dioica Agglutinin Is a Complex Mixture of Isolectins 1

    Science.gov (United States)

    Van Damme, Els J. M.; Broekaert, Willem F.; Peumans, Willy J.

    1988-01-01

    Rhizomes of stinging nettle (Urtica dioica) contain a complex mixture of isolectins. Ion exchange chromatography with a high resolution fast protein liquid chromatography system revealed six isoforms which exhibit identical agglutination properties and carbohydrate-binding specificity and in addition have the same molecular structure and virtually identical biochemical properties. However, since the U. dioica agglutinin isolectins differ definitely with respect to their amino acid composition, it is likely that at least some of them are different polypeptides coded for by different genes. Images Fig. 3 PMID:16665952

  5. Whey Peptide-Iron Complexes Increase the Oxidative Stability of Oil-in-Water Emulsions in Comparison to Iron Salts.

    Science.gov (United States)

    Caetano-Silva, Maria Elisa; Barros Mariutti, Lilian Regina; Bragagnolo, Neura; Bertoldo-Pacheco, Maria Teresa; Netto, Flavia Maria

    2018-02-28

    Food fortification with iron may favor lipid oxidation in both food matrices and the human body. This study aimed at evaluating the effect of peptide-iron complexation on lipid oxidation catalyzed by iron, using oil-in-water (O/W) emulsions as a model system. The extent of lipid oxidation of emulsions containing iron salts (FeSO 4 or FeCl 2 ) or iron complexes (peptide-iron complexes or ferrous bisglycinate) was evaluated during 7 days, measured as primary (peroxide value) and secondary products (TBARS and volatile compounds). Both salts catalyzed lipid oxidation, leading to peroxide values 2.6- to 4.6-fold higher than the values found for the peptide-iron complexes. The addition of the peptide-iron complexes resulted in the formation of lower amounts of secondary volatiles of lipid oxidation (up to 78-fold) than those of iron salts, possibly due to the antioxidant activity of the peptides and their capacity to keep iron apart from the lipid phase, since the iron atom is coordinated and takes part in a stable structure. The peptide-iron complexes showed potential to reduce the undesirable sensory changes in food products and to decrease the side effects related to free iron and the lipid damage of cell membranes in the organism, due to the lower reactivity of iron in the complexed form.

  6. Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex

    DEFF Research Database (Denmark)

    Stryhn, A; Andersen, P S; Pedersen, L O

    1996-01-01

    Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide...... each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T...... cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody....

  7. Insights into aquatic toxicities of the antibiotics oxytetracycline and ciprofloxacin in the presence of metal: Complexation versus mixture

    International Nuclear Information System (INIS)

    Zhang Yu; Cai Xiyun; Lang Xianming; Qiao Xianliang; Li Xuehua; Chen Jingwen

    2012-01-01

    Co-contamination of ligand-like antibiotics (e.g., tetracyclines and quinolones) and heavy metals prevails in the environment, and thus the complexation between them is involved in environmental risks of antibiotics. To understand toxicological significance of the complex, effects of metal coordination on antibiotics' toxicity were investigated. The complexation of two antibiotics, oxytetracycline and ciprofloxacin, with three heavy metals, copper, zinc, and cadmium, was verified by spectroscopic techniques. The antibiotics bound metals via multiple coordination sites and rendered a mixture of various complexation speciations. Toxicity analysis indicated that metal coordination did modify the toxicity of the antibiotics and that antibiotic, metal, and their complex acted primarily as concentration addition. Comparison of EC 50 values revealed that the complex commonly was highest toxic and predominately correlated in toxicity to the mixture. Finally, environmental scenario analysis demonstrated that ignoring complexation would improperly classify environmental risks of the antibiotics. - Highlights: ► The complex of antibiotic with metal is a mixture of various complexation modes. ► Antibiotic and metal act as various combined interactions when their complexation is ignored. ► Antibiotic, metal, and their complex act as concentration addition interaction. ► Complex commonly is the highest toxicant. ► Neglecting complexation renders improper classification of risks for antibiotics. - Antibiotic, heavy metal and their complex act primarily as concentration addition interaction and the complex commonly is highest toxic.

  8. Insights into aquatic toxicities of the antibiotics oxytetracycline and ciprofloxacin in the presence of metal: Complexation versus mixture

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Zhang [Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024 (China); Cai Xiyun, E-mail: xiyuncai@dlut.edu.cn [Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024 (China); Xianming, Lang [Liaoning Academy of Environmental Sciences, Shenyang 110031 (China); Xianliang, Qiao; Xuehua, Li; Jingwen, Chen [Key Laboratory of Industrial Ecology and Environmental Engineering (Ministry of Education), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024 (China)

    2012-07-15

    Co-contamination of ligand-like antibiotics (e.g., tetracyclines and quinolones) and heavy metals prevails in the environment, and thus the complexation between them is involved in environmental risks of antibiotics. To understand toxicological significance of the complex, effects of metal coordination on antibiotics' toxicity were investigated. The complexation of two antibiotics, oxytetracycline and ciprofloxacin, with three heavy metals, copper, zinc, and cadmium, was verified by spectroscopic techniques. The antibiotics bound metals via multiple coordination sites and rendered a mixture of various complexation speciations. Toxicity analysis indicated that metal coordination did modify the toxicity of the antibiotics and that antibiotic, metal, and their complex acted primarily as concentration addition. Comparison of EC{sub 50} values revealed that the complex commonly was highest toxic and predominately correlated in toxicity to the mixture. Finally, environmental scenario analysis demonstrated that ignoring complexation would improperly classify environmental risks of the antibiotics. - Highlights: Black-Right-Pointing-Pointer The complex of antibiotic with metal is a mixture of various complexation modes. Black-Right-Pointing-Pointer Antibiotic and metal act as various combined interactions when their complexation is ignored. Black-Right-Pointing-Pointer Antibiotic, metal, and their complex act as concentration addition interaction. Black-Right-Pointing-Pointer Complex commonly is the highest toxicant. Black-Right-Pointing-Pointer Neglecting complexation renders improper classification of risks for antibiotics. - Antibiotic, heavy metal and their complex act primarily as concentration addition interaction and the complex commonly is highest toxic.

  9. Evaluating polymer degradation with complex mixtures using a simplified surface area method.

    Science.gov (United States)

    Steele, Kandace M; Pelham, Todd; Phalen, Robert N

    2017-09-01

    Chemical-resistant gloves, designed to protect workers from chemical hazards, are made from a variety of polymer materials such as plastic, rubber, and synthetic rubber. One material does not provide protection against all chemicals, thus proper polymer selection is critical. Standardized testing, such as chemical degradation tests, are used to aid in the selection process. The current methods of degradation ratings based on changes in weight or tensile properties can be expensive and data often do not exist for complex chemical mixtures. There are hundreds of thousands of chemical products on the market that do not have chemical resistance data for polymer selection. The method described in this study provides an inexpensive alternative to gravimetric analysis. This method uses surface area change to evaluate degradation of a polymer material. Degradation tests for 5 polymer types against 50 complex mixtures were conducted using both gravimetric and surface area methods. The percent change data were compared between the two methods. The resulting regression line was y = 0.48x + 0.019, in units of percent, and the Pearson correlation coefficient was r = 0.9537 (p ≤ 0.05), which indicated a strong correlation between percent weight change and percent surface area change. On average, the percent change for surface area was about half that of the weight change. Using this information, an equivalent rating system was developed for determining the chemical degradation of polymer gloves using surface area.

  10. Investigation of quantitative separation of thorium, uranium, neptunium and plutonium from complex radiochemical mixtures

    International Nuclear Information System (INIS)

    Ushatskij, V.N.; Preobrazhenskaya, L.D.; Kolychev, V.B.; Gugel', E.S.

    1979-01-01

    Quantitative separation of actinides and their radiochemical purification with the aid of TBP with subsequent separation of thorium and quantitative separation of U, Np and Pu with the aid of D2EHPA have been studied. The method has been developed for quantitative extraction-chromatographic separation and radiochemical purification of nanogram amounts of U, Pu and microgram amounts of Th and Np from complex radiochemical mixtures containing both fragment radioisotopes and non-radioactive macrocomponents ( Fe,Al,Mg,Mn, Na and others). The method calls for application of one-extraction-chromatographic column with TBP and one column with D2EHPA. Thorium is separated at the first stage since it does not form complexes in a chloride solution during washing of the sorption column with 6. OM HCl. Npsup((4)) and Pusup((3)) required for separation are stabilized with the aid of hydrazine and hydroxylamine mixture. The yield of each of the above-cited actinide elements during the complete two-stage separation and at the stage of their separation varies within the range of 98.5-99.3%

  11. Modeling the surface tension of complex, reactive organic-inorganic mixtures

    Science.gov (United States)

    Schwier, A. N.; Viglione, G. A.; Li, Z.; McNeill, V. Faye

    2013-11-01

    Atmospheric aerosols can contain thousands of organic compounds which impact aerosol surface tension, affecting aerosol properties such as heterogeneous reactivity, ice nucleation, and cloud droplet formation. We present new experimental data for the surface tension of complex, reactive organic-inorganic aqueous mixtures mimicking tropospheric aerosols. Each solution contained 2-6 organic compounds, including methylglyoxal, glyoxal, formaldehyde, acetaldehyde, oxalic acid, succinic acid, leucine, alanine, glycine, and serine, with and without ammonium sulfate. We test two semi-empirical surface tension models and find that most reactive, complex, aqueous organic mixtures which do not contain salt are well described by a weighted Szyszkowski-Langmuir (S-L) model which was first presented by Henning et al. (2005). Two approaches for modeling the effects of salt were tested: (1) the Tuckermann approach (an extension of the Henning model with an additional explicit salt term), and (2) a new implicit method proposed here which employs experimental surface tension data obtained for each organic species in the presence of salt used with the Henning model. We recommend the use of method (2) for surface tension modeling of aerosol systems because the Henning model (using data obtained from organic-inorganic systems) and Tuckermann approach provide similar modeling results and goodness-of-fit (χ2) values, yet the Henning model is a simpler and more physical approach to modeling the effects of salt, requiring less empirically determined parameters.

  12. Effect of positively charged short peptides on stability of cubic phases of monoolein/dioleoylphosphatidic acid mixtures.

    Science.gov (United States)

    Masum, Shah Md; Li, Shu Jie; Awad, Tarek S; Yamazaki, Masahito

    2005-06-07

    To elucidate the stability and phase transition of cubic phases of biomembranes with infinite periodic minimal surface is indispensable from biological and physicochemical aspects. In this report, we investigated the effect of positively charged peptide-3K (LLKKK) and poly(L-lysine) on the phase stability of monoolein (MO) membranes containing negatively charged dioleoylphosphatidic acid (DOPA) (i.e., DOPA/MO membranes) using small-angle X-ray scattering. At first, the effect of peptide-3K on 10% DOPA/90% MO membrane in excess water, which is in the Q229 phase, was investigated. At 3.4 mM peptide-3K, a Q229 to Q230 phase transition occurred, and at >3.4 mM peptide-3K, the membrane was in the Q230 phase. Poly(L-lysine) (M(w) 1K-4K) also induced the Q230 phase, but peptide-2K (LLKK) could not induce it in the same membrane. We also investigated the effect of peptide-3K on the multilamellar vesicle (MLV) of 25% DOPA/75% MO membrane, which is in L(alpha) phase. In the absence of peptide, the spacing of MLV was very large (11.3 nm), but at > or = 8 mM peptide-3K, it greatly decreased to a constant value (5.2 nm), irrespective of the peptide concentration, indicating that peptide-3K and the membranes form an electrostatically stabilized aggregation with low water content. Poly(L-lysine) also decreased greatly the spacing of the 25% DOPA/75% MO MLV, indicating the formation of a similar aggregation. To compare the effects of peptide-3K and poly(L-lysine) with that of osmotic stress on stability of the cubic phase, we investigated the effect of poly(ethylene glycol) with molecular weight 7500 (PEG-6K) on the phase stability of 10% DOPA/90% MO membrane. With an increase in PEG-6K concentration, i.e., with an increase in osmotic stress, the most stable phase changed as follows; Q229 (Schwartz's P surface) --> Q224 (D) --> Q230 (G). On the basis of these results, we discuss the mechanism of the effects of the positively charged short peptides (peptide-3K) and poly

  13. Persistent activation of DNA damage signaling in response to complex mixtures of PAHs in air particulate matter

    Energy Technology Data Exchange (ETDEWEB)

    Jarvis, Ian W.H., E-mail: Ian.Jarvis@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Bergvall, Christoffer, E-mail: Christoffer.Bergvall@anchem.su.se [Department of Analytical Chemistry, Stockholm University, Svante Arrhenius väg 16, SE-106 91 Stockholm (Sweden); Bottai, Matteo, E-mail: Matteo.Bottai@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Westerholm, Roger, E-mail: Roger.Westerholm@anchem.su.se [Department of Analytical Chemistry, Stockholm University, Svante Arrhenius väg 16, SE-106 91 Stockholm (Sweden); Stenius, Ulla, E-mail: Ulla.Stenius@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden); Dreij, Kristian, E-mail: Kristian.Dreij@ki.se [Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm (Sweden)

    2013-02-01

    Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are present in air particulate matter (PM) and have been associated with many adverse human health effects including cancer and respiratory disease. However, due to their complexity, the risk of exposure to mixtures is difficult to estimate. In the present study the effects of binary mixtures of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and complex mixtures of PAHs in urban air PM extracts on DNA damage signaling was investigated. Applying a statistical model to the data we observed a more than additive response for binary mixtures of BP and DBP on activation of DNA damage signaling. Persistent activation of checkpoint kinase 1 (Chk1) was observed at significantly lower BP equivalent concentrations in air PM extracts than BP alone. Activation of DNA damage signaling was also more persistent in air PM fractions containing PAHs with more than four aromatic rings suggesting larger PAHs contribute a greater risk to human health. Altogether our data suggests that human health risk assessment based on additivity such as toxicity equivalency factor scales may significantly underestimate the risk of exposure to complex mixtures of PAHs. The data confirms our previous findings with PAH-contaminated soil (Niziolek-Kierecka et al., 2012) and suggests a possible role for Chk1 Ser317 phosphorylation as a biological marker for future analyses of complex mixtures of PAHs. -- Highlights: ► Benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP) and air PM PAH extracts were compared. ► Binary mixture of BP and DBP induced a more than additive DNA damage response. ► Air PM PAH extracts were more potent than toxicity equivalency factor estimates. ► Larger PAHs (> 4 rings) contribute more to the genotoxicity of PAHs in air PM. ► Chk1 is a sensitive marker for persistent activation of DNA damage signaling from PAH mixtures.

  14. Persistent activation of DNA damage signaling in response to complex mixtures of PAHs in air particulate matter

    International Nuclear Information System (INIS)

    Jarvis, Ian W.H.; Bergvall, Christoffer; Bottai, Matteo; Westerholm, Roger; Stenius, Ulla; Dreij, Kristian

    2013-01-01

    Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are present in air particulate matter (PM) and have been associated with many adverse human health effects including cancer and respiratory disease. However, due to their complexity, the risk of exposure to mixtures is difficult to estimate. In the present study the effects of binary mixtures of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and complex mixtures of PAHs in urban air PM extracts on DNA damage signaling was investigated. Applying a statistical model to the data we observed a more than additive response for binary mixtures of BP and DBP on activation of DNA damage signaling. Persistent activation of checkpoint kinase 1 (Chk1) was observed at significantly lower BP equivalent concentrations in air PM extracts than BP alone. Activation of DNA damage signaling was also more persistent in air PM fractions containing PAHs with more than four aromatic rings suggesting larger PAHs contribute a greater risk to human health. Altogether our data suggests that human health risk assessment based on additivity such as toxicity equivalency factor scales may significantly underestimate the risk of exposure to complex mixtures of PAHs. The data confirms our previous findings with PAH-contaminated soil (Niziolek-Kierecka et al., 2012) and suggests a possible role for Chk1 Ser317 phosphorylation as a biological marker for future analyses of complex mixtures of PAHs. -- Highlights: ► Benzo[a]pyrene (BP), dibenzo[a,l]pyrene (DBP) and air PM PAH extracts were compared. ► Binary mixture of BP and DBP induced a more than additive DNA damage response. ► Air PM PAH extracts were more potent than toxicity equivalency factor estimates. ► Larger PAHs (> 4 rings) contribute more to the genotoxicity of PAHs in air PM. ► Chk1 is a sensitive marker for persistent activation of DNA damage signaling from PAH mixtures.

  15. Application of tritium-labeled complex technical mixture of PCB congeners as radiotracer in ecological investigations

    International Nuclear Information System (INIS)

    Kim, A.A.

    2005-01-01

    The quantitative analysis of PCBs (polychlorinated biphenyls) is enough difficult analytical task. According to IUPAC nomenclature it is described more than 200 possible PCB congeners from which about 150 congeners are present at an environment. At the same time in environment PCBs are present as complex technical mixes of congeners (trade names include Apirolio, Aroclor, Clophen, Fenchlor, Kanechlor, Phenoclor, Pyralene, Pyranol, Pyroclor, Santotherm FR, and Sovol). In routine practice of gas chromatography analysis the quantitative measurement of PCB marker congeners is applied. In the most samples containing PCBs, there are often several dozens of different congeners. For practical reasons, all of them are not always measured, but the most important congeners are used as indicators. In Belgian chicken incident, only seven abundant congeners were usually measured: congeners with IUPAC numbers 28, 52, 101, 118, 138, 153, and 180 (2,4,4 '-TriCB, 2,2 ', 5,5 '-TCB, 2,2 ', 4,5,5 '-PeCB, 2,3 ', 4,4 ', 5-PeCB, 2,2 ', 3,4,4 ', 5 '-HxCB, 2,2 ', 4,4 ', 5,5 '-HxCB, 2,2 ', 3,4,4 ', 5,5 '-HpCB, respectively). The seven congeners are estimated to constitute about one third of all PCBs in the contaminated feed. Such measurement can give the big error in a quantitative estimation of PCBs contents in a sample. As alternative method of quantitative measurement PCBs the radiochemical method with use radioactive labeled PCBs as radiotracer can be used. We had been developed a method of synthesis of labeled by thermally activated tritium a complex technical mixture of PCBs congeners - Sovol. We had been showed a basic opportunity of receiving of uniformly tritium labeled mixture of PCBs congeners without structural failure of mixture. The tritium labeled preparation of complex technical mixture of PCBs congeners (Sovol) has been received. On the basis of received tritium labeled preparation of PCBs (Sovol) we had been developed the following quantitative and qualitative radiochemical

  16. Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

    Science.gov (United States)

    Upadhya, Archana; Sangave, Preeti C

    2016-10-01

    Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R 8 ) and CHK 6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH 3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  17. Characterization of the interaction forces in a drug carrier complex of doxorubicin with a drug-binding peptide.

    Science.gov (United States)

    Gocheva, Gergana; Ilieva, Nina; Peneva, Kalina; Ivanova, Anela

    2018-04-01

    Polypeptide-based materials are used as building blocks for drug delivery systems aimed at toxicity decrease in chemotherapeutics. A molecular-level approach is adopted for investigating the non-covalent interactions between doxorubicin and a recently synthesized drug-binging peptide as a key part of a system for delivery to neoplastic cells. Molecular dynamics simulations in aqueous solution at room and body temperature are applied to investigate the structure and the binding modes within the drug-peptide complex. The tryptophans are outlined as the main chemotherapeutic adsorption sites, and the importance of their placement in the peptide sequence is highlighted. The drug-peptide binging energy is evaluated by density functional theory calculations. Principal component analysis reveals comparable importance of several types of interaction for the binding strength. π-Stacking is dominant, but other factors are also significant: intercalation, peptide backbone stacking, electrostatics, dispersion, and solvation. Intra- and intermolecular H-bonding also stabilizes the complexes. The influence of solvent molecules on the binding energy is mild. The obtained data characterize the drug-to-peptide attachment as a mainly attractive collective process with interactions spanning a broad range of values. These results explain with atomistic detail the experimentally registered doxorubicin-binging ability of the peptide and outline the complex as a prospective carrying unit that can be employed in design of drug delivery systems. © 2017 John Wiley & Sons A/S.

  18. Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent

    Science.gov (United States)

    Wadzinski, Tyler J.; Steinauer, Angela; Hie, Liana; Pelletier, Guillaume; Schepartz, Alanna; Miller, Scott J.

    2018-06-01

    Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

  19. Natural antimicrobial peptide complexes in the fighting of antibiotic resistant biofilms: Calliphora vicina medicinal maggots.

    Directory of Open Access Journals (Sweden)

    Natalia Gordya

    Full Text Available Biofilms, sedimented microbial communities embedded in a biopolymer matrix cause vast majority of human bacterial infections and many severe complications such as chronic inflammatory diseases and cancer. Biofilms' resistance to the host immunity and antibiotics makes this kind of infection particularly intractable. Antimicrobial peptides (AMPs are a ubiquitous facet of innate immunity in animals. However, AMPs activity was studied mainly on planktonic bacteria and little is known about their effects on biofilms. We studied structure and anti-biofilm activity of AMP complex produced by the maggots of blowfly Calliphora vicina living in environments extremely contaminated by biofilm-forming germs. The complex exhibits strong cell killing and matrix destroying activity against human pathogenic antibiotic resistant Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii biofilms as well as non-toxicity to human immune cells. The complex was found to contain AMPs from defensin, cecropin, diptericin and proline-rich peptide families simultaneously expressed in response to bacterial infection and encoded by hundreds mRNA isoforms. All the families combine cell killing and matrix destruction mechanisms, but the ratio of these effects and antibacterial activity spectrum are specific to each family. These molecules dramatically extend the list of known anti-biofilm AMPs. However, pharmacological development of the complex as a whole can provide significant advantages compared with a conventional one-component approach. In particular, a similar level of activity against biofilm and planktonic bacteria (MBEC/MIC ratio provides the complex advantage over conventional antibiotics. Available methods of the complex in situ and in vitro biosynthesis make this idea practicable.

  20. Natural antimicrobial peptide complexes in the fighting of antibiotic resistant biofilms: Calliphora vicina medicinal maggots.

    Science.gov (United States)

    Gordya, Natalia; Yakovlev, Andrey; Kruglikova, Anastasia; Tulin, Dmitry; Potolitsina, Evdokia; Suborova, Tatyana; Bordo, Domenico; Rosano, Camillo; Chernysh, Sergey

    2017-01-01

    Biofilms, sedimented microbial communities embedded in a biopolymer matrix cause vast majority of human bacterial infections and many severe complications such as chronic inflammatory diseases and cancer. Biofilms' resistance to the host immunity and antibiotics makes this kind of infection particularly intractable. Antimicrobial peptides (AMPs) are a ubiquitous facet of innate immunity in animals. However, AMPs activity was studied mainly on planktonic bacteria and little is known about their effects on biofilms. We studied structure and anti-biofilm activity of AMP complex produced by the maggots of blowfly Calliphora vicina living in environments extremely contaminated by biofilm-forming germs. The complex exhibits strong cell killing and matrix destroying activity against human pathogenic antibiotic resistant Escherichia coli, Staphylococcus aureus and Acinetobacter baumannii biofilms as well as non-toxicity to human immune cells. The complex was found to contain AMPs from defensin, cecropin, diptericin and proline-rich peptide families simultaneously expressed in response to bacterial infection and encoded by hundreds mRNA isoforms. All the families combine cell killing and matrix destruction mechanisms, but the ratio of these effects and antibacterial activity spectrum are specific to each family. These molecules dramatically extend the list of known anti-biofilm AMPs. However, pharmacological development of the complex as a whole can provide significant advantages compared with a conventional one-component approach. In particular, a similar level of activity against biofilm and planktonic bacteria (MBEC/MIC ratio) provides the complex advantage over conventional antibiotics. Available methods of the complex in situ and in vitro biosynthesis make this idea practicable.

  1. Amphiphilic Peptide Interactions with Complex Biological Membranes : Effect of peptide properties on antimicrobial and anti-inflammatory effects

    OpenAIRE

    Singh, Shalini

    2016-01-01

    With increasing problem of resistance development in bacteria against conventional antibiotics, as well as problems associated with diseases either triggered or enhanced by infection, there is an urgent need to identify new types of effective therapeutics for the treatment of infectious diseases and its consequences. Antimicrobial and anti-inflammatory peptides have attracted considerable interest as potential new antibiotics in this context. While antimicrobial function of such peptides is b...

  2. Ubiquitination regulates MHC class II-peptide complex retention and degradation in dendritic cells

    OpenAIRE

    Walseng, Even; Furuta, Kazuyuki; Bosch, Berta; Weih, Karis A.; Matsuki, Yohei; Bakke, Oddmund; Ishido, Satoshi; Roche, Paul A.

    2010-01-01

    The expression and turnover of MHC class II-peptide complexes (pMHC-II) on the surface of dendritic cells (DCs) is essential for their ability to activate CD4 T cells efficiently. The half-life of surface pMHC-II is significantly greater in activated (mature) DCs than in resting (immature) DCs, but the molecular mechanism leading to this difference remains unknown. We now show that ubiquitination of pMHC-II by the E3 ubiquitin ligase membrane-associated RING-CH 1 (March-I) regulates surface e...

  3. Conformation of an Shc-derived phosphotyrosine-containing peptide complexed with the Grb2 SH2 domain

    International Nuclear Information System (INIS)

    Ogura, Kenji; Tsuchiya, Shigeo; Terasawa, Hiroaki; Yuzawa, Satoru; Hatanaka, Hideki; Mandiyan, Valsan; Schlessinger, Joseph; Inagaki, Fuyuhiko

    1997-01-01

    We have determined the structure of an Shc-derived phosphotyrosine-containing peptide complexed with Grb2 SH2 based on intra-and intermolecular NOE correlations observed by a series of isotope-filtered NMR experiments using a PFG z-filter. In contrast to an extended conformation of phosphotyrosine-containing peptides bound to Src, Syp and PLC γ SH2s, the Shc-derived peptide formed a turn at the +1 and +2 positions next to the phosphotyrosine residue. Trp 121 , located at the EF1 site of Grb2 SH2, blocked the peptide binding in an extended conformation. The present study confirms that each phosphotyrosine-containing peptide binds to the cognate SH2 with a specific conformation, which gives the structural basis for the binding specificity between SH2s and target proteins

  4. Effects of complex organic mixtures of coal liquid on cardiovascular function

    International Nuclear Information System (INIS)

    Springer, D.L.; Sasser, L.B.; Zangar, R.C.; Mahlum, D.D.

    1986-01-01

    The most common diseases in the US are those involving the cardiovascular system. Exposure to certain environmental chemicals and complex mixtures may be involved in some aspects of cardiovascular disease. They have previously reported that high-boiling coal liquids resulted in several affects related to the cardiovascular system of the rate when exposed via whole-body inhalation to the mixture. The most striking observation was a dose dependent elevation in arterial blood pressure for heavy distillate (HD) exposed rates compared to control animals at 2 weeks. They also noted an increase in heart rate and plasma and blood volume. Additional rats were evaluated 6 weeks after exposure, to determine whether these effects represented permanent changes in cardiovascular function, and the effects appeared to be reversible after this longer period. During the past year they have completed the assays of the studies initiated previously and have addressed some possible factors which could explain the effects that they observed. Electrolytes in plasma of rats exposed to the HD were analyzed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). Plasma aldosterone and angiotensin were measured by radioimmunoassays, and plasma cholesterol, triglycerides, and high-density lipoprotein were evaluated with an Abbott VP bichromatic chemistry analyzer. In addition, a comparison of the blood pressure of control rats and rats fed a restricted diet were made, to determine if the anorexia which resulted from HD exposure could be responsible for the changes they observed in cardiovascular function

  5. Complex-shaped ceramic composites obtained by machining compact polymer-filler mixtures

    Directory of Open Access Journals (Sweden)

    Rosa Maria da Rocha

    2005-06-01

    Full Text Available Research in the preparation of ceramics from polymeric precursors is giving rise to increased interest in ceramic technology because it allows the use of several promising polymer forming techniques. In this work ceramic composite pieces were obtained by pyrolysis of a compacted mixture of a polysiloxane resin and alumina/silicon powder. The mixture consists of 60 vol% of the polymer phase and 40 vol% of the filler in a 1:1 ratio for alumina/silicon, which was hot pressed to crosslink the polymer, thus forming a compact body. This green body was trimmed into different geometries and pyrolised in nitrogen atmosphere at temperatures up to 1600 °C. X-ray diffraction analysis indicated the formation of phases such as mullite and Si2ON2 during pyrolysis, that result from reactions between fillers, polymer decomposition products and nitrogen atmosphere. The porosity was found to be less than 20% and the mass loss around 10%. The complex geometry was maintained after pyrolysis and shrinkage was approximately 8%, proving pyrolisis to be a suitable process to form near-net-shaped bulk ceramic components.

  6. Atmospheric pressure chemical ionization Fourier transform ion cyclotron resonance mass spectrometry for complex thiophenic mixture analysis

    KAUST Repository

    Hourani, Nadim

    2013-10-01

    Rationale Polycyclic aromatic sulfur heterocycles (PASHs) are detrimental species for refining processes in petroleum industry. Current mass spectrometric Methods that determine their composition are often preceded by derivatization and dopant addition approaches. Different ionization Methods have different impact on the molecular assignment of complex PASHs. The analysis of such species under atmospheric pressure chemical ionization (APCI) is still considered limited due to uncontrolled ion generation with low- and high-mass PASHs. Methods The ionization behavior of a model mixture of five selected PASH standards was investigated using an APCI source with nitrogen as the reagent gas. A complex thiophenic fraction was separated from a vacuum gas oil (VGO) and injected using the same method. The samples were analyzed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). RESULTS PASH model analytes were successfully ionized and mainly [M + H]+ ions were produced. The same ionization pattern was observed for the real thiophenic sample. It was found that S1 class species were the major sulfur-containing species found in the VGO sample. These species indicated the presence of alkylated benzothiophenic (BT), dibenzothiophenic (DBT) and benzonaphthothiophenic (BNT) series that were detected by APCI-FTICR MS. CONCLUSIONS This study provides an established APCI-FTICR MS method for the analysis of complex PASHs. PASHs were detected without using any derivatization and without fragmentation. The method can be used for the analysis of S-containing crude oil samples. © 2013 John Wiley & Sons, Ltd.

  7. Dissociation of Calmodulin-Target Peptide Complexes by the Lipid Mediator Sphingosylphosphorylcholine

    Science.gov (United States)

    Kovacs, Erika; Tóth, Judit; Vértessy, Beáta G.; Liliom, Károly

    2010-01-01

    Previously we have identified the lipid mediator sphingosylphosphorylcholine (SPC) as the first potentially endogenous inhibitor of the ubiquitous Ca2+ sensor calmodulin (CaM) (Kovacs, E., and Liliom, K. (2008) Biochem. J. 410, 427–437). Here we give mechanistic insight into CaM inhibition by SPC, based on fluorescence stopped-flow studies with the model CaM-binding domain melittin. We demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. Furthermore, we present kinetic evidence that both species compete for the same target site on CaM, and thus SPC can be considered as a competitive inhibitor of CaM-target peptide interactions. We also show that SPC disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, inositol 1,4,5-trisphosphate receptor type 1, and the plasma membrane Ca2+ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca2+ signaling, we hypothesize that SPC could lead to Ca2+ mobilization in vivo. Hence, we suggest that the action of the sphingolipid on CaM might explain the previously recognized phenomenon that SPC liberates Ca2+ from intracellular stores. Moreover, we demonstrate that unlike traditional synthetic CaM inhibitors, SPC disrupts the complex between not only the Ca2+-saturated but also the apo form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors. PMID:19910470

  8. Design, synthesis and DNA interactions of a chimera between a platinum complex and an IHF mimicking peptide.

    Science.gov (United States)

    Rao, Harita; Damian, Mariana S; Alshiekh, Alak; Elmroth, Sofi K C; Diederichsen, Ulf

    2015-12-28

    Conjugation of metal complexes with peptide scaffolds possessing high DNA binding affinity has shown to modulate their biological activities and to enhance their interaction with DNA. In this work, a platinum complex/peptide chimera was synthesized based on a model of the Integration Host Factor (IHF), an architectural protein possessing sequence specific DNA binding and bending abilities through its interaction with a minor groove. The model peptide consists of a cyclic unit resembling the minor grove binding subdomain of IHF, a positively charged lysine dendrimer for electrostatic interactions with the DNA phosphate backbone and a flexible glycine linker tethering the two units. A norvaline derived artificial amino acid was designed to contain a dimethylethylenediamine as a bidentate platinum chelating unit, and introduced into the IHF mimicking peptides. The interaction of the chimeric peptides with various DNA sequences was studied by utilizing the following experiments: thermal melting studies, agarose gel electrophoresis for plasmid DNA unwinding experiments, and native and denaturing gel electrophoresis to visualize non-covalent and covalent peptide-DNA adducts, respectively. By incorporation of the platinum metal center within the model peptide mimicking IHF we have attempted to improve its specificity and DNA targeting ability, particularly towards those sequences containing adjacent guanine residues.

  9. Versatile polyion complex micelles for peptide and siRNA vectorization to engineer tolerogenic dendritic cells.

    Science.gov (United States)

    Mebarek, Naila; Vicente, Rita; Aubert-Pouëssel, Anne; Quentin, Julie; Mausset-Bonnefont, Anne-Laure; Devoisselle, Jean-Marie; Jorgensen, Christian; Bégu, Sylvie; Louis-Plence, Pascale

    2015-05-01

    Dendritic cells (DCs) are professional antigen-presenting cells that play a critical role in maintaining the balance between immunity and tolerance and, as such are a promising immunotherapy tool to induce immunity or to restore tolerance. The main challenge to harness the tolerogenic properties of DCs is to preserve their immature phenotype. We recently developed polyion complex micelles, formulated with double hydrophilic block copolymers of poly(methacrylic acid) and poly(ethylene oxide) blocks and able to entrap therapeutic molecules, which did not induce DC maturation. In the current study, the intrinsic destabilizing membrane properties of the polymers were used to optimize endosomal escape property of the micelles in order to propose various strategies to restore tolerance. On the first hand, we showed that high molecular weight (Mw) copolymer-based micelles were efficient to favor the release of the micelle-entrapped peptide into the endosomes, and thus to improve peptide presentation by immature (i) DCs. On the second hand, we put in evidence that low Mw copolymer-based micelles were able to favor the cytosolic release of micelle-entrapped small interfering RNAs, dampening the DCs immunogenicity. Therefore, we demonstrate the versatile use of polyionic complex micelles to preserve tolerogenic properties of DCs. Altogether, our results underscored the potential of such micelle-loaded iDCs as a therapeutic tool to restore tolerance in autoimmune diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Peptide-mediated vectorization of metal complexes: conjugation strategies and biomedical applications.

    Science.gov (United States)

    Soler, Marta; Feliu, Lidia; Planas, Marta; Ribas, Xavi; Costas, Miquel

    2016-08-16

    The rich chemical and structural versatility of transition metal complexes provides numerous novel paths to be pursued in the design of molecules that exert particular chemical or physicochemical effects that could operate over specific biological targets. However, the poor cell permeability of metallodrugs represents an important barrier for their therapeutic use. The conjugation between metal complexes and a functional peptide vector can be regarded as a versatile and potential strategy to improve their bioavailability and accumulation inside cells, and the site selectivity of their effect. This perspective lies in reviewing the recent advances in the design of metallopeptide conjugates for biomedical applications. Additionally, we highlight the studies where this approach has been directed towards the incorporation of redox active metal centers into living organisms for modulating the cellular redox balance, as a tool with application in anticancer therapy.

  11. The shock synthesis of complex organics from impacts into cometary analogue mixtures

    Science.gov (United States)

    Price, M.; Wozniakiewicz, P.; Cole, M.; Martins, Z.; Burchell, M.

    2014-07-01

    Introduction: If amino acids are required for the evolution of life, what was their source? Many different theories abound as to the source of amino acids on the early Earth including exogenous delivery from comets/asteroids (for example, glycine was found recently on comet Wild-2 [1]), formation in the protoplanetary nebula [2], or UV catalysed reactions of gases [3]. An alternative explanation is that amino acids can be shock-synthesised during the impact on an icy body onto a rocky body (or, equivalently, the impact of rocky body onto an icy surface). This theory is supported by computer simulations [4] and by very recent experimental data, which demonstrated the formation of simple (including abiotic) amino acids from shocks into ice mixtures mimicking the composition of comets and the surfaces of the icy Jovian and Saturnian satellites. Although the results from these experiments are fundamentally important, the yield of synthesised amino acids was low (nano-grams of material), complicating their detection and identification. In order to increase the collected yield of complex organics, and aid in their detection and identification, we have implemented a new collection technique within our hypervelocity impact facility. Experimental Methodology: Figure 1A) shows a low-resolution high-speed photograph of an impact plasma generated from an impact of a stainless-steel sphere into a mixture of water, CO_{2}, ammonia, and methanol ices. The plasma has an intense blue colour, and lasted for < 1 msec (the frame-rate of the camera). It is during and within this flash that complex organics are most likely synthesised, and thus to maximise the collection of these materials, we have implemented a new collection mechanism. Figure 1B) shows the prototype collection mechanism. Here an aluminium cold-plate (˜150 K) is placed in front of the target holder containing the ice mixtures. The plate has a central hole which allows the projectile to pass through to impact the ice

  12. Surface complexation modeling of Cu(II adsorption on mixtures of hydrous ferric oxide and kaolinite

    Directory of Open Access Journals (Sweden)

    Schaller Melinda S

    2008-09-01

    Full Text Available Abstract Background The application of surface complexation models (SCMs to natural sediments and soils is hindered by a lack of consistent models and data for large suites of metals and minerals of interest. Furthermore, the surface complexation approach has mostly been developed and tested for single solid systems. Few studies have extended the SCM approach to systems containing multiple solids. Results Cu adsorption was measured on pure hydrous ferric oxide (HFO, pure kaolinite (from two sources and in systems containing mixtures of HFO and kaolinite over a wide range of pH, ionic strength, sorbate/sorbent ratios and, for the mixed solid systems, using a range of kaolinite/HFO ratios. Cu adsorption data measured for the HFO and kaolinite systems was used to derive diffuse layer surface complexation models (DLMs describing Cu adsorption. Cu adsorption on HFO is reasonably well described using a 1-site or 2-site DLM. Adsorption of Cu on kaolinite could be described using a simple 1-site DLM with formation of a monodentate Cu complex on a variable charge surface site. However, for consistency with models derived for weaker sorbing cations, a 2-site DLM with a variable charge and a permanent charge site was also developed. Conclusion Component additivity predictions of speciation in mixed mineral systems based on DLM parameters derived for the pure mineral systems were in good agreement with measured data. Discrepancies between the model predictions and measured data were similar to those observed for the calibrated pure mineral systems. The results suggest that quantifying specific interactions between HFO and kaolinite in speciation models may not be necessary. However, before the component additivity approach can be applied to natural sediments and soils, the effects of aging must be further studied and methods must be developed to estimate reactive surface areas of solid constituents in natural samples.

  13. Peptide pool immunization and CD8+ T cell reactivity

    DEFF Research Database (Denmark)

    Rasmussen, Susanne B; Harndahl, Mikkel N; Buus, Anette Stryhn

    2013-01-01

    Mice were immunized twice with a pool of five peptides selected among twenty 8-9-mer peptides for their ability to form stable complexes at 37°C with recombinant H-2K(b) (half-lives 10-15h). Vaccine-induced immunity of splenic CD8(+) T cells was studied in a 24h IFNγ Elispot assay. Surprisingly...... peptides induced normal peptide immunity i.e. the specific T cell reactivity in the Elispot culture was strictly dependent on exposure to the immunizing peptide ex vivo. However, immunization with two of the peptides, a VSV- and a Mycobacterium-derived peptide, resulted in IFNγ spot formation without...... peptide in the Elispot culture. Immunization with a mixture of the VSV-peptide and a "normal" peptide also resulted in IFNγ spot formation without addition of peptide to the assay culture. Peptide-tetramer staining of CD8(+) T cells from mice immunized with a mixture of VSV-peptide and "normal" peptide...

  14. Fixed-Point Algorithms for the Blind Separation of Arbitrary Complex-Valued Non-Gaussian Signal Mixtures

    Directory of Open Access Journals (Sweden)

    Douglas Scott C

    2007-01-01

    Full Text Available We derive new fixed-point algorithms for the blind separation of complex-valued mixtures of independent, noncircularly symmetric, and non-Gaussian source signals. Leveraging recently developed results on the separability of complex-valued signal mixtures, we systematically construct iterative procedures on a kurtosis-based contrast whose evolutionary characteristics are identical to those of the FastICA algorithm of Hyvarinen and Oja in the real-valued mixture case. Thus, our methods inherit the fast convergence properties, computational simplicity, and ease of use of the FastICA algorithm while at the same time extending this class of techniques to complex signal mixtures. For extracting multiple sources, symmetric and asymmetric signal deflation procedures can be employed. Simulations for both noiseless and noisy mixtures indicate that the proposed algorithms have superior finite-sample performance in data-starved scenarios as compared to existing complex ICA methods while performing about as well as the best of these techniques for larger data-record lengths.

  15. Identifying technical aliases in SELDI mass spectra of complex mixtures of proteins

    Science.gov (United States)

    2013-01-01

    Background Biomarker discovery datasets created using mass spectrum protein profiling of complex mixtures of proteins contain many peaks that represent the same protein with different charge states. Correlated variables such as these can confound the statistical analyses of proteomic data. Previously we developed an algorithm that clustered mass spectrum peaks that were biologically or technically correlated. Here we demonstrate an algorithm that clusters correlated technical aliases only. Results In this paper, we propose a preprocessing algorithm that can be used for grouping technical aliases in mass spectrometry protein profiling data. The stringency of the variance allowed for clustering is customizable, thereby affecting the number of peaks that are clustered. Subsequent analysis of the clusters, instead of individual peaks, helps reduce difficulties associated with technically-correlated data, and can aid more efficient biomarker identification. Conclusions This software can be used to pre-process and thereby decrease the complexity of protein profiling proteomics data, thus simplifying the subsequent analysis of biomarkers by decreasing the number of tests. The software is also a practical tool for identifying which features to investigate further by purification, identification and confirmation. PMID:24010718

  16. Experimental purification of paclitaxel from a complex mixture of taxanes using a simulated moving bed

    Directory of Open Access Journals (Sweden)

    M. A. Cremasco

    2009-03-01

    Full Text Available A laboratory-scale simulated moving bed (SMB was designed and tested for the separation of paclitaxel, a powerful anti-cancer agent known as Taxol@, from impurities of a plant tissue culture (PTC broth. The innovative strategy of a pseudo-binary model, where mixtures A and B were treated as single solutes A and B, was used in the linear standing wave analysis to fix the SMB operating parameters for a multicomponent and complex system. Linear standing wave design was used to specify the zone flow rates and the switching time for the laboratory-scale SMB unit, with two steps of separation. The SMB consists of four packed columns, where each column is 12.5 cm in length and 1.5 cm in diameter. Two sequential separation steps were used to recover paclitaxel from a small feed batch (less than one liter. Placlitaxel was recovered from the complex plant tissue culture broth in 82% yield and 72% purity.

  17. Analysis of peptide mixtures for proteomics research using LC–ESI-MS with a simple microgradient device

    Czech Academy of Sciences Publication Activity Database

    Lenobel, R.; Řehulková, H.; Šebela, M.; Franc, V.; Kahle, Vladislav; Moravcová, Dana; Řehulka, P.

    2015-01-01

    Roč. 33, č. 6 (2015), s. 420-428 ISSN 1527-5949 R&D Projects: GA MV VG20112015021 Institutional support: RVO:68081715 Keywords : LC-ESI-MS * proteomics * peptide Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 0.519, year: 2014 http://hdl.handle.net/11104/0249120

  18. Characterization of the pharmacokinetics of gasoline using PBPK modeling with a complex mixtures chemical lumping approach.

    Science.gov (United States)

    Dennison, James E; Andersen, Melvin E; Yang, Raymond S H

    2003-09-01

    Gasoline consists of a few toxicologically significant components and a large number of other hydrocarbons in a complex mixture. By using an integrated, physiologically based pharmacokinetic (PBPK) modeling and lumping approach, we have developed a method for characterizing the pharmacokinetics (PKs) of gasoline in rats. The PBPK model tracks selected target components (benzene, toluene, ethylbenzene, o-xylene [BTEX], and n-hexane) and a lumped chemical group representing all nontarget components, with competitive metabolic inhibition between all target compounds and the lumped chemical. PK data was acquired by performing gas uptake PK studies with male F344 rats in a closed chamber. Chamber air samples were analyzed every 10-20 min by gas chromatography/flame ionization detection and all nontarget chemicals were co-integrated. A four-compartment PBPK model with metabolic interactions was constructed using the BTEX, n-hexane, and lumped chemical data. Target chemical kinetic parameters were refined by studies with either the single chemical alone or with all five chemicals together. o-Xylene, at high concentrations, decreased alveolar ventilation, consistent with respiratory irritation. A six-chemical interaction model with the lumped chemical group was used to estimate lumped chemical partitioning and metabolic parameters for a winter blend of gasoline with methyl t-butyl ether and a summer blend without any oxygenate. Computer simulation results from this model matched well with experimental data from single chemical, five-chemical mixture, and the two blends of gasoline. The PBPK model analysis indicated that metabolism of individual components was inhibited up to 27% during the 6-h gas uptake experiments of gasoline exposures.

  19. Structure of the superantigen staphylococcal enterotoxin B in complex with TCR and peptide-MHC demonstrates absence of TCR-peptide contacts.

    Science.gov (United States)

    Rödström, Karin E J; Elbing, Karin; Lindkvist-Petersson, Karin

    2014-08-15

    Superantigens are immune-stimulatory toxins produced by Staphylococcus aureus, which are able to interact with host immune receptors to induce a massive release of cytokines, causing toxic shock syndrome and possibly death. In this article, we present the x-ray structure of staphylococcal enterotoxin B (SEB) in complex with its receptors, the TCR and MHC class II, forming a ternary complex. The structure, in combination with functional analyses, clearly shows how SEB adopts a wedge-like position when binding to the β-chain of TCR, allowing for an interaction between the α-chain of TCR and MHC. Furthermore, the binding mode also circumvents contact between TCR and the peptide presented by MHC, which enables SEB to initiate a peptide-independent activation of T cells. Copyright © 2014 by The American Association of Immunologists, Inc.

  20. Hepatoma targeting peptide conjugated bio-reducible polymer complexed with oncolytic adenovirus for cancer gene therapy.

    Science.gov (United States)

    Choi, Joung-Woo; Kim, Hyun Ah; Nam, Kihoon; Na, Youjin; Yun, Chae-Ok; Kim, SungWan

    2015-12-28

    Despite adenovirus (Ad) vector's numerous advantages for cancer gene therapy, such as high ability of endosomal escape, efficient nuclear entry mechanism, and high transduction, and therapeutic efficacy, tumor specific targeting and antiviral immune response still remain as a critical challenge in clinical setting. To overcome these obstacles and achieve cancer-specific targeting, we constructed tumor targeting bioreducible polymer, an arginine grafted bio-reducible polymer (ABP)-PEG-HCBP1, by conjugating PEGylated ABP with HCBP1 peptides which has high affinity and selectivity towards hepatoma. The ABP-PEG-HCBP1-conjugated replication incompetent GFP-expressing ad, (Ad/GFP)-ABP-PEG-HCBP1, showed a hepatoma cancer specific uptake and transduction compared to either naked Ad/GFP or Ad/GFP-ABP. Competition assays demonstrated that Ad/GFP-ABP-PEG-HCBP1-mediated transduction was specifically inhibited by HCBP1 peptide rather than coxsackie and adenovirus receptor specific antibody. In addition, ABP-PEG-HCBP1 can protect biological activity of Ad against serum, and considerably reduced both innate and adaptive immune response against Ad. shMet-expressing oncolytic Ad (oAd; RdB/shMet) complexed with ABP-PEG-HCBP1 delivered oAd efficiently into hepatoma cancer cells. The oAd/ABP-PEG-HCBP1 demonstrated enhanced cancer cell killing efficacy in comparison to oAd/ABP complex. Furthermore, Huh7 and HT1080 cancer cells treated with oAd/shMet-ABP-PEG-HCBP1 complex had significantly decreased Met and VEGF expression in hepatoma cancer, but not in non-hepatoma cancer. In sum, these results suggest that HCBP1-conjugated bioreducible polymer could be used to deliver oncolytic Ad safely and efficiently to treat hepatoma. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Transition Metal Donor-Peptide-Acceptor Complexes: From Intramolecular Electron Transfer Reactions to the Study of Reactive Intermediates

    Energy Technology Data Exchange (ETDEWEB)

    Isied, Stephan S.

    2003-03-11

    The trans-polyproline (PII) oligomers (Figure 1) are unusually rigid peptide structures which have been extensively studied by our group for peptide mediated intramolecular electron transfer (ET) at long distances. We have previously studied ET across a series of metal ion donor (D) acceptor (A) oligoproline peptides with different distances, driving forces and reorganizational energies. The majority of these experiments involve generating the ET intermediate using pulse radiolysis methods, although more recently photochemical methods are also used. Results of these studies showed that ET across peptides can vary by more than twelve orders of magnitude. Using ruthenium bipyridine donors, ET reaction rate constants across several proline residues (n = 4 - 9) occurred in the millisecond (ms) to {micro}s timescale, thus limiting the proline peptide conformational motions to only minor changes (far smaller than the large changes that occur on the ms to sec timescale, such as trans to cis proline isomerization). The present report describes our large data base of experimental results for D-peptide-A complexes in terms of a model where the involvement of both superexchange and hopping (hole and electron) mechanisms account for the long range ET rate constants observed. Our data shows that the change from superexchange to hopping mechanisms occurs at different distances depending on the type of D and A and their interactions with the peptides. Our model is also consistent with generalized models for superexchange and hopping which have been put forward by a number of theoretical groups to account for long range ET phenomena.

  2. Application of the cubic-plus-association (CPA) equation of state to complex mixtures with aromatic hydrocarbons

    DEFF Research Database (Denmark)

    Folas, Georgios; Kontogeorgis, Georgios; Michelsen, Michael Locht

    2006-01-01

    The cubic-plus-association (CPA) equation of state is applied to phase equilibria of mixtures containing alcohols, glycols, water, and aromatic or olefinic hydrocarbons. Previously, CPA has been successfully used for mixtures containing various associating compounds (alcohols, glycols, amines......, organic acids, and water) and aliphatic hydrocarbons. We show in this work that the model can be satisfactorily extended to complex vapor-liquid-liquid equilibria with aromatic or olefinic hydrocarbons. The solvation between aromatics/olefinics and polar compounds is accounted for. This is particularly...... important for mixtures containing water and glycols, but less so for mixtures with alcohols. For water/hydrocarbons, a single binary interaction parameter which accounts for the solvation is fitted to the experimental liquid-liquid equilibria (LLE) data. The interaction parameter of the physical term...

  3. Fast 2D NMR Spectroscopy for In vivo Monitoring of Bacterial Metabolism in Complex Mixtures

    Directory of Open Access Journals (Sweden)

    Rupashree Dass

    2017-07-01

    Full Text Available The biological toolbox is full of techniques developed originally for analytical chemistry. Among them, spectroscopic experiments are very important source of atomic-level structural information. Nuclear magnetic resonance (NMR spectroscopy, although very advanced in chemical and biophysical applications, has been used in microbiology only in a limited manner. So far, mostly one-dimensional 1H experiments have been reported in studies of bacterial metabolism monitored in situ. However, low spectral resolution and limited information on molecular topology limits the usability of these methods. These problems are particularly evident in the case of complex mixtures, where spectral peaks originating from many compounds overlap and make the interpretation of changes in a spectrum difficult or even impossible. Often a suite of two-dimensional (2D NMR experiments is used to improve resolution and extract structural information from internuclear correlations. However, for dynamically changing sample, like bacterial culture, the time-consuming sampling of so-called indirect time dimensions in 2D experiments is inefficient. Here, we propose the technique known from analytical chemistry and structural biology of proteins, i.e., time-resolved non-uniform sampling. The method allows application of 2D (and multi-D experiments in the case of quickly varying samples. The indirect dimension here is sparsely sampled resulting in significant reduction of experimental time. Compared to conventional approach based on a series of 1D measurements, this method provides extraordinary resolution and is a real-time approach to process monitoring. In this study, we demonstrate the usability of the method on a sample of Escherichia coli culture affected by ampicillin and on a sample of Propionibacterium acnes, an acne causing bacterium, mixed with a dose of face tonic, which is a complicated, multi-component mixture providing complex NMR spectrum. Through our experiments

  4. Treatment of complex biological mixtures with pulsed electric fields An energy transfer characterization

    International Nuclear Information System (INIS)

    Schrive, Luc

    2004-01-01

    Sewage sludge from waste water treatment plants is a complex biological mixture and a problematic by-product because of valorisation restrictions. In order to limit its production, pulsed electric fields (PEF) were studied because of their biological effects and their potentially physico-chemical action. This work demonstrated a paradoxical phenomenon: cell lysis triggered a respirometric activation followed by a delayed lethality. This phenomenon was related to the leakage of internal compounds which were immediately bio-assimilated. At high energy expense, the plasmic membrane permeabilization led to cell death. Practically, with the technical configuration of the equipment, no hydrolysis was detected. This limitation decreases the interest for excess sludge reduction, but for the same reason, PEF cold sterilization technique can be assessed as a promising process. The representation of the electric energy transfer from electrodes to cell was exchanged by the study of mass transfer from the biological cell to the surrounding media under an electromotive force. Thus, the survival rate was modelled by a Sherwood number taking account of electrical, biological and hydraulic parameters. (author) [fr

  5. Complex Mixture Analysis of Organic Compounds in Yogurt by NMR Spectroscopy

    Science.gov (United States)

    Lu, Yi; Hu, Fangyu; Miyakawa, Takuya; Tanokura, Masaru

    2016-01-01

    NMR measurements do not require separation and chemical modification of samples and therefore rapidly and directly provide non-targeted information on chemical components in complex mixtures. In this study, one-dimensional (1H, 13C, and 31P) and two-dimensional (1H-13C and 1H-31P) NMR spectroscopy were conducted to analyze yogurt without any pretreatment. 1H, 13C, and 31P NMR signals were assigned to 10 types of compounds. The signals of α/β-lactose and α/β-galactose were separately observed in the 1H NMR spectra. In addition, the signals from the acyl chains of milk fats were also successfully identified but overlapped with many other signals. Quantitative difference spectra were obtained by subtracting the diffusion ordered spectroscopy (DOSY) spectra from the quantitative 1H NMR spectra. This method allowed us to eliminate interference on the overlaps; therefore, the correct intensities of signals overlapped with those from the acyl chains of milk fat could be determined directly without separation. Moreover, the 1H-31P HMBC spectra revealed for the first time that N-acetyl-d-glucosamine-1-phosphate is contained in yogurt. PMID:27322339

  6. Complex mixture-associated hormesis and toxicity: the case of leather tanning industry.

    Science.gov (United States)

    Pagano, Giovanni; Castello, Giuseppe; Gallo, Marialuisa; Borriello, Ilaria; Guida, Marco

    2008-01-01

    A series of studies investigated the toxicities of tannery-derived complex mixtures, i.e. vegetable tannin (VT) from Acacia sp. or phenol-based synthetic tannin (ST), and waste-water from tannin-based vs. chromium-based tanneries. Toxicity was evaluated by multiple bioassays including developmental defects and loss of fertilization rate in sea urchin embryos and sperm (Paracentrotus lividus and Sphaerechinus granularis), and algal growth inhibition (Dunaliella tertiolecta and Selenastrum capricornutum). Both VT and ST water extracts resulted in hormetic effects at concentrations ranging 0.1 to 0.3%, and toxicity at levels > or =1%, both in sea urchin embryo and sperm, and in algal growth bioassays. When comparing tannin-based tannery wastewater (TTW) vs. chromium-based tannery effluent (CTE), a hormesis to toxicity trend was observed for TTW both in terms of developmental and fertilization toxicity in sea urchins, and in algal growth inhibition, with hormetic effects at 0.1 to 0.2% TTW, and toxicity at TTW levels > or =1%. Unlike TTW, CTE showed a monotonic toxicity increase from the lowest tested level (0.1%) and CTE toxicity at higher levels was significantly more severe than TTW-induced toxicity. The results support the view that leather production utilizing tannins might be regarded as a more environmentally friendly procedure than chromium-based tanning process.

  7. Dynamic study of excited state hydrogen-bonded complexes of harmane in cyclohexane-toluene mixtures.

    Science.gov (United States)

    Carmona, Carmen; Balón, Manuel; Galán, Manuel; Guardado, Pilar; Muñoz, María A

    2002-09-01

    Photoinduced proton transfer reactions of harmane or 1-methyl-9H-pyrido[3,4-b]indole (HN) in the presence of the proton donor hexafluoroisopropanol (HFIP) in cyclohexane-toluene mixtures (CY-TL; 10% vol/vol of TL) have been studied. Three excited state species have been identified: a 1:2 hydrogen-bonded proton transfer complex (PTC), between the pyridinic nitrogen of the substrate and the proton donor, a hydrogen-bonded cation-like exciplex (CL*) with a stoichiometry of at least 1:3 and a zwitterionic exciplex (Z*). Time-resolved fluorescence measurements evidence that upon excitation of ground state PTC, an excited state equilibrium is established between PTC* and the cationlike exciplex, CL*, lambdaem approximately/= 390 nm. This excited state reaction is assisted by another proton donor molecule. Further reaction of CL* with an additional HFIP molecule produces the zwitterionic species, Z*, lambda(em) approximately/= 500 nm. From the analysis of the multiexponential decays, measured at different emission wavelengths and as a function of HFIP concentration, the mechanism of these excited state reactions has been established. Thus, three rate constants and three reciprocal lifetimes have been determined. The simultaneous study of 1,9-dimethyl-9H-pyrido[3,4-b]indole (MHN) under the same experimental conditions has helped to understand the excited state kinetics of these processes.

  8. Associations between complex OHC mixtures and thyroid and cortisol hormone levels in East Greenland polar bears.

    Science.gov (United States)

    Bechshøft, T Ø; Sonne, C; Dietz, R; Born, E W; Muir, D C G; Letcher, R J; Novak, M A; Henchey, E; Meyer, J S; Jenssen, B M; Villanger, G D

    2012-07-01

    The multivariate relationship between hair cortisol, whole blood thyroid hormones, and the complex mixtures of organohalogen contaminant (OHC) levels measured in subcutaneous adipose of 23 East Greenland polar bears (eight males and 15 females, all sampled between the years 1999 and 2001) was analyzed using projection to latent structure (PLS) regression modeling. In the resulting PLS model, most important variables with a negative influence on cortisol levels were particularly BDE-99, but also CB-180, -201, BDE-153, and CB-170/190. The most important variables with a positive influence on cortisol were CB-66/95, α-HCH, TT3, as well as heptachlor epoxide, dieldrin, BDE-47, p,p'-DDD. Although statistical modeling does not necessarily fully explain biological cause-effect relationships, relationships indicate that (1) the hypothalamic-pituitary-adrenal (HPA) axis in East Greenland polar bears is likely to be affected by OHC-contaminants and (2) the association between OHCs and cortisol may be linked with the hypothalamus-pituitary-thyroid (HPT) axis. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Identification of unresolved complex mixtures (UCMs) of hydrocarbons in commercial fish oil supplements.

    Science.gov (United States)

    Reid, Anna-Jean M; Budge, Suzanne M

    2015-01-01

    Heightened awareness of the health benefits of fish oil consumption has led to a great increase in the number of fish oil supplements available to the consumer. Therefore manufacturers are continually looking for ways to distinguish their products from those of competitors. Minimally refined or virgin fish oils provide a unique feature; however, petroleum hydrocarbon contamination from oil spills is a reality in the world's oceans. The question arises whether oil produced from fish species caught in these polluted areas is free of petroleum hydrocarbons, with particular interest in unresolved complex mixtures (UCMs). This study investigates the presence of UCMs in commercially available fish oil supplements advertised as being virgin, as well as refined. Weathered petroleum hydrocarbons in the form of a UCM were found at 523 µg g(-1) in a virgin Alaskan salmon oil supplement. Supplements that were refined were free of this contamination. Fish used in the production of fish oil supplements appear to have accumulated petrogenic hydrocarbons in their tissues which were not removed by minimal oil refining. Further study is required to determine if there are any health implications associated with long-term consumption of these contaminated supplements. © 2014 Society of Chemical Industry.

  10. Simultaneous discovery, estimation and prediction analysis of complex traits using a bayesian mixture model.

    Directory of Open Access Journals (Sweden)

    Gerhard Moser

    2015-04-01

    Full Text Available Gene discovery, estimation of heritability captured by SNP arrays, inference on genetic architecture and prediction analyses of complex traits are usually performed using different statistical models and methods, leading to inefficiency and loss of power. Here we use a Bayesian mixture model that simultaneously allows variant discovery, estimation of genetic variance explained by all variants and prediction of unobserved phenotypes in new samples. We apply the method to simulated data of quantitative traits and Welcome Trust Case Control Consortium (WTCCC data on disease and show that it provides accurate estimates of SNP-based heritability, produces unbiased estimators of risk in new samples, and that it can estimate genetic architecture by partitioning variation across hundreds to thousands of SNPs. We estimated that, depending on the trait, 2,633 to 9,411 SNPs explain all of the SNP-based heritability in the WTCCC diseases. The majority of those SNPs (>96% had small effects, confirming a substantial polygenic component to common diseases. The proportion of the SNP-based variance explained by large effects (each SNP explaining 1% of the variance varied markedly between diseases, ranging from almost zero for bipolar disorder to 72% for type 1 diabetes. Prediction analyses demonstrate that for diseases with major loci, such as type 1 diabetes and rheumatoid arthritis, Bayesian methods outperform profile scoring or mixed model approaches.

  11. Improved Separation of Complex Polycyclic Aromatic Hydrocarbon Mixtures Using Novel Column Combinations in GC×GC/ToF-MS

    Science.gov (United States)

    Manzano, Carlos; Hoh, Eunha; Simonich, Staci L. Massey

    2012-01-01

    Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are difficult to resolve because of the high degree of overlap in compound vapor pressures, boiling points and mass spectral fragmentation patterns. The objective of this research was to improve the separation of complex PAH mixtures (including 97 different parent, alkyl-, nitro-, oxy-, thio-, chloro-, bromo-, and high molecular weight PAHs) using GC×GC/ToF-MS by maximizing the orthogonality of different GC column combinations and improving the separation of PAHs from the sample matrix interferences, including unresolved complex mixtures (UCM). Four different combinations of non-polar, polar, liquid crystal and nano-stationary phase columns were tested. Each column combination was optimized and evaluated for orthogonality using a method based on conditional entropy that considers the quantitative peak distribution in the entire two-dimensional space. Finally, an atmospheric particulate matter with diameter column in the first dimension and a 1.2 m × 0.10 mm × 0.10 µm NSP-35 nano-stationary phase column in the second dimension. In addition, the use of this column combination in GC×GC/ToF-MS resulted in significantly shorter analysis times (176 min) for complex PAH mixtures compared to one-dimensional GC/MS (257 min), as well as potentially reduced sample preparation time. PMID:22769970

  12. Introducing Students to Gas Chromatography-Mass Spectrometry Analysis and Determination of Kerosene Components in a Complex Mixture

    Science.gov (United States)

    Pacot, Giselle Mae M.; Lee, Lyn May; Chin, Sung-Tong; Marriott, Philip J.

    2016-01-01

    Gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS (GC-MS/MS) are useful in many separation and characterization procedures. GC-MS is now a common tool in industry and research, and increasingly, GC-MS/MS is applied to the measurement of trace components in complex mixtures. This report describes an upper-level undergraduate experiment…

  13. Tumour promotion by complex mixtures of polyhalogenated aromatic hydrocarbons (PHAHs) and the applicability of the toxic equivalency factor (TEF) concept

    NARCIS (Netherlands)

    Plas, van der S.A.

    2000-01-01

    The aim of the project described in this thesis consisted of two main objectives, first, to examine the tumour promotion potential of complex, environmentally relevant mixtures of polychlorinated biphenyls (PCBs), polychlorinated dibenzo- p -dioxins (PCDDs) and

  14. Polarographic reduction of Yb/sup +3/ benzoate and salicylate complexes in aqueous-nonaqueous mixtures at D. M. E

    Energy Technology Data Exchange (ETDEWEB)

    Zutshi, K; Gupta, K C [Rajasthan Univ., Jaipur (India). Dept. of Chemistry

    1977-01-01

    The reduction of Yb/sup +3/ and Yb/sup +3/-benzoate and salicylate complexes was studied polarographically at constant ionic strength at 25 +- 0.02/sup 0/C in aqueous-nonaqueous mixtures. The reduction was found to be diffusion-controlled, but the electrode process was irreversible in all cases. The kinetic parameters were determined by Koutecky's method.

  15. Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Cordwell, Stuart J; Roepstorff, Peter

    2002-01-01

    The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed......-phase (RP) microcolumns prior to mass spectrometric analysis have resulted in increased signal-to-noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation...

  16. DPPH radical scavenging activity of a mixture of fatty acids and peptide-containing compounds in a protein hydrolysate of Jatropha curcas seed cake.

    Science.gov (United States)

    Phengnuam, Thanyarat; Goroncy, Alexander K; Rutherfurd, Shane M; Moughan, Paul J; Suntornsuk, Worapot

    2013-12-04

    Jatropha curcas, a tropical plant, has great potential commercial relevance as its seeds have high oil content. The seeds can be processed into high-quality biofuel producing seed cake as a byproduct. The seed cake, however, has not gotten much attention toward its potential usefulness. This work was aimed to determine the antioxidant activity of different fractions of a protein hydrolysate from J. curcas seed cake and to elucidate the molecular structures of the antioxidants. Seed cake was first processed into crude protein isolate and the protein was hydrolyzed by Neutrase. The hydrolysate obtained from 1 h of Neutrase hydrolysis showed the strongest antioxidant activity against DPPH radical (2,2-diphenyl-1-picrylhydrazyl). After a purification series of protein hydrolysate by liquid chromatography, chemicals acting as DPPH radical inhibitors were found to be a mixture of fatty acids, fatty acid derivatives, and a small amount of peptides characterized by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

  17. Simultaneous Determination of Binding Constants for Multiple Carbohydrate Hosts in Complex Mixtures

    DEFF Research Database (Denmark)

    Meier, Sebastian; Beeren, Sophie

    2014-01-01

    We describe a simple method for the simultaneous determination of association constants for a guest binding to seven different hosts in a mixture of more than 20 different oligosaccharides. If the binding parameters are known for one component in the mixture, a single NMR titration suffices...

  18. Rapid Quadrupole-Time-of-Flight Mass Spectrometry Method Quantifies Oxygen-Rich Lignin Compound in Complex Mixtures

    Science.gov (United States)

    Boes, Kelsey S.; Roberts, Michael S.; Vinueza, Nelson R.

    2018-03-01

    Complex mixture analysis is a costly and time-consuming task facing researchers with foci as varied as food science and fuel analysis. When faced with the task of quantifying oxygen-rich bio-oil molecules in a complex diesel mixture, we asked whether complex mixtures could be qualitatively and quantitatively analyzed on a single mass spectrometer with mid-range resolving power without the use of lengthy separations. To answer this question, we developed and evaluated a quantitation method that eliminated chromatography steps and expanded the use of quadrupole-time-of-flight mass spectrometry from primarily qualitative to quantitative as well. To account for mixture complexity, the method employed an ionization dopant, targeted tandem mass spectrometry, and an internal standard. This combination of three techniques achieved reliable quantitation of oxygen-rich eugenol in diesel from 300 to 2500 ng/mL with sufficient linearity (R2 = 0.97 ± 0.01) and excellent accuracy (percent error = 0% ± 5). To understand the limitations of the method, it was compared to quantitation attained on a triple quadrupole mass spectrometer, the gold standard for quantitation. The triple quadrupole quantified eugenol from 50 to 2500 ng/mL with stronger linearity (R2 = 0.996 ± 0.003) than the quadrupole-time-of-flight and comparable accuracy (percent error = 4% ± 5). This demonstrates that a quadrupole-time-of-flight can be used for not only qualitative analysis but also targeted quantitation of oxygen-rich lignin molecules in complex mixtures without extensive sample preparation. The rapid and cost-effective method presented here offers new possibilities for bio-oil research, including: (1) allowing for bio-oil studies that demand repetitive analysis as process parameters are changed and (2) making this research accessible to more laboratories. [Figure not available: see fulltext.

  19. Enhanced transdermal delivery with less irritation by magainin pore-forming peptide with a N-lauroylsarcosine and sorbitan monolaurate mixture.

    Science.gov (United States)

    Lee, Haerin; Park, Juhyun; Kim, Yeu-Chun

    2018-02-01

    Transdermal drug delivery is advantageous over other conventional drug administration routes. However, it can be inefficient because of the natural barrier of the stratum corneum which is the uppermost layer of the skin. A previous study verified that the treatment of magainin pore-forming peptide with N-lauroylsarcosine (NLS) on human skin can increase skin permeability by 47-fold. However, NLS is well known as a potential skin irritant. The irritation potential of NLS is known to decrease when mixed with sorbitan monolaurate (S20). Encouraged by these results, we combined S20 with magainin-NLS to enhance transdermal drug transport with less skin irritation. In this study, nine groups with magainin and NLS:S20 mixtures at different concentrations and weight fractions were screened to maximize their synergistic effect. To quantify the efficacy to toxicity ratio of each formulation, we defined the ratio as the "enhancement ratio/irritation potential (ER/IP)." The ER was observed by Franz cell diffusion of the target drug fluorescein, and the IP was measured by the cytotoxicity of the NIH/3T3 mouse fibroblast cell line. As a result, the magainin with the NLS:S20 mixture increased the permeability of porcine skin as well as decreased the toxicity. Among the various combinations, a formulation of 2% (w/v) NLS:S20 with a weight fraction of 0.6:0.4 had the largest ER/IP. ATR-FTIR spectroscopy of the formulations and skin was done to analyze the interactions in the formulations themselves and between the formulations and the skin. Both the intercellular lipidic route and transcellular route through the stratum corneum protein were involved in the delivery of fluorescein. This study turned pore-forming peptides into an efficient and safe penetration enhancer by combining them with other chemical penetration enhancers. Moreover, this discovery could be a possible method for enabling the transdermal delivery of macromolecules.

  20. Data on coffee composition and mass spectrometry analysis of mixtures of coffee related carbohydrates, phenolic compounds and peptides

    Directory of Open Access Journals (Sweden)

    Ana S.P. Moreira

    2017-08-01

    Full Text Available The data presented here are related to the research paper entitled “Transglycosylation reactions, a main mechanism of phenolics incorporation in coffee melanoidins: inhibition by Maillard reaction” (Moreira et al., 2017 [1]. Methanolysis was applied in coffee fractions to quantify glycosidically-linked phenolics in melanoidins. Moreover, model mixtures mimicking coffee beans composition were roasted and analyzed using mass spectrometry-based approaches to disclose the regulatory role of proteins in transglycosylation reactions extension. This article reports the detailed chemical composition of coffee beans and derived fractions. In addition, it provides gas chromatography–mass spectrometry (GC–MS chromatograms and respective GC–MS spectra of silylated methanolysis products obtained from phenolic compounds standards, as well as the detailed identification of all compounds observed by electrospray mass spectrometry (ESI-MS analysis of roasted model mixtures, paving the way for the identification of the same type of compounds in other samples.

  1. Crystal structure of importin-{alpha} complexed with a classic nuclear localization sequence obtained by oriented peptide library screening

    Energy Technology Data Exchange (ETDEWEB)

    Takeda, A.A.S.; Fontes, M.R.M. [UNESP, Universidade Estadual Paulista, Botucatu, SP (Brazil); Yang, S.N.Y. [University of Melbourne, Melbourne (Australia); Harris, J.M. [Queensland University of Technology, Brisbane (Australia); Jans, D.A. [Monash University, Clayton (Australia); Kobe, B. [University of Queensland, Brisbane, QU (Australia)

    2012-07-01

    Full text: Importin-{alpha} (Imp{alpha}) plays a role in the classical nuclear import pathway, binding to cargo proteins with activities in the nucleus. Different Imp{alpha} paralogs responsible for specific cargos can be found in a single organism. The cargos contain nuclear localization sequences (NLSs), which are characterized by one or two clusters of basic amino acids (monopartite and bipartite NLSs, respectively). In this work we present the crystal structure of Imp{alpha} from M. musculus (residues 70-529, lacking the auto inhibitory domain) bound to a NLS peptide (pepTM). The peptide corresponds to the optimal sequence obtained by an oriented peptide library experiment designed to probe the specificity of the major NLS binding site. The peptide library used five degenerate positions and identified the sequence KKKRR as the optimal sequence for binding to this site for mouse Imp{alpha} (70-529). The protein was obtained using an E. coli expression system and purified by affinity chromatography followed by an ion exchange chromatography. A single crystal of Imp{alpha} -pepTM complex was grown by the hanging drop method. The data were collected using the Synchrotron Radiation Source LNLS, Brazil and processed to 2.3. Molecular replacement techniques were used to determine the crystal structure. Electron density corresponding to the peptide was present in both major and minor binding sites The peptide is bound to Imp{alpha} similar as the simian virus 40 (SV40) large tumour (T)-antigen NLS. Binding assays confirmed that the peptide bound to Imp{alpha} with low nM affinities. This is the first time that structural information has been linked to an oriented peptide library screening approach for importin-{alpha}; the results will contribute to understanding of the sequence determinants of classical NLSs, and may help identify as yet unidentified classical NLSs in novel proteins. (author)

  2. T cell responses affected by aminopeptidase N (CD13)-mediated trimming of major histocompatibility complex class II-bound peptides

    DEFF Research Database (Denmark)

    Larsen, S L; Pedersen, L O; Buus, S

    1996-01-01

    Endocytosed protein antigens are believed to be fragmented in what appears to be a balance between proteolysis and MHC-mediated epitope protection, and the resulting peptide-MHC complexes are transported to the surface of the antigen-presenting cells (APC) and presented to T cells. The events tha...

  3. Availability of endogenous peptides limits expression of an M3a-Ld major histocompatibility complex class I chimera

    Science.gov (United States)

    1994-01-01

    Taking advantage of our understanding of the peptide specificity of the major histocompatibility complex class I-b molecule M3a, we sought to determine why these molecules are poorly represented on the cell surface. To this end we constructed a chimeric molecule with the alpha 1 and alpha 2 domains of M3a and alpha 3 of Ld thereby allowing use of available monoclonal antibodies to quantify surface expression. Transfected, but not control, B10.CAS2 (H-2M3b) cells were lysed readily by M3a-restricted monoclonal cytotoxic T lymphocytes. Thus, the chimera bound, trafficked, and presented endogenous mitochondrial peptides. However, despite high levels of M3a-Ld mRNA, transfectants were negative by surface staining. This finding was consistent with inefficient trafficking to the cell surface. Incubation at 26 degrees C, thought to permit trafficking of unoccupied heavy (H) chains, resulted in detectable cell surface expression of chimeric molecules. Incubation with exogenous peptide at 26 degrees C (but not at 37 degrees C) greatly enhanced expression of M3a-Ld molecules in a dose- dependent manner, suggesting stabilization of unoccupied molecules. Stable association of beta 2-microglobulin with the chimeric H chain was observed in labeled cell lysates only in the presence of exogenous specific peptide, indicating that peptide is required for the formation of a ternary complex. These results indicate that surface expression of M3a-Ld is limited largely by the steady-state availability of endogenous peptides. Since most known M3a-binding peptides are N- formylated, native M3a may normally be expressed at high levels only during infection by intracellular bacteria. PMID:8270862

  4. Delivering Transmembrane Peptide Complexes to the Gas Phase Using Nanodiscs and Electrospray Ionization

    Science.gov (United States)

    Li, Jun; Richards, Michele R.; Kitova, Elena N.; Klassen, John S.

    2017-10-01

    The gas-phase conformations of dimers of the channel-forming membrane peptide gramicidin A (GA), produced from isobutanol or aqueous solutions of GA-containing nanodiscs (NDs), are investigated using electrospray ionization-ion mobility separation-mass spectrometry (ESI-IMS-MS) and molecular dynamics (MD) simulations. The IMS arrival times measured for (2GA + 2Na)2+ ions from isobutanol reveal three different conformations, with collision cross-sections (Ω) of 683 Å2 (conformation 1, C1), 708 Å2 (C2), and 737 Å2 (C3). The addition of NH4CH3CO2 produced (2GA + 2Na)2+ and (2GA + H + Na)2+ ions, with Ω similar to those of C1, C2, and C3, as well as (2GA + 2H)2+, (2GA + 2NH4)2+, and (2GA + H + NH4)2+ ions, which adopt a single conformation with a Ω similar to that of C2. These results suggest that the nature of the charging agents, imparted by the ESI process, can influence dimer conformation in the gas phase. Notably, the POPC NDs produced exclusively (2GA + 2NH4)2+ dimer ions; the DMPC NDs produced both (2GA + 2H)2+ and (2GA + 2NH4)2+ dimer ions. While the Ω of (2GA + 2H)2+ is similar to that of C2, the (2GA + 2NH4)2+ ions from NDs adopt a more compact structure, with a Ω of 656 Å2. It is proposed that this compact structure corresponds to the ion conducting single stranded head-to-head helical GA dimer. These findings highlight the potential of NDs, combined with ESI, for transferring transmembrane peptide complexes directly from lipid bilayers to the gas phase. [Figure not available: see fulltext.

  5. Selective Sensing of Tyrosine Phosphorylation in Peptides Using Terbium(III Complexes

    Directory of Open Access Journals (Sweden)

    Jun Sumaoka

    2016-01-01

    Full Text Available Phosphorylation of tyrosine residues in proteins, as well as their dephosphorylation, is closely related to various diseases. However, this phosphorylation is usually accompanied by more abundant phosphorylation of serine and threonine residues in the proteins and covers only 0.05% of the total phosphorylation. Accordingly, highly selective detection of phosphorylated tyrosine in proteins is an urgent subject. In this review, recent developments in this field are described. Monomeric and binuclear TbIII complexes, which emit notable luminescence only in the presence of phosphotyrosine (pTyr, have been developed. There, the benzene ring of pTyr functions as an antenna and transfers its photoexcitation energy to the TbIII ion as the emission center. Even in the coexistence of phosphoserine (pSer and phosphothreonine (pThr, pTyr can be efficintly detected with high selectivity. Simply by adding these TbIII complexes to the solutions, phosphorylation of tyrosine in peptides by protein tyrosine kinases and dephosphorylation by protein tyrosine phosphatases can be successfully visualized in a real-time fashion. Furthermore, the activities of various inhibitors on these enzymes are quantitatively evaluated, indicating a strong potential of the method for efficient screening of eminent inhibitors from a number of candidates.

  6. Ion-Exchange Sample Displacement Chromatography as a Method for Fast and Simple Isolation of Low- and High-Abundance Proteins from Complex Biological Mixtures

    Directory of Open Access Journals (Sweden)

    Martina Srajer Gajdosik

    2014-01-01

    Full Text Available Sample displacement chromatography (SDC in reversed phase and ion-exchange modes was introduced at the end of 1980s. This chromatographic method was first used for preparative purification of synthetic peptides, and subsequently adapted for protein fractionation, mainly in anion-exchange mode. In the past few years, SDC has been successfully used for enrichment of low- and medium-abundance proteins from complex biological fluids on both monolithic and bulk chromatographic supports. If aqueous mobile phase is used with the application of mild chromatographic conditions, isolated proteins are not denatured and can also keep their biological activity. In this paper, the use of SDC in anion-exchange mode on a high-capacity chromatographic resin for separation of proteins from complex biological mixtures such as human plasma is demonstrated. By use of three and more columns coupled in series during sample application, and subsequent parallel elution of detached columns, additional separation of bound proteins was achieved. Highly enriched human serum albumin fraction and a number of physiologically active medium- and low-abundance proteins could be fractionated and detected by electrospray ionization tandem mass spectrometry (ESI-MS/MS and matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS. The use of the aforementioned columns that can be sanitized with 1 M sodium hydroxide for further application of SDC in biotechnology and food technology was discussed.

  7. Separation of rare earth mixtures by gas chromatography using dipivaloylmethane as complexing agent

    International Nuclear Information System (INIS)

    Golubtsova, V.Yu.; Luchinkin, V.V.; Martynenko, L.I.; Murav'eva, I.A.; Sokolov, D.N.

    1981-01-01

    Possibility of using dipivaloylmethave for quantitative separation of rare earth element mixtures under the regime of chromatography for preparative and analytical purposes, is studied. Introduction of β-diketone surplus into the chromatographic solution is shown to remove the necessity of column conditioning. It is stated that chelate solution should have concentration above the threshold one. The developed method is applicable for quantitative separation of some rare earth mixtures for preparative purposes, as well as for the analysis of rare earth mixtures, containing components in equivalent quantities [ru

  8. Comparative Analysis of Stress Induced Gene Expression in Caenorhabditis elegans following Exposure to Environmental and Lab Reconstituted Complex Metal Mixture.

    Directory of Open Access Journals (Sweden)

    Ranjeet Kumar

    Full Text Available Metals are essential for many physiological processes and are ubiquitously present in the environment. However, high metal concentrations can be harmful to organisms and lead to physiological stress and diseases. The accumulation of transition metals in the environment due to either natural processes or anthropogenic activities such as mining results in the contamination of water and soil environments. The present study used Caenorhabditis elegans to evaluate gene expression as an indicator of physiological response, following exposure to water collected from three different locations downstream of a Swedish mining site and a lab reconstituted metal mixture. Our results indicated that the reconstituted metal mixture exerted a direct stress response in C. elegans whereas the environmental waters elicited either a diminished or abrogated response. This suggests that it is not sufficient to use the biological effects observed from laboratory mixtures to extrapolate the effects observed in complex aquatic environments and apply this to risk assessment and intervention.

  9. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  10. New horizons in mouse immunoinformatics: reliable in silico prediction of mouse class I histocompatibility major complex peptide binding affinity.

    Science.gov (United States)

    Hattotuwagama, Channa K; Guan, Pingping; Doytchinova, Irini A; Flower, Darren R

    2004-11-21

    Quantitative structure-activity relationship (QSAR) analysis is a main cornerstone of modern informatic disciplines. Predictive computational models, based on QSAR technology, of peptide-major histocompatibility complex (MHC) binding affinity have now become a vital component of modern day computational immunovaccinology. Historically, such approaches have been built around semi-qualitative, classification methods, but these are now giving way to quantitative regression methods. The additive method, an established immunoinformatics technique for the quantitative prediction of peptide-protein affinity, was used here to identify the sequence dependence of peptide binding specificity for three mouse class I MHC alleles: H2-D(b), H2-K(b) and H2-K(k). As we show, in terms of reliability the resulting models represent a significant advance on existing methods. They can be used for the accurate prediction of T-cell epitopes and are freely available online ( http://www.jenner.ac.uk/MHCPred).

  11. Syntheses and structures of technetium(V) and rhenium(V) oxo complexes of peptide having KYC-sequence

    International Nuclear Information System (INIS)

    Takayama, T.; Suzuki, K.; Sekine, T.; Kudo, H.

    2000-01-01

    Technetium(V) and rhenium(V) oxo complexes of a peptide having a KYC-sequence such as KYCAR (H 3 L 5 ) and KYCAREPPTRTNAYQGQG-NH 2 (H 3 L 18 ) were synthesized, and structures of the complexes were characterized by spectroscopic techniques. All of the complexes were synthesized by the ligand exchange reaction of [(n-C 4 H 9 ) 4 N][MOCl 4 ] (M = 99 Tc, Re) with peptide in methanol or dimethylformamide solution. These complexes have a square pyramidal structure with an oxo ligand at the apical position. The peptide is coordinated to a metal atom through N amine of lysine. S thiol of cysteine, and N amide of tyrosine and cysteine in the equatorial plane. A lysine (CH 2 ) 4 NH 2 group of the L 5 ligand has the syn conformation with respect to metal-oxo bonding in the complex. The syn isomer was selectively formed in the ligand exchange reaction. The conversion of the syn isomer to the anti isomer was observed only for syn-[ReO(L 5 )], in which the coordination of water to the trans position of the oxo ligand was involved. (orig.)

  12. Syntheses and structures of technetium(V) and rhenium(V) oxo complexes of peptide having KYC-sequence

    Energy Technology Data Exchange (ETDEWEB)

    Takayama, T.; Suzuki, K.; Sekine, T.; Kudo, H. [Dept. of Chemistry, Tohoku Univ., Sendai (Japan)

    2000-07-01

    Technetium(V) and rhenium(V) oxo complexes of a peptide having a KYC-sequence such as KYCAR (H{sub 3}L{sup 5}) and KYCAREPPTRTNAYQGQG-NH{sub 2} (H{sub 3}L{sup 18}) were synthesized, and structures of the complexes were characterized by spectroscopic techniques. All of the complexes were synthesized by the ligand exchange reaction of [(n-C{sub 4}H{sub 9}){sub 4}N][MOCl{sub 4}] (M = {sup 99}Tc, Re) with peptide in methanol or dimethylformamide solution. These complexes have a square pyramidal structure with an oxo ligand at the apical position. The peptide is coordinated to a metal atom through N{sub amine} of lysine. S{sub thiol} of cysteine, and N{sub amide} of tyrosine and cysteine in the equatorial plane. A lysine (CH{sub 2}){sub 4}NH{sub 2} group of the L{sup 5} ligand has the syn conformation with respect to metal-oxo bonding in the complex. The syn isomer was selectively formed in the ligand exchange reaction. The conversion of the syn isomer to the anti isomer was observed only for syn-[ReO(L{sup 5})], in which the coordination of water to the trans position of the oxo ligand was involved. (orig.)

  13. Chronic toxicity of unresolved complex mixtures (UCM) of hydrocarbons in marine sediments

    Energy Technology Data Exchange (ETDEWEB)

    Scarlett, A.; Galloway, T.S. [Plymouth Univ., Drake Circus (United Kingdom). School of Biological Sciences; Rowland, S.J. [Plymouth Univ., Drake Circus (United Kingdom). School of Earth, Ocean and Environmental Sciences

    2007-08-15

    Background, Aim and Scope: Unresolved complex mixtures (UCM) of hydrocarbons, containing many thousands of compounds which cannot be resolved by conventional gas chromatography (GC), are common contaminants of sediments but little is known of their potential to affect sediment-dwelling organisms. Evidence exists for reduced health status in mussels, arising from aqueous exposure to aromatic UCM components acting through a narcotic mode of action. However, UCM contaminants in sediments may not be sufficiently bioavailable to elicit toxic effects. The aim of our study was therefore to measure the sublethal effects of chronic exposure to model UCM-dominated oils at environmentally realistic concentrations and compare this to effects produced by a UCM containing weathered crude oil. A further aim was to determine which, if any, fractions of the oils were responsible for any observed toxicity. Materials and Methods: Whole oils were spiked into estuarine sediment to give nominal concentrations of 500 {mu}g g-1 dry weight. Juveniles of the estuarine amphipod Corophium volutator were exposed to the contaminated sediment for 35 days and their survival, growth rate and reproductive success quantified. Using an effect-directed fractionation approach, the oils were fractionated into aliphatic and two aromatic fractions by open column chromatography and their toxicity assessed by further chronic exposures using juvenile C. volutator. Results: The growth rates of amphipods were reduced following exposure to the oils although this was only statistically significant for the weathered oil; reproductive success was reduced by all oil exposures. Sediment spiked with UCM fractions also caused reduced growth and reproduction but no particular fraction was found to be responsible for the observed toxicity. Survivorship was not affected by any oil or fraction. Discussion: The study showed that chronic exposure to sediments contaminated by UCM-dominated oils could have population level

  14. Collision-induced dissociation of noncovalent complexes between vancomycin antibiotics and peptide ligand stereoisomers: evidence for molecular recognition in the gas phase

    DEFF Research Database (Denmark)

    Jørgensen, Thomas J. D.; Delforge, D; Remacle, J

    1999-01-01

    In solution, the antibiotics of the vancomycin group bind stereospecifically to peptides with the C-terminal sequence: -L-Lys-D-Ala-D-Ala, Substitution by a L-Ala at either of the two C-terminal residues causes a dramatic decrease in the binding affinity to the antibiotics. This solution behavior...... is clearly reflected in electrospray ionization (ESI) mass spectra obtained from equimolar mixtures of an antibiotic, an isotopically labelled peptide ligand and an unlabelled peptide stereoisomer. Using collision-induced dissociation (CID) we have probed the gas phase stability of isomeric (1:1) noncovalent...

  15. Preparation and bioavailability of calcium-chelating peptide complex from tilapia skin hydrolysates.

    Science.gov (United States)

    Chen, Jun; Qiu, Xujian; Hao, Gengxin; Zhang, Meng; Weng, Wuyin

    2017-11-01

    With the continuous improvement in material life, the generation of fish by-products and the market demand for calcium supplements have been increasing in China. Therefore a calcium-chelating peptide complex (CPC) from tilapia skins was prepared and its effect on calcium (Ca)-deficient mice was investigated. The molecular weight distribution of CPC mainly ranged from 2000 to 180 Da, and its contents of complete amino acids and free amino acids were 85.30 and 8.67% (w/w) respectively. Scanning electron microscopy images and Fourier transform infrared data revealed that Ca crystals were bound with gelatin hydrolysates via interaction between Ca ions and NH/CN groups. When Ca-deficient mice were fed CPC and CaCO 3 respectively for 4 weeks, no significant differences in serum biochemistry or bone mineral density were found. However, the length, weight, Ca content and hydroxyproline content of the femur, Ca absorption and body weight gain of mice fed CPC were significantly higher than those of mice fed CaCO 3 . It is concluded that the prepared CPC could promote bone formation via better bioavailability of Ca and an increase in bone collagen. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  16. Analysis of skin derived peptides from the Cuyaba Dwarf Frog Physalaemus nattereri by off-line LC MALDI MS/MS

    DEFF Research Database (Denmark)

    Castro, Mariana S; Pires Júnior, Osmindo Rodrigues; Fontes, Wagner

    2017-01-01

    We have investigated the potential for analysis of the complex peptide mixtures secreted from frog skin using off-line LC-MALDI MS/MS. Since only limited information about the sequence of such peptides is available, de novo sequencing followed by Blast search was needed. An automated workflow has...... for future studies of peptides secreted from frogs....

  17. Improved Resolution of Hydrocarbon Structures and Constitutional Isomers in Complex Mixtures Using Gas Chromatography-Vacuum Ultraviolet-Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Isaacman, Gabriel [Univ. of California, Berkeley, CA (United States); Wilson, Kevin R. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Chan, Arthur W. H. [Univ. of California, Berkeley, CA (United States); Worton, David R. [Univ. of California, Berkeley, CA (United States). Aerosol Dynamics Inc., Berkeley, CA (United States); Kimmel, Joel R. [Aerodyne Research, Inc., Billerica, MA (United States); Univ. of Colorado, Boulder, CO (United States). Tofwerk AG, Thun (Switzerland); Nah, Theodora [Univ. of California, Berkeley, CA (United States); Hohaus, Thorsten [Aerodyne Research, Inc., Billerica, MA (United States); Gonin, Marc [Tofwerk AG, Thun (Switzerland); Kroll, Jesse H. [Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Worsnop, Douglas R. [Aerodyne Research, Inc., Billerica, MA (United States); Goldstein, Allen H. [Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2012-01-30

    Understanding the composition of complex hydrocarbon mixtures is important for environmental studies in a variety of fields, but many prevalent compounds cannot be confidently identified using traditional gas chromatography/mass spectrometry (GC/MS) techniques. In this study, we use vacuum-ultraviolet (VUV) ionization to elucidate the structures of a traditionally “unresolved complex mixture” by separating components by GC retention time, tR, and mass-to-charge ratio, m/z, which are used to determine carbon number, NC, and the number of rings and double bonds, NDBE. Constitutional isomers are resolved on the basis of tR, enabling the most complete quantitative analysis to date of structural isomers in an environmentally relevant hydrocarbon mixture. Unknown compounds are classified in this work by carbon number, degree of saturation, presence of rings, and degree of branching, providing structural constraints. The capabilities of this analysis are explored using diesel fuel, in which constitutional isomer distribution patterns are shown to be reproducible between carbon numbers and follow predictable rules. Nearly half of the aliphatic hydrocarbon mass is shown to be branched, suggesting branching is more important in diesel fuel than previously shown. Lastly, the classification of unknown hydrocarbons and the resolution of constitutional isomers significantly improves resolution capabilities for any complex hydrocarbon mixture.

  18. Structure of the complex between teicoplanin and a bacterial cell-wall peptide: use of a carrier-protein approach

    International Nuclear Information System (INIS)

    Economou, Nicoleta J.; Zentner, Isaac J.; Lazo, Edwin; Jakoncic, Jean; Stojanoff, Vivian; Weeks, Stephen D.; Grasty, Kimberly C.; Cocklin, Simon; Loll, Patrick J.

    2013-01-01

    Using a carrier-protein strategy, the structure of teicoplanin bound to its bacterial cell-wall target has been determined. The structure reveals the molecular determinants of target recognition, flexibility in the antibiotic backbone and intrinsic radiation sensitivity of teicoplanin. Multidrug-resistant bacterial infections are commonly treated with glycopeptide antibiotics such as teicoplanin. This drug inhibits bacterial cell-wall biosynthesis by binding and sequestering a cell-wall precursor: a d-alanine-containing peptide. A carrier-protein strategy was used to crystallize the complex of teicoplanin and its target peptide by fusing the cell-wall peptide to either MBP or ubiquitin via native chemical ligation and subsequently crystallizing the protein–peptide–antibiotic complex. The 2.05 Å resolution MBP–peptide–teicoplanin structure shows that teicoplanin recognizes its ligand through a combination of five hydrogen bonds and multiple van der Waals interactions. Comparison of this teicoplanin structure with that of unliganded teicoplanin reveals a flexibility in the antibiotic peptide backbone that has significant implications for ligand recognition. Diffraction experiments revealed an X-ray-induced dechlorination of the sixth amino acid of the antibiotic; it is shown that teicoplanin is significantly more radiation-sensitive than other similar antibiotics and that ligand binding increases radiosensitivity. Insights derived from this new teicoplanin structure may contribute to the development of next-generation antibacterials designed to overcome bacterial resistance

  19. Optical Coherence Tomography Noise Reduction Using Anisotropic Local Bivariate Gaussian Mixture Prior in 3D Complex Wavelet Domain

    OpenAIRE

    Rabbani, Hossein; Sonka, Milan; Abramoff, Michael D.

    2013-01-01

    In this paper, MMSE estimator is employed for noise-free 3D OCT data recovery in 3D complex wavelet domain. Since the proposed distribution for noise-free data plays a key role in the performance of MMSE estimator, a priori distribution for the pdf of noise-free 3D complex wavelet coefficients is proposed which is able to model the main statistical properties of wavelets. We model the coefficients with a mixture of two bivariate Gaussian pdfs with local parameters which are able to capture th...

  20. Binding stability of peptides on major histocompatibility complex class I proteins: role of entropy and dynamics

    Science.gov (United States)

    Gul, Ahmet; Erman, Burak

    2018-03-01

    Prediction of peptide binding on specific human leukocyte antigens (HLA) has long been studied with successful results. We herein describe the effects of entropy and dynamics by investigating the binding stabilities of 10 nanopeptides on various HLA Class I alleles using a theoretical model based on molecular dynamics simulations. The fluctuational entropies of the peptides are estimated over a temperature range of 310-460 K. The estimated entropies correlate well with experimental binding affinities of the peptides: peptides that have higher binding affinities have lower entropies compared to non-binders, which have significantly larger entropies. The computation of the entropies is based on a simple model that requires short molecular dynamics trajectories and allows for approximate but rapid determination. The paper draws attention to the long neglected dynamic aspects of peptide binding, and provides a fast computation scheme that allows for rapid scanning of large numbers of peptides on selected HLA antigens, which may be useful in defining the right peptides for personal immunotherapy.

  1. Evaluating Complex Mixtures in the Zebrafish Embryo by Reconstituting Field Water Samples: A Metal Pollution Case Study

    Directory of Open Access Journals (Sweden)

    Ellen D. G. Michiels

    2017-03-01

    Full Text Available Accurately assessing the toxicity of complex, environmentally relevant mixtures remains an important challenge in ecotoxicology. The goal was to identify biological effects after exposure to environmental water samples and to determine whether the observed effects could be explained by the waterborne metal mixture found in the samples. Zebrafish embryos were exposed to water samples of five different sites originating from two Flemish (Mol and Olen, Belgium metal contaminated streams: “Scheppelijke Nete” (SN and “Kneutersloop” (K, and a ditch (D, which is the contamination source of SN. Trace metal concentrations, and Na, K, Mg and Ca concentrations were measured using ICP-MS and were used to reconstitute site-specific water samples. We assessed whether the effects that were observed after exposure to environmental samples could be explained by metal mixture toxicity under standardized laboratory conditions. Exposure to “D” or “reconstituted D” water caused 100% mortality. SN and reconstituted SN water caused similar effects on hatching, swim bladder inflation, growth and swimming activity. A canonical discriminant analysis confirmed a high similarity between both exposure scenarios, indicating that the observed toxicity was indeed primarily caused by metals. The applied workflow could be a valuable approach to evaluate mixture toxicity that limits time and costs while maintaining biological relevance.

  2. Cyanine-based probe\\tag-peptide pair fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M. Uljana; Cao, Haishi

    2013-01-15

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  3. Multidimensional profiling of components in complex mixtures of natural products for metabolic analysis, proof of concept: application to Quillaja saponins.

    Science.gov (United States)

    Bankefors, Johan; Nord, Lars I; Kenne, Lennart

    2010-02-01

    A method for separation and detection of major and minor components in complex mixtures has been developed, utilising two-dimensional high-performance liquid chromatography (2D-HPLC) combined with electrospray ionisation ion-trap multiple-stage mass spectrometry (ESI-ITMS(n)). Chromatographic conditions were matched with mass spectrometric detection to maximise the number of components that could be separated. The described procedure has proven useful to discern several hundreds of saponin components when applied to Quillaja saponaria Molina bark extracts. The discrimination of each saponin component relies on the fact that three coordinates (x, y, z) for each component can be derived from the retention time of the two chromatographic steps (x, y) and the m/z-values from the multiple-stage mass spectrometry (z(n), n=1, 2, ...). Thus an improved graphical representation was obtained by combining retention times from the two-stage separation with +MS(1) (z(1)) and the additional structural information from the second mass stage +MS(2) (z(2), z(3)) corresponding to the main fragment ions. By this approach three-dimensional plots can be made that reveal both the chromatographic and structural properties of a specific mixture which can be useful in fingerprinting of complex mixtures. 2009 Elsevier B.V. All rights reserved.

  4. Simulation study of the production of biodiesel using feedstock mixtures of fatty acids in complex reactive distillation columns

    International Nuclear Information System (INIS)

    Cossio-Vargas, E.; Hernandez, S.; Segovia-Hernandez, J.G.; Cano-Rodriguez, M.I.

    2011-01-01

    Biodiesel can be produced from a number of natural, renewable sources, but vegetable oils are the main feedstocks. The current manufacturing biodiesel processes, however, have several disadvantages: expensive separation of products from the reaction mixture, and high costs due to relatively complex processes involving one to two reactors and several separation units. Therefore, to solve these problems, in recent years several researchers have developed a sustainable biodiesel production process based on reactive distillation. In this paper the production of biodiesel using feedstock mixtures of fatty acids is explored using reactive distillation sequences with thermal coupling. The results indicate that the complex reactive distillation sequences can produce a mixture of esters as bottoms product that can be used as biodiesel. In particular, the thermally coupled distillation sequence involving a side rectifier can handle the reaction and complete separation in accordance with process intensification principles. -- Highlights: ► Production of biodiesel using thermally coupled distillation sequences without reboilers. ► Esterification of fatty organic acids using reactive distillation. ► Carnot’s factor in reactive distillation.

  5. Structure of a SARS coronavirus-derived peptide bound to the human major histocompatibility complex class I molecule HLA-B*1501

    DEFF Research Database (Denmark)

    Røder, Gustav; Kristensen, Ole; Kastrup, Jette S

    2008-01-01

    , the crystal structure of HLA-B*1501 in complex with a SARS coronavirus-derived nonapeptide (VQQESSFVM) has been determined at high resolution (1.87 A). The peptide is deeply anchored in the B and F pockets, but with the Glu4 residue pointing away from the floor in the peptide-binding groove, making...

  6. Cryotrapping assisted mass spectrometry for the analysis of complex gas mixtures

    International Nuclear Information System (INIS)

    Ferreira, Jose A.; Tabares, Francisco L.

    2007-01-01

    A simple method is described for the unambiguous identification of the individual components in a gas mixture showing strong overlapping of their mass spectrometric cracking patterns. The method, herein referred to as cryotrapping assisted mass spectrometry, takes advantage of the different vapor pressure values of the individual components at low temperature (78 K for liquid nitrogen traps), and thus of the different depletion efficiencies and outgassing patterns during the fast cooling and slow warming up of the trap, respectively. Examples of the use of this technique for gas mixtures with application to plasma enhanced chemical vapor deposition of carbon and carbon-nitrogen hard films are shown. Detection of traces of specific C 3 hydrocarbons ( 2 containing deposition plasmas are addressed as representative examples of specific applications of the technique

  7. Band-selective excited ultrahigh resolution PSYCHE-TOCSY: fast screening of organic molecules and complex mixtures.

    Science.gov (United States)

    Kakita, Veera Mohana Rao; Vemulapalli, Sahithya Phani Babu; Bharatam, Jagadeesh

    2016-04-01

    Precise assignments of (1) H atomic sites and establishment of their through-bond COSY or TOCSY connectivity are crucial for molecular structural characterization by using (1) H NMR spectroscopy. However, this exercise is often hampered by signal overlap, primarily because of (1) H-(1) H scalar coupling multiplets, even at typical high magnetic fields. The recent developments in homodecoupling strategies for effectively suppressing the coupling multiplets into nice singlets (pure-shift), particularly, Morris's advanced broadband pure-shift yielded by chirp excitation (PSYCHE) decoupling and ultrahigh resolution PSYCHE-TOCSY schemes, have shown new possibilities for unambiguous structural elucidation of complex organic molecules. The superior broadband PSYCHE-TOCSY exhibits enhanced performance over the earlier TOCSY methods, which however warrants prolonged experimental times due to the requirement of large number of dwell increments along the indirect dimension. Herein, we present fast and band-selective analog of the broadband PSYCHE-TOCSY, which is useful for analyzing complex organic molecules that exhibit characteristic yet crowded spectral regions. The simple pulse scheme relies on band-selective excitation (BSE) followed by PSYCHE homodecoupling in the indirect dimension. The BSE-PSYCHE-TOCSY has been exemplified for Estradiol and a complex carbohydrate mixture comprised of six constituents of closely comparable molecular weights. The experimental times are greatly reduced viz., ~20 fold for Estradiol and ~10 fold for carbohydrate mixture, with respect to the broadband PSYCHE-TOCSY. Furthermore, unlike the earlier homonuclear band-selective decoupling, the BSE-PSYCHE-decoupling provides fully decoupled pure-shift spectra for all the individual chemical sites within the excited band. The BSE-PSYCHE-TOCSY is expected to have significant potential for quick screening of complex organic molecules and mixtures at ultrahigh resolution. Copyright © 2015 John Wiley

  8. Two putative subunits of a peptide pump encoded in the human major histocompatability complex class 2 region

    International Nuclear Information System (INIS)

    Bahram, S.; Arnold, D.; Bresnahan, M.; Strominger, J.L.; Spies, T.

    1991-01-01

    The class 2 region of the human major histocompatibility complex (MHC) may encode several genes controlling the processing of endogenous antigen and the presentation of peptide epitopes by MHC class 1 molecules to cytotoxic T lymphocytes. A previously described peptide supply factor (PSF1) is a member of the multidrug-resistance family of transporters and may pump cytosolic peptides into the membrane-bound compartment where class 1 molecules assemble. A second transporter gene, PSF2, was identified 10 kilobases (kb) from PSF1, near the class 2 DOB gene. The complete sequences of PSF1 and PSF2 were determined from cDNA clones. The translation products are closely related in sequence and predicted secondary structure. Both contain a highly conserved ATP-binding fold and share 25% homology in a hydrophobic domain with a tentative number of eight membrane-spanning segments. Based on the principle dimeric organization of these two domains in other transporters, PSF1 and PSF2 may function as complementary subunits, independently as homodimers, or both. Taken together with previous genetic evidence, the coregulation of PSF1 and PSF2 by γ interferon and the to-some-degree coordinate transcription of these genes suggest a common role in peptide-loading of class 1 molecules, although a distinct function of PSF2 cannot be ruled out

  9. Anxiolytic-Like Effect of a Salmon Phospholipopeptidic Complex Composed of Polyunsaturated Fatty Acids and Bioactive Peptides

    Directory of Open Access Journals (Sweden)

    Nabila Belhaj

    2013-10-01

    Full Text Available A phospholipopeptidic complex obtained by the enzymatic hydrolysis of salmon heads in green conditions; exert anxiolytic-like effects in a time and dose-dependent manner, with no affection of locomotor activity. This study focused on the physico-chemical properties of the lipidic and peptidic fractions from this natural product. The characterization of mineral composition, amino acid and fatty acids was carried out. Stability of nanoemulsions allowed us to realize a behavioral study conducted with four different tests on 80 mice. This work highlighted the dose dependent effects of the natural complex and its various fractions over a period of 14 days compared to a conventional anxiolytic. The intracellular redox status of neural cells was evaluated in order to determine the free radicals scavenging potential of these products in the central nervous system (CNS, after mice sacrifice. The complex peptidic fraction showed a strong scavenging property and similar results were found for the complex as well as its lipidic fraction. For the first time, the results of this study showed the anxiolytic-like and neuroprotective properties of a phospholipopeptidic complex extracted from salmon head. The applications on anxiety disorders might be relevant, depending on the doses, the fraction used and the chronicity of the supplementation.

  10. An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide-Calcium Complex.

    Science.gov (United States)

    Sun, Na; Jin, Ziqi; Li, Dongmei; Yin, Hongjie; Lin, Songyi

    2017-11-08

    The binding mode between the pentapeptide (DHTKE) from egg white hydrolysates and calcium ions was elucidated upon its structural and thermodynamics characteristics. The present study demonstrated that the DHTKE peptide could spontaneously bind calcium with a 1:1 stoichiometry, and that the calcium-binding site corresponded to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Moreover, the effect of the DHTKE-calcium complex on improving the calcium absorption was investigated in vitro using Caco-2 cells. Results showed that the DHTKE-calcium complex could facilitate the calcium influx into the cytosol and further improve calcium absorption across Caco-2 cell monolayers by more than 7 times when compared to calcium-free control. This study facilitates the understanding about the binding mechanism between peptides and calcium ions as well as suggests a potential application of egg white peptides as nutraceuticals to improve calcium absorption.

  11. Biomarkers of Cytotoxic, Genotoxic and Apoptotic Effects in Cyprinus carpio Exposed to Complex Mixture of Contaminants from Hospital Effluents.

    Science.gov (United States)

    Olvera-Néstor, Corina G; Morales-Avila, Enrique; Gómez-Olivan, Leobardo M; Galár-Martínez, Marcela; García-Medina, Sandra; Neri-Cruz, Nadia

    2016-03-01

    Hospital wastewater is an important source of emerging contaminants. Recent studies emphasize the importance of assessing the effects of mixtures of contaminants rather than environmental risk of their individual components, as well as the determination of intrinsic toxicity of wastewater. Mixtures of pollutants has possible interactions that have notable environmental side effects. The aim of this study is an attempt to characterize biomarkers in Cyprinus carpio related to the exposure to a complex mixture of contaminants found in hospital wastewater. Results of a particular hospital effluent show the presence of traces of heavy metals, high chlorine concentration and emerging contaminants such as non-steroidal anti-inflammatory drugs. The LC50 was of 5.49 % at 96 h. The cytotoxic, genotoxic and apoptotic biomarkers increase when fishes were exposed to wastewater (1/10 CL50) from hospital wastewater. This study emphasizes the importance of identifying and quantifying the effects of contaminants as pharmaceuticals, disinfectants and surfactants in order to design and implement an ecotoxicological plan.

  12. Validated univariate and multivariate spectrophotometric methods for the determination of pharmaceuticals mixture in complex wastewater

    Science.gov (United States)

    Riad, Safaa M.; Salem, Hesham; Elbalkiny, Heba T.; Khattab, Fatma I.

    2015-04-01

    Five, accurate, precise, and sensitive univariate and multivariate spectrophotometric methods were developed for the simultaneous determination of a ternary mixture containing Trimethoprim (TMP), Sulphamethoxazole (SMZ) and Oxytetracycline (OTC) in waste water samples collected from different cites either production wastewater or livestock wastewater after their solid phase extraction using OASIS HLB cartridges. In univariate methods OTC was determined at its λmax 355.7 nm (0D), while (TMP) and (SMZ) were determined by three different univariate methods. Method (A) is based on successive spectrophotometric resolution technique (SSRT). The technique starts with the ratio subtraction method followed by ratio difference method for determination of TMP and SMZ. Method (B) is successive derivative ratio technique (SDR). Method (C) is mean centering of the ratio spectra (MCR). The developed multivariate methods are principle component regression (PCR) and partial least squares (PLS). The specificity of the developed methods is investigated by analyzing laboratory prepared mixtures containing different ratios of the three drugs. The obtained results are statistically compared with those obtained by the official methods, showing no significant difference with respect to accuracy and precision at p = 0.05.

  13. Inflammatory and Vasoactive Effects of Serum Following Inhalation of Varied Complex Mixtures.

    Science.gov (United States)

    Aragon, Mario J; Chrobak, Izabela; Brower, Jeremy; Roldan, Luis; Fredenburgh, Laura E; McDonald, Jacob D; Campen, Matthew J

    2016-04-01

    Chronic cardiovascular disease is associated with air pollution exposure in epidemiology and toxicology studies. Inhaled toxicants can induce changes in serum bioactivity that impact endothelial inflammatory gene expression in vitro and impair vasorelaxation ex vivo, which are common precursors to atherosclerosis. Comparisons between single pollutants and common combustion mixtures, in terms of driving such serum inflammatory and vasoactive effects, have not been characterized. Healthy C57BL/6 mice were exposed to a single 6-h period of contrasting pollutant atmospheres: road dust, mixed vehicle emissions (MVE; a combination of gasoline and diesel engine emissions) particulate matter, mixed vehicle emissions gases, road dust plus ozone, road dust plus MVE, and hardwood smoke. Serum obtained from mice 24 h after these exposures was used as a stimulus to assess inflammatory potential in two assays: incubated with primary murine cerebrovascular endothelial cells for 4 h to measure inflammatory gene expression or applied to naïve aortic rings in an ex vivo myographic preparation. Road dust and wood smoke exposures were most potent at inducing inflammatory gene expression, while MVE atmospheres and wood smoke were most potent at impairing vasorelaxation to acetylcholine. Responses are consistent with recent reports on MVE toxicity, but reveal novel serum bioactivity related to wood smoke and road dust. These studies suggest that the compositional changes in serum and resultant bioactivity following inhalation exposure to pollutants may be highly dependent on the composition of mixtures.

  14. Peptide aptamer-assisted immobilization of green fluorescent protein for creating biomolecule-complexed carbon nanotube device

    Science.gov (United States)

    Nii, Daisuke; Nozawa, Yosuke; Miyachi, Mariko; Yamanoi, Yoshinori; Nishihara, Hiroshi; Tomo, Tatsuya; Shimada, Yuichiro

    2017-10-01

    Carbon nanotubes are a novel material for next-generation applications. In this study, we generated carbon nanotube and green fluorescent protein (GFP) conjugates using affinity binding peptides. The carbon nanotube-binding motif was introduced into the N-terminus of the GFP through molecular biology methods. Multiple GFPs were successfully aligned on a single-walled carbon nanotube via the molecular recognition function of the peptide aptamer, which was confirmed through transmission electron microscopy and optical analysis. Fluorescence spectral analysis results also suggested that the carbon nanotube-GFP complex was autonomously formed with orientation and without causing protein denaturation during immobilization. This simple process has a widespread potential for fabricating carbon nanotube-biomolecule hybrid devices.

  15. Selective Gene Delivery for Integrating Exogenous DNA into Plastid and Mitochondrial Genomes Using Peptide-DNA Complexes.

    Science.gov (United States)

    Yoshizumi, Takeshi; Oikawa, Kazusato; Chuah, Jo-Ann; Kodama, Yutaka; Numata, Keiji

    2018-05-14

    Selective gene delivery into organellar genomes (mitochondrial and plastid genomes) has been limited because of a lack of appropriate platform technology, even though these organelles are essential for metabolite and energy production. Techniques for selective organellar modification are needed to functionally improve organelles and produce transplastomic/transmitochondrial plants. However, no method for mitochondrial genome modification has yet been established for multicellular organisms including plants. Likewise, modification of plastid genomes has been limited to a few plant species and algae. In the present study, we developed ionic complexes of fusion peptides containing organellar targeting signal and plasmid DNA for selective delivery of exogenous DNA into the plastid and mitochondrial genomes of intact plants. This is the first report of exogenous DNA being integrated into the mitochondrial genomes of not only plants, but also multicellular organisms in general. This fusion peptide-mediated gene delivery system is a breakthrough platform for both plant organellar biotechnology and gene therapy for mitochondrial diseases in animals.

  16. Deinococcus Mn2+-peptide complex: A novel approach to alphavirus vaccine development.

    Science.gov (United States)

    Gayen, Manoshi; Gupta, Paridhi; Morazzani, Elaine M; Gaidamakova, Elena K; Knollmann-Ritschel, Barbara; Daly, Michael J; Glass, Pamela J; Maheshwari, Radha K

    2017-06-22

    Over the last ten years, Chikungunya virus (CHIKV), an Old World alphavirus has caused numerous outbreaks in Asian and European countries and the Americas, making it an emerging pathogen of great global health importance. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, on the other hand, has been developed as a bioweapon in the past due to its ease of preparation, aerosol dispersion and high lethality in aerosolized form. Currently, there are no FDA approved vaccines against these viruses. In this study, we used a novel approach to develop inactivated vaccines for VEEV and CHIKV by applying gamma-radiation together with a synthetic Mn-decapeptide-phosphate complex (MnDpPi), based on manganous-peptide-orthophosphate antioxidants accumulated in the extremely radiation-resistant bacterium Deinococcus radiodurans. Classical gamma-irradiated vaccine development approaches are limited by immunogenicity-loss due to oxidative damage to the surface proteins at the high doses of radiation required for complete virus-inactivation. However, addition of MnDpPi during irradiation process selectively protects proteins, but not the nucleic acids, from the radiation-induced oxidative damage, as required for safe and efficacious vaccine development. Previously, this approach was used to develop a bacterial vaccine. In the present study, we show that this approach can successfully be applied to protecting mice against viral infections. Irradiation of VEEV and CHIKV in the presence of MnDpPi resulted in substantial epitope preservation even at supra-lethal doses of gamma-rays (50,000Gy). Irradiated viruses were found to be completely inactivated and safe in vivo (neonatal mice). Upon immunization, VEEV inactivated in the presence of MnDpPi resulted in drastically improved protective efficacy. Thus, the MnDpPi-based gamma-inactivation approach described here can readily be applied to developing vaccines against any pathogen of interest in a fast and cost

  17. Synthesis of Reusable Silica Nanosphere-Supported Pt(IV Complex for Formation of Disulfide Bonds in Peptides

    Directory of Open Access Journals (Sweden)

    Xiaonan Hou

    2017-02-01

    Full Text Available Some peptide-based drugs, including oxytocin, vasopressin, ziconotide, pramlintide, nesiritide, and octreotide, contain one intramolecular disulfide bond. A novel and reusable monodispersed silica nanosphere-supported Pt(IV complex (SiO2@TPEA@Pt(IV; TPEA: N-[3-(trimethoxysilylpropyl]ethylenediamine was synthesized via a four-step procedure and was used for the formation of intramolecular disulfide bonds in peptides. Transmission electron microscopy (TEM and chemical mapping results for the Pt(II intermediates and for SiO2@TPEA@Pt(IV show that the silica nanospheres possess a monodisperse spherical structure and contain uniformly-distributed Si, O, C, N, Cl, and Pt. The valence state of Pt on the silica nanospheres was characterized by X-ray photoelectron spectroscopy (XPS. The Pt(IV loaded on SiO2@TPEA@Pt(IV was 0.15 mmol/g, as determined by UV-VIS spectrometry. The formation of intramolecular disulfides in six dithiol-containing peptides of variable lengths by the use of SiO2@TPEA@Pt(IV was investigated, and the relative oxidation yields were determined by high-performance liquid chromatography (HPLC. In addition, peptide 1 (Ac-CPFC-NH2 was utilized to study the reusability of SiO2@TPEA@Pt(IV. No significant decrease in the relative oxidation yield was observed after ten reaction cycles. Moreover, the structure of SiO2@TPEA@Pt(IV after being used for ten cycles was determined to be similar to its initial one, demonstrating the cycling stability of the complex.

  18. Influence of structural and surface properties of whey-derived peptides on zinc-chelating capacity, and in vitro gastric stability and bioaccessibility of the zinc-peptide complexes.

    Science.gov (United States)

    Udechukwu, M Chinonye; Downey, Brianna; Udenigwe, Chibuike C

    2018-02-01

    Gastrointestinal stability of zinc-peptide complexes is essential for zinc delivery. As peptide surface charge can influence their metal complex stability, we evaluated the zinc-chelating capacity and stability of zinc complexes of whey protein hydrolysates (WPH), produced with Everlase (WPH-Ever; ζ-potential, -39mV) and papain (WPH-Pap; ζ-potential, -7mV), during simulated digestion. WPH-Ever had lower amount of zinc-binding amino acids but showed higher zinc-chelating capacity than WPH-Pap. This is attributable to the highly anionic surface charge of WPH-Ever for electrostatic interaction with zinc. Release of zinc during peptic digestion was lower for WPH-Ever-zinc, and over 50% of zinc remained bound in both peptide complexes after peptic-pancreatic digestion. Fourier transform infrared spectroscopy suggests the involvement of carboxylate ion, and sidechain carbon-oxygen of aspartate/glutamate and serine/threonine in zinc-peptide complexation. The findings indicate that strong zinc chelation can promote gastric stability and impede intestinal release, for peptides intended for use as dietary zinc carriers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Tetraspan microdomains distinct from lipid rafts enrich select peptide-MHC class II complexes

    Czech Academy of Sciences Publication Activity Database

    Kropshofer, H.; Spindeldreher, S.; Rohn, T. A.; Platania, N.; Grygar, C.; Daniel, N.; Wolpl, A.; Langen, H.; Hořejší, Václav; Vogt, A. B.

    2002-01-01

    Roč. 3, č. 1 (2002), s. 61-68 ISSN 1529-2908 Institutional research plan: CEZ:AV0Z5052915 Keywords : MHC II * tetraspan microdomains * peptide presentation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 27.868, year: 2002

  20. Peptide-Functionalized Luminescent Iridium Complexes for Lifetime Imaging of CXCR4 Expression

    NARCIS (Netherlands)

    Kuil, J.; Steunenberg, P.; Chin, P.T.K.; Oldenburg, J.; Jalink, K.; Velders, A.H.; Leeuwen, F.W.B. van

    2011-01-01

    The chemokine receptor 4 (CXCR4) is over-expressed in 23 types of cancer in which it plays a role in, among others, the metastatic spread. For this reason it is a potential biomarker for the field of diagnostic oncology. The antagonistic Ac-TZ14011 peptide, which binds to CXCR4, has been conjugated

  1. Quantitative Quantum Mechanical Spectral Analysis (qQMSA) of (1)H NMR spectra of complex mixtures and biofluids.

    Science.gov (United States)

    Tiainen, Mika; Soininen, Pasi; Laatikainen, Reino

    2014-05-01

    The quantitative interpretation of (1)H NMR spectra of mixtures like the biofluids is a demanding task due to spectral complexity and overlap. Complications may arise also from water suppression, T2-editing, protein interactions, relaxation differences of the species, experimental artifacts and, furthermore, the spectra may contain unknown components and macromolecular background which cannot be easily separated from baseline. In this work, tools and strategies for quantitative Quantum Mechanical Spectral Analysis (qQMSA) of (1)H NMR spectra from complex mixtures were developed and systematically assessed. In the present approach, the signals of well-defined, stoichiometric components are described by a QM model, while the background is described by a multiterm baseline function and the unknown signals using optimizable and adjustable lines, regular multiplets or any spectral structures which can be composed from spectral lines. Any prior knowledge available from the spectrum can also be added to the model. Fitting strategies for weak and strongly overlapping spectral systems were developed and assessed using two basic model systems, the metabolite mixtures without and with macromolecular (serum) background. The analyses show that if the spectra are measured in high-throughput manner, the consistent absolute quantification demands some calibration to compensate the different response factors of the protons and compounds. On the other hand, the results show that also the T2-edited spectra can be measured so that they obey well the QM rules. In general, qQMSA exploits and interprets the spectral information in maximal way taking full advantage from the QM properties of the spectra and, at the same time, offers chemical confidence which means that individual components can be identified with high confidence on the basis of their accurate spectral parameters. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Optical Coherence Tomography Noise Reduction Using Anisotropic Local Bivariate Gaussian Mixture Prior in 3D Complex Wavelet Domain.

    Science.gov (United States)

    Rabbani, Hossein; Sonka, Milan; Abramoff, Michael D

    2013-01-01

    In this paper, MMSE estimator is employed for noise-free 3D OCT data recovery in 3D complex wavelet domain. Since the proposed distribution for noise-free data plays a key role in the performance of MMSE estimator, a priori distribution for the pdf of noise-free 3D complex wavelet coefficients is proposed which is able to model the main statistical properties of wavelets. We model the coefficients with a mixture of two bivariate Gaussian pdfs with local parameters which are able to capture the heavy-tailed property and inter- and intrascale dependencies of coefficients. In addition, based on the special structure of OCT images, we use an anisotropic windowing procedure for local parameters estimation that results in visual quality improvement. On this base, several OCT despeckling algorithms are obtained based on using Gaussian/two-sided Rayleigh noise distribution and homomorphic/nonhomomorphic model. In order to evaluate the performance of the proposed algorithm, we use 156 selected ROIs from 650 × 512 × 128 OCT dataset in the presence of wet AMD pathology. Our simulations show that the best MMSE estimator using local bivariate mixture prior is for the nonhomomorphic model in the presence of Gaussian noise which results in an improvement of 7.8 ± 1.7 in CNR.

  3. Optical Coherence Tomography Noise Reduction Using Anisotropic Local Bivariate Gaussian Mixture Prior in 3D Complex Wavelet Domain

    Directory of Open Access Journals (Sweden)

    Hossein Rabbani

    2013-01-01

    Full Text Available In this paper, MMSE estimator is employed for noise-free 3D OCT data recovery in 3D complex wavelet domain. Since the proposed distribution for noise-free data plays a key role in the performance of MMSE estimator, a priori distribution for the pdf of noise-free 3D complex wavelet coefficients is proposed which is able to model the main statistical properties of wavelets. We model the coefficients with a mixture of two bivariate Gaussian pdfs with local parameters which are able to capture the heavy-tailed property and inter- and intrascale dependencies of coefficients. In addition, based on the special structure of OCT images, we use an anisotropic windowing procedure for local parameters estimation that results in visual quality improvement. On this base, several OCT despeckling algorithms are obtained based on using Gaussian/two-sided Rayleigh noise distribution and homomorphic/nonhomomorphic model. In order to evaluate the performance of the proposed algorithm, we use 156 selected ROIs from 650 × 512 × 128 OCT dataset in the presence of wet AMD pathology. Our simulations show that the best MMSE estimator using local bivariate mixture prior is for the nonhomomorphic model in the presence of Gaussian noise which results in an improvement of 7.8 ± 1.7 in CNR.

  4. NMR assignments of SPOC domain of the human transcriptional corepressor SHARP in complex with a C-terminal SMRT peptide.

    Science.gov (United States)

    Mikami, Suzuka; Kanaba, Teppei; Ito, Yutaka; Mishima, Masaki

    2013-10-01

    The transcriptional corepressor SMRT/HDAC1-associated repressor protein (SHARP) recruits histone deacetylases. Human SHARP protein is thought to function in processes involving steroid hormone responses and the Notch signaling pathway. SHARP consists of RNA recognition motifs (RRMs) in the N-terminal region and the spen paralog and ortholog C-terminal (SPOC) domain in the C-terminal region. It is known that the SPOC domain binds the LSD motif in the C-terminal tail of corepressors silencing mediator for retinoid and thyroid receptor (SMRT)/nuclear receptor corepressor (NcoR). We are interested in delineating the mechanism by which the SPOC domain recognizes the LSD motif of the C-terminal tail of SMRT/NcoR. To this end, we are investigating the tertiary structure of the SPOC/SMRT peptide using NMR. Herein, we report on the (1)H, (13)C and (15)N resonance assignments of the SPOC domain in complex with a SMRT peptide, which contributes towards a structural understanding of the SPOC/SMRT peptide and its molecular recognition.

  5. Cyanine-based probe\\tag-peptide pair for fluorescence protein imaging and fluorescence protein imaging methods

    Science.gov (United States)

    Mayer-Cumblidge, M Uljana [Richland, WA; Cao, Haishi [Richland, WA

    2010-08-17

    A molecular probe comprises two arsenic atoms and at least one cyanine based moiety. A method of producing a molecular probe includes providing a molecule having a first formula, treating the molecule with HgOAc, and subsequently transmetallizing with AsCl.sub.3. The As is liganded to ethanedithiol to produce a probe having a second formula. A method of labeling a peptide includes providing a peptide comprising a tag sequence and contacting the peptide with a biarsenical molecular probe. A complex is formed comprising the tag sequence and the molecular probe. A method of studying a peptide includes providing a mixture containing a peptide comprising a peptide tag sequence, adding a biarsenical probe to the mixture, and monitoring the fluorescence of the mixture.

  6. Complex mixtures: Relevance of combined exposure to substances at low dose levels

    NARCIS (Netherlands)

    Leeman, W.R.; Krul, L.; Houben, G.F.

    2013-01-01

    Upon analysis of chemically complex food matrices a forest of peaks is likely to be found. Identification of these peaks and concurrent determination of the toxicological relevance upon exposure is very time consuming, expensive and often requires animal studies. Recently, a safety assessment

  7. Deinococcus Mn2+ -Peptide Complex: A Novel Approach to Alphavirus Vaccine Development

    Science.gov (United States)

    2016-08-05

    vaccines, ionizing radiation (IR)-induced destruction of a virus’ genome is desired, while radiation - induced damage to epitopes is...development of irradiation-based approaches to vaccine production [1-3]. During ionizing radiation (IR) exposure, the energy of the photons induces direct...specifically protect proteins from the far more damaging indirect effects of gamma (γ)-rays in aqueous preparations. Mn2+-peptide antioxidants that

  8. Effect of secondary structure on the interactions of peptide T4 LYS (11-36) in mixtures of aqueous sodium chloride and 2,2,2,-Trifluoroethanol

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Camille O.; Spiegelberg, Susanne; Prausnitz, John M.; Blanch, Harvey W.

    2001-10-01

    The potential of mean force for protein-protein interactions is key to the development of a statistical-mechanical model for salt-induced protein precipitation and crystallization, and for understanding certain disease states, including cataract formation and {beta}-amyloid pathology in Alzheimer's disease. Fluorescence anisotropy provides a method for quantitative characterization of intermolecular interactions due to reversible association. Monomer-dimer equilibria for the peptide T4 LYS(11-36) were studied by fluorescence anisotropy. This peptide, derived from the {beta}-sheet region of the T4 lysozyme molecule, has the potential to form amyloid fibrils. 2,2,2-trifluoroethanol (TFE) induces a change in peptide secondary structure, and was used in aqueous solutions at concentrations from 0 to 50% (v/v) at 25 and 37 C to examine the role of peptide conformation on peptide-peptide interactions. The association constant for dimerization increased with rising TFE concentration and with falling temperature. The peptide-peptide potential of mean force was computed from these association constants. Circular-dichroism measurements showed that the secondary structure of the peptide plays an important role in these strong attractive interactions due to intermolecular hydrogen-bond formation and hydrophobic interactions.

  9. Non-complexed four cascade enzyme mixture: simple purification and synergetic co-stabilization.

    Directory of Open Access Journals (Sweden)

    Suwan Myung

    Full Text Available Cell-free biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it was essential to produce (purified enzymes at low costs and stabilize them for a long time so to decrease biocatalyst costs. We studied the stability of the four recombinant enzyme mixtures, all of which originated from thermophilic microorganisms: triosephosphate isomerase (TIM from Thermus thermophiles, fructose bisphosphate aldolase (ALD from Thermotoga maritima, fructose bisphosphatase (FBP from T. maritima, and phosphoglucose isomerase (PGI from Clostridium thermocellum. It was found that TIM and ALD were very stable at evaluated temperature so that they were purified by heat precipitation followed by gradient ammonia sulfate precipitation. In contrast, PGI was not stable enough for heat treatment. In addition, the stability of a low concentration PGI was enhanced by more than 25 times in the presence of 20 mg/L bovine serum albumin or the other three enzymes. At a practical enzyme loading of 1000 U/L for each enzyme, the half-life time of free PGI was prolong to 433 h in the presence of the other three enzymes, resulting in a great increase in the total turn-over number of PGI to 6.2×10(9 mole of product per mole of enzyme. This study clearly suggested that the presence of other proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme PGI due to in vitro macromolecular crowding effect. Also, this result could be used to explain why not all enzymes isolated from thermophilic microorganisms are stable in vitro because of a lack of the macromolecular crowding environment.

  10. Prediction and measurement of complexation of radionuclide mixtures by α-isosaccharinic, gluconic and picolinic acids

    International Nuclear Information System (INIS)

    Nick Evans; Peter Warwick; Monica Felipe-Sotelo

    2012-01-01

    The purpose of this study was to investigate the effects of competition between cobalt, europium and strontium for isosaccharinate, gluconate and picolinate. Systems where results indicated that competitive effects were significant have been identified. Thermodynamic calculations were performed for each system for comparison with the experimental results. Some exceptions may be due to precipitation of some species, or presence of species not in databases, or formation of mixed-metal complexes, or sorption to the solid phase(s). In some of the experiments, the complexity of the systems studied caused difficulty in identifying consistent trends. By concentrating on the results for simpler systems (i.e. for solubilities in the presence and absence of organic complexants and with just one competing metal ion), the evidence for competition effects has been investigated. Evidence for solubility enhancement due to organic ligands was apparent in the data for the systems Co with gluconate and Eu with isosaccharinate and gluconate. Of these above cases, the systems in which the effects of the competing ion are consistent with competition were limited to the cases of Eu with isosaccharinate and Sr as the competing ion, and Eu with gluconate and either Co or Sr as the competing ion. (author)

  11. Complex mixtures of dissolved pesticides show potential aquatic toxicity in a synoptic study of Midwestern U.S. streams

    Science.gov (United States)

    Nowell, Lisa H.; Moran, Patrick W.; Schmidt, Travis S.; Norman, Julia E.; Nakagaki, Naomi; Shoda, Megan E.; Mahler, Barbara J.; Van Metre, Peter C.; Stone, Wesley W.; Sandstrom, Mark W.; Hladik, Michelle L.

    2018-01-01

    complex pesticide mixtures yet reported in discrete water samples in the U.S. and, using multiple lines of evidence, predicts that pesticides were potentially toxic to nontarget aquatic life in about half of the sampled streams.

  12. Investigation of complexing in solutions of salt mixture In(NO3)3-NaVO3

    International Nuclear Information System (INIS)

    Nakhodnova, A.N.; Listratenko, I.V.

    1987-01-01

    Spectrophotometry, conductometry and pH-metry are used to investigate properties and composition of the solid phases of isomolar series of In(NO 3 ) 3 -NaVO 3 salt mixture solutions and series of solutions having constant concentration of one of the components and varied of the other. Results of investigation are presented. It is stated that in the investigated solution series in weakly acid media HPA with the ratios [In 3+ ]:[V 5+ ] being equal to 11:1, 6:1, and 1:9, are formed. Composition of the complexes is mainly defined by the ratio of the components in In(NO 3 ) 3 and NaVO 3 salt mixture solutions and the medium acidity. Compounds of Na 2 OxIn 2 O 3 x2.5V 2 O 5 x8.5H 2 O and Cs 2 OxIn 2 O 3 x6V 2 O 5 x6.5H 2 O empirical formulae are separated. Results of IR spectroscopy, derivatography and X-ray phase analysis of the corresponding salts are presented

  13. Investigation of complexing in solutions of salt mixture In(NO/sub 3/)/sub 3/-NaVO/sub 3/

    Energy Technology Data Exchange (ETDEWEB)

    Nakhodnova, A N; Listratenko, I V

    1987-05-01

    Spectrophotometry, conductometry and pH-metry are used to investigate properties and composition of the solid phases of isomolar series of In(NO/sub 3/)/sub 3/-NaVO/sub 3/ salt mixture solutions and series of solutions having constant concentration of one of the components and varied of the other. Results of investigation are presented. It is stated that in the investigated solution series in weakly acid media HPA with the ratios (In/sup 3+/):(V/sup 5+/) being equal to 11:1, 6:1, and 1:9, are formed. Composition of the complexes is mainly defined by the ratio of the components in In(NO/sub 3/)/sub 3/ and NaVO/sub 3/ salt mixture solutions and the medium acidity. Compounds of Na/sub 2/OxIn/sub 2/O/sub 3/x2.5V/sub 2/O/sub 5/x8.5H/sub 2/O and Cs/sub 2/OxIn/sub 2/O/sub 3/x6V/sub 2/O/sub 5/x6.5H/sub 2/O empirical formulae are separated. Results of IR spectroscopy, derivatography and X-ray phase analysis of the corresponding salts are presented.

  14. Methods of analysis for complex organic aerosol mixtures from urban emission sources of particulate carbon

    International Nuclear Information System (INIS)

    Mazurek, M.A.; Hildemann, L.M.; Simoneit, B.R.T.

    1990-10-01

    Organic aerosols comprise approximately 30% by mass of the total fine particulate matter present in urban atmospheres. The chemical composition of such aerosols is complex and reflects input from multiple sources of primary emissions to the atmosphere, as well as from secondary production of carbonaceous aerosol species via photochemical reactions. To identify discrete sources of fine carbonaceous particles in urban atmospheres, analytical methods must reconcile both bulk chemical and molecular properties of the total carbonaceous aerosol fraction. This paper presents an overview of the analytical protocol developed and used in a study of the major sources of fine carbon particles emitted to an urban atmosphere. 23 refs., 1 fig., 2 tabs

  15. Influence of organic component on geometry and stability of the Dy(3) complexes with benzoic and aminobenzoic acids in water-80 vol.% DMSO(DMFA) mixtures

    International Nuclear Information System (INIS)

    Kondrashina, Yu.G.; Mustafina, A.R.; Devyatov, F.V.; Vul'fson, S.G.; Kazanskij Gosudarstvennyj Univ., Kazan

    1995-01-01

    Data of pH-metric and magnetooptical analyses were used to evaluate stability and structure of benzoate and aminobenzoate dysprosium (3) complexes in water and water - 80 vol.% DMSO (DMFA) mixtures. Factors, dictating change of complex structure and stability when passing from water to organic water solvents, are discussed. 19 refs.; 2 figs.; 1 tab

  16. Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor*

    Science.gov (United States)

    Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H.; Rudolph, Rainer; Reedtz-Runge, Steffen

    2010-01-01

    GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr13 to Val33 when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor. PMID:19861722

  17. Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor.

    Science.gov (United States)

    Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H; Rudolph, Rainer; Reedtz-Runge, Steffen

    2010-01-01

    GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

  18. Effect of hydraulic retention time on the biodegradation of complex phenolic mixture from simulated coal wastewater in hybrid UASB reactors

    Energy Technology Data Exchange (ETDEWEB)

    Ramakrishnan, Anushuya [Centre for Environmental Science and Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076 (India); Gupta, Sudhir Kumar [Centre for Environmental Science and Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076 (India)], E-mail: skgupta@iitb.ac.in

    2008-05-01

    This study describes the feasibility of anaerobic treatment of complex phenolics mixture from a simulated synthetic coal wastewater using four identical 13.5 L (effective volume) bench scale hybrid up-flow anaerobic sludge blanket (HUASB) (combining UASB + anaerobic filter) reactors at four different hydraulic retention times (HRT) under mesophilic (27 {+-} 5 {sup o}C) conditions. Synthetic coal wastewater with an average chemical oxygen demand (COD) of 2240 mg/L and phenolics concentration of 752 mg/L was used as substrate. The phenolics contained phenol (490 mg/L); m-, o-, p-cresols (123.0, 58.6, 42 mg/L); 2,4-, 2,5-, 3,4- and 3,5-dimethyl phenols (6.3, 6.3, 4.4 and 21.3 mg/L) as major phenolic compounds. The study demonstrated that at optimum HRT, 24 h, and phenolic loading rate of 0.75 g COD/(m{sup 3}-d), the phenolics and COD removal efficiency of the reactors were 96% and 86%, respectively. Bio-kinetic models were applied to data obtained from experimental studies in hybrid UASB reactor. Grau second-order multi-component substrate removal model was best fitted to the hybrid UASB reactor. The second-order substrate removal rate constant (k{sub 2(s)}) was found as 1.72 h{sup -1} for the hybrid reactor treating complex phenolic mixture. Morphological examination of the sludge revealed rod-type Methanothrix-like, cells to be dominant on the surface.

  19. Effect of hydraulic retention time on the biodegradation of complex phenolic mixture from simulated coal wastewater in hybrid UASB reactors

    International Nuclear Information System (INIS)

    Ramakrishnan, Anushuya; Gupta, Sudhir Kumar

    2008-01-01

    This study describes the feasibility of anaerobic treatment of complex phenolics mixture from a simulated synthetic coal wastewater using four identical 13.5 L (effective volume) bench scale hybrid up-flow anaerobic sludge blanket (HUASB) (combining UASB + anaerobic filter) reactors at four different hydraulic retention times (HRT) under mesophilic (27 ± 5 o C) conditions. Synthetic coal wastewater with an average chemical oxygen demand (COD) of 2240 mg/L and phenolics concentration of 752 mg/L was used as substrate. The phenolics contained phenol (490 mg/L); m-, o-, p-cresols (123.0, 58.6, 42 mg/L); 2,4-, 2,5-, 3,4- and 3,5-dimethyl phenols (6.3, 6.3, 4.4 and 21.3 mg/L) as major phenolic compounds. The study demonstrated that at optimum HRT, 24 h, and phenolic loading rate of 0.75 g COD/(m 3 -d), the phenolics and COD removal efficiency of the reactors were 96% and 86%, respectively. Bio-kinetic models were applied to data obtained from experimental studies in hybrid UASB reactor. Grau second-order multi-component substrate removal model was best fitted to the hybrid UASB reactor. The second-order substrate removal rate constant (k 2(s) ) was found as 1.72 h -1 for the hybrid reactor treating complex phenolic mixture. Morphological examination of the sludge revealed rod-type Methanothrix-like, cells to be dominant on the surface

  20. Reduced gas seepages in ophiolitic complexes: Evidences for multiple origins of the H2-CH4-N2 gas mixtures

    Science.gov (United States)

    Vacquand, Christèle; Deville, Eric; Beaumont, Valérie; Guyot, François; Sissmann, Olivier; Pillot, Daniel; Arcilla, Carlo; Prinzhofer, Alain

    2018-02-01

    This paper proposes a comparative study of reduced gas seepages occurring in ultrabasic to basic rocks outcropping in ophiolitic complexes based on the study of seepages from Oman, the Philippines, Turkey and New Caledonia. This study is based on analyses of the gas chemical composition, noble gases contents, stable isotopes of carbon, hydrogen and nitrogen. These seepages are mostly made of mixtures of three main components which are H2, CH4 and N2 in various proportions. The relative contents of the three main gas components show 4 distinct types of gas mixtures (H2-rich, N2-rich, N2-H2-CH4 and H2-CH4). These types are interpreted as reflecting different zones of gas generation within or below the ophiolitic complexes. In the H2-rich type, associated noble gases display signatures close to the value of air. In addition to the atmospheric component, mantle and crustal contributions are present in the N2-rich, N2-H2-CH4 and H2-CH4 types. H2-bearing gases are either associated with ultra-basic (pH 10-12) spring waters or they seep directly in fracture systems from the ophiolitic rocks. In ophiolitic contexts, ultrabasic rocks provide an adequate environment with available Fe2+ and alkaline conditions that favor H2 production. CH4 is produced either directly by reaction of dissolved CO2 with basic-ultrabasic rocks during the serpentinization process or in a second step by H2-CO2 interaction. H2 is present in the gas when no more carbon is available in the system to generate CH4. The N2-rich type is notably associated with relatively high contents of crustal 4He and in this gas type N2 is interpreted as issued mainly from sediments located below the ophiolitic units.

  1. Structural Analysis of Der p 1–Antibody Complexes and Comparison with Complexes of Proteins or Peptides with Monoclonal Antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Osinski, Tomasz; Pomés, Anna; Majorek, Karolina A.; Glesner, Jill; Offermann, Lesa R.; Vailes, Lisa D.; Chapman, Martin D.; Minor, Wladek; Chruszcz, Maksymilian (INDOOR); (UV); (SC)

    2015-05-29

    Der p 1 is a major allergen from the house dust mite, Dermatophagoides pteronyssinus, that belongs to the papain-like cysteine protease family. To investigate the antigenic determinants of Der p 1, we determined two crystal structures of Der p 1 in complex with the Fab fragments of mAbs 5H8 or 10B9. Epitopes for these two Der p 1–specific Abs are located in different, nonoverlapping parts of the Der p 1 molecule. Nevertheless, surface area and identity of the amino acid residues involved in hydrogen bonds between allergen and Ab are similar. The epitope for mAb 10B9 only showed a partial overlap with the previously reported epitope for mAb 4C1, a cross-reactive mAb that binds Der p 1 and its homolog Der f 1 from Dermatophagoides farinae. Upon binding to Der p 1, the Fab fragment of mAb 10B9 was found to form a very rare α helix in its third CDR of the H chain. To provide an overview of the surface properties of the interfaces formed by the complexes of Der p 1–10B9 and Der p 1–5H8, along with the complexes of 4C1 with Der p 1 and Der f 1, a broad analysis of the surfaces and hydrogen bonds of all complexes of Fab–protein or Fab–peptide was performed. This work provides detailed insight into the cross-reactive and specific allergen–Ab interactions in group 1 mite allergens. The surface data of Fab–protein and Fab–peptide interfaces can be used in the design of conformational epitopes with reduced Ab binding for immunotherapy.

  2. Estimation of yohimbine base in complex mixtures by quantitative HPTLC application.

    Science.gov (United States)

    Adel-Kader, Maged Saad; Alwahebi, Naif Wahebi Hamadan; Alam, Prawez

    2017-01-01

    The indole alkaloid Yohimbine has been used for over two centuries in the treatment of erectly dysfunction. Several formulations containing yohimbine salts, yohimbe bark power or extract are marketed worldwide. Determination of the amount of yohimbine in such formulation is a challenging task due to their complex nature. Extraction followed by acid-base purification resulted in a relatively pure alkaloids containing fractions. The exact amounts of yohimbine free base in different formulations were determined by densitometric HPTLC validated methods using silica gel TLC plates. Standard curve for yohimbine was generated using yohimbine hydrochloride subjected to the same acid-base treatment as the used samples. All formulations found to contain yohimbine though some with less concentration than the labeled amount.

  3. Differential gene expression patterns in developing sexually dimorphic rat brain regions exposed to antiandrogenic, estrogenic, or complex endocrine disruptor mixtures: glutamatergic synapses as target.

    Science.gov (United States)

    Lichtensteiger, Walter; Bassetti-Gaille, Catherine; Faass, Oliver; Axelstad, Marta; Boberg, Julie; Christiansen, Sofie; Rehrauer, Hubert; Georgijevic, Jelena Kühn; Hass, Ulla; Kortenkamp, Andreas; Schlumpf, Margret

    2015-04-01

    The study addressed the question whether gene expression patterns induced by different mixtures of endocrine disrupting chemicals (EDCs) administered in a higher dose range, corresponding to 450×, 200×, and 100× high-end human exposure levels, could be characterized in developing brain with respect to endocrine activity of mixture components, and which developmental processes were preferentially targeted. Three EDC mixtures, A-Mix (anti-androgenic mixture) with 8 antiandrogenic chemicals (di-n-butylphthalate, diethylhexylphthalate, vinclozolin, prochloraz, procymidone, linuron, epoxiconazole, and DDE), E-Mix (estrogenic mixture) with 4 estrogenic chemicals (bisphenol A, 4-methylbenzylidene camphor, 2-ethylhexyl 4-methoxycinnamate, and butylparaben), a complex mixture, AEP-Mix, containing the components of A-Mix and E-Mix plus paracetamol, and paracetamol alone, were administered by oral gavage to rat dams from gestation day 7 until weaning. General developmental endpoints were not affected by EDC mixtures or paracetamol. Gene expression was analyzed on postnatal day 6, during sexual brain differentiation, by exon microarray in medial preoptic area in the high-dose group, and by real-time RT-PCR in medial preoptic area and ventromedial hypothalamus in all dose groups. Expression patterns were mixture, sex, and region specific. Effects of the analgesic drug paracetamol, which exhibits antiandrogenic activity in peripheral systems, differed from those of A-Mix. All mixtures had a strong, mixture-specific impact on genes encoding for components of excitatory glutamatergic synapses and genes controlling migration and pathfinding of glutamatergic and GABAergic neurons, as well as genes linked with increased risk of autism spectrum disorders. Because development of glutamatergic synapses is regulated by sex steroids also in hippocampus, this may represent a general target of ECD mixtures.

  4. Quantification of HLA-DM-Dependent Major Histocompatibility Complex of Class II Immunopeptidomes by the Peptide Landscape Antigenic Epitope Alignment Utility

    Directory of Open Access Journals (Sweden)

    Miguel Álvaro-Benito

    2018-05-01

    Full Text Available The major histocompatibility complex of class II (MHCII immunopeptidome represents the repertoire of antigenic peptides with the potential to activate CD4+ T cells. An understanding of how the relative abundance of specific antigenic epitopes affects the outcome of T cell responses is an important aspect of adaptive immunity and offers a venue to more rationally tailor T cell activation in the context of disease. Recent advances in mass spectrometric instrumentation, computational power, labeling strategies, and software analysis have enabled an increasing number of stratified studies on HLA ligandomes, in the context of both basic and translational research. A key challenge in the case of MHCII immunopeptidomes, often determined for different samples at distinct conditions, is to derive quantitative information on consensus epitopes from antigenic peptides of variable lengths. Here, we present the design and benchmarking of a new algorithm [peptide landscape antigenic epitope alignment utility (PLAtEAU] allowing the identification and label-free quantification (LFQ of shared consensus epitopes arising from series of nested peptides. The algorithm simplifies the complexity of the dataset while allowing the identification of nested peptides within relatively short segments of protein sequences. Moreover, we apply this algorithm to the comparison of the ligandomes of cell lines with two different expression levels of the peptide-exchange catalyst HLA-DM. Direct comparison of LFQ intensities determined at the peptide level is inconclusive, as most of the peptides are not significantly enriched due to poor sampling. Applying the PLAtEAU algorithm for grouping of the peptides into consensus epitopes shows that more than half of the total number of epitopes is preferentially and significantly enriched for each condition. This simplification and deconvolution of the complex and ambiguous peptide-level dataset highlights the value of the PLAt

  5. Proteomics strategy for identifying candidate bioactive proteins in complex mixtures: application to the platelet releasate.

    LENUS (Irish Health Repository)

    O'Connor, Roisin

    2010-01-01

    Proteomic approaches have proven powerful at identifying large numbers of proteins, but there are fewer reports of functional characterization of proteins in biological tissues. Here, we describe an experimental approach that fractionates proteins released from human platelets, linking bioassay activity to identity. We used consecutive orthogonal separation platforms to ensure sensitive detection: (a) ion-exchange of intact proteins, (b) SDS-PAGE separation of ion-exchange fractions and (c) HPLC separation of tryptic digests coupled to electrospray tandem mass spectrometry. Migration of THP-1 monocytes in response to complete or fractionated platelet releasate was assessed and located to just one of the forty-nine ion-exchange fractions. Over 300 proteins were identified in the releasate, with a wide range of annotated biophysical and biochemical properties, in particular platelet activation, adhesion, and wound healing. The presence of PEDF and involucrin, two proteins not previously reported in platelet releasate, was confirmed by western blotting. Proteins identified within the fraction with monocyte promigratory activity and not in other inactive fractions included vimentin, PEDF, and TIMP-1. We conclude that this analytical platform is effective for the characterization of complex bioactive samples.

  6. Evaluating the Similarity of Complex Drinking-Water Disinfection By-Product Mixtures: Overview of the Issues

    Science.gov (United States)

    The Presentation describes the advantages and challenges of working with Whole Mixtures, discusses an exploratory approach for evaluating sufficient similarity, and challenges of applying such approaches to other environmental mixtures.

  7. Complex mixtures in industrial workspaces: lessons for indoor air quality evaluations.

    Science.gov (United States)

    Lippy, B E; Turner, R W

    1991-11-01

    Acceptable occupational exposure levels for hundreds of airborne concentrations of dusts, vapors, fumes, and gases have been set by consensus organizations and regulatory bodies for decades. These levels have established tremendous precedent and are tempting reference values in the relatively new field of indoor air quality evaluations where validated criteria are greatly needed. The American Conference of Government Industrial Hygienists (ACGIH) has been the most visible and productive group setting these guidelines for industrial exposure. The ACGIH Chemical Substances Committee has published an annual list of threshold limit values (TLVs) for more than 40 years. Currently the list covers more than 400 substances. In 1989, the Occupational Safety and Health Administration (OSHA) published updated permissible exposure limits (PELs) for approximately 600 substances. Most PELs before this update were adopted from the 1968 ACGIH list of TLVs and consensus standards of the American Standards Association. This OSHA update has resulted in reductions of 212 PELs and the addition of 164 new levels. The magnitude of the problem of protecting workers can be seen by the small fraction that the OSHA PELs represent of the more than 60,000 entries in the National Institute for Occupational Safety and Health's Registry of Toxic Effects of Chemical Substances. None of these levels, whether guidelines or regulatory requirements, are established based on any possible synergistic effect with other chemicals. The only guidance given by the ACGIH for synergistic effects is that such cases must be determined individually. Clearly, there are major drawbacks in using occupational standards and guidelines for evaluating the health effects of chemical agents that can be found in office settings, often in concentrations orders of magnitude less than what is routinely measured in the workplace. These guidelines are even less valuable when the concern is the complex mixing of chemicals in

  8. A complicated complex: Ion channels, voltage sensing, cell membranes and peptide inhibitors.

    Science.gov (United States)

    Zhang, Alan H; Sharma, Gagan; Undheim, Eivind A B; Jia, Xinying; Mobli, Mehdi

    2018-04-21

    Voltage-gated ion channels (VGICs) are specialised ion channels that have a voltage dependent mode of action, where ion conduction, or gating, is controlled by a voltage-sensing mechanism. VGICs are critical for electrical signalling and are therefore important pharmacological targets. Among these, voltage-gated sodium channels (Na V s) have attracted particular attention as potential analgesic targets. Na V s, however, comprise several structurally similar subtypes with unique localisations and distinct functions, ranging from amplification of action potentials in nociception (e.g. Na V 1.7) to controlling electrical signalling in cardiac function (Na V 1.5). Understanding the structural basis of Na V function is therefore of great significance, both to our knowledge of electrical signalling and in development of subtype and state selective drugs. An important tool in this pursuit has been the use of peptides from animal venoms as selective Na V modulators. In this review, we look at peptides, particularly from spider venoms, that inhibit Na V s by binding to the voltage sensing domain (VSD) of this channel, known as gating modifier toxins (GMT). In the first part of the review, we look at the structural determinants of voltage sensing in VGICs, the gating cycle and the conformational changes that accompany VSD movement. Next, the modulation of the analgesic target Na V 1.7 by GMTs is reviewed to develop bioinformatic tools that, based on sequence information alone, can identify toxins that are likely to inhibit this channel. The same approach is also used to define VSD sequences, other than that from Na V 1.7, which are likely to be sensitive to this class of toxins. The final section of the review focuses on the important role of the cellular membrane in channel modulation and also how the lipid composition affects measurements of peptide-channel interactions both in binding kinetics measurements in solution and in cell-based functional assays. Copyright © 2018

  9. Purification, crystallization and preliminary X-ray diffraction analysis of the Fyn SH2 domain and its complex with a phosphotyrosine peptide

    International Nuclear Information System (INIS)

    Huculeci, Radu; Buts, Lieven; Lenaerts, Tom; Nuland, Nico A. J. van; Garcia-Pino, Abel

    2012-01-01

    The SH2 domain of the human protein tyrosine kinase Fyn has been expressed, purified and crystallized in the unbound state and in complex with a high-affinity phosphotyrosine peptide. SH2 domains are widespread protein-binding modules that recognize phosphotyrosines and play central roles in intracellular signalling pathways. The SH2 domain of the human protein tyrosine kinase Fyn has been expressed, purified and crystallized in the unbound state and in complex with a high-affinity phosphotyrosine peptide. X-ray data were collected to a resolution of 2.00 Å for the unbound form and 1.40 Å for the protein in complex with the phosphotyrosine peptide

  10. Electroplex emission from a layer of a mixture of a europium complex and tris(8-quinolinolato) aluminum

    Science.gov (United States)

    Cao, Hong; Gao, Xicun; Huang, Chun-Hui

    2000-07-01

    With a europium complex, tris(α-thenoyltrifluoroacetonato) bis(triphenylphosphine oxide) europium (Eu(TTA) 3(TPPO) 2), as the light-emitting layer, N, N'-diphenyl- N, N'-di( m-tolyl)-benzidine (TPD) as the hole transport layer and tris(8-quinolinolato) aluminum (ALQ) as the electron transport layer, the triple-layer electroluminescent (EL) device emits red light characteristic of Eu 3+ emission. However, as the mixture of Eu(TTA) 3(TPPO) 2 and ALQ is co-evaporated as the light-emitting layer to form a bilayer EL device, a new wide-banded emission peaked at c. 640 nm was obtained. This emission is neither from ALQ nor from the europium complex. The photoluminescence (PL) of the thin film on quartz substrate evaporated from one mixed solid powder of Eu(TTA) 3(TPPO) 2 and ALQ is composed of distinct PL emissions of Eu(TTA) 3(TPPO) 2 and ALQ, denying an exciplex formation mechanism. It is impossible to form a host-guest system. We propose that the EL emission peaked at c. 640 nm is from an electroplex route: a transition between the lowest unoccupied molecular orbital (LUMO) of Eu(TTA) 3(TPPO) 2 and the highest occupied molecular orbital (HOMO) of ALQ.

  11. Prediction of Antimicrobial Peptides Based on Sequence Alignment and Support Vector Machine-Pairwise Algorithm Utilizing LZ-Complexity

    Directory of Open Access Journals (Sweden)

    Xin Yi Ng

    2015-01-01

    Full Text Available This study concerns an attempt to establish a new method for predicting antimicrobial peptides (AMPs which are important to the immune system. Recently, researchers are interested in designing alternative drugs based on AMPs because they have found that a large number of bacterial strains have become resistant to available antibiotics. However, researchers have encountered obstacles in the AMPs designing process as experiments to extract AMPs from protein sequences are costly and require a long set-up time. Therefore, a computational tool for AMPs prediction is needed to resolve this problem. In this study, an integrated algorithm is newly introduced to predict AMPs by integrating sequence alignment and support vector machine- (SVM- LZ complexity pairwise algorithm. It was observed that, when all sequences in the training set are used, the sensitivity of the proposed algorithm is 95.28% in jackknife test and 87.59% in independent test, while the sensitivity obtained for jackknife test and independent test is 88.74% and 78.70%, respectively, when only the sequences that has less than 70% similarity are used. Applying the proposed algorithm may allow researchers to effectively predict AMPs from unknown protein peptide sequences with higher sensitivity.

  12. Structure of the Paramyxovirus Parainfluenza Virus 5 Nucleoprotein in Complex with an Amino-Terminal Peptide of the Phosphoprotein

    Energy Technology Data Exchange (ETDEWEB)

    Aggarwal, Megha; Leser, George P.; Kors, Christopher A.; Lamb, Robert A.; Sundquist, Wesley I.

    2017-12-13

    Parainfluenza virus 5 (PIV5) belongs to the familyParamyxoviridae, which consists of enveloped viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). Paramyxovirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N0P) derived from the N terminus of P (P50) at 2.65 Å. The PIV5 N0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N0P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxovirus replication.

    IMPORTANCEParamyxovirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled parainfluenza virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as

  13. Quantification of Multiple Components of Complex Aluminum-Based Adjuvant Mixtures by Using Fourier Transform Infrared Spectroscopy and Partial Least Squares Modeling.

    Science.gov (United States)

    Dowling, Quinton M; Kramer, Ryan M

    2017-01-01

    Fourier transform infrared (FTIR) spectroscopy is widely used in the pharmaceutical industry for process monitoring, compositional quantification, and characterization of critical quality attributes in complex mixtures. Advantages over other spectroscopic measurements include ease of sample preparation, quantification of multiple components from a single measurement, and the ability to quantify optically opaque samples. This method describes the use of a multivariate model for quantifying a TLR4 agonist (GLA) adsorbed onto aluminum oxyhydroxide (Alhydrogel ® ) using FTIR spectroscopy that may be adapted to quantify other complex aluminum based adjuvant mixtures.

  14. Headspace versus direct immersion solid phase microextraction in complex matrixes: investigation of analyte behavior in multicomponent mixtures.

    Science.gov (United States)

    Gionfriddo, Emanuela; Souza-Silva, Érica A; Pawliszyn, Janusz

    2015-08-18

    This work aims to investigate the behavior of analytes in complex mixtures and matrixes with the use of solid-phase microextraction (SPME). Various factors that influence analyte uptake such as coating chemistry, extraction mode, the physicochemical properties of analytes, and matrix complexity were considered. At first, an aqueous system containing analytes bearing different hydrophobicities, molecular weights, and chemical functionalities was investigated by using commercially available liquid and solid porous coatings. The differences in the mass transfer mechanisms resulted in a more pronounced occurrence of coating saturation in headspace mode. Contrariwise, direct immersion extraction minimizes the occurrence of artifacts related to coating saturation and provides enhanced extraction of polar compounds. In addition, matrix-compatible PDMS-modified solid coatings, characterized by a new morphology that avoids coating fouling, were compared to their nonmodified analogues. The obtained results indicate that PDMS-modified coatings reduce artifacts associated with coating saturation, even in headspace mode. This factor, coupled to their matrix compatibility, make the use of direct SPME very practical as a quantification approach and the best choice for metabolomics studies where wide coverage is intended. To further understand the influence on analyte uptake on a system where additional interactions occur due to matrix components, ex vivo and in vivo sampling conditions were simulated using a starch matrix model, with the aim of mimicking plant-derived materials. Our results corroborate the fact that matrix handling can affect analyte/matrix equilibria, with consequent release of high concentrations of previously bound hydrophobic compounds, potentially leading to coating saturation. Direct immersion SPME limited the occurrence of the artifacts, which confirms the suitability of SPME for in vivo applications. These findings shed light into the implementation of in

  15. The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides

    DEFF Research Database (Denmark)

    Buus, S; Sette, A; Colon, S M

    1987-01-01

    The capacity of purified I-Ad, I-Ed, I-Ak, and I-Ek to bind to protein derived peptides that have been previously reported to be T cell immunogens has been examined. For each of the 12 peptides studied strong binding to the relevant Ia restriction element was observed. All the peptides bound more...... than one Ia molecule; however, for 11 of 12 peptides, the dominant binding was to the restriction element, whereas in one instance the dominant binding was to a nonrestriction element. When the peptides were used to inhibit the presentation of antigen by prefixed accessory cells to T cells......, an excellent correlation was found between the capacity of a peptide to inhibit the binding of an antigen to purified Ia and the capacity of the peptide to inhibit accessory cell presentation of the antigen. Thus, the binding of peptide to purified Ia is immunologically relevant, and Ia seems to be the only...

  16. Pan-specific prediction of peptide-MHC Class I complex stability, a correlate of T cell immunogenicity

    DEFF Research Database (Denmark)

    Rasmussen, Michael; Fenoy, Emilio; Harndahl, Mikkel

    2016-01-01

    Binding of peptides to MHC class I (MHC-I) molecules is the most selective event in the processing and presentation of Ags to CTL, and insights into the mechanisms that govern peptide-MHC-I binding should facilitate our understanding of CTL biology. Peptide-MHC-I interactions have traditionally b...

  17. Preliminary crystallographic characterization of the Grb2 SH2 domain in complex with a FAK-derived phosphotyrosyl peptide

    International Nuclear Information System (INIS)

    Chen, Hsiao-Hsin; Chen, Cuei-Wen; Chang, Yu-Yung; Shen, Tang-Long; Hsu, Chun-Hua

    2010-01-01

    Crystals of the Grb2 SH2 domain in complex with a phosphotyrosyl peptide corresponding to residues 921–930 of focal adhesion kinase (FAK) have been obtained using the sitting-drop vapour-diffusion technique. Data have been collected to 2.49 Å resolution. Growth factor receptor-bound protein 2 (Grb2) is an adaptor protein with a single SH2 domain that specifically binds to focal adhesion kinase (FAK) when residue Tyr925 of FAK is phosphorylated. The Grb2–FAK interaction is associated with cellular integrin-activated signal transduction events leading to the activation of the Ras-MAPK pathway. Crystals of the Grb2 SH2 domain in complex with a phosphopeptide corresponding to residues 921–930 of FAK have been obtained using the sitting-drop vapour-diffusion technique. The crystals belonged to space group P3 1 21, with unit-cell parameters a = b = 102.7, c = 127.6 Å, α = β = 90.0, γ = 120.0°. A diffraction data set was collected from a flash-cooled crystal at 100 K to 2.49 Å resolution using synchrotron radiation. Structure determination by molecular replacement and analysis of the detailed structure of the complex are currently in progress

  18. Peptide biomarkers used for the selective breeding of a complex polygenic trait in honey bees.

    Science.gov (United States)

    Guarna, M Marta; Hoover, Shelley E; Huxter, Elizabeth; Higo, Heather; Moon, Kyung-Mee; Domanski, Dominik; Bixby, Miriam E F; Melathopoulos, Andony P; Ibrahim, Abdullah; Peirson, Michael; Desai, Suresh; Micholson, Derek; White, Rick; Borchers, Christoph H; Currie, Robert W; Pernal, Stephen F; Foster, Leonard J

    2017-08-21

    We present a novel way to select for highly polygenic traits. For millennia, humans have used observable phenotypes to selectively breed stronger or more productive livestock and crops. Selection on genotype, using single-nucleotide polymorphisms (SNPs) and genome profiling, is also now applied broadly in livestock breeding programs; however, selection on protein/peptide or mRNA expression markers has not yet been proven useful. Here we demonstrate the utility of protein markers to select for disease-resistant hygienic behavior in the European honey bee (Apis mellifera L.). Robust, mechanistically-linked protein expression markers, by integrating cis- and trans- effects from many genomic loci, may overcome limitations of genomic markers to allow for selection. After three generations of selection, the resulting marker-selected stock outperformed an unselected benchmark stock in terms of hygienic behavior, and had improved survival when challenged with a bacterial disease or a parasitic mite, similar to bees selected using a phenotype-based assessment for this trait. This is the first demonstration of the efficacy of protein markers for industrial selective breeding in any agricultural species, plant or animal.

  19. Endocrine-Disrupting Chemicals and Oil and Natural Gas Operations: Potential Environmental Contamination and Recommendations to Assess Complex Environmental Mixtures.

    Science.gov (United States)

    Kassotis, Christopher D; Tillitt, Donald E; Lin, Chung-Ho; McElroy, Jane A; Nagel, Susan C

    2016-03-01

    Hydraulic fracturing technologies, developed over the last 65 years, have only recently been combined with horizontal drilling to unlock oil and gas reserves previously deemed inaccessible. Although these technologies have dramatically increased domestic oil and natural gas production, they have also raised concerns for the potential contamination of local water supplies with the approximately 1,000 chemicals that are used throughout the process, including many known or suspected endocrine-disrupting chemicals. We discuss the need for an endocrine component to health assessments for drilling-dense regions in the context of hormonal and antihormonal activities for chemicals used. We discuss the literature on a) surface and groundwater contamination by oil and gas extraction operations, and b) potential human exposure, particularly in the context of the total hormonal and antihormonal activities present in surface and groundwater from natural and anthropogenic sources; we also discuss initial analytical results and critical knowledge gaps. In light of the potential for environmental release of oil and gas chemicals that can disrupt hormone receptor systems, we recommend methods for assessing complex hormonally active environmental mixtures. We describe a need for an endocrine-centric component for overall health assessments and provide information supporting the idea that using such a component will help explain reported adverse health trends as well as help develop recommendations for environmental impact assessments and monitoring programs.

  20. Endocrine-disrupting chemicals and oil and natural gas operations: Potential environmental contamination and recommendations to assess complex environmental mixtures

    Science.gov (United States)

    Kassotis, Christopher D.; Tillitt, Donald E.; Lin, Chung-Ho; McElroy, Jane A.; Nagel, Susan C.

    2016-01-01

    Background: Hydraulic fracturing technologies, developed over the last 65 years, have only recently been combined with horizontal drilling to unlock oil and gas reserves previously deemed inaccessible. While these technologies have dramatically increased domestic oil and natural gas production, they have also raised concerns for the potential contamination of local water supplies with the approximately 1,000 chemicals used throughout the process, including many known or suspected endocrine-disrupting chemicals.Objectives: We discuss the need for an endocrine component to health assessments for drilling-dense regions in the context of hormonal and anti-hormonal activities for chemicals used.Methods: We discuss the literature on 1) surface and ground water contamination by oil and gas extraction operations, and 2) potential human exposure, particularly in context of the total hormonal and anti-hormonal activities present in surface and ground water from natural and anthropogenic sources, with initial analytical results and critical knowledge gaps discussed.Discussion: In light of the potential for environmental release of oil and gas chemicals that can disrupt hormone receptor systems, we recommend methods for assessing complex hormonally active environmental mixtures.Conclusions: We describe a need for an endocrine-centric component for overall health assessments and provide supporting information that using this may help explain reported adverse health trends as well as help develop recommendations for environmental impact assessments and monitoring programs.

  1. Direct elution of sup(99m)Tc complexes from neutron irradiation produced 99Mo incorporated in a MoCl2-MoBr2 mixture

    International Nuclear Information System (INIS)

    Ganzerli Valentini, M.T.; Stella, R.; Genova, N.

    1987-01-01

    A novel type of sup(99m)Tc generator, enabling radiopharmaceutical preparation without reductant addition, was prepared and tested. Neutron activated product 99 Mo, in the form of dichloride cluster, was incorporated into inactive molybdenum dichloride-dibromide mixture (MCB) that was left to settle over an activated alumina layer. Direct elution with aqueous ligands such as salicylate or iminodiacetate derivatives in the pH range 6.5-7.5 yielded chemically stable sup(99m)Tc complexes accompanied by small amounts of secondary products (mostly sup(99m)TcO - 4 ). Pentavalent oxidation state in the salicylate complex and tervalent in the iminodiacetate (IDA) and in the N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetate (HIDA) complexes were assigned to the element after comparison with reference complexes. The anion exchange version of reverse-phase HPLC was used to resolve the eluted product mixture. (author)

  2. Bayesian demosaicing using Gaussian scale mixture priors with local adaptivity in the dual tree complex wavelet packet transform domain

    Science.gov (United States)

    Goossens, Bart; Aelterman, Jan; Luong, Hiep; Pizurica, Aleksandra; Philips, Wilfried

    2013-02-01

    In digital cameras and mobile phones, there is an ongoing trend to increase the image resolution, decrease the sensor size and to use lower exposure times. Because smaller sensors inherently lead to more noise and a worse spatial resolution, digital post-processing techniques are required to resolve many of the artifacts. Color filter arrays (CFAs), which use alternating patterns of color filters, are very popular because of price and power consumption reasons. However, color filter arrays require the use of a post-processing technique such as demosaicing to recover full resolution RGB images. Recently, there has been some interest in techniques that jointly perform the demosaicing and denoising. This has the advantage that the demosaicing and denoising can be performed optimally (e.g. in the MSE sense) for the considered noise model, while avoiding artifacts introduced when using demosaicing and denoising sequentially. In this paper, we will continue the research line of the wavelet-based demosaicing techniques. These approaches are computationally simple and very suited for combination with denoising. Therefore, we will derive Bayesian Minimum Squared Error (MMSE) joint demosaicing and denoising rules in the complex wavelet packet domain, taking local adaptivity into account. As an image model, we will use Gaussian Scale Mixtures, thereby taking advantage of the directionality of the complex wavelets. Our results show that this technique is well capable of reconstructing fine details in the image, while removing all of the noise, at a relatively low computational cost. In particular, the complete reconstruction (including color correction, white balancing etc) of a 12 megapixel RAW image takes 3.5 sec on a recent mid-range GPU.

  3. Sodiation as a tool for enhancing the diagnostic value of MALDI-TOF/TOF-MS spectra of complex astaxanthin ester mixtures from Haematococcus pluvialis

    NARCIS (Netherlands)

    Weesepoel, Y.J.A.; Vincken, J.P.; Pop, R.M.; Liu, K.; Gruppen, H.

    2013-01-01

    The microalga Haematococcus pluvialis produces the pigment astaxanthin mainly in esterified form with a multitude of fatty acids, which results in a complex mixture of carotenol mono- and diesters. For rapid fingerprinting of these esters, matrix-assisted laser desorption ionization time of flight

  4. Genotoxicity but not the AhR-mediated activity of PAHs is inhibited by other components of complex mixtures of ambient air pollutants

    Czech Academy of Sciences Publication Activity Database

    Líbalová, Helena; Krčková, S.; Uhlířová, Kateřina; Milcová, Alena; Schmuczerová, Jana; Cigánek, M.; Kléma, J.; Machala, M.; Šrám, Radim; Topinka, Jan

    2014-01-01

    Roč. 225, č. 3 (2014), s. 350-357 ISSN 0378-4274 R&D Projects: GA ČR GAP503/11/0142 Institutional support: RVO:68378041 Keywords : air pollutin * DNA adducts * complex mixtures Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.262, year: 2014

  5. Solution structure of the human Grb7-SH2 domain/erbB2 peptide complex and structural basis for Grb7 binding to ErbB2

    International Nuclear Information System (INIS)

    Ivancic, Monika; Daly, Roger J.; Lyons, Barbara A.

    2003-01-01

    The solution structure of the hGrb7-SH2 domain in complex with a ten amino acid phosphorylated peptide ligand representative of the erbB2 receptor tyrosine kinase (pY1139) is presented as determined by nuclear magnetic resonance methods. The hGrb7-SH2 domain structure reveals the Src homology 2 domain topology consisting of a central β-sheet capped at each end by an α-helix. The presence of a four residue insertion in the region between β-strand E and the EF loop and resulting influences on the SH2 domain/peptide complex structure are discussed. The binding conformation of the erbB2 peptide is in a β-turn similar to that found in phosphorylated tyrosine peptides bound to the Grb2-SH2 domain. To our knowledge this is only the second example of an SH2 domain binding its naturally occurring ligands in a turn, instead of extended, conformation. Close contacts between residues responsible for binding specificity in hGrb7-SH2 and the erbB2 peptide are characterized and the potential effect of mutation of these residues on the hGrb7-SH2 domain structure is discussed

  6. Crystallization and Preliminary Diffraction Analysis of the CAL PDZ Domain in Complex with a Selective Peptide Inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    J Amacher; P Cushing; J Weiner; D Madden

    2011-12-31

    Cystic fibrosis (CF) is associated with loss-of-function mutations in the CF transmembrane conductance regulator (CFTR), which regulates epithelial fluid and ion homeostasis. The CFTR cytoplasmic C-terminus interacts with a number of PDZ (PSD-95/Dlg/ZO-1) proteins that modulate its intracellular trafficking and chloride-channel activity. Among these, the CFTR-associated ligand (CAL) has a negative effect on apical-membrane expression levels of the most common disease-associated mutant {Delta}F508-CFTR, making CAL a candidate target for the treatment of CF. A selective peptide inhibitor of the CAL PDZ domain (iCAL36) has recently been developed and shown to stabilize apical expression of {Delta}F508-CFTR, enhancing net chloride-channel activity, both alone and in combination with the folding corrector corr-4a. As a basis for structural studies of the CAL-iCAL36 interaction, a purification protocol has been developed that increases the oligomeric homogeneity of the protein. Here, the cocrystallization of the complex in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 35.9, b = 47.7, c = 97.3 {angstrom}, is reported. The crystals diffracted to 1.4 {angstrom} resolution. Based on the calculated Matthews coefficient (1.96 {angstrom}{sup 3} Da{sup -1}), it appears that the asymmetric unit contains two complexes.

  7. Identification of novel peptide ligands for the cancer-specific receptor mutation EFGRvIII using a mixture-based synthetic combinatorial library

    DEFF Research Database (Denmark)

    Denholt, Charlotte Lund; Hansen, Paul Robert; Pedersen, Nina

    2009-01-01

    We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in se...

  8. Deconvolution of mixture spectra and increased throughput of Peptide identification by utilization of intensified complementary ions formed in tandem mass spectrometry

    DEFF Research Database (Denmark)

    Kryuchkov, Fedor; Verano-Braga, Thiago; Hansen, Thomas Aarup

    2013-01-01

    -resolution orbitrap mass spectrometry, an increase in the number of peptide identifications was obtained relative to the original CAD MS/MS spectra when intensified golden complementary (+18.6%) and CAD complementary pairs (+17.2%) were submitted to the Mascot search engine. This also exceeded the results obtained...

  9. A framework for the use of single-chemical transcriptomics data in predicting the hazards associated with complex mixtures of polycyclic aromatic hydrocarbons.

    Science.gov (United States)

    Labib, Sarah; Williams, Andrew; Kuo, Byron; Yauk, Carole L; White, Paul A; Halappanavar, Sabina

    2017-07-01

    The assumption of additivity applied in the risk assessment of environmental mixtures containing carcinogenic polycyclic aromatic hydrocarbons (PAHs) was investigated using transcriptomics. MutaTMMouse were gavaged for 28 days with three doses of eight individual PAHs, two defined mixtures of PAHs, or coal tar, an environmentally ubiquitous complex mixture of PAHs. Microarrays were used to identify differentially expressed genes (DEGs) in lung tissue collected 3 days post-exposure. Cancer-related pathways perturbed by the individual or mixtures of PAHs were identified, and dose-response modeling of the DEGs was conducted to calculate gene/pathway benchmark doses (BMDs). Individual PAH-induced pathway perturbations (the median gene expression changes for all genes in a pathway relative to controls) and pathway BMDs were applied to models of additivity [i.e., concentration addition (CA), generalized concentration addition (GCA), and independent action (IA)] to generate predicted pathway-specific dose-response curves for each PAH mixture. The predicted and observed pathway dose-response curves were compared to assess the sensitivity of different additivity models. Transcriptomics-based additivity calculation showed that IA accurately predicted the pathway perturbations induced by all mixtures of PAHs. CA did not support the additivity assumption for the defined mixtures; however, GCA improved the CA predictions. Moreover, pathway BMDs derived for coal tar were comparable to BMDs derived from previously published coal tar-induced mouse lung tumor incidence data. These results suggest that in the absence of tumor incidence data, individual chemical-induced transcriptomics changes associated with cancer can be used to investigate the assumption of additivity and to predict the carcinogenic potential of a mixture.

  10. Improved resolution of hydrocarbon structures and constitutional isomers in complex mixtures using Gas Chromatography-Vacuum Ultraviolet-Mass Spectrometry (GC-VUV-MS)

    Energy Technology Data Exchange (ETDEWEB)

    Aerosol Dynamics Inc; Aerodyne Research, Inc.,; Tofwerk AG, Thun; Isaacman, Gabriel; Wilson, Kevin R.; Chan, Arthur W. H.; Worton, David R.; Kimmel, Joel R.; Nah, Theodora; Hohaus, Thorsten; Gonin, Marc; Kroll, Jesse H.; Worsnop, Doug R.; Goldstein, Allen H.

    2011-09-13

    Understanding the composition of complex hydrocarbon mixtures is important for environmental studies in a variety of fields, but many prevalent compounds cannot be confidently identified using traditional gas chromatography-mass spectrometry (GC-MS) techniques. This work uses vacuum-ultraviolet (VUV) ionization to elucidate the structures of a traditionally"unresolved complex mixture" by separating components by GC retention time, tR, and mass-to-charge ratio, m/Q, which are used to determine carbon number, NC, and the number of rings and double bonds, NDBE. Constitutional isomers are resolved based on tR, enabling the most complete quantitative analysis to date of structural isomers in an environmentally-relevant hydrocarbon mixture. Unknown compounds are classified in this work by carbon number, degree of saturation, presence of rings, and degree of branching, providing structural constraints. The capabilities of this analysis are explored using diesel fuel, in which constitutional isomer distribution patterns are shown to be reproducible between carbon numbers and follow predictable rules. Nearly half of the aliphatic hydrocarbon mass is shown to be branched, suggesting branching is more important in diesel fuel than previously shown. The classification of unknown hydrocarbons and the resolution of constitutional isomers significantly improves resolution capabilities for any complex hydrocarbon mixture.

  11. PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

    Science.gov (United States)

    Kostova, Maya B; Brennen, William Nathaniel; Lopez, David; Anthony, Lizamma; Wang, Hao; Platz, Elizabeth; Denmeade, Samuel R

    2018-08-01

    Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter

  12. Widespread contamination of wildflower and bee-collected pollen with complex mixtures of neonicotinoids and fungicides commonly applied to crops.

    Science.gov (United States)

    David, Arthur; Botías, Cristina; Abdul-Sada, Alaa; Nicholls, Elizabeth; Rotheray, Ellen L; Hill, Elizabeth M; Goulson, Dave

    2016-03-01

    There is considerable and ongoing debate as to the harm inflicted on bees by exposure to agricultural pesticides. In part, the lack of consensus reflects a shortage of information on field-realistic levels of exposure. Here, we quantify concentrations of neonicotinoid insecticides and fungicides in the pollen of oilseed rape, and in pollen of wildflowers growing near arable fields. We then compare this to concentrations of these pesticides found in pollen collected by honey bees and in pollen and adult bees sampled from bumble bee colonies placed on arable farms. We also compared this with levels found in bumble bee colonies placed in urban areas. Pollen of oilseed rape was heavily contaminated with a broad range of pesticides, as was the pollen of wildflowers growing nearby. Consequently, pollen collected by both bee species also contained a wide range of pesticides, notably including the fungicides carbendazim, boscalid, flusilazole, metconazole, tebuconazole and trifloxystrobin and the neonicotinoids thiamethoxam, thiacloprid and imidacloprid. In bumble bees, the fungicides carbendazim, boscalid, tebuconazole, flusilazole and metconazole were present at concentrations up to 73nanogram/gram (ng/g). It is notable that pollen collected by bumble bees in rural areas contained high levels of the neonicotinoids thiamethoxam (mean 18ng/g) and thiacloprid (mean 2.9ng/g), along with a range of fungicides, some of which are known to act synergistically with neonicotinoids. Pesticide exposure of bumble bee colonies in urban areas was much lower than in rural areas. Understanding the effects of simultaneous exposure of bees to complex mixtures of pesticides remains a major challenge. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Distribution of rhodopin and spirilloxanthin between LH1 and LH2 complexes when incorporating carotenoid mixture into the membrane of purple sulfur bacterium Allochromatium minutissimum in vitro.

    Science.gov (United States)

    Bolshakov, M A; Ashikhmin, A A; Makhneva, Z K; Moskalenko, A A

    2016-11-01

    Carotenoid mixture enriched by rhodopin and spirilloxanthin was incorporated in LH2 and LH1 complexes from Allochromatium (Alc.) minutissimum in vitro. The maximum incorporating level was ~95%. Rhodopin (56.4%) and spirilloxanthin (13.8%) were incorporated into the LH1 complex, in contrast to the control complex, which contained primarily spirilloxanthin (66.8%). After incorporating, the LH2 complex contained rhodopin (66.7%) and didehydrorhodopin (14.6%), which was close to their content in the control (67.4 and 20.5%, respectively). Thus, it was shown that carotenoids from the total pool are not selectively incorporated into LH2 and LH1 complexes in vitro in the proportion corresponding to the carotenoid content in the complexes in vivo.

  14. Structure of HLA-A*0301 in complex with a peptide of proteolipid protein: insights into the role of HLA-A alleles in susceptibility to multiple sclerosis

    International Nuclear Information System (INIS)

    McMahon, Róisín M.; Friis, Lone; Siebold, Christian; Friese, Manuel A.; Fugger, Lars; Jones, E. Yvonne

    2011-01-01

    The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-ray crystallography to 2.7 Å resolution. The structure of the human major histocompatability (MHC) class I molecule HLA-A*0301 (HLA-A3) in complex with a nonameric peptide (KLIETYFSK) has been determined by X-ray crystallography to 2.7 Å resolution. HLA-A3 is a predisposing allele for multiple sclerosis (MS), an autoimmune disease of the central nervous system. The KLIETYFSK peptide is a naturally processed epitope of proteolipid protein, a myelin protein and candidate target for immune-mediated myelin destruction in MS. Comparison of the structure of HLA-A3 with that of HLA-A2, an MHC class I molecule which is protective against MS, indicates that both MHC class I molecules present very similar faces for T-cell receptor recognition whilst differing in the specificity of their peptide-binding grooves. These characteristics may underlie the opposing (predisposing versus protective) associations that they exhibit both in humans and in mouse models of MS-like disease. Furthermore, subtle alterations within the peptide-binding groove of HLA-A3 and other A3-like MHC class I molecules, members of the so-called A3 superfamily, may be sufficient to alter their presentation of autoantigen peptides such as KLIETYFSK. This in turn may modulate their contribution to the associated risk of autoimmune disease

  15. Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

    Science.gov (United States)

    Tabernero, L; Chang, C Y; Ohringer, S L; Lau, W F; Iwanowicz, E J; Han, W C; Wang, T C; Seiler, S M; Roberts, D G; Sack, J S

    1995-02-10

    The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

  16. High-performance liquid chromatography coupled with post-column dual-bioactivity assay for simultaneous screening of xanthine oxidase inhibitors and free radical scavengers from complex mixture.

    Science.gov (United States)

    Li, D Q; Zhao, J; Li, S P

    2014-06-06

    Xanthine oxidase (XO) can catalyze hypoxanthine and xanthine to generate uric acid and reactive oxygen species (ROS), including superoxide anion radical (O₂(•-)) and hydrogen peroxide. XO inhibitors and free radical scavengers are beneficial to the treatment of gout and many related diseases. In the present study, an on-line high-performance liquid chromatography (HPLC) coupled with post-column dual-bioactivity assay was established and successfully applied to simultaneously screening of XO inhibitors and free radical scavengers from a complex mixture, Oroxylum indicum extract. The integrated system of HPLC separation, bioactivity screening and mass spectrometry identification was proved to be simple and effective for rapid and sensitive screening of individual bioactive compounds in complex mixtures. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Preparation and crystallization of the Grb7 SH2 domain in complex with the G7-18NATE nonphosphorylated cyclic inhibitor peptide

    International Nuclear Information System (INIS)

    Yap, Min Y.; Wilce, Matthew C. J.; Clayton, Daniel J.; Perlmutter, Patrick; Aguilar, Marie-Isabel; Wilce, Jacqueline A.

    2010-01-01

    The preparation and successful crystallization of the Grb7 SH2 domain in complex with the specific cyclic peptide inhibitor G7-18NATE are reported. This structure is anticipated to reveal the basis of the binding affinity and specificity and to assist with the development of second-generation inhibitors of Grb7, which is involved in cancer progression. Grb7 is an adapter protein that is involved in signalling pathways that mediate eukaryotic cell proliferation and migration. Its overexpression in several cancer types has implicated it in cancer progression and led to the development of the G7-18NATE cyclic peptide inhibitor. Here, the preparation of crystals of G7-18NATE in complex with its Grb7 SH2 domain target is reported. Crystals of the complex were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant at room temperature. X-ray diffraction data were collected from crystals to 2.4 Å resolution using synchrotron X-ray radiation at 100 K. The diffraction was consistent with space group P2 1 , with unit-cell parameters a = 52.7, b = 79.1, c = 54.7 Å, α = γ = 90.0, β = 104.4°. The structure of the G7-18NATE peptide in complex with its target will facilitate the rational development of Grb7-targeted cancer therapeutics

  18. Simultaneous alignment and clustering of peptide data using a Gibbs sampling approach

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Lund, Ole; Nielsen, Morten

    2013-01-01

    Motivation: Proteins recognizing short peptide fragments play a central role in cellular signaling. As a result of high-throughput technologies, peptide-binding protein specificities can be studied using large peptide libraries at dramatically lower cost and time. Interpretation of such large...... peptide datasets, however, is a complex task, especially when the data contain multiple receptor binding motifs, and/or the motifs are found at different locations within distinct peptides.Results: The algorithm presented in this article, based on Gibbs sampling, identifies multiple specificities...... of unaligned peptide datasets of variable length. Example applications described in this article include mixtures of binders to different MHC class I and class II alleles, distinct classes of ligands for SH3 domains and sub-specificities of the HLA-A*02:01 molecule.Availability: The Gibbs clustering method...

  19. Study on complex formation of dicyclohexyl-18-crown-6 with Mg2+, Ca2+ and Sr2+ in acetonitrile-water binary mixtures by conductometry

    OpenAIRE

    Mallika Sanyal

    2017-01-01

    The complexation reactions between Mg2+, Ca2+ and Sr2+ cations and dicyclohexyl-18-crown-6 (DCH 18C6) have been studied in acetonitrile–water binary mixtures at different temperatures by conductometry. The formation constants of the resulting 1:1 (M:L) complexes for all the three cations were determined from computer fitting of the molar conductance versus mole ratio data. The results show that the selectivity order of DCH 18C6 for the metal cations in the acetonitrile-water binary solvent at...

  20. Expression, refolding and crystallizations of the Grb2-like (GADS) C-terminal SH3 domain complexed with a SLP-76 motif peptide

    International Nuclear Information System (INIS)

    Faravelli, Alessandro; Dimasi, Nazzareno

    2005-01-01

    Several crystals of the Grb2-like C-terminal SH3 domain in complex with a motif peptide from the SLP-76 protein were obtained and characterized. The Grb2-like adaptor protein GADS is composed of an N-terminal SH3 domain, an SH2 domain, a proline-rich region and a C-terminal SH3 domain. GADS interacts through its C-terminal SH3 domain with the adaptor protein SLP-76, thus recruiting this protein and other associated molecules to the linker for activation of T-cell (LAT) protein. The DNA encoding the C-terminal SH3 domain of GADS (GADS-cSH3) was assembled synthetically using a recursive PCR technique and the protein was overexpressed in Escherichia coli, refolded and purified. Several crystals of this domain in complex with the SLP-76 peptide were obtained and characterized

  1. The influence of olfactory concept on the probability of detecting sub- and peri-threshold odorants in a complex mixture

    NARCIS (Netherlands)

    Bult, J.H.F.; Schifferstein, H.N.J.; Roozen, J.P.; Voragen, A.G.J.; Kroeze, J.H.A.

    2001-01-01

    The headspace of apple juice was analysed to obtain an ecologically relevant stimulus model mixture of apple volatiles. Two sets of volatiles were made up: a set of eight supra-threshold volatiles (MIX) and a set of three sub-threshold volatiles. These sets were used to test the hypothesis that

  2. CD147 is a regulatory subunit of the gamma-secretase complex inAlzheimer's disease amyloid beta-peptide production

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Shuxia; Zhou, Hua; Walian, Peter J.; Jap, Bing K.

    2005-04-06

    {gamma}-secretase is a membrane protein complex that cleaves the {beta}-amyloid precursor protein (APP) within the transmembrane region, following prior processing by {beta}-secretase, producing amyloid {beta}-peptides (A{beta}{sub 40} and A{beta}{sub 42}). Errant production of A{beta}-peptides that substantially increases A{beta}{sub 42} production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1 (Psn-1), which contains the proteolytic active site, and three other membrane proteins, nicastrin (Nct), APH-1, and PEN-2 are required to form the core of the active {gamma}-secretase complex. Here, we report the purification of the native {gamma}-secretase complexes from HeLa cell membranes and the identification of an additional {gamma}-secretase complex subunit, CD147, a transmembrane glycoprotein with two immunoglobulin-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through co-immunoprecipitation studies of the purified protein from HeLa cells and solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of A{beta} peptides without changing the expression level of the other {gamma}-secretase components or APP substrates while CD147 overexpression had no statistically significant effect on amyloid {beta}-peptide production, other {gamma}-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the {gamma}-secretase complex directly down-modulates the production of A{beta}-peptides. {gamma}-secretase was first recognized through its role in the production of the A{beta} peptides that are pathogenic in Alzheimer's disease (AD) (1). {gamma}-secretase is a membrane protein complex with unusual aspartyl protease activity that cleaves a variety of type I membrane proteins

  3. Rare Earth Electrochemical Property Measurements and Phase Diagram Development in a Complex Molten Salt Mixture for Molten Salt Recycle

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jinsuo; Guo, Shaoqiang

    2018-03-30

    novel process using a galvanic couple between a cathode basket made of stainless steel and a Gd rod is investigated in LiCl-KCl-UCl3-MgCl2. The process shows rapid reduction of UCl3 to U and MgCl2 to Mg without any co-reduction of LaCl3, NdCl3, or CeCl3 because of the higher standard potential of Gd3+/Gd. Electrolysis of the molten salt results in a perturbation of the composition, which in turn can affect the equilibrium phase behavior. Studies on phase behavior in more complex salt mixtures as will be encountered in real electrorefiner salt drawdown are very limited. In present research, the solidus and liquidus temperatures for four quaternary LiCl-KCl-CsCl-RECl3 (RE = La, Nd, Ce, and Gd) salt systems are analyzed by differential scanning calorimetry (DSC) measurement. The presence of CsCl causes a slight depression in the liquidus temperature for all of the four rare earth chloride mixtures when compared to the corresponding ternary system liquidus temperatures in the absence of any CsCl. Thermodynamic assessment for the LiCl-KCl-LnCl3 ternary system(here Gd as a reprehensive of Ln) has been carried out by CALPHAD method using two-sublattice model. From the optimized phase diagram, the solubility of GdCl3 in LiCl-KCl eutectic is obtained. This model can also be applied to other salt system to evaluate the thermodynamic properties of other REs in pyroprocessing salt systems with more components. In the end, an electrolysis model is developed to predict the electrolysis process for RE drawdown from LiCl-KCl salt. The model considers both the diffusion in electrolyte and Faraday process on the electrode surface and a surface layer is introduced to account for the fact that diffusion current is not necessarily equal to the current due to the Butler-Volmer equation. Using the fundamental data obtained from this study, the proposed model is validated by chronoamperometry and chronopotentiometry experimental data.

  4. Enzyme-coupled nanoparticles-assisted laser desorption ionization mass spectrometry for searching for low-mass inhibitors of enzymes in complex mixtures.

    Science.gov (United States)

    Salwiński, Aleksander; Da Silva, David; Delépée, Raphaël; Maunit, Benoît

    2014-04-01

    In this report, enzyme-coupled magnetic nanoparticles (EMPs) were shown to be an effective affinity-based tool for finding specific interactions between enzymatic targets and the low-mass molecules in complex mixtures using classic MALDI-TOF apparatus. EMPs used in this work act as nonorganic matrix enabling ionization of small molecules without any interference in the low-mass range (enzyme-coupled nanoparticles-assisted laser desorption ionization MS, ENALDI MS) and simultaneously carry the superficial specific binding sites to capture inhibitors present in a studied mixture. We evaluated ENALDI approach in two complementary variations: 'ion fading' (IF-ENALDI), based on superficial adsorption of inhibitors and 'ion hunting' (IH-ENALDI), based on selective pre-concentration of inhibitors. IF-ENALDI was applied for two sets of enzyme-inhibitor pairs: tyrosinase-glabridin and trypsin-leupeptin and for the real plant sample: Sparrmannia discolor leaf and stem methanol extract. The efficacy of IH-ENALDI was shown for the pair of trypsin-leupeptin. Both ENALDI approaches pose an alternative for bioassay-guided fractionation, the common method for finding inhibitors in the complex mixtures.

  5. Automated 2D peptide separation on a 1D nano-LC-MS system

    DEFF Research Database (Denmark)

    Taylor, Paul; Nielsen, Peter A; Trelle, Morten Beck

    2009-01-01

    the on-line separation of highly complex peptide mixtures directly coupled with mass spectrometry-based identification. Here, we present a variation of the traditional MudPIT protocol, combining highly sensitive chromatography using a nanoflow liquid chromatography system (nano-LC) with a two...

  6. Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase

    International Nuclear Information System (INIS)

    Trewhella, J.; Blumenthal, D.K.; Rokop, S.E.; Seeger, P.A.

    1990-01-01

    Small-angle X-ray and neutron scattering have been used to study the solution structures of calmodulin complexed with synthetic peptides corresponding to residues 342-366 and 301-326, designated PhK5 and PhK13, respectively, in the regulatory domain of the catalytic subunit of skeletal muscle phosphorylase kinase. The scattering data show that binding of PhK5 to calmodulin induces a dramatic contraction of calmodulin, similar to that previously observed when calmodulin is complexed with the calmodulin-binding domain peptide from rabbit skeletal muscle myosin light chain kinase. In contrast, calmodulin remains extended upon binding PhK13. In the presence of both peptides, calmodulin also remains extended. Apparently, the presence of PhK13 inhibits calmodulin from undergoing the PhK5-induced contraction. These data indicate that there is a fundamentally different type of calmodulin-target enzyme interaction in the case of the catalytic subunit of phosphorylase kinase compared with that for myosin light chain kinase

  7. Humidity control as a strategy for lattice optimization applied to crystals of HLA-A*1101 complexed with variant peptides from dengue virus

    International Nuclear Information System (INIS)

    Chotiyarnwong, Pojchong; Stewart-Jones, Guillaume B.; Tarry, Michael J.; Dejnirattisai, Wanwisa; Siebold, Christian; Koch, Michael; Stuart, David I.; Harlos, Karl; Malasit, Prida; Screaton, Gavin; Mongkolsapaya, Juthathip; Jones, E. Yvonne

    2007-01-01

    Crystals of an MHC class I molecule bound to naturally occurring peptide variants from the dengue virus NS3 protein contained high levels of solvent and required optimization of cryoprotectant and dehydration protocols for each complex to yield well ordered diffraction, a process facilitated by the use of a free-mounting system. T-cell recognition of the antigenic peptides presented by MHC class I molecules normally triggers protective immune responses, but can result in immune enhancement of disease. Cross-reactive T-cell responses may underlie immunopathology in dengue haemorrhagic fever. To analyze these effects at the molecular level, the functional MHC class I molecule HLA-A*1101 was crystallized bound to six naturally occurring peptide variants from the dengue virus NS3 protein. The crystals contained high levels of solvent and required optimization of the cryoprotectant and dehydration protocols for each complex to yield well ordered diffraction, a process that was facilitated by the use of a free-mounting system

  8. 3D analysis of the TCR/pMHCII complex formation in monkeys vaccinated with the first peptide inducing sterilizing immunity against human malaria.

    Directory of Open Access Journals (Sweden)

    Manuel A Patarroyo

    Full Text Available T-cell receptor gene rearrangements were studied in Aotus monkeys developing high antibody titers and sterilizing immunity against the Plasmodium falciparum malaria parasite upon vaccination with the modified synthetic peptide 24112, which was identified in the Merozoite Surface Protein 2 (MSP-2 and is known to bind to HLA-DRbeta1*0403 molecules with high capacity. Spectratyping analysis showed a preferential usage of Vbeta12 and Vbeta6 TCR gene families in 67% of HLA-DRbeta1*0403-like genotyped monkeys. Docking of peptide 24112 into the HLA-DRbeta1*0401-HA peptide-HA1.7TCR complex containing the VDJ rearrangements identified in fully protected monkeys showed a different structural signature compared to nonprotected monkeys. These striking results show the exquisite specificity of the TCR/pMHCII complex formation needed for inducing sterilizing immunity and provide important hints for a logical and rational methodology to develop multiepitopic, minimal subunit-based synthetic vaccines against infectious diseases, among them malaria.

  9. Humidity control as a strategy for lattice optimization applied to crystals of HLA-A*1101 complexed with variant peptides from dengue virus

    Energy Technology Data Exchange (ETDEWEB)

    Chotiyarnwong, Pojchong [Department of Immunology, Division of Medicine, Hammersmith Hospital, Imperial College, London (United Kingdom); Medical Molecular Biology Unit, Faculty of Medicine, Siriraj Hospital, Mahidol University (Thailand); Stewart-Jones, Guillaume B.; Tarry, Michael J. [Division of Structural Biology and Oxford Protein Production Facility (OPPF), The Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Headington, Oxford OX3 7BN (United Kingdom); Dejnirattisai, Wanwisa [Department of Immunology, Division of Medicine, Hammersmith Hospital, Imperial College, London (United Kingdom); Medical Molecular Biology Unit, Faculty of Medicine, Siriraj Hospital, Mahidol University (Thailand); Siebold, Christian; Koch, Michael; Stuart, David I.; Harlos, Karl [Division of Structural Biology and Oxford Protein Production Facility (OPPF), The Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Headington, Oxford OX3 7BN (United Kingdom); Malasit, Prida [Medical Molecular Biology Unit, Faculty of Medicine, Siriraj Hospital, Mahidol University (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani, Bangkok (Thailand); Screaton, Gavin [Department of Immunology, Division of Medicine, Hammersmith Hospital, Imperial College, London (United Kingdom); Mongkolsapaya, Juthathip, E-mail: j.mongkolsapaya@imperial.ac.uk [Department of Immunology, Division of Medicine, Hammersmith Hospital, Imperial College, London (United Kingdom); Medical Molecular Biology Unit, Faculty of Medicine, Siriraj Hospital, Mahidol University (Thailand); Jones, E. Yvonne, E-mail: j.mongkolsapaya@imperial.ac.uk [Division of Structural Biology and Oxford Protein Production Facility (OPPF), The Henry Wellcome Building for Genomic Medicine, Roosevelt Drive, Headington, Oxford OX3 7BN (United Kingdom); Department of Immunology, Division of Medicine, Hammersmith Hospital, Imperial College, London (United Kingdom)

    2007-05-01

    Crystals of an MHC class I molecule bound to naturally occurring peptide variants from the dengue virus NS3 protein contained high levels of solvent and required optimization of cryoprotectant and dehydration protocols for each complex to yield well ordered diffraction, a process facilitated by the use of a free-mounting system. T-cell recognition of the antigenic peptides presented by MHC class I molecules normally triggers protective immune responses, but can result in immune enhancement of disease. Cross-reactive T-cell responses may underlie immunopathology in dengue haemorrhagic fever. To analyze these effects at the molecular level, the functional MHC class I molecule HLA-A*1101 was crystallized bound to six naturally occurring peptide variants from the dengue virus NS3 protein. The crystals contained high levels of solvent and required optimization of the cryoprotectant and dehydration protocols for each complex to yield well ordered diffraction, a process that was facilitated by the use of a free-mounting system.

  10. Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.

    Science.gov (United States)

    Bommakanti, R K; Bokoch, G M; Tolley, J O; Schreiber, R E; Siemsen, D W; Klotz, K N; Jesaitis, A J

    1992-04-15

    Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.

  11. Prediction of peptide drift time in ion mobility mass spectrometry from sequence-based features

    KAUST Repository

    Wang, Bing; Zhang, Jun; Chen, Peng; Ji, Zhiwei; Deng, Shuping; Li, Chi

    2013-01-01

    Background: Ion mobility-mass spectrometry (IMMS), an analytical technique which combines the features of ion mobility spectrometry (IMS) and mass spectrometry (MS), can rapidly separates ions on a millisecond time-scale. IMMS becomes a powerful tool to analyzing complex mixtures, especially for the analysis of peptides in proteomics. The high-throughput nature of this technique provides a challenge for the identification of peptides in complex biological samples. As an important parameter, peptide drift time can be used for enhancing downstream data analysis in IMMS-based proteomics.Results: In this paper, a model is presented based on least square support vectors regression (LS-SVR) method to predict peptide ion drift time in IMMS from the sequence-based features of peptide. Four descriptors were extracted from peptide sequence to represent peptide ions by a 34-component vector. The parameters of LS-SVR were selected by a grid searching strategy, and a 10-fold cross-validation approach was employed for the model training and testing. Our proposed method was tested on three datasets with different charge states. The high prediction performance achieve demonstrate the effectiveness and efficiency of the prediction model.Conclusions: Our proposed LS-SVR model can predict peptide drift time from sequence information in relative high prediction accuracy by a test on a dataset of 595 peptides. This work can enhance the confidence of protein identification by combining with current protein searching techniques. 2013 Wang et al.; licensee BioMed Central Ltd.

  12. Prediction of peptide drift time in ion mobility mass spectrometry from sequence-based features

    KAUST Repository

    Wang, Bing

    2013-05-09

    Background: Ion mobility-mass spectrometry (IMMS), an analytical technique which combines the features of ion mobility spectrometry (IMS) and mass spectrometry (MS), can rapidly separates ions on a millisecond time-scale. IMMS becomes a powerful tool to analyzing complex mixtures, especially for the analysis of peptides in proteomics. The high-throughput nature of this technique provides a challenge for the identification of peptides in complex biological samples. As an important parameter, peptide drift time can be used for enhancing downstream data analysis in IMMS-based proteomics.Results: In this paper, a model is presented based on least square support vectors regression (LS-SVR) method to predict peptide ion drift time in IMMS from the sequence-based features of peptide. Four descriptors were extracted from peptide sequence to represent peptide ions by a 34-component vector. The parameters of LS-SVR were selected by a grid searching strategy, and a 10-fold cross-validation approach was employed for the model training and testing. Our proposed method was tested on three datasets with different charge states. The high prediction performance achieve demonstrate the effectiveness and efficiency of the prediction model.Conclusions: Our proposed LS-SVR model can predict peptide drift time from sequence information in relative high prediction accuracy by a test on a dataset of 595 peptides. This work can enhance the confidence of protein identification by combining with current protein searching techniques. 2013 Wang et al.; licensee BioMed Central Ltd.

  13. Formation and electrochemical investigation of ordered cobalt coordinated peptide monolayers on gold substrates

    International Nuclear Information System (INIS)

    Wang Xinxin; Nagata, Kenji; Higuchi, Masahiro

    2012-01-01

    The monolayers composed of cobalt coordinated peptides were prepared on gold substrates by two different approaches. One was the self-assembly method, which was used to prepare a peptide monolayer on the gold substrate via the spontaneous attachment of peptides owing to the interaction between gold and sulfur at the N-terminal of the peptide. The other one was the stepwise polymerization method that was utilized to fabricate the unidirectionally arranged peptide monolayer by the stepwise condensation of amino acids from the initiator fixed on the gold substrate. Leu 2 Ala(4-Pyri)Leu 6 Ala(4-Pyri)Leu 6 sequence was chosen as the cobalt coordinated peptide. The 4-pyridyl alanines, Ala(4-Pyri)s, were introduced as ligands for cobalt to the leucine-rich sequential peptide. The complexation between cobalt and pyridyl groups of the peptide induced the formation of a stable α-helical bundle, which oriented perpendicularly to the substrate surface. In the case of the monolayer fabricated by the stepwise polymerization method, the direction of the peptide macro-dipole moment aligned unidirectionally, and the cobalt complexes were fixed in the monolayer to form the ordered arrangement. On the other hand, the peptides prepared by the self-assembly method formed the mixture of parallel and antiparallel packing owing to the dipole-dipole interaction. The spatial location of the cobalt complexes in the monolayer prepared by the self-assembly method was distorted, compared with that in the monolayer fabricated by the stepwise polymerization method. The vectorial electron flow through the peptide monolayer was achieved by the regular alignment of the peptide macro-dipole moment and the cobalt complexes in the monolayer fabricated by the stepwise polymerization method. - Highlights: ► We fabricated ordered Co coordinated peptide monolayers on the gold substrates. ► The Co complexes in peptide monolayer formed an ordered arrangement of the peptide. ► The peptide macro

  14. In vitro safety and efficacy evaluations of a complex botanical mixture of Eugenia dysenterica DC. (Myrtaceae): Prospects for developing a new dermocosmetic product.

    Science.gov (United States)

    Moreira, Larissa Cleres; de Ávila, Renato Ivan; Veloso, Danillo Fabrini Maciel Costa; Pedrosa, Tatiana Nascimento; Lima, Emerson Silva; do Couto, Renê Oliveira; Lima, Eliana Martins; Batista, Aline Carvalho; de Paula, José Realino; Valadares, Marize Campos

    2017-12-01

    In the context of developing a new natural product-based cosmetic, the in vitro efficacy and safety evaluations of a complex botanical mixture based on Eugenia dysenterica leaf hydroalcoholic extract (EDE) (2.5-1000μg/mL) were carried out. Chromatographic analysis demonstrated the presence of the tannin (ellagic acid) and flavonoids (quercetin and gallic acid) which characterize the EDE as a polyphenol-rich mixture. Using HFF-1 fibroblasts, it was shown that EDE promoted cell regeneration after UVA exposure. It also led to the inhibition of the collagenase, elastase and tyrosinase enzymes, which are involved in skin-related disorders. In terms of toxicological evaluation, the EDE was classified as non-phototoxic through the 3T3 Neutral Red Uptake Phototoxicity Test (OECD N° 432, 2004) and non-eye irritant by Bovine Corneal Opacity and Permeability (OECD N° 437, 2013) assay, in conjunction with corneal histomorphometric analysis. Furthermore, the EDE has no skin sensitization potential as demonstrated by a two-out-of-three prediction model [protein-binding/haptenization (OECD N° 442C, 2015), keratinocyte and dendritic cell activations]. In addition, it was shown that the EDE seems to be non-genotoxic through the cytokinesis-block micronucleus assay (OECD N° 487, 2014) using HepG2 cells. When considered together, these findings support the use of EDE botanical mixture in cosmetic/pharmaceutical products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Co-formation of hydroperoxides and ultra-fine particles during the reactions of ozone with a complex VOC mixture under simulated indoor conditions

    DEFF Research Database (Denmark)

    Fan, Z.H.; Weschler, Charles J.; Han, IK

    2005-01-01

    In this study we examined the co-formation of hydrogen peroxide and other hydroperoxides (collectively presented as H2O2*) as well as submicron particles, including ultra-fine particles (UFP), resulting from the reactions of ozone (O-3) with a complex mixture of volatile organic compounds (VOCs...... higher than typical indoor levels. When O-3 was added to a 25-m(3) controlled environmental facility (CEF) containing the 23 VOC mixture, both H2O2* and submicron particles were formed. The 2-h average concentration of H2O2* was 1.89 +/- 0.30ppb, and the average total particle number concentration was 46...... to achieve saturated concentrations of the condensable organics. When the 2 terpenes were removed from the O-3/23 VOCs mixture, no H2O2* or particles were formed, indicating that the reactions of O-3 With the two terpenes were the key processes contributing to the formation of H2O2* and submicron particles...

  16. Improved Efficacy of Synthesizing *MIII-Labeled DOTA Complexes in Binary Mixtures of Water and Organic Solvents. A Combined Radio- and Physicochemical Study.

    Science.gov (United States)

    Pérez-Malo, Marylaine; Szabó, Gergely; Eppard, Elisabeth; Vagner, Adrienn; Brücher, Ernő; Tóth, Imre; Maiocchi, Alessandro; Suh, Eul Hyun; Kovács, Zoltán; Baranyai, Zsolt; Rösch, Frank

    2018-05-21

    Typically, the synthesis of radiometal-based radiopharmaceuticals is performed in buffered aqueous solutions. We found that the presence of organic solvents like ethanol increased the radiolabeling yields of [ 68 Ga]Ga-DOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacatic acid). In the present study, the effect of organic cosolvents [ethanol (EtOH), isopropyl alcohol, and acetonitrile] on the radiolabeling yields of the macrocyclic chelator DOTA with several trivalent radiometals (gallium-68, scandium-44, and lutetium-177) was systematically investigated. Various binary water (H 2 O)/organic solvent mixtures allowed the radiolabeling of DOTA at a significantly lower temperature than 95 °C, which is relevant for the labeling of sensitive biological molecules. Simultaneously, much lower amounts of the chelators were required. This strategy may have a fundamental impact on the formulation of trivalent radiometal-based radiopharmaceuticals. The equilibrium properties and formation kinetics of [M(DOTA)] - (M III = Ga III , Ce III , Eu III , Y III , and Lu III ) complexes were investigated in H 2 O/EtOH mixtures (up to 70 vol % EtOH). The protonation constants of DOTA were determined by pH potentiometry in H 2 O/EtOH mixtures (0-70 vol % EtOH, 0.15 M NaCl, 25 °C). The log K 1 H and log K 2 H values associated with protonation of the ring N atoms decreased with an increase of the EtOH content. The formation rates of [M(DOTA)] - complexes increase with an increase of the pH and [EtOH]. Complexation occurs through rapid formation of the diprotonated [M(H 2 DOTA)] + intermediates, which are in equilibrium with the kinetically active monoprotonated [M(HDOTA)] intermediates. The rate-controlling step is deprotonation (and rearrangement) of the monoprotonated intermediate, which occurs through H 2 O ( *M(HL) k H 2 O ) and OH - ( *M(HL) k OH ) assisted reaction pathways. The rate constants are essentially independent of the EtOH concentration, but the M(HL) k H2O

  17. Enzyme-polyelectrolyte complexes in water-ethanol mixtures: negatively charged groups artificially introduced into alpha-chymotrypsin provide additional activation and stabilization effects.

    Science.gov (United States)

    Kudryashova, E V; Gladilin, A K; Vakurov, A V; Heitz, F; Levashov, A V; Mozhaev, V V

    1997-07-20

    Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

  18. Simulation of Major Histocompatibility Complex (MHC Structure and Peptide Loading into an MHC Binding Pocket with Teachers’Hands

    Directory of Open Access Journals (Sweden)

    Mojtaba Sankian

    2013-10-01

    Full Text Available Molecular understanding of three-dimensional (3D peptide: MHC models require both basic knowledge of computational modeling and skilled visual perception, which are not possessed by all students. The present model aims to simulate MHC molecular structure with the hands and make a profound impression on the students.

  19. Crystal structures of two peptide-HLA-B*1501 complexes; structural characterization of the HLA-B62 supertype

    DEFF Research Database (Denmark)

    Roder, G; Blicher, Thomas; Justesen, Sune Frederik Lamdahl

    2006-01-01

    MHC class I molecules govern human cytotoxic T cell responses. Their specificity determines which peptides they sample from the intracellular protein environment and then present to human cytotoxic T cells. More than 1100 different MHC class I proteins have been found in human populations...

  20. Ion-solvent interactions and the complex behaviour of U(IV) and U(VI) with chloro-ligands in ethanol-water mixtures

    International Nuclear Information System (INIS)

    Kim, J.I.; Duschner, H.; Hashimoto, T.; Born, H.J.

    1975-01-01

    The complex chemical behaviour of U(IV) and U(VI) in amphiprotic solutions, especially in mixtures of solvents, was investigated using ion exchange and solvent extraction methods. The experimental data was used, on the one hand, in order to obtain complexing constants in dependence of ligands and their concentration as well as of the agent and to classify these in a universal scale of ion activities with water as reference point, and on the other hand, to explain the interactions between central atom, ligand and solvating molecule. One aim of these investigations is to understand the basic mechanisms in adjusting the equilibrium between two different phases in the separation chemistry of these elements. (orig./LH) [de

  1. Crystal structure of the G3BP2 NTF2-like domain in complex with a canonical FGDF motif peptide.

    Science.gov (United States)

    Kristensen, Ole

    2015-11-06

    The crystal structure of the NTF2-like domain of the human Ras GTPase SH3 Binding Protein (G3BP), isoform 2, was determined at a resolution of 2.75 Å in complex with a peptide containing a FGDF sequence motif. The overall structure of the protein is highly similar to the homodimeric N-terminal domains of the G3BP1 and Rasputin proteins. Recently, a subset of G3BP interacting proteins was recognized to share a common sequence motif, FGDF. The most studied binding partners, USP10 and viral nsP3, interfere with essential G3BP functions related to assembly of cellular stress granules. Reported molecular modeling suggested that FGDF-motif containing peptides bind in an extended conformation into a hydrophobic groove on the surface of the G3BP NTF2-like domain in a manner similar to the known binding of FxFG nucleoporin repeats. The results in this paper provide evidence for a different binding mode. The FGDF peptide binds and changes conformation of the protruding N-terminal residues by providing hydrophobic interactions to a symmetry related molecule that facilitated crystallization of the G3BP2 isoform. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Peptides as catalysts in the RNA world

    DEFF Research Database (Denmark)

    Wieczorek, Rafal; Dörr, Mark; Luisi, Pier Luigi

    The emergence of RNA chains from prebiotic soup is considered a stumbling block in the RNA world theory (Orgel 2004). Both the activation of RNA monomers and their subsequent oligomerization is hard to achieve in accepted early Earth conditions, thus putting doubt on the prebiotic plausibility...... chemistry and the RNA world. Prebiotic soup likely contained complex mixtures of various molecules. Interaction of peptides and nucleotides shows that we should give more consideration to systems chemistry approach in the origin-of-life research. Gorlero M, Wieczorek R, Adamala K, Giorgi A, Schininà ME...

  3. A functional monomer is not enough: principal component analysis of the influence of template complexation in pre-polymerization mixtures on imprinted polymer recognition and morphology.

    Science.gov (United States)

    Golker, Kerstin; Karlsson, Björn C G; Rosengren, Annika M; Nicholls, Ian A

    2014-11-10

    In this report, principal component analysis (PCA) has been used to explore the influence of template complexation in the pre-polymerization phase on template molecularly imprinted polymer (MIP) recognition and polymer morphology. A series of 16 bupivacaine MIPs were studied. The ethylene glycol dimethacrylate (EGDMA)-crosslinked polymers had either methacrylic acid (MAA) or methyl methacrylate (MMA) as the functional monomer, and the stoichiometry between template, functional monomer and crosslinker was varied. The polymers were characterized using radioligand equilibrium binding experiments, gas sorption measurements, swelling studies and data extracted from molecular dynamics (MD) simulations of all-component pre-polymerization mixtures. The molar fraction of the functional monomer in the MAA-polymers contributed to describing both the binding, surface area and pore volume. Interestingly, weak positive correlations between the swelling behavior and the rebinding characteristics of the MAA-MIPs were exposed. Polymers prepared with MMA as a functional monomer and a polymer prepared with only EGDMA were found to share the same characteristics, such as poor rebinding capacities, as well as similar surface area and pore volume, independent of the molar fraction MMA used in synthesis. The use of PCA for interpreting relationships between MD-derived descriptions of events in the pre-polymerization mixture, recognition properties and morphologies of the corresponding polymers illustrates the potential of PCA as a tool for better understanding these complex materials and for their rational design.

  4. A Functional Monomer Is Not Enough: Principal Component Analysis of the Influence of Template Complexation in Pre-Polymerization Mixtures on Imprinted Polymer Recognition and Morphology

    Directory of Open Access Journals (Sweden)

    Kerstin Golker

    2014-11-01

    Full Text Available In this report, principal component analysis (PCA has been used to explore the influence of template complexation in the pre-polymerization phase on template molecularly imprinted polymer (MIP recognition and polymer morphology. A series of 16 bupivacaine MIPs were studied. The ethylene glycol dimethacrylate (EGDMA-crosslinked polymers had either methacrylic acid (MAA or methyl methacrylate (MMA as the functional monomer, and the stoichiometry between template, functional monomer and crosslinker was varied. The polymers were characterized using radioligand equilibrium binding experiments, gas sorption measurements, swelling studies and data extracted from molecular dynamics (MD simulations of all-component pre-polymerization mixtures. The molar fraction of the functional monomer in the MAA-polymers contributed to describing both the binding, surface area and pore volume. Interestingly, weak positive correlations between the swelling behavior and the rebinding characteristics of the MAA-MIPs were exposed. Polymers prepared with MMA as a functional monomer and a polymer prepared with only EGDMA were found to share the same characteristics, such as poor rebinding capacities, as well as similar surface area and pore volume, independent of the molar fraction MMA used in synthesis. The use of PCA for interpreting relationships between MD-derived descriptions of events in the pre-polymerization mixture, recognition properties and morphologies of the corresponding polymers illustrates the potential of PCA as a tool for better understanding these complex materials and for their rational design.

  5. Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2

    International Nuclear Information System (INIS)

    Ozorowski, Gabriel; Milton, Saskia; Luecke, Hartmut

    2013-01-01

    Structure of a 20-amino-acid peptide of AHNAK bound asymmetrically to the AnxA2–S100A10A heterotetramer (1:2:2 symmetry) provides insights into the atomic level interactions that govern this membrane-repair scaffolding complex. AHNAK, a large 629 kDa protein, has been implicated in membrane repair, and the annexin A2–S100A10 heterotetramer [(p11) 2 (AnxA2) 2 )] has high affinity for several regions of its 1002-amino-acid C-terminal domain. (p11) 2 (AnxA2) 2 is often localized near the plasma membrane, and this C2-symmetric platform is proposed to be involved in the bridging of membrane vesicles and trafficking of proteins to the plasma membrane. All three proteins co-localize at the intracellular face of the plasma membrane in a Ca 2+ -dependent manner. The binding of AHNAK to (p11) 2 (AnxA2) 2 has been studied previously, and a minimal binding motif has been mapped to a 20-amino-acid peptide corresponding to residues 5654–5673 of the AHNAK C-terminal domain. Here, the 2.5 Å resolution crystal structure of this 20-amino-acid peptide of AHNAK bound to the AnxA2–S100A10 heterotetramer (1:2:2 symmetry) is presented, which confirms the asymmetric arrangement first described by Rezvanpour and coworkers and explains why the binding motif has high affinity for (p11) 2 (AnxA2) 2 . Binding of AHNAK to the surface of (p11) 2 (AnxA2) 2 is governed by several hydrophobic interactions between side chains of AHNAK and pockets on S100A10. The pockets are large enough to accommodate a variety of hydrophobic side chains, allowing the consensus sequence to be more general. Additionally, the various hydrogen bonds formed between the AHNAK peptide and (p11) 2 (AnxA2) 2 most often involve backbone atoms of AHNAK; as a result, the side chains, particularly those that point away from S100A10/AnxA2 towards the solvent, are largely interchangeable. While the structure-based consensus sequence allows interactions with various stretches of the AHNAK C-terminal domain, comparison

  6. Structure of a C-terminal AHNAK peptide in a 1:2:2 complex with S100A10 and an acetylated N-terminal peptide of annexin A2

    Energy Technology Data Exchange (ETDEWEB)

    Ozorowski, Gabriel [University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697-3900 (United States); Milton, Saskia [University of California, Irvine, Irvine, CA 92697-3900 (United States); Luecke, Hartmut, E-mail: hudel@uci.edu [University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697-3900 (United States); University of California, Irvine, Irvine, CA 92697 (United States); University of California, Irvine, Irvine, CA 92697 (United States)

    2013-01-01

    Structure of a 20-amino-acid peptide of AHNAK bound asymmetrically to the AnxA2–S100A10A heterotetramer (1:2:2 symmetry) provides insights into the atomic level interactions that govern this membrane-repair scaffolding complex. AHNAK, a large 629 kDa protein, has been implicated in membrane repair, and the annexin A2–S100A10 heterotetramer [(p11){sub 2}(AnxA2){sub 2})] has high affinity for several regions of its 1002-amino-acid C-terminal domain. (p11){sub 2}(AnxA2){sub 2} is often localized near the plasma membrane, and this C2-symmetric platform is proposed to be involved in the bridging of membrane vesicles and trafficking of proteins to the plasma membrane. All three proteins co-localize at the intracellular face of the plasma membrane in a Ca{sup 2+}-dependent manner. The binding of AHNAK to (p11){sub 2}(AnxA2){sub 2} has been studied previously, and a minimal binding motif has been mapped to a 20-amino-acid peptide corresponding to residues 5654–5673 of the AHNAK C-terminal domain. Here, the 2.5 Å resolution crystal structure of this 20-amino-acid peptide of AHNAK bound to the AnxA2–S100A10 heterotetramer (1:2:2 symmetry) is presented, which confirms the asymmetric arrangement first described by Rezvanpour and coworkers and explains why the binding motif has high affinity for (p11){sub 2}(AnxA2){sub 2}. Binding of AHNAK to the surface of (p11){sub 2}(AnxA2){sub 2} is governed by several hydrophobic interactions between side chains of AHNAK and pockets on S100A10. The pockets are large enough to accommodate a variety of hydrophobic side chains, allowing the consensus sequence to be more general. Additionally, the various hydrogen bonds formed between the AHNAK peptide and (p11){sub 2}(AnxA2){sub 2} most often involve backbone atoms of AHNAK; as a result, the side chains, particularly those that point away from S100A10/AnxA2 towards the solvent, are largely interchangeable. While the structure-based consensus sequence allows interactions with various

  7. Turn-on fluorescence sensor based on single-walled-carbon-nanohorn-peptide complex for the detection of thrombin.

    Science.gov (United States)

    Zhu, Shuyun; Liu, Zhongyuan; Hu, Lianzhe; Yuan, Yali; Xu, Guobao

    2012-12-14

    Proteases play a central role in several widespread diseases. Thus, there is a great need for the fast and sensitive detection of various proteolytic enzymes. Herein, we have developed a carbon nanotube (CNT)-based protease biosensing platform that uses peptides as a fluorescence probe for the first time. Single-walled carbon nanohorns (SWCNHs) and thrombin were used to demonstrate this detection strategy. SWCNHs can adsorb a fluorescein-based dye (FAM)-labeled peptide (FAM-pep) and quench the fluorescence of FAM. In contrast, thrombin can cleave FAM-pep on SWCNHs and recover the fluorescence of FAM, which allows the sensitive detection of thrombin. This biosensor has a high sensitivity and selectivity toward thrombin, with a detection limit of 100 pM. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. An SH2 domain model of STAT5 in complex with phospho-peptides define ``STAT5 Binding Signatures''

    Science.gov (United States)

    Gianti, Eleonora; Zauhar, Randy J.

    2015-05-01

    The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

  9. An SH2 domain model of STAT5 in complex with phospho-peptides define "STAT5 Binding Signatures".

    Science.gov (United States)

    Gianti, Eleonora; Zauhar, Randy J

    2015-05-01

    The signal transducer and activator of transcription 5 (STAT5) is a member of the STAT family of proteins, implicated in cell growth and differentiation. STAT activation is regulated by phosphorylation of protein monomers at conserved tyrosine residues, followed by binding to phospho-peptide pockets and subsequent dimerization. STAT5 is implicated in the development of severe pathological conditions, including many cancer forms. However, nowadays a few STAT5 inhibitors are known, and only one crystal structure of the inactive STAT5 dimer is publicly available. With a view to enabling structure-based drug design, we have: (1) analyzed phospho-peptide binding pockets on SH2 domains of STAT5, STAT1 and STAT3; (2) generated a model of STAT5 bound to phospho-peptides; (3) assessed our model by docking against a class of known STAT5 inhibitors (Müller et al. in ChemBioChem 9:723-727, 2008); (4) used molecular dynamics simulations to optimize the molecular determinants responsible for binding and (5) proposed unique "Binding Signatures" of STAT5. Our results put in place the foundations to address STAT5 as a target for rational drug design, from sequence, structural and functional perspectives.

  10. Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

    Science.gov (United States)

    Brown, D R; Bailey, J M; Oliveri, A N; Levin, E D; Di Giulio, R T

    2016-01-01

    Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in

  11. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs....... The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered sin.-le-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins......, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA...

  12. Mixed ligand complexes of essential metal ions with L-glutamine and succinic acid in SLS-water mixtures

    Directory of Open Access Journals (Sweden)

    Bindu Hima Gandham

    2012-01-01

    Full Text Available Speciation of mixed ligand complexes of Co(II, Ni(II and Cu(II with L-glutamine and succinic acid was studied in varying amounts (0.0-2.5% w/v of sodium lauryl sulphate in aqueous solutions maintaining an ionic strength of 0.16 mol L-1 (NaCl at 303.0 K. Titrations were carried out in the presence of different relative concentrations (M : L : X = 1 : 2 : 2, 1 : 4 : 2, 1 : 2 : 4 of metal (M to L-glutamine (L to succinic acid (X with sodium hydroxide. Stability constants of ternary complexes were refined with MINIQUAD75. The best-fit chemical models were selected based on statistical parameters and residual analysis. The species detected were ML2X, MLX, MLXH and MLXH2 for Co(II, Ni(II and Cu(II. Extra stability of ternary complexes compared to their binary complexes was believed to be due to electrostatic interactions of the side chains of ligands, charge neutralization, chelate effect, stacking interactions and hydrogen bonding. The species distribution with pH at different compositions of SLS and plausible equilibria for the formation of species were also presented.

  13. Kinetic stability of the dysprosium(3) complex with tetraazaporphine in acetic acid-water and acetic acid-methanol mixtures

    International Nuclear Information System (INIS)

    Khelevina, O.G.; Vojnov, A.A.

    1999-01-01

    Water-soluble dysprosium tetraazaporphine with acetylacetonate-ion as extraligand is synthesized for the first time. Its kinetic stability in acetic acid solutions is investigated. It is shown that the complex is dissociated with formation of free tetraazaporphine. Kinetic parameters of dissociation reaction are determined [ru

  14. Algorithms for GPU-based molecular dynamics simulations of complex fluids: Applications to water, mixtures, and liquid crystals.

    Science.gov (United States)

    Kazachenko, Sergey; Giovinazzo, Mark; Hall, Kyle Wm; Cann, Natalie M

    2015-09-15

    A custom code for molecular dynamics simulations has been designed to run on CUDA-enabled NVIDIA graphics processing units (GPUs). The double-precision code simulates multicomponent fluids, with intramolecular and intermolecular forces, coarse-grained and atomistic models, holonomic constraints, Nosé-Hoover thermostats, and the generation of distribution functions. Algorithms to compute Lennard-Jones and Gay-Berne interactions, and the electrostatic force using Ewald summations, are discussed. A neighbor list is introduced to improve scaling with respect to system size. Three test systems are examined: SPC/E water; an n-hexane/2-propanol mixture; and a liquid crystal mesogen, 2-(4-butyloxyphenyl)-5-octyloxypyrimidine. Code performance is analyzed for each system. With one GPU, a 33-119 fold increase in performance is achieved compared with the serial code while the use of two GPUs leads to a 69-287 fold improvement and three GPUs yield a 101-377 fold speedup. © 2015 Wiley Periodicals, Inc.

  15. Resonance ionization and time-of-flight mass spectrometry for the analysis of trace substances in complex gas mixtures

    International Nuclear Information System (INIS)

    Nagel, Holger; Weickhardt, Christian; Boesl, Ulrich; Frey, Ruediger

    1995-01-01

    The analysis of mixtures of technical gases still comprises a lot of problems: the large number of components with very different and often rapidly varying concentrations makes great demands on analytical methods. By use of conventional analytical methods, signals of trace substances may interfere with signals of main components, whereas small signals representing low concentrations are covered by signals of main substances.The resonant-enhanced multiphoton ionization (REMPI) makes use of excited intermediate states of molecules. As these states are characteristic of each substance, one or more components of interest can be ionized with high efficiency without interference of other molecules by using a special laser-wavelength. The combination of the above mentioned ionization method with a reflectron time-of-flight mass spectrometer permits a very fast and sensitive detection of preselected trace substances.As ionization processes of higher order strongly depend on the laser intensity, there is no direct relation between ion signals and concentrations of exhaust components. Quantitative assessments are based on an especially developed calibration technique that makes use of internal standards. Applied under environmental aspects, this new analytical method helps to analyze a large number of components extracted from exhaust gases of combustion engines with high time resolution (<20 ms motor synchronously), high sensitivity (1 ppm) and high quantitative accuracy (more than 10%). A preliminary list of detectable compounds contains 30 substances

  16. X-hitting: A new algorithm for novelty detection and dereplication by UV spectra of complex mixtures of natural products

    DEFF Research Database (Denmark)

    Hansen, Michael Edberg; Smedsgaard, Jørn; Larsen, Thomas Ostenfeld

    2005-01-01

    A major challenge in lead discovery is to detect well-known and trivial compounds rapidly, a process known as dereplication, so that isolation, structure elucidation, and pharmacological investigations can be focused on novel compounds. In this paper, we present a new algorithm, X-hitting, based...... on cross sample comparison of full UV spectra from HPLC analysis of highly complex natural product extracts/samples. X-Hitting allows automatic identification of known compounds but more important also allows finding of potentially new or similar compounds. We demonstrate this new algorithm by automatic...... identification of known structures, a task we call cross-hitting, and tentative identification of potentially new bioactive compounds, a task we call new-hitting, in HPLC data from analysis of fungal extracts. Both tasks are illustrated using 18 important reference compounds and complex fungal extracts obtained...

  17. A complementary mobile phase approach based on the peak count concept oriented to the full resolution of complex mixtures.

    Science.gov (United States)

    Ortín, A; Torres-Lapasió, J R; García-Álvarez-Coque, M C

    2011-08-26

    Situations of minimal resolution are often found in liquid chromatography, when samples that contain a large number of compounds, or highly similar in terms of structure and/or polarity, are analysed. This makes full resolution with a single separation condition (e.g., mobile phase, gradient or column) unfeasible. In this work, the optimisation of the resolution of such samples in reversed-phase liquid chromatography is approached using two or more isocratic mobile phases with a complementary resolution behaviour (complementary mobile phases, CMPs). Each mobile phase is dedicated to the separation of a group of compounds. The CMPs are selected in such a way that, when the separation is considered globally, all the compounds in the sample are satisfactorily resolved. The search of optimal CMPs can be carried out through a comprehensive examination of the mobile phases in a selected domain. The computation time of this search has been reported to be substantially reduced by application of a genetic algorithm with local search (LOGA). A much simpler approach is here described, which is accessible to non-experts in programming, and offers solutions of the same quality as LOGA, with a similar computation time. The approach makes a sequential search of CMPs based on the peak count concept, which is the number of peaks exceeding a pre-established resolution threshold. The new approach is described using as test sample a mixture of 30 probe compounds, 23 of them with an ionisable character, and the pH and organic solvent contents as experimental factors. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Fingerprinting of complex mixtures with the use of high performance liquid chromatography, inductively coupled plasma atomic emission spectroscopy and chemometrics

    International Nuclear Information System (INIS)

    Ni Yongnian; Peng Yunyan; Kokot, Serge

    2008-01-01

    The molecular and metal profile fingerprints were obtained from a complex substance, Atractylis chinensis DC-a traditional Chinese medicine (TCM), with the use of the high performance liquid chromatography (HPLC) and inductively coupled plasma atomic emission spectroscopy (ICP-AES) techniques. This substance was used in this work as an example of a complex biological material, which has found application as a TCM. Such TCM samples are traditionally processed by the Bran, Cut, Fried and Swill methods, and were collected from five provinces in China. The data matrices obtained from the two types of analysis produced two principal component biplots, which showed that the HPLC fingerprint data were discriminated on the basis of the methods for processing the raw TCM, while the metal analysis grouped according to the geographical origin. When the two data matrices were combined into a one two-way matrix, the resulting biplot showed a clear separation on the basis of the HPLC fingerprints. Importantly, within each different grouping the objects separated according to their geographical origin, and they ranked approximately in the same order in each group. This result suggested that by using such an approach, it is possible to derive improved characterisation of the complex TCM materials on the basis of the two kinds of analytical data. In addition, two supervised pattern recognition methods, K-nearest neighbors (KNNs) method, and linear discriminant analysis (LDA), were successfully applied to the individual data matrices-thus, supporting the PCA approach

  19. Comprehensive Metabolite Identification Strategy Using Multiple Two-Dimensional NMR Spectra of a Complex Mixture Implemented in the COLMARm Web Server

    Energy Technology Data Exchange (ETDEWEB)

    Bingol, Kerem [Environmental; Li, Da-Wei; Zhang, Bo; Brüschweiler, Rafael

    2016-12-06

    Identification of metabolites in complex mixtures represents a key step in metabolomics. A new strategy is introduced, which is implemented in a new public web server, COLMARm, that permits the co-analysis of up to three 2D NMR spectra, namely 13C-1H HSQC, 1H-1H TOCSY, and 13C-1H HSQC-TOCSY for the comprehensive, accurate, and efficient performance of this task. The highly versatile and interactive nature of COLMARm permits its application to a wide range of metabolomics samples independent of the magnetic field. Database query is performed using the HSQC spectrum and the top metabolite hits are then validated against the TOCSY-type experiment(s) by superimposing the expected cross-peaks on the mixture spectrum. In this way the user can directly accept or reject candidate metabolites by taking advantage of the complementary spectral information offered by these experiments and their different sensitivities. The power of COLMARm is demonstrated for a human serum sample uncovering the existence of 14 metabolites that hitherto were not identified by NMR.

  20. Peptides derived from specific interaction sites of the fibroblast growth factor 2 - FGF receptor complexes induce receptor activation and signaling

    DEFF Research Database (Denmark)

    Manfè, Valentina; Kochoyan, Artur; Bock, Elisabeth

    2010-01-01

    J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06718.x Abstract Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic...... biologic actions by binding, dimerizing, and activating FGF receptors (FGFRs). The present study reports the physiologic impact of various FGF2-FGFR1 contact sites employing three different synthetic peptides, termed canofins, designed based on structural analysis of the interactions between FGF2 and FGFR1...

  1. Application of high performance liquid chromatography for the profiling of complex chemical mixtures with the aid of chemometrics.

    Science.gov (United States)

    Ni, Yongnian; Zhang, Liangsheng; Churchill, Jane; Kokot, Serge

    2007-06-15

    In this paper, chemometrics methods were applied to resolve the high performance liquid chromatography (HPLC) fingerprints of complex, many-component substances to compare samples from a batch from a given manufacturer, or from those of different producers. As an example of such complex substances, we used a common Chinese traditional medicine, Huoxiang Zhengqi Tincture (HZT) for this research. Twenty-one samples, each representing a separate HZT production batch from one of three manufacturers were analyzed by HPLC with the aid of a diode array detector (DAD). An Agilent Zorbax Eclipse XDB-C18 column with an Agilent Zorbax high pressure reliance cartridge guard-column were used. The mobile phase consisted of water (A) and methanol (B) with a gradient program of 25-65% (v/v, B) during 0-30min, 65-55% (v/v, B) during 30-35min and 55-100% (v/v, B) during 35-60min (flow rate, 1.0mlmin(-1); injection volume, 20mul; and column temperature-ambient). The detection wavelength was adjusted for maximum sensitivity at different time periods. A peak area matrix with 21objectsx14HPLC variables was obtained by sampling each chromatogram at 14 common retention times. Similarities were then calculated to discriminate the batch-to-batch samples and also, a more informative multi-criteria decision making methodology (MCDM), PROMETHEE and GAIA, was applied to obtain more information from the chromatograms in order to rank and compare the complex HZT profiles. The results showed that with the MCDM analysis, it was possible to match and discriminate correctly the batch samples from the three different manufacturers. Fourier transform infrared (FT-IR) spectra taken from samples from several batches were compared by the common similarity method with the HPLC results. It was found that the FT-IR spectra did not discriminate the samples from the different batches.

  2. Predicting membrane flux decline from complex mixtures using flow-field flow fractionation measurements and semi-empirical theory.

    Science.gov (United States)

    Pellegrino, J; Wright, S; Ranvill, J; Amy, G

    2005-01-01

    Flow-Field Flow Fractionation (FI-FFF) is an idealization of the cross flow membrane filtration process in that, (1) the filtration flux and crossflow velocity are constant from beginning to end of the device, (2) the process is a relatively well-defined laminar-flow hydrodynamic condition, and (3) the solutes are introduced as a pulse-input that spreads due to interactions with each other and the membrane in the dilute-solution limit. We have investigated the potential for relating FI-FFF measurements to membrane fouling. An advection-dispersion transport model was used to provide 'ideal' (defined as spherical, non-interacting solutes) solute residence time distributions (RTDs) for comparison with 'real' RTDs obtained experimentally at different cross-field velocities and solution ionic strength. An RTD moment analysis based on a particle diameter probability density function was used to extract "effective" characteristic properties, rather than uniquely defined characteristics, of the standard solute mixture. A semi-empirical unsteady-state, flux decline model was developed that uses solute property parameters. Three modes of flux decline are included: (1) concentration polarization, (2) cake buildup, and (3) adsorption on/in pores, We have used this model to test the hypothesis-that an analysis of a residence time distribution using FI-FFF can describe 'effective' solute properties or indices that can be related to membrane flux decline in crossflow membrane filtration. Constant flux filtration studies included the changes of transport hydrodynamics (solvent flux to solute back diffusion (J/k) ratios), solution ionic strength, and feed water composition for filtration using a regenerated cellulose ultrafiltration membrane. Tests of the modeling hypothesis were compared with experimental results from the filtration measurements using several correction parameters based on the mean and variance of the solute RTDs. The corrections used to modify the boundary layer

  3. Antimicrobial Peptides in Reptiles

    Science.gov (United States)

    van Hoek, Monique L.

    2014-01-01

    Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development. PMID:24918867

  4. Chemical imaging techniques for the analysis of complex mixtures: New application to the characterization of ritual matters on African wooden statuettes

    International Nuclear Information System (INIS)

    Mazel, Vincent; Richardin, Pascale; Touboul, David; Brunelle, Alain; Walter, Philippe; Laprevote, Olivier

    2006-01-01

    Chemical imaging techniques, based on the combination of microscopy and spectroscopy, are well suited to study both the composition and the spatial organization of heterogeneous complex mixtures of organic and mineral matter. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), followed by scanning electron microscopy with energy dispersive X-ray analysis (SEM-EDX) and Fourier transform infrared microscopy (FTIR microscopy) have been applied to non-destructive analysis of micro-samplings of ritual matters deposited on the surface of African wooden statuettes. With a very careful preparation, using ultramicrotomy on embedded samples, it was possible to perform successively all the measurements on a single fragment. Comparison and superposition of the different chemical images, obtained on a sample from a significant actual artefact, have allowed us to identify minerals (clays, quartz and calcium carbonate), proteins, starch, urate salts and lipids and to map their spatial distribution

  5. Reduced gas seepages in serpentinized peridotite complexes: Evidences for multiple origins of the H2-CH4-N2 gas mixtures

    Science.gov (United States)

    Deville, E.; Vacquand, C.; Beaumont, V.; Francois, G.; Sissmann, O.; Pillot, D.; Arcilla, C. A.; Prinzhofer, A.

    2017-12-01

    A comparative study of reduced gas seepages associated to serpentinized ultrabasic rocks was conducted in the ophiolitic complexes of Oman, the Philippines, Turkey and New Caledonia. This study is based on analyzes of the gas chemical composition, noble gases contents, and stable isotopes of carbon, hydrogen and nitrogen. These gas seepages are mostly made of mixtures of three main components which are H2, CH4 and N2 in various proportions. The relative contents of the three main gas components show 4 distinct families of gas mixtures (H2-rich, N2-rich, N2-H2-CH4 and H2-CH4). These families are interpreted as reflecting different zones of gas generation within or below the ophiolitic complexes. In the H2-rich family associated noble gases display signatures close to the value of air. In addition to the atmospheric component, mantle and crustal contributions are present in the N2-rich, N2-H2-CH4 and H2-CH4 families. H2-bearing gases are either associated to ultra-basic (pH 10-12) spring waters or they seep directly in fracture systems from the ophiolitic rocks. In ophiolitic contexts, ultrabasic rocks provide an adequate environment with available Fe2+ and high pH conditions that favor H2 production. CH4 is produced either directly by reaction of dissolved CO2 with basic-ultrabasic rocks during the serpentinization process or in a second step by H2-CO2 interaction. H2 is present in the gas when no more carbon is available in the system to generate CH4 (conditions of strong carbon restriction). The N2-rich family is associated with relatively high contents of crustal 4He. In this family N2 is interpreted as issued mainly from sediments located below the ophiolitic units.

  6. Final summarizing report on Grant DE-SC0001014 "Separation of Highly Complex Mixtures by Two-dimension Liquid Chromatography"

    Energy Technology Data Exchange (ETDEWEB)

    Guiochon, Georges [Univ. of Tennessee, Knoxville, TN (United States)

    2013-09-16

    The goal of our research was a fundamental investigation of methods available for the coupling of two separate chromatographic separations that would considerably enhance the individual separation power of each of these two separations. This gain arises from the combination of two independent retention mechanisms, one of them separating the components that coelute on the other column, making possible the separation of many more compounds in a given time. The two separation mechanisms used must be very different. This is possible because many retention mechanisms are available, using different kinds of molecular interactions, hydrophobic or hydrophilic interactions, polar interactions, hydrogen bonding, complex formation, ionic interactions, steric exclusion. Two methods can be used, allowing separations to be performed in space (spreading the bands of sample components on a plate covered with stationary phase layer) or in time (eluting the sample components through a column and detecting the bands leaving the column). Both offer a wide variety of possible combinations and were studied.

  7. Collision-Induced Infrared Absorption by Collisional Complexes in Dense Hydrogen-Helium Gas Mixtures at Thousands of Kelvin

    Science.gov (United States)

    Abel, Martin; Frommhold, Lothar; Li, Xiaoping; Hunt, Katharine L. C.

    2011-06-01

    The interaction-induced absorption by collisional pairs of H{_2} molecules is an important opacity source in the atmospheres of the outer planets and cool stars. The emission spectra of cool white dwarf stars differ significantly in the infrared from the expected blackbody spectra of their cores, which is largely due to absorption by collisional H{_2}-H{_2}, H{_2}-He, and H{_2}-H complexes in the stellar atmospheres. Using quantum-chemical methods we compute the atmospheric absorption from hundreds to thousands of kelvin. Laboratory measurements of interaction-induced absorption spectra by H{_2} pairs exist only at room temperature and below. We show that our results reproduce these measurements closely, so that our computational data permit reliable modeling of stellar atmosphere opacities even for the higher temperatures. L. Frommhold, Collision-Induced Absorption in Gases, Cambridge University Press, Cambridge, New York, 1993 and 2006 Xiaoping Li, Katharine L. C. Hunt, Fei Wang, Martin Abel, and Lothar Frommhold, "Collision-Induced Infrared Absorption by Molecular Hydrogen Pairs at Thousands of Kelvin", International Journal of Spectroscopy, vol. 2010, Article ID 371201, 11 pages, 2010. doi: 10.1155/2010/371201 M. Abel, L. Frommhold, X. Li, and K. L. C. Hunt, "Collision-induced absorption by H{_2} pairs: From hundreds to thousands of Kelvin," J. Phys. Chem. A, published online, DOI: 10.1021/jp109441f L. Frommhold, M. Abel, F. Wang, M. Gustafsson, X. Li, and K. L. C. Hunt, "Infrared atmospheric emission and absorption by simple molecular complexes, from first principles", Mol. Phys. 108, 2265, 2010

  8. The production and crystallization of the human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 complexed with deamidated gliadin peptides implicated in coeliac disease

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, Kate N.; Reid, Hugh H.; Borg, Natalie A.; Broughton, Sophie E.; Huyton, Trevor [The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Anderson, Robert P. [Autoimmunity and Transplantation Division, Walter and Eliza Hall Institute, 1G Royal Parade, Parkville, Victoria 3050 (Australia); Department of Gastroenterology, The Royal Melbourne Hospital, Grattan Street, Parkville, Victoria 3050 (Australia); McCluskey, James [Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010 (Australia); Rossjohn, Jamie, E-mail: jamie.rossjohn@med.monash.edu.au [The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia)

    2007-12-01

    The production and crystallization of human leukocyte antigen class II molecules HLA-DQ2 and HLA-DQ8 in complex with deamidated gliadin peptides is reported. Crystals of HLA-DQ2{sup PQPELPYPQ} diffracted to 3.9 Å, while the HLA-DQ8{sup EGSFQPSQE} crystals diffracted to 2.1 Å, allowing structure determination by molecular replacement. The major histocompatibility complex (MHC) class II molecules HLA-DQ2 and HLA-DQ8 are key risk factors in coeliac disease, as they bind deamidated gluten peptides that are subsequently recognized by CD4{sup +} T cells. Here, the production and crystallization of both HLA-DQ2 and HLA-DQ8 in complex with the deamidated gliadin peptides DQ2 α-I (PQPELPYPQ) and DQ8 α-I (EGSFQPSQE), respectively, are reported.

  9. Identification of prenatal toxic components of complex PAH mixtures derived from fossil fuel combustion employing rodent embryo culture systems

    International Nuclear Information System (INIS)

    Irvin, T.R.; Akgerman, A.

    1991-01-01

    Many adverse health effects caused by combustion-generated toxins have been recognized for some time. Acute pulmonary toxicity among urban populations has been repeatedly recorded during periods of high smoke, soot, and organo-particulate pollution. The combustion of coals and petroleum-derived fuels results in emission of particulate and organic vapor-phase components to the atmosphere. Isolation of these particle-absorbed compounds and subsequent toxicological testing has further indicated the importance of chronic, low-level exposure to airborne combustion-generated toxins in the etiology of many forms of human cancer; particulate phases of these emissions have been found to contain polycyclic aromatic hydrocarbons and heterocyclic organic compounds, absorbed onto the particle matrix, which possess potent carcinogenic and mutagenic properties. In this paper, the authors define a postimplantation rat embryo culture system constructed to identify prenatal toxic components of complex polycyclic aromatic hydrocarbon soots. Employing this culture system, we also describe its application to identify prenatal toxic components of diesel soot particulates

  10. Impact of clonal competition for peptide-MHC complexes on the CD8[superscript +] T-cell repertoire selection in a persistent viral infection

    Energy Technology Data Exchange (ETDEWEB)

    Wynn, Katherine K.; Fulton, Zara; Cooper, Leanne; Silins, Sharon L.; Gras, Stephanie; Archbold, Julia K.; Tynan, Fleur E.; Miles, John J.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie; Khanna, Rajiv (Monash); (Queensland Inst. of Med. Rsrch.); (Melbourne)

    2008-04-29

    CD8{sup +} T-cell responses to persistent viral infections are characterized by the accumulation of an oligoclonal T-cell repertoire and a reduction in the naive T-cell pool. However, the precise mechanism for this phenomenon remains elusive. Here we show that human cytomegalovirus (HCMV)-specific CD8{sup +} T cells recognizing distinct epitopes from the pp65 protein and restricted through an identical HLA class I allele (HLA B*3508) exhibited either a highly conserved public T-cell repertoire or a private, diverse T-cell response, which was uniquely altered in each donor following in vitro antigen exposure. Selection of a public T-cell receptor (TCR) was coincident with an atypical major histocompatibility complex (MHC)-peptide structure, in that the epitope adopted a helical conformation that bulged from the peptide-binding groove, while a diverse TCR profile was observed in response to the epitope that formed a flatter, more 'featureless' landscape. Clonotypes with biased TCR usage demonstrated more efficient recognition of virus-infected cells, a greater CD8 dependency, and were more terminally differentiated in their phenotype when compared with the T cells expressing diverse TCR. These findings provide new insights into our understanding on how the biology of antigen presentation in addition to the structural features of the pMHC-I might shape the T-cell repertoire and its phenotype.

  11. A novel BLyS antagonist peptide designed based on the 3-D complex structure of BCMA and BLyS

    International Nuclear Information System (INIS)

    Sun Jian; Feng Jiannan; Li Yan; Shen Beifen

    2006-01-01

    B lymphocyte stimulator (BLyS) is a member of tumor necrosis factor (TNF) family. Because of its roles in autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and Sjogren syndrome (SS), BLyS antagonists have been tested to treat SLE- and RA-like symptoms in mice and obtained optimistic results. So far, reported BLyS antagonists were mostly decoyed BLyS receptors or anti-BLyS antibodies. In this study, a novel BLyS antagonist peptide, PT, was designed based on the modeling 3-D complex structure of BCMA and BLyS. The interaction mode of PT with BLyS was analyzed theoretically. The results of competitive ELISA demonstrated that PT could inhibit the binding of BCMA-Fc and anti-BLyS antibody to BLyS in vitro. In addition, PT could partly block the proliferating activity of BLyS on mice splenocytes. The BLyS antagonizing activity of PT was significant (p < 0.05). This study highlights the possibility of using BLyS antagonist peptide to neutralize BLyS activity. Further optimization of PT with computer-guided molecular design method to enhance its biopotency may be useful in developing new BLyS antagonists to treat BLyS-related autoimmune diseases

  12. Stability of binary complexes of Pb(II, Cd(II and Hg(II with maleic acid in TX100-water mixtures

    Directory of Open Access Journals (Sweden)

    M. Ramanaiah

    2014-09-01

    Full Text Available Binary complexes of maleic acid with toxic metal ions such as Pb(II, Cd(II and Hg(II have been studied in 0.0-2.5% v/v tritonX-100 (TX100 - water media at 303 K at an ionic strength of 0.16 M. The active forms of the ligand are LH2, LH- and L2-. The derived ‘best fit’ chemical speciation models are based on crystallographic R-factors, χ2 and Skewness and Kurtosis factors. The predominant species formed are of the type ML2, ML2H and ML3. The trend in variation of complex stability constants with change in the mole fraction of the medium is explained on the basis of prevailing electrostatic and non-electrostatic forces. The species distribution as a function of pH at different compositions of TX100-water mixtures and plausible speciation equilibria are presented and discussed. DOI: http://dx.doi.org/10.4314/bcse.v28i3.7

  13. Characterisation of metals in the electronic waste of complex mixtures of end-of-life ICT products for development of cleaner recovery technology.

    Science.gov (United States)

    Sun, Z H I; Xiao, Y; Sietsma, J; Agterhuis, H; Visser, G; Yang, Y

    2015-01-01

    Recycling of valuable metals from electronic waste, especially complex mixtures of end-of-life information and communication technology (ICT) products, is of great difficulty due to their complexity and heterogeneity. One of the important reasons is the lack of comprehensive characterisation on such materials, i.e. accurate compositions, physical/chemical properties. In the present research, we focus on developing methodologies for the characterisation of metals in an industrially processed ICT waste. The morphology, particle size distribution, compositional distribution, occurrence, liberation as well as the thermo-chemical properties of the ICT waste were investigated with various characterisation techniques, including X-ray Fluorescence Spectrometry (XRF), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) with energy dispersed spectroscopy (EDS). Due to the high heterogeneity of the material, special sample preparation procedures were introduced to minimise the discrepancies during compositional analyses. As a result, a clearer overview of the ICT waste has been reached. This research provides better understanding of the extractability of each metal and improves the awareness of potential obstacles for extraction. It will lead to smarter decisions during further development of a clean and effective recovery process. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Characterisation of metals in the electronic waste of complex mixtures of end-of-life ICT products for development of cleaner recovery technology

    International Nuclear Information System (INIS)

    Sun, Z.H.I.; Xiao, Y.; Sietsma, J.; Agterhuis, H.; Visser, G.; Yang, Y.

    2015-01-01

    Highlights: • New characterisation methodology has been established to understand an industrially processed ICT waste. • Particle size distribution, composition, thermal–chemical behaviour and occurrence of metals were considered. • The characterisation provides direct guidelines for values recovery from the waste. - Abstract: Recycling of valuable metals from electronic waste, especially complex mixtures of end-of-life information and communication technology (ICT) products, is of great difficulty due to their complexity and heterogeneity. One of the important reasons is the lack of comprehensive characterisation on such materials, i.e. accurate compositions, physical/chemical properties. In the present research, we focus on developing methodologies for the characterisation of metals in an industrially processed ICT waste. The morphology, particle size distribution, compositional distribution, occurrence, liberation as well as the thermo-chemical properties of the ICT waste were investigated with various characterisation techniques, including X-ray Fluorescence Spectrometry (XRF), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) with energy dispersed spectroscopy (EDS). Due to the high heterogeneity of the material, special sample preparation procedures were introduced to minimise the discrepancies during compositional analyses. As a result, a clearer overview of the ICT waste has been reached. This research provides better understanding of the extractability of each metal and improves the awareness of potential obstacles for extraction. It will lead to smarter decisions during further development of a clean and effective recovery process

  15. Characterisation of metals in the electronic waste of complex mixtures of end-of-life ICT products for development of cleaner recovery technology

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Z.H.I. [Department of Materials Science and Engineering, TU Delft, 2628 CD Delft (Netherlands); Xiao, Y. [Ironmaking Department, R and D, Tata Steel, 1970 CA IJmuiden (Netherlands); Sietsma, J. [Department of Materials Science and Engineering, TU Delft, 2628 CD Delft (Netherlands); Agterhuis, H.; Visser, G. [Business Development, Van Gansewinkel Groep BV, 5657 DH Eindhoven (Netherlands); Yang, Y. [Department of Materials Science and Engineering, TU Delft, 2628 CD Delft (Netherlands)

    2015-01-15

    Highlights: • New characterisation methodology has been established to understand an industrially processed ICT waste. • Particle size distribution, composition, thermal–chemical behaviour and occurrence of metals were considered. • The characterisation provides direct guidelines for values recovery from the waste. - Abstract: Recycling of valuable metals from electronic waste, especially complex mixtures of end-of-life information and communication technology (ICT) products, is of great difficulty due to their complexity and heterogeneity. One of the important reasons is the lack of comprehensive characterisation on such materials, i.e. accurate compositions, physical/chemical properties. In the present research, we focus on developing methodologies for the characterisation of metals in an industrially processed ICT waste. The morphology, particle size distribution, compositional distribution, occurrence, liberation as well as the thermo-chemical properties of the ICT waste were investigated with various characterisation techniques, including X-ray Fluorescence Spectrometry (XRF), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) with energy dispersed spectroscopy (EDS). Due to the high heterogeneity of the material, special sample preparation procedures were introduced to minimise the discrepancies during compositional analyses. As a result, a clearer overview of the ICT waste has been reached. This research provides better understanding of the extractability of each metal and improves the awareness of potential obstacles for extraction. It will lead to smarter decisions during further development of a clean and effective recovery process.

  16. Peptide Vaccines for Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Rory C. F. De Brito

    2018-05-01

    Full Text Available Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

  17. Peptide Vaccines for Leishmaniasis.

    Science.gov (United States)

    De Brito, Rory C F; Cardoso, Jamille M De O; Reis, Levi E S; Vieira, Joao F; Mathias, Fernando A S; Roatt, Bruno M; Aguiar-Soares, Rodrigo Dian D O; Ruiz, Jeronimo C; Resende, Daniela de M; Reis, Alexandre B

    2018-01-01

    Due to an increase in the incidence of leishmaniases worldwide, the development of new strategies such as prophylactic vaccines to prevent infection and decrease the disease have become a high priority. Classic vaccines against leishmaniases were based on live or attenuated parasites or their subunits. Nevertheless, the use of whole parasite or their subunits for vaccine production has numerous disadvantages. Therefore, the use of Leishmania peptides to design more specific vaccines against leishmaniases seems promising. Moreover, peptides have several benefits in comparison with other kinds of antigens, for instance, good stability, absence of potentially damaging materials, antigen low complexity, and low-cost to scale up. By contrast, peptides are poor immunogenic alone, and they need to be delivered correctly. In this context, several approaches described in this review are useful to solve these drawbacks. Approaches, such as, peptides in combination with potent adjuvants, cellular vaccinations, adenovirus, polyepitopes, or DNA vaccines have been used to develop peptide-based vaccines. Recent advancements in peptide vaccine design, chimeric, or polypeptide vaccines and nanovaccines based on particles attached or formulated with antigenic components or peptides have been increasingly employed to drive a specific immune response. In this review, we briefly summarize the old, current, and future stands on peptide-based vaccines, describing the disadvantages and benefits associated with them. We also propose possible approaches to overcome the related weaknesses of synthetic vaccines and suggest future guidelines for their development.

  18. Cellular Uptake and Photo-Cytotoxicity of a Gadolinium(III-DOTA-Naphthalimide Complex “Clicked” to a Lipidated Tat Peptide

    Directory of Open Access Journals (Sweden)

    William I. O’Malley

    2016-02-01

    Full Text Available A new bifunctional macrocyclic chelator featuring a conjugatable alkynyl-naphthalimide fluorophore pendant group has been prepared and its Gd(III complex coupled to a cell-penetrating lipidated azido-Tat peptide derivative using Cu(I-catalysed “click” chemistry. The resulting fluorescent conjugate is able to enter CAL-33 tongue squamous carcinoma cells, as revealed by confocal microscopy, producing a very modest anti-proliferative effect (IC50 = 93 µM. Due to the photo-reactivity of the naphthalimide moiety, however, the conjugate’s cytotoxicity is significantly enhanced (IC50 = 16 µM upon brief low-power UV-A irradiation.

  19. Peptide dendrimers

    Czech Academy of Sciences Publication Activity Database

    Niederhafner, Petr; Šebestík, Jaroslav; Ježek, Jan

    2005-01-01

    Roč. 11, - (2005), 757-788 ISSN 1075-2617 R&D Projects: GA ČR(CZ) GA203/03/1362 Institutional research plan: CEZ:AV0Z40550506 Keywords : multiple antigen peptides * peptide dendrimers * synthetic vaccine * multipleantigenic peptides Subject RIV: CC - Organic Chemistry Impact factor: 1.803, year: 2005

  20. Novel peptide-based platform for the dual presentation of biologically active peptide motifs on biomaterials.

    Science.gov (United States)

    Mas-Moruno, Carlos; Fraioli, Roberta; Albericio, Fernando; Manero, José María; Gil, F Javier

    2014-05-14

    Biofunctionalization of metallic materials with cell adhesive molecules derived from the extracellular matrix is a feasible approach to improve cell-material interactions and enhance the biointegration of implant materials (e.g., osseointegration of bone implants). However, classical biomimetic strategies may prove insufficient to elicit complex and multiple biological signals required in the processes of tissue regeneration. Thus, newer strategies are focusing on installing multifunctionality on biomaterials. In this work, we introduce a novel peptide-based divalent platform with the capacity to simultaneously present distinct bioactive peptide motifs in a chemically controlled fashion. As a proof of concept, the integrin-binding sequences RGD and PHSRN were selected and introduced in the platform. The biofunctionalization of titanium with this platform showed a positive trend towards increased numbers of cell attachment, and statistically higher values of spreading and proliferation of osteoblast-like cells compared to control noncoated samples. Moreover, it displayed statistically comparable or improved cell responses compared to samples coated with the single peptides or with an equimolar mixture of the two motifs. Osteoblast-like cells produced higher levels of alkaline phosphatase on surfaces functionalized with the platform than on control titanium; however, these values were not statistically significant. This study demonstrates that these peptidic structures are versatile tools to convey multiple biofunctionality to biomaterials in a chemically defined manner.

  1. Crystal structure of a polyhistidine-tagged recombinant catalytic subunit of cAMP-dependent protein kinase complexed with the peptide inhibitor PKI(5-24) and adenosine.

    Science.gov (United States)

    Narayana, N; Cox, S; Shaltiel, S; Taylor, S S; Xuong, N

    1997-04-15

    The crystal structure of the hexahistidine-tagged mouse recombinant catalytic subunit (H6-rC) of cAMP-dependent protein kinase (cAPK), complexed with a 20-residue peptide inhibitor from the heat-stable protein kinase inhibitor PKI(5-24) and adenosine, was determined at 2.2 A resolution. Novel crystallization conditions were required to grow the ternary complex crystals. The structure was refined to a final crystallographic R-factor of 18.2% with good stereochemical parameters. The "active" enzyme adopts a "closed" conformation as found in rC:PKI(5-24) [Knighton et al. (1991a,b) Science 253, 407-414, 414-420] and packs in a similar manner with the peptide providing a major contact surface. This structure clearly defines the subsites of the unique nucleotide binding site found in the protein kinase family. The adenosine occupies a mostly hydrophobic pocket at the base of the cleft between the two lobes and is completely buried. The missing triphosphate moiety of ATP is filled with a water molecule (Wtr 415) which replaces the gamma-phosphate of ATP. The glycine-rich loop between beta1 and beta2 helps to anchor the phosphates while the ribose ring is buried beneath beta-strand 2. Another ordered water molecule (Wtr 375) is pentacoordinated with polar atoms from adenosine, Leu 49 in beta-strand 1, Glu 127 in the linker strand between the two lobes, Tyr 330, and a third water molecule, Wtr 359. The conserved nucleotide fold can be defined as a lid comprised of beta-strand 1, the glycine-rich loop, and beta-strand 2. The adenine ring is buried beneath beta-strand 1 and the linker strand (120-127) that joins the small and large lobes. The C-terminal tail containing Tyr 330, a segment that lies outside the conserved core, covers this fold and anchors it in a closed conformation. The main-chain atoms of the flexible glycine-rich loop (residues 50-55) in the ATP binding domain have a mean B-factor of 41.4 A2. This loop is quite mobile, in striking contrast to the other

  2. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

    Cardiac-derived peptide hormones were identified more than 25 years ago. An astonishing amount of clinical studies have established cardiac natriuretic peptides and their molecular precursors as useful markers of heart disease. In contrast to the clinical applications, the biogenesis of cardiac...... peptides has only been elucidated during the last decade. The cellular synthesis including amino acid modifications and proteolytic cleavages has proven considerably more complex than initially perceived. Consequently, the elimination phase of the peptide products in circulation is not yet well....... An inefficient post-translational prohormone maturation will also affect the biology of the cardiac natriuretic peptide system. This review aims at summarizing the myocardial synthesis of natriuretic peptides focusing on B-type natriuretic peptide, where new data has disclosed cardiac myocytes as highly...

  3. Grouting mixture

    Energy Technology Data Exchange (ETDEWEB)

    Klyusov, A A; Bakshutov, V S; Kulyavtsev, V A

    1980-10-23

    A grouting mixture is proposed for low-temperature boreholes. The mixture contains cement, beta gypsum polyhydrate, and calcium chloride, so as to increase the water resistance and strength properties of expanding brick at conditions from 20 to -5/sup 0/ C, the components are in the following ratios: (by wt.-%): cement, 77.45-88.06; beta gypsum polyhydrate, 9.79-19.36; calcium chloride, 2.15-3.19. Grouting mortar for cold boreholes serves as the cement.

  4. Chemical characterization of organic aerosol above a mid-latitude forest reveals a complex mixture of highly-functionalized chemical species and diverse structural features with temporal variability

    Science.gov (United States)

    Gentner, D. R.; Ditto, J.; Barnes, E.; Khare, P.

    2017-12-01

    Highly-functionalized organic compounds are known to be a major component of the complex mixture of the particle-phase compounds that comprise organic aerosol, yet little is known about the identity of many of these compounds, and their formation pathways and roles in atmospheric processes are poorly understood. We present results from the comprehensive chemical speciation of PM10 organic aerosols collected in July 2016 at the remote mid-latitude forest field site during PROPHET. Samples were analyzed via liquid and gas chromatography coupled with a quadrupole time-of-flight tandem mass spectrometry (MS×MS) following electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). 8 hr samples were collected during day- and night-time sampling periods rather than more typical 24-hour samples. This analysis of the organic aerosol yielded over 12,000 unique compounds for which we have high accuracy molecular masses, formulas, and additional information on structural features using MS×MS. O:C ratios were 0.3 on average, yet the top 10% of compounds ranged 0.7-2.3. 70% and 69% of day- and night-time samples were nitrogen-containing, whereas 26% and 24% contained sulfur, respectively. Within these broader molecular categories, we observed a wide variety of molecular features that reveal a diversity of functional groups and moieties. In this presentation, we present the results of our speciation, temporal variability, connections to air parcel back trajectories and other bulk properties, and potential formation pathways.

  5. Complex organic pollutant mixtures originating from industrial and municipal emissions in surface waters of the megacity Jakarta-an example of a water pollution problem in emerging economies.

    Science.gov (United States)

    Dsikowitzky, Larissa; Hagemann, Lukas; Dwiyitno; Ariyani, Farida; Irianto, Hari Eko; Schwarzbauer, Jan

    2017-12-01

    During the last decades, the global industrial production partly shifted from industrialized nations to emerging and developing countries. In these upcoming economies, the newly developed industrial centers are generally located in densely populated areas, resulting in the discharge of often only partially treated industrial and municipal wastewaters into the surface waters. There is a huge gap of knowledge about the composition of the complex organic pollutant mixtures occurring in such heavily impacted areas. Therefore, we applied a non-target screening to comprehensively assess river pollution in a large industrial area located in the megacity Jakarta. More than 100 structurally diverse organic contaminants were identified, some of which were reported here for the first time as environmental contaminants. The concentrations of paper manufacturing chemicals in river water-for example, of the endocrine-disrupting compound bisphenol A (50-8000 ng L -1 )-were as high as in pure untreated paper industry wastewaters. The non-target screening approach is the adequate tool for the identification of water contaminants in the new global centers of industrial manufacturing-as the first crucial step towards the evaluation of as yet unrecognized environmental risks.

  6. Seasonal and size-related variation of subcellular biomarkers in quagga mussels (Dreissena bugensis) inhabiting sites affected by moderate contamination with complex mixtures of pollutants.

    Science.gov (United States)

    Ács, A; Vehovszky, Á; Győri, J; Farkas, A

    2016-07-01

    The size-related differences in subcellular biomarker responses were assessed in Dreissena bugensis mussels inhabiting harbours moderately affected by pollution with complex mixtures of heavy metals and polycyclic aromatic hydrocarbons (PAHs). Adult D. bugensis samples were collected from three harbours of Lake Balaton (Hungary) characterized by moderate shipping activity, and as reference site, from a highly protected remote area of the lake. Biomarkers of exposure (metallothioneins (MTs), ethoxyresorufin-o-deethylase (EROD)), oxidative stress (lipid peroxidation (LPO), DNA strand breaks (DNAsb)) and possible endocrine disruption (vitellogenin-like proteins (VTG)) were analysed in whole-tissue homogenates of differently sized groups of mussels in relation to environmental parameters and priority pollutants (heavy metals and polycyclic aromatic hydrocarbons). Integrated biomarker response (IBR) indices were calculated for biomarker responses gained through in situ measurements to signalize critical sites and to better distinguish natural tendencies from biological effects of contaminants. Biomarker responses showed close positive correlation in case of MT, EROD, LPO, and DNAsb and negative correlation with VTG levels with mussel shell length in autumn, when higher levels of biomarkers appeared, possibly due to natural lifecycle changes of animals.

  7. Speciation of binary complexes of Pb(II and Cd(II with L-asparagine in dimethyl sulfoxide - water mixtures

    Directory of Open Access Journals (Sweden)

    C. N. Rao

    2016-02-01

    Full Text Available Chemical speciation of L-Asparagine complexes of Pb(II and Cd(II in presence of (0-50% v/v dimethyl sulfoxide(DMSO-water mixtures has been studied potentiometrically at 303.0 K and at an ionic strength of 0.16 mol L-1. The models containing different number of species were refined by using the computer program MINIQUAD75. The number of species in the models is chosen based on exhaustive modeling. The predominant species formed are of the type ML2, ML2H, and ML2H2. The best fit chemical models were chosen based on statistical parameters. The convenience of the models is ascertained by studying the effect of errors in concentrations of ingredients. The trend in variation of stability constants with change in the composition of medium is explained on the basis of predominant electrostatic and non-electrostatic forces. Chemical speciation was discussed based on the distribution diagrams. DOI: http://dx.doi.org/10.4314/bcse.v30i1.6

  8. Application of High-Resolution Ultrasonic Spectroscopy for analysis of complex formulations. Compressibility of solutes and solute particles in liquid mixtures

    International Nuclear Information System (INIS)

    Buckin, V

    2012-01-01

    The paper describes key aspects of interpretation of compressibility of solutes in liquid mixtures obtained through high-resolution measurements of ultrasonic parameters. It examines the fundamental relationships between the characteristics of solutes and the contributions of solutes to compressibility of liquid mixtures expressed through apparent adiabatic compressibility of solutes, and adiabatic compressibility of solute particles. In addition, it analyses relationships between the adiabatic compressibility of solutes and the measured ultrasonic characteristics of mixtures. Especial attention is given to the effects of solvents on the measured adiabatic compressibility of solutes and on concentration increment of ultrasonic velocity of solutes in mixtures.

  9. Direct elution of sup(99m)Tc complexes from neutron irradiation produced /sup 99/Mo incorporated in a MoCl/sub 2/-MoBr/sub 2/ mixture

    Energy Technology Data Exchange (ETDEWEB)

    Ganzerli Valentini, M.T.; Stella, R.; Genova, N.

    1987-01-01

    A novel type of sup(99m)Tc generator, enabling radiopharmaceutical preparation without reductant addition, was prepared and tested. Neutron activated product /sup 99/Mo, in the form of dichloride cluster, was incorporated into inactive molybdenum dichloride-dibromide mixture (MCB) that was left to settle over an activated alumina layer. Direct elution with aqueous ligands such as salicylate or iminodiacetate derivatives in the pH range 6.5-7.5 yielded chemically stable sup(99m)Tc complexes accompanied by small amounts of secondary products (mostly sup(99m)TcO/sup -//sub 4/). Pentavalent oxidation state in the salicylate complex and tervalent in the iminodiacetate (IDA) and in the N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetate (HIDA) complexes were assigned to the element after comparison with reference complexes. The anion exchange version of reverse-phase HPLC was used to resolve the eluted product mixture.

  10. Drastic Effect of the Peptide Sequence on the Copper-Binding Properties of Tripeptides and the Electrochemical Behaviour of Their Copper(II) Complexes.

    Science.gov (United States)

    Mena, Silvia; Mirats, Andrea; Caballero, Ana B; Guirado, Gonzalo; Barrios, Leoní A; Teat, Simon J; Rodriguez-Santiago, Luis; Sodupe, Mariona; Gamez, Patrick

    2018-04-06

    The binding and electrochemical properties of the complexes Cu II -HAH, Cu II -HWH, Cu II -Ac-HWH, Cu II -HHW, and Cu II -WHH have been studied by using NMR and UV/Vis spectroscopies, CV, and density functional calculations. The results obtained highlight the importance of the peptidic sequence on the coordination properties and, consequently, on the redox properties of their Cu II complexes. For Cu II -HAH and Cu II -HWH, no cathodic processes are observed up to -1.2 V; that is, the complexes exhibit very high stability towards copper reduction. This behaviour is associated with the formation of very stable square-planar (5,5,6)-membered chelate rings (ATCUN motif), which enclose two deprotonated amides. In contrast, for non-ATCUN Cu II -Ac-HWH, Cu II -HHW complexes, simulations seem to indicate that only one deprotonated amide is enclosed in the coordination sphere. In these cases, the main electrochemical feature is a reductive irreversible one electron-transfer process from Cu II to Cu I , accompanied with structural changes of the metal coordination sphere and reprotonation of the amide. Finally, for Cu II -WHH, two major species have been detected: one at low pH (10) with an ATCUN motif, both species coexisting at intermediate pH. The present study shows that the use of CV, using glassy carbon as a working electrode, is an ideal and rapid tool for the determination of the redox properties of Cu II metallopeptides. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Unraveling the Molecular Complexity of O-Glycosylated Endogenous (N-Terminal) pro-B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure.

    Science.gov (United States)

    Halfinger, Bernhard; Hammerer-Lercher, Angelika; Amplatz, Benno; Sarg, Bettina; Kremser, Leopold; Lindner, Herbert H

    2017-01-01

    Currently, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF). Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unprocessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. There is a clear and urgent need to identify the glycosylation sites in the endogenously circulating peptides requested by the community to gain further insights into the different naturally occurring forms. The glycosylation sites of (NT-) proBNP (NT-proBNP and/or proBNP) were characterized in leftovers of heparinized plasma samples of severe HF patients (NT-proBNP: >10000 ng/L) by using tandem immunoaffinity purification, sequential exoglycosidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-flow liquid chromatography electrospray multistage mass spectrometry. We describe 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients. Differentially glycosylated variants were detected based on highly accurate mass determination and multistage mass spectrometry. Remarkably, for each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable. Our results directly demonstrate for the first time a rather complex distribution of the endogenously circulating glycoforms by mass spectrometric analysis in HF patients, and show 9 glycosites in human (NT-) proBNP. This information may also have an impact on commercial immunoassays applying antibodies specific for the central region of (NT-) proBNP, which detect mostly nonglycosylated forms. © 2016 American Association for Clinical Chemistry.

  12. Preparation of crystals for characterizing the Grb7 SH2 domain before and after complex formation with a bicyclic peptide antagonist.

    Science.gov (United States)

    Ambaye, Nigus D; Gunzburg, Menachem J; Traore, Daouda A K; Del Borgo, Mark P; Perlmutter, Patrick; Wilce, Matthew C J; Wilce, Jacqueline A

    2014-02-01

    Human growth factor receptor-bound protein 7 (Grb7) is an adapter protein involved in cell growth, migration and proliferation. It is now recognized that Grb7 is an emerging therapeutic target in specific cancer subtypes. Recently, the discovery of a bicyclic peptide inhibitor that targets the Grb7 SH2 domain, named G7-B1, was reported. In an attempt to probe the foundation of its interaction with Grb7, the crystallization and preliminary data collection of both the apo and G7-B1-bound forms of the Grb7 SH2 domain are reported here. Diffraction-quality crystals were obtained using the hanging-drop vapour-diffusion method. After several rounds of microseeding, crystals of the apo Grb7 SH2 domain were obtained that diffracted to 1.8 Å resolution, while those of the G7-B1-Grb7 SH2 domain complex diffracted to 2.2 Å resolution. The apo Grb7 SH2 domain crystallized in the trigonal space group P63, whereas the G7-B1-Grb7 SH2 domain complex crystallized in the monoclinic space group P21. The experimental aspects of crystallization, crystal optimization and data collection and the preliminary data are reported.

  13. Cocaine- and amphetamine-regulated transcript peptide increases mitochondrial respiratory chain complex II activity and protects against oxygen-glucose deprivation in neurons.

    Science.gov (United States)

    Sha, Dujuan; Wang, Luna; Zhang, Jun; Qian, Lai; Li, Qiming; Li, Jin; Qian, Jian; Gu, Shuangshuang; Han, Ling; Xu, Peng; Xu, Yun

    2014-09-25

    The mechanisms of ischemic stroke, a main cause of disability and death, are complicated. Ischemic stroke results from the interaction of various factors including oxidative stress, a key pathological mechanism that plays an important role during the acute stage of ischemic brain injury. This study demonstrated that cocaine- and amphetamine-regulated transcript (CART) peptide, specifically CART55-102, increased the survival rate, but decreased the mortality of neurons exposed to oxygen-glucose deprivation (OGD), in a dose-dependent manner. The above-mentioned effects of CART55-102 were most significant at 0.4nM. These results indicated that CART55-102 suppressed neurotoxicity and enhanced neuronal survival after oxygen-glucose deprivation. CART55-102 (0.4nM) significantly diminished reactive oxygen species levels and markedly increased the activity of mitochondrial respiratory chain complex II in oxygen-glucose deprived neurons. In summary, CART55-102 suppressed oxidative stress in oxygen-glucose deprived neurons, possibly through elevating the activity of mitochondrial respiratory chain complex II. This result provides evidence for the development of CART55-102 as an antioxidant drug. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

    2017-01-24

    The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

  15. Mixtures and interactions

    NARCIS (Netherlands)

    Groten, J.P.

    2000-01-01

    Drinking water can be considered as a complex mixture that consists of tens, hundreds or thousands of chemicals of which the composition is qualitatively and quantitatively not fully known. From a public health point of view it is most relevant to answer the question of whether chemicals in drinking

  16. Characterizing oxidative flow reactor SOA production and OH radical exposure from laboratory experiments of complex mixtures (engine exhaust) and simple precursors (monoterpenes)

    Science.gov (United States)

    Michael Link, M. L.; Friedman, B.; Ortega, J. V.; Son, J.; Kim, J.; Park, G.; Park, T.; Kim, K.; Lee, T.; Farmer, D.

    2016-12-01

    Recent commercialization of the Oxidative Flow Reactor (OFR, occasionally described in the literature as a "Potential Aerosol Mass") has created the opportunity for many researchers to explore the mechanisms behind OH-driven aerosol formation on a wide range of oxidative timescales (hours to weeks) in both laboratory and field measurements. These experiments have been conducted in both laboratory and field settings, including simple (i.e. single component) and complex (multi-component) precursors. Standard practices for performing OFR experiments, and interpreting data from the measurements, are still being developed. Measurement of gas and particle phase chemistry, from oxidation products generated in the OFR, through laboratory studies on single precursors and the measurement of SOA from vehicle emissions on short atmospheric timescales represent two very different experiments in which careful experimental design is essential for exploring reaction mechanisms and SOA yields. Two parameters essential in experimental design are (1) the role of seed aerosol in controlling gas-particle partitioning and SOA yields, and (2) the accurate determination of OH exposure during any one experiment. We investigated the role of seed aerosol surface area in controlling the observed SOA yields and gas/particle composition from the OH-initiated oxidation of four monoterpenes using an aerosol chemical ionization time-of-flight mass spectrometer and scanning mobility particle sizer. While the OH exposure during laboratory experiments is simple to constrain, complex mixtures such as diesel exhaust have high estimated OH reactivity values, and thus require careful consideration. We developed methods for constraining OH radical exposure in the OFR during vehicle exhaust oxidation experiments. We observe changes in O/C ratios and highly functionalized species over the temperature gradient employed in the aerosol-CIMS measurement. We relate this observed, speciated chemistry to the

  17. Inhibition of peptide bond formation by pleuromutilins: the structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin.

    Science.gov (United States)

    Schlünzen, Frank; Pyetan, Erez; Fucini, Paola; Yonath, Ada; Harms, Jörg M

    2004-12-01

    Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuromutilins on the ribosome has not been determined on a molecular level. The 3.5 A structure of the 50S ribosomal subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S ribosomal subunit with its tricyclic mutilin core positioned in a tight pocket at the A-tRNA binding site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P-tRNA binding site. Thereby, tiamulin directly inhibits peptide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.

  18. ER stress stimulates production of the key antimicrobial peptide, cathelicidin, by forming a previously unidentified intracellular S1P signaling complex.

    Science.gov (United States)

    Park, Kyungho; Ikushiro, Hiroko; Seo, Ho Seong; Shin, Kyong-Oh; Kim, Young Il; Kim, Jong Youl; Lee, Yong-Moon; Yano, Takato; Holleran, Walter M; Elias, Peter; Uchida, Yoshikazu

    2016-03-08

    We recently identified a previously unidentified sphingosine-1-phosphate (S1P) signaling mechanism that stimulates production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), in mammalian cells exposed to external perturbations, such as UVB irradiation and other oxidative stressors that provoke subapoptotic levels of endoplasmic reticulum (ER) stress, independent of the well-known vitamin D receptor-dependent mechanism. ER stress increases cellular ceramide and one of its distal metabolites, S1P, which activates NF-κB followed by C/EBPα activation, leading to CAMP production, but in a S1P receptor-independent fashion. We now show that S1P activates NF-κB through formation of a previously unidentified signaling complex, consisting of S1P, TRAF2, and RIP1 that further associates with three stress-responsive proteins; i.e., heat shock proteins (GRP94 and HSP90α) and IRE1α. S1P specifically interacts with the N-terminal domain of heat shock proteins. Because this ER stress-initiated mechanism is operative in both epithelial cells and macrophages, it appears to be a universal, highly conserved response, broadly protective against diverse external perturbations that lead to increased ER stress. Finally, these studies further illuminate how ER stress and S1P orchestrate critical stress-specific signals that regulate production of one protective response by stimulating production of the key innate immune element, CAMP.

  19. The T-Cell Receptor Can Bind to the Peptide-Bound Major Histocompatibility Complex and Uncomplexed β2-Microglobulin through Distinct Binding Sites

    DEFF Research Database (Denmark)

    Merkle, Patrick S.; Irving, Melita; Hongjian, Song

    2017-01-01

    from molecular dynamics simulations. Using a biological assay based on TCR gene-engineered primary human T cells, we did not observe a significant effect of β2m on T-cell cytotoxicity, suggesting an alternate role for β2m binding. Overall, we show that binding of β2m to the TCR occurs in vitro and......T-Cell receptor (TCR)-mediated recognition of the peptide-bound major histocompatibility complex (pMHC) initiates an adaptive immune response against antigen-presenting target cells. The recognition events take place at the TCR-pMHC interface, and their effects on TCR conformation and dynamics...... are controversial. Here, we have measured the time-resolved hydrogen/deuterium exchange (HDX) of a soluble TCR in the presence and absence of its cognate pMHC by mass spectrometry to delineate the impact of pMHC binding on solution-phase structural dynamics in the TCR. Our results demonstrate that while TCR...

  20. Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

    Science.gov (United States)

    Saito, Kazuki; Nakato, Mamiko; Mizuguchi, Takaaki; Wada, Shinji; Uchimura, Hiromasa; Kataoka, Hiroshi; Yokoyama, Shigeyuki; Hirota, Hiroshi; Kiso, Yoshiaki

    2014-03-01

    To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, β-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 μM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Synthesis and in vitro/in vivo evaluation of novel mono- and trivalent technetium-99m labeled ghrelin peptide complexes as potential diagnostic radiopharmaceuticals

    International Nuclear Information System (INIS)

    Koźmiński, Przemysław; Gniazdowska, Ewa

    2015-01-01

    Introduction: Ghrelin is an endogenous hormone present in blood. It is released from the oxyntic cells (X/A-like cells) of the stomach and fundus and can exist in two forms: as an acylated and des-acylated ghrelin. Ghrelin is an endogenous ligand of the growth hormone receptor (growth hormone secretagogue receptor, GHS-R). Overexpression of GHS-R1a receptor was identified in cells of different types of tumors (e.g. pituitary adenoma, neuroendocrine tumors of the thyroid, lung, breast, gonads, prostate, stomach, colorectal, endocrine and non-endocrine pancreatic tumors). This fact suggests that gamma radionuclide labeled ghrelin peptide may be considered as a potential diagnostic radiopharmaceutical. Methods: Ghrelin peptide labeled with mono- and trivalent technetium-99m complexes, 99m Tc-Lys-GHR, has been prepared on the n.c.a. scale. The physicochemical (stability, charge, shape, lipophilicity) and biological (receptor affinity, biodistribution) properties of the conjugates have been studied relevant to use the conjugates as receptor-based diagnostic radiopharmaceuticals. Results: The obtained conjugates [ 99m Tc(CO) 3 L N,O (CN-Lys-GHR)] + , 99m Tc(CO) 3 L S,O (CN-Lys-GHR) and 99m Tc(NS 3 )(CN-Lys-GHR) show different shape, charge, lipophilicity and two of them, 99m Tc(CO) 3 L S,O (CN-Lys-GHR) and 99m Tc(NS 3 )(CN-Lys-GHR), high stability in neutral aqueous solutions, even in the presence of excess concentration of histidine/cysteine competitive standard ligands or human serum. The in vitro binding affinity of 99m Tc-Lys-GHR conjugates with respect to growth hormone secretagogue receptor (GHS-R1a) present on DU-145 cells was in the range of IC 50 from 45 to 54 nM. The conjugate 99m Tc(CO) 3 L S,O (CN-Lys-GHR) exhibited excretion route by the liver and kidney in comparable degree, while the more lipophilic conjugate 99m Tc(NS 3 )(CN-Lys-GHR)—mainly by the liver. Conclusions: Basing on the results concerning physicochemical and biochemical properties, the

  2. A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently

    DEFF Research Database (Denmark)

    Sylvester-Hvid, C; Buus, S

    1999-01-01

    of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule...... electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained....

  3. Cnidarian Neurotoxic Peptides Affecting Central Nervous System Targets.

    Science.gov (United States)

    Lazcano-Pérez, Fernando; Hernández-Guzmán, Ulises; Sánchez-Rodríguez, Judith; Arreguín-Espinosa, Roberto

    2016-01-01

    Natural products from animal venoms have been used widely in the discovery of novel molecules with particular biological activities that enable their use as potential drug candidates. The phylum Cnidaria (jellyfish, sea anemones, corals zoanthids, hydrozoans, etc.) is the most ancient venomous phylum on earth. Its venoms are composed of a complex mixture of peptidic compounds with neurotoxic and cytolitic properties that have shown activity on mammalian systems despite the fact that they are naturally targeted against fish and invertebrate preys, mainly crustaceans. For this reason, cnidarian venoms are an interesting and vast source of molecules with a remarkable activity on central nervous system, targeting mainly voltage-gated ion channels, ASIC channels, and TRPV1 receptors. In this brief review, we list the amino acid sequences of most cnidarian neurotoxic peptides reported to date. Additionally, we propose the inclusion of a new type of voltage-gated sea anemone sodium channel toxins based on the most recent reports.

  4. Optimization of Reversed-Phase Peptide Liquid Chromatography Ultraviolet Mass Spectrometry Analyses Using an Automated Blending Methodology

    Science.gov (United States)

    Chakraborty, Asish B.; Berger, Scott J.

    2005-01-01

    The balance between chromatographic performance and mass spectrometric response has been evaluated using an automated series of experiments where separations are produced by the real-time automated blending of water with organic and acidic modifiers. In this work, the concentration effects of two acidic modifiers (formic acid and trifluoroacetic acid) were studied on the separation selectivity, ultraviolet, and mass spectrometry detector response, using a complex peptide mixture. Peptide retention selectivity differences were apparent between the two modifiers, and under the conditions studied, trifluoroacetic acid produced slightly narrower (more concentrated) peaks, but significantly higher electrospray mass spectrometry suppression. Trifluoroacetic acid suppression of electrospray signal and influence on peptide retention and selectivity was dominant when mixtures of the two modifiers were analyzed. Our experimental results indicate that in analyses where the analyzed components are roughly equimolar (e.g., a peptide map of a recombinant protein), the selectivity of peptide separations can be optimized by choice and concentration of acidic modifier, without compromising the ability to obtain effective sequence coverage of a protein. In some cases, these selectivity differences were explored further, and a rational basis for differentiating acidic modifier effects from the underlying peptide sequences is described. PMID:16522853

  5. Using Big Data Analytics to Address Mixtures Exposure

    Science.gov (United States)

    The assessment of chemical mixtures is a complex issue for regulators and health scientists. We propose that assessing chemical co-occurrence patterns and prevalence rates is a relatively simple yet powerful approach in characterizing environmental mixtures and mixtures exposure...

  6. Assessing Exposures to Magnetic Resonance Imaging’s Complex Mixture of Magnetic Fields for In Vivo, In Vitro, and Epidemiologic Studies of Health Effects for Staff and Patients

    Directory of Open Access Journals (Sweden)

    Jennifer Frankel

    2018-03-01

    Full Text Available A complex mixture of electromagnetic fields is used in magnetic resonance imaging (MRI: static, low-frequency, and radio frequency magnetic fields. Commonly, the static magnetic field ranges from one to three Tesla. The low-frequency field can reach several millitesla and with a time derivative of the order of some Tesla per second. The radiofrequency (RF field has a magnitude in the microtesla range giving rise to specific absorption rate values of a few Watts per kilogram. Very little attention has been paid to the case where there is a combined exposure to several different fields at the same time. Some studies have shown genotoxic effects in cells after exposure to an MRI scan while others have not demonstrated any effects. A typical MRI exam includes muliple imaging sequences of varying length and intensity, to produce different types of images. Each sequence is designed with a particular purpose in mind, so one sequence can, for example, be optimized for clearly showing fat water contrast, while another is optimized for high-resolution detail. It is of the utmost importance that future experimental studies give a thorough description of the exposure they are using, and not just a statement such as “An ordinary MRI sequence was used.” Even if the sequence is specified, it can differ substantially between manufacturers on, e.g., RF pulse height, width, and duty cycle. In the latest SCENIHR opinion, it is stated that there is very little information regarding the health effects of occupational exposure to MRI fields, and long-term prospective or retrospective cohort studies on workers are recommended as a high priority. They also state that MRI is increasingly used in pediatric diagnostic imaging, and a cohort study into the effects of MRI exposure on children is recommended as a high priority. For the exposure assessment in epidemiological studies, there is a clear difference between patients and staff and further work is needed on this

  7. Nature of unresolved complex mixture in size-distributed emissions from residential wood combustion as measured by thermal desorption-gas chromatography-mass spectrometry

    Science.gov (United States)

    Hays, Michael D.; Smith, N. Dean; Dong, Yuanji

    2004-08-01

    Unresolved complex mixture (UCM) is an analytical artifact of gas chromatographs of combustion source-related fine aerosol extracts. In this study the UCM is examined in size-resolved fine aerosol emissions from residential wood combustion. The aerosols are sorted by size in an electrical low-pressure impactor (ELPI) and subsequently analyzed by thermal desorption/gas chromatography/mass spectrometry (TD/GC/MS). A semiquantitative system for predicting the branched alkane, cycloalkane, alkylbenzene, C3-, C4-, C5-alkylbenzene, methylnaphthalene, C3-, C4-, C5-alkylnaphthalene, methylphenanthrene C2-, C3-alkylphenanthrene, and dibenzothiophene concentrations in the UCM is introduced. Analysis by TD/GS/MS detects UCM on each ELPI stage for all six combustion tests. The UCM baseline among the different fuel types is variable. In particular, the UCM of Pseudotsuga sp. is enriched in later-eluting compounds of lower volatility. A high level of reproducibility is achieved in determining UCM areas. UCM fractions (UCM ion area/total extracted ion chromatograph area) by individual ELPI stage return a mean relative standard deviation of 19.1% over the entire combustion test set, indicating a highly consistent UCM fraction across the ELPI size boundaries. Among the molecular ions investigated, branched alkane (m/z 57) and dibenzothiophene (m/z 212 and 226) constituents are most abundant in UCM emissions from RWC, collectively accounting for 64-95% of the targeted chemical species. The total UCM emissions span 446-756 mg/kg of dry biomass burned and correspond to an upper limit of 7.1% of the PM2.5 mass. The UCM emissions are primarily accumulation mode (0.1 μm ≤ aerodynamic diameter (da) ≤ 1 μm), with a geometric mean diameter (dg) range of 120.3-518.4 nm. UCM in PM2.5 is chemically asymmetric (shifted to finer da), typically clustering at da ≤ 1 μm. Measurable shifts in dg and changes in distribution widths (σg) on an intratest basis suggest that the particle density

  8. Detachable strong cation exchange monolith, integrated with capillary zone electrophoresis and coupled with pH gradient elution, produces improved sensitivity and numbers of peptide identifications during bottom-up analysis of complex proteomes.

    Science.gov (United States)

    Zhang, Zhenbin; Yan, Xiaojing; Sun, Liangliang; Zhu, Guijie; Dovichi, Norman J

    2015-04-21

    A detachable sulfonate-silica hybrid strong cation-exchange monolith was synthesized in a fused silica capillary, and used for solid phase extraction with online pH gradient elution during capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) proteomic analysis. Tryptic digests were prepared in 50 mM formic acid and loaded onto the strong cation-exchange monolith. Fractions were eluted using a series of buffers with lower concentration but higher pH values than the 50 mM formic acid background electrolyte. This combination of elution and background electrolytes results in both sample stacking and formation of a dynamic pH junction and allows use of relatively large elution buffer volumes while maintaining reasonable peak efficiency and resolution. A series of five pH bumps were applied to elute E. coli tryptic peptides from the monolith, followed by analysis using CZE coupled to an LTQ-Orbitrap Velos mass spectrometer; 799 protein groups and 3381 peptides were identified from 50 ng of the digest in a 2.5 h analysis, which approaches the identification rate for this organism that was obtained with an Orbitrap Fusion. We attribute the improved numbers of peptide and protein identifications to the efficient fractionation by the online pH gradient elution, which decreased the complexity of the sample in each elution step and improved the signal intensity of low abundance peptides. We also performed a comparative analysis using a nanoACQUITY UltraPerformance LCH system. Similar numbers of protein and peptide identifications were produced by the two methods. Protein identifications showed significant overlap between the two methods, whereas peptide identifications were complementary.

  9. IMMUNOLOGICAL CHARACTERISTIC OF SYNTHETIC PEPTIDES SIMILAR TO ACTUAL HIV ANTIGEN DETERMINANTS

    Directory of Open Access Journals (Sweden)

    S. V. Korobova

    2016-01-01

    complex process and may be influenced by several factors, such as antibody titer, isotype, or antibody structure. Probably, to induce neutralizing antibodies by this peptide mixture, it is necessary to choose appropriate adjutants and immunization schedule. Moreover, it was shown that peptides could increase in vitro virus infectivity in pseudovirus-based model, using the CAP 45.2.00.G3, QH209.14M.ENV.A2, QD435.100M.ENV.E1 molecular clone. These viral isolates belong to different HIV-1 subtypes.

  10. Prospective Power Calculations for the Four Lab Study of A Multigenerational Reproductive/Developmental Toxicity Rodent Bioassay Using A Complex Mixture of Disinfection By-Products in the Low-Response Region

    Directory of Open Access Journals (Sweden)

    Jane Ellen Simmons

    2011-10-01

    Full Text Available In complex mixture toxicology, there is growing emphasis on testing environmentally representative doses that improve the relevance of results for health risk assessment, but are typically much lower than those used in traditional toxicology studies. Traditional experimental designs with typical sample sizes may have insufficient statistical power to detect effects caused by environmentally relevant doses. Proper study design, with adequate statistical power, is critical to ensuring that experimental results are useful for environmental health risk assessment. Studies with environmentally realistic complex mixtures have practical constraints on sample concentration factor and sample volume as well as the number of animals that can be accommodated. This article describes methodology for calculation of statistical power for non-independent observations for a multigenerational rodent reproductive/developmental bioassay. The use of the methodology is illustrated using the U.S. EPA’s Four Lab study in which rodents were exposed to chlorinated water concentrates containing complex mixtures of drinking water disinfection by-products. Possible experimental designs included two single-block designs and a two-block design. Considering the possible study designs and constraints, a design of two blocks of 100 females with a 40:60 ratio of control:treated animals and a significance level of 0.05 yielded maximum prospective power (~90% to detect pup weight decreases, while providing the most power to detect increased prenatal loss.

  11. Prospective Power Calculations for the Four Lab Study of A Multigenerational Reproductive/Developmental Toxicity Rodent Bioassay Using A Complex Mixture of Disinfection By-Products in the Low-Response Region

    Science.gov (United States)

    Dingus, Cheryl A.; Teuschler, Linda K.; Rice, Glenn E.; Simmons, Jane Ellen; Narotsky, Michael G.

    2011-01-01

    In complex mixture toxicology, there is growing emphasis on testing environmentally representative doses that improve the relevance of results for health risk assessment, but are typically much lower than those used in traditional toxicology studies. Traditional experimental designs with typical sample sizes may have insufficient statistical power to detect effects caused by environmentally relevant doses. Proper study design, with adequate statistical power, is critical to ensuring that experimental results are useful for environmental health risk assessment. Studies with environmentally realistic complex mixtures have practical constraints on sample concentration factor and sample volume as well as the number of animals that can be accommodated. This article describes methodology for calculation of statistical power for non-independent observations for a multigenerational rodent reproductive/developmental bioassay. The use of the methodology is illustrated using the U.S. EPA’s Four Lab study in which rodents were exposed to chlorinated water concentrates containing complex mixtures of drinking water disinfection by-products. Possible experimental designs included two single-block designs and a two-block design. Considering the possible study designs and constraints, a design of two blocks of 100 females with a 40:60 ratio of control:treated animals and a significance level of 0.05 yielded maximum prospective power (~90%) to detect pup weight decreases, while providing the most power to detect increased prenatal loss. PMID:22073030

  12. FIELD IMPLEMENTATION OF A WINSOR TYPE I SURFACTANT/ALCOHOL MIXTURE FOR IN SITU SOLUBILIZATION OF A COMPLEX LNAPL AS A SINGLE-PHASE MICROEMULSION

    Science.gov (United States)

    A Winsor Type I surfactant/alcohol mixture was used as an in situ flushing agent to solubilize a muticomponent nonaqueous phase liquid (NAPL) as a single-phase microemulsion (SPME) in a hydraulically isolated test cell at Hill Air Force Base (AFB), Utah. The surfactant (polyoxye...

  13. SOT Risk Assessment: Complex mixtures of anti-androgens at concentrations below individual chemical effect levels produces reproductive tract malformations in the male rat

    Science.gov (United States)

    This product is an invited webinar to the Society of Toxicology Risk Assessment Specialty Section (co-hosted by the Mixtures Specialty Section) as part of their monthly webinar series. The webinar is scheduled for 3:00PM on Wednesday September 13th.

  14. Membrane manufacture for peptide separations

    KAUST Repository

    Kim, Dooli; Salazar Moya, Octavio Ruben; Nunes, Suzana Pereira

    2016-01-01

    Nanostructured polymeric membranes are key tools in biomedical applications such as hemodialysis, protein separations, in the food industry, and drinking water supply from seawater. Despite of the success in different separation processes, membrane manufacture itself is at risk, since the most used solvents are about to be banned in many countries due to environmental and health issues. We propose for the first time the preparation of polyethersulfone membranes based on dissolution in the ionic liquid 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DEP). We obtained a series of membranes tailored for separation of solutes with molecular weight of 30, 5, 1.3, and 1.25 kg mol-1 with respective water permeances of 140, 65, 30 and 20 Lm-2h-1bar-1. We demonstrate their superior efficiency in the separation of complex mixtures of peptides with molecular weights in the range of 800 to 3500 gmol-1. Furthermore, the thermodynamics and kinetics of phase separation leading to the pore formation in the membranes were investigated. The rheology of the solutions and the morphology of the prepared membranes were examed and compared to those of polyethersulfone in organic solvents currently used for membrane manufacture.

  15. Membrane manufacture for peptide separations

    KAUST Repository

    Kim, Dooli

    2016-06-07

    Nanostructured polymeric membranes are key tools in biomedical applications such as hemodialysis, protein separations, in the food industry, and drinking water supply from seawater. Despite of the success in different separation processes, membrane manufacture itself is at risk, since the most used solvents are about to be banned in many countries due to environmental and health issues. We propose for the first time the preparation of polyethersulfone membranes based on dissolution in the ionic liquid 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DEP). We obtained a series of membranes tailored for separation of solutes with molecular weight of 30, 5, 1.3, and 1.25 kg mol-1 with respective water permeances of 140, 65, 30 and 20 Lm-2h-1bar-1. We demonstrate their superior efficiency in the separation of complex mixtures of peptides with molecular weights in the range of 800 to 3500 gmol-1. Furthermore, the thermodynamics and kinetics of phase separation leading to the pore formation in the membranes were investigated. The rheology of the solutions and the morphology of the prepared membranes were examed and compared to those of polyethersulfone in organic solvents currently used for membrane manufacture.

  16. Toxicology of Chemical Mixtures: A Review of Mixtures Assessment

    National Research Council Canada - National Science Library

    Bjarnason, Stephen

    2004-01-01

    .... Recent advances in disciplines such as genomics, proteomics, metabonomics and physiologically-based pharmacokinetic modeling should assist in the hazard assessment of complex chemical mixtures. However, the process of regulatory assessment of these types of exposures will remain both complex and difficult.

  17. Diversity of peptidic and proteinaceous toxins from social Hymenoptera venoms.

    Science.gov (United States)

    Dos Santos-Pinto, José Roberto Aparecido; Perez-Riverol, Amilcar; Lasa, Alexis Musacchio; Palma, Mario Sergio

    2018-06-15

    Among venomous animals, Hymenoptera have been suggested as a rich source of natural toxins. Due to their broad ecological diversity, venom from Hymenoptera insects (bees, wasps and ants) have evolved differentially thus widening the types and biological functions of their components. To date, insect toxinology analysis have scarcely uncovered the complex composition of bee, wasp and ant venoms which include low molecular weight compounds, highly abundant peptides and proteins, including several allergens. In Hymenoptera, these complex mixtures of toxins represent a potent arsenal of biological weapons that are used for self-defense, to repel intruders and to capture prey. Consequently, Hymenoptera venom components have a broad range of pharmacological targets and have been extensively studied, as promising sources of new drugs and biopesticides. In addition, the identification and molecular characterization of Hymenoptera venom allergens have allowed for the rational design of component-resolved diagnosis of allergy, finally improving the outcome of venom immunotherapy (VIT). Until recently, a limited number of Hymenoptera venoms had been unveiled due to the technical limitations of the approaches used to date. Nevertheless, the application of novel techniques with high dynamic range has significantly increased the number of identified peptidic and proteinaceous toxins. Considering this, the present review summarizes the current knowledge about the most representative Hymenoptera venom peptides and proteins which are under study for a better understanding of the insect-caused envenoming process and the development of new drugs and biopesticides. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Peptide separation by capillary electrophoresis with ultraviolet detection: Some simple approaches to enhance detection sensitivity and resolution

    International Nuclear Information System (INIS)

    Surugau, Noumie L.

    2011-01-01

    Capillary electrophoresis (CE) is one of the leading separation technologies for analysis of water-soluble analytes. CE has many advantages over the more established methods such as liquid chromatography and gel electrophoresis particularly in rapid analysis, require very little sample, use less or no toxic organic solvent, high peak efficiency and ease of automation. Despite the many attractive advantages of CE, CE users continue to seek improvements particularly on detection sensitivity, resolution and selectivity. This paper presented several simple approaches to improve detection sensitivity using simple sample pre-concentration called field-enhanced sample injection (FESI) and chromatographic-based ZipTip C 18 pre-concentrator. Also, some improvements in the resolution of complex peptides mixture when using two strategies namely, capillary coating and manipulation of the hydrophobicity of peptides using perfluorinated acids as background electrolyte (BGE), which have anionic conjugate base forms with hydrophobic character. As test compounds, standard peptide mixture and proteins digests were used for these studies. The results showed that FESI has significantly enhanced the detection signal of peptide standards and bovine serum albumin (BSA) tryptic digests. As for the use of ZipTip C 18 pre-concentrator, selective enhancement in detection signal was particularly notable on the late migrating peptides. Coating the capillary proved to have little changes on the CE of peptides when used in conjunction with acidic BGE. Electropherograms of BSA tryptic peptides in pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA) showed interesting profile, with notable resolution improvement for peptides with close similarity in electrophoretic mobilities. (author)

  19. Next-generation detection of antigen-responsive T cells using DNA barcode-labeled peptide-major histocompatibility complex I multimers

    DEFF Research Database (Denmark)

    Bentzen, Amalie Kai; Marquard, Andrea Marion; Lyngaa, Rikke Birgitte

    2016-01-01

    sample using >1000 different peptide-MHC multimers labeled with individual DNA barcodes.After isolation of MHC multimer binding T cells their recognition are revealed by amplification andsequencing of the MHC multimer-associated DNA barcodes. The relative frequency of the sequencedDNA barcodes...... originating from a given peptide-MHC motif relates to the size of the antigenresponsiveT cell population. We have demonstrated the use of large panels of >1000 DNA barcodedMHC multimers for detection of rareT cell populations of virus and cancer-restricted origin in various tissues and compared...

  20. Peptide aldehyde inhibitors of bacterial peptide deformylases.

    Science.gov (United States)

    Durand, D J; Gordon Green, B; O'Connell, J F; Grant, S K

    1999-07-15

    Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides. Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gram-negative Escherichia coli and gram-positive Bacillus subtilis. The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E. coli and B. subtilis PDF with Ki values of 26.0 and 55.6 microM, respectively. Cobalt-substituted E. coli and B. subtilis deformylases were also inhibited by these aldehydes with Ki values for calpeptin of 9.5 and 12.4 microM, respectively. Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex. In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B. subtilis Co(II)-PDF activity with the loss of the active site metal. The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity. Copyright 1999 Academic Press.

  1. Characterization of angiotensin-I converting enzyme inhibiting peptide from Venerupis philippinarum with nano-liquid chromatography in combination with orbitrap mass spectrum detection and molecular docking

    Science.gov (United States)

    Shi, Lei; Wu, Tizhi; Sheng, Naijuan; Yang, Li; Wang, Qian; Liu, Rui; Wu, Hao

    2017-06-01

    The complexity and diversity of peptide mixture from protein hydrolysates make their characterization difficult. In this study, a method combining nano LC-MS/MS with molecular docking was applied to identifying and characterizing a peptide with angiotensin-I converting enzyme (ACE-I) inhibiting activity from Venerupis philippinarum hydrolysate. Firstly, ethanol supernatant of V. philippinarum hydrolysate was separated into active fractions with chromatographic methods such as ion-exchange chromatography and high performance liquid chromatography in combination. Then seven peptides from active fraction were identified according to the searching result of the MS/MS spectra against protein databases. Peptides were synthesized and subjected to ACE-I-inhibition assay. The peptide NTLTLIDTGIGMTK showed the highest potency with an IC50 of 5.75 μmol L-1. The molecular docking analysis showed that the ACE-I inhibiting peptide NTLTLIDTGIGMTK bond with residues Glu123, Glu403, Arg522, Glu376, Gln281 and Asn285 of ACE-I. Therefore, active peptides could be identified with the present method rather than the traditional purification and identification strategies. It may also be feasible to identify other food-derived peptides which target other enzymes and receptors with the method developed in this study.

  2. Unbiased Selective Isolation of Protein N-Terminal Peptides from Complex Proteome Samples Using Phospho Tagging PTAG) and TiO2-based Depletion

    NARCIS (Netherlands)

    Mommen, G.P.M.; Waterbeemd, van de B.; Meiring, H.D.; Kersten, G.; Heck, A.J.R.; Jong, de A.P.J.M.

    2012-01-01

    A positional proteomics strategy for global N-proteome analysis is presented based on phospho tagging (PTAG) of internal peptides followed by depletion by titanium dioxide (TiO2) affinity chromatography. Therefore, N-terminal and lysine amino groups are initially completely dimethylated with

  3. Deciphering the structure of isomeric oligosaccharides in a complex mixture by tandem mass spectrometry: Photon activation with vacuum ultra-violet brings unique information and enables definitive structure assignment

    Energy Technology Data Exchange (ETDEWEB)

    Ropartz, David, E-mail: David.Ropartz@nantes.inra.fr [INRA, UR1268 Biopolymers Interactions Assemblies, F-44316 Nantes (France); Lemoine, Jérôme [Institut des Sciences Analytiques, UMR 5280, Université Lyon 1-CNRS, Université de Lyon, F-69622 Villeurbanne cedex (France); Giuliani, Alexandre [Synchrotron SOLEIL, L’Orme des Merisiers, F-91190 Gif-sur-Yvette (France); UAR 1008 CEPIA, INRA, F-44316 Nantes (France); Bittebière, Yann [INRA, UR1268 Biopolymers Interactions Assemblies, F-44316 Nantes (France); Enjalbert, Quentin [Institut des Sciences Analytiques, UMR 5280, Université Lyon 1-CNRS, Université de Lyon, F-69622 Villeurbanne cedex (France); Institut Lumière Matière, UMR5306, Université Lyon 1-CNRS, Université de Lyon, F-69622 Villeurbanne cedex (France); Antoine, Rodolphe; Dugourd, Philippe [Institut Lumière Matière, UMR5306, Université Lyon 1-CNRS, Université de Lyon, F-69622 Villeurbanne cedex (France); Ralet, Marie-Christine; Rogniaux, Hélène [INRA, UR1268 Biopolymers Interactions Assemblies, F-44316 Nantes (France)

    2014-01-07

    Graphical abstract: -- Highlights: •A complex mixture of methylated oligogalacturonans was fractionated by IP-RP-UHPLC. •Synchrotron-radiation in VUV range was used as an activation process for tandem MS. •VUV activation brought rich structural information compared to LE-CAD. •Resolution of more than 35 structures, including isomers, was successfully completed. -- Abstract: Carbohydrates have a wide variety of structures whose complexity and heterogeneity challenge the field of analytical chemistry. Tandem mass spectrometry, with its remarkable sensitivity and high information content, provides key advantages to addressing the structural elucidation of polysaccharides. Yet, classical fragmentation by collision-activated dissociation (CAD) in many cases fails to reach a comprehensive structural determination, especially when isomers have to be differentiated. In this work, for the first time, vacuum ultra-violet (VUV) synchrotron radiation is used as the activation process in tandem mass spectrometry of large oligosaccharides. Compared to low energy CAD (LE-CAD), photon activated dissociation brought more straightforward and valuable structural information. The outstanding feature was that complete series of informative ions were produced, with only minor neutral losses. Moreover, systematic fragmentation rules could be drawn thus facilitating the definitive assignments of fragment identities. As a result, most of the structures present in a complex mixture of oligogalacturonans could be comprehensively resolved, including many isomers differing in the position of methyl groups along the galacturonic acid backbone.

  4. Toward the prediction of class I and II mouse major histocompatibility complex-peptide-binding affinity: in silico bioinformatic step-by-step guide using quantitative structure-activity relationships.

    Science.gov (United States)

    Hattotuwagama, Channa K; Doytchinova, Irini A; Flower, Darren R

    2007-01-01

    Quantitative structure-activity relationship (QSAR) analysis is a cornerstone of modern informatics. Predictive computational models of peptide-major histocompatibility complex (MHC)-binding affinity based on QSAR technology have now become important components of modern computational immunovaccinology. Historically, such approaches have been built around semiqualitative, classification methods, but these are now giving way to quantitative regression methods. We review three methods--a 2D-QSAR additive-partial least squares (PLS) and a 3D-QSAR comparative molecular similarity index analysis (CoMSIA) method--which can identify the sequence dependence of peptide-binding specificity for various class I MHC alleles from the reported binding affinities (IC50) of peptide sets. The third method is an iterative self-consistent (ISC) PLS-based additive method, which is a recently developed extension to the additive method for the affinity prediction of class II peptides. The QSAR methods presented here have established themselves as immunoinformatic techniques complementary to existing methodology, useful in the quantitative prediction of binding affinity: current methods for the in silico identification of T-cell epitopes (which form the basis of many vaccines, diagnostics, and reagents) rely on the accurate computational prediction of peptide-MHC affinity. We have reviewed various human and mouse class I and class II allele models. Studied alleles comprise HLA-A*0101, HLA-A*0201, HLA-A*0202, HLA-A*0203, HLA-A*0206, HLA-A*0301, HLA-A*1101, HLA-A*3101, HLA-A*6801, HLA-A*6802, HLA-B*3501, H2-K(k), H2-K(b), H2-D(b) HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, I-A(b), I-A(d), I-A(k), I-A(S), I-E(d), and I-E(k). In this chapter we show a step-by-step guide into predicting the reliability and the resulting models to represent an advance on existing methods. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method

  5. Deciding which chemical mixtures risk assessment methods work best for what mixtures

    International Nuclear Information System (INIS)

    Teuschler, Linda K.

    2007-01-01

    The most commonly used chemical mixtures risk assessment methods involve simple notions of additivity and toxicological similarity. Newer methods are emerging in response to the complexities of chemical mixture exposures and effects. Factors based on both science and policy drive decisions regarding whether to conduct a chemical mixtures risk assessment and, if so, which methods to employ. Scientific considerations are based on positive evidence of joint toxic action, elevated human exposure conditions or the potential for significant impacts on human health. Policy issues include legislative drivers that may mandate action even though adequate toxicity data on a specific mixture may not be available and risk assessment goals that impact the choice of risk assessment method to obtain the amount of health protection desired. This paper discusses three important concepts used to choose among available approaches for conducting a chemical mixtures risk assessment: (1) additive joint toxic action of mixture components; (2) toxicological interactions of mixture components; and (3) chemical composition of complex mixtures. It is proposed that scientific support for basic assumptions used in chemical mixtures risk assessment should be developed by expert panels, risk assessment methods experts, and laboratory toxicologists. This is imperative to further develop and refine quantitative methods and provide guidance on their appropriate applications. Risk assessors need scientific support for chemical mixtures risk assessment methods in the form of toxicological data on joint toxic action for high priority mixtures, statistical methods for analyzing dose-response for mixtures, and toxicological and statistical criteria for determining sufficient similarity of complex mixtures

  6. Peptide chemistry toolbox - Transforming natural peptides into peptide therapeutics.

    Science.gov (United States)

    Erak, Miloš; Bellmann-Sickert, Kathrin; Els-Heindl, Sylvia; Beck-Sickinger, Annette G

    2018-06-01

    The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Identification of neurotensin-related peptides in human thymic epithelial cell membranes and relationship with major histocompatibility complex class I molecules.

    Science.gov (United States)

    Vanneste, Y; Thome, A N; Vandersmissen, E; Charlet, C; Franchimont, D; Martens, H; Lhiaubet, A M; Schimpff, R M; Rostène, W; Geenen, V

    1997-06-01

    This study shows the expression at the cell surface of human thymic epithelial cells (TEC) of a neurotensin (NT)-like immunoreactivity. NT radio-immunoassay (RIA) revealed that cultured human TEC contain +/-5 ng immunoreactive (ir) NT/10(6) cells, of which 5% is associated with plasma cell membranes. HPLC analysis of NT-ir present in human TEC showed a major peak of NT-ir corresponding to NT1-13. NT-ir was not detected in the supernatant of human TEC cultures. Using an affinity column prepared with a anti-MHC class I monoclonal antibody, NT-ir-related peptides were retained on the column and eluted together with MHC class I-related proteins. According to the elution time on HPLC of these peptides, they correspond to intact NT1-13, as well as to smaller fragments of NT1-13.

  8. Diversification of the celiac disease α-gliadin complex in wheat: a 33-mer peptide with six overlapping epitopes, evolved following polyploidization.

    Science.gov (United States)

    Ozuna, Carmen V; Iehisa, Julio C M; Giménez, María J; Alvarez, Juan B; Sousa, Carolina; Barro, Francisco

    2015-06-01

    The gluten proteins from wheat, barley and rye are responsible both for celiac disease (CD) and for non-celiac gluten sensitivity, two pathologies affecting up to 6-8% of the human population worldwide. The wheat α-gliadin proteins contain three major CD immunogenic peptides: p31-43, which induces the innate immune response; the 33-mer, formed by six overlapping copies of three highly stimulatory epitopes; and an additional DQ2.5-glia-α3 epitope which partially overlaps with the 33-mer. Next-generation sequencing (NGS) and Sanger sequencing of α-gliadin genes from diploid and polyploid wheat provided six types of α-gliadins (named 1-6) with strong differences in their frequencies in diploid and polyploid wheat, and in the presence and abundance of these CD immunogenic peptides. Immunogenic variants of the p31-43 peptide were found in most of the α-gliadins. Variants of the DQ2.5-glia-α3 epitope were associated with specific types of α-gliadins. Remarkably, only type 1 α-gliadins contained 33-mer epitopes. Moreover, the full immunodominant 33-mer fragment was only present in hexaploid wheat at low abundance, probably as the result of allohexaploidization events from subtype 1.2 α-gliadins found only in Aegilops tauschii, the D-genome donor of hexaploid wheat. Type 3 α-gliadins seem to be the ancestral type as they are found in most of the α-gliadin-expressing Triticeae species. These findings are important for reducing the incidence of CD by the breeding/selection of wheat varieties with low stimulatory capacity of T cells. Moreover, advanced genome-editing techniques (TALENs, CRISPR) will be easier to implement on the small group of α-gliadins containing only immunogenic peptides. © 2015 Society for Experimental Biology and John Wiley & Sons Ltd.

  9. Chemical speciation of Pb(II, Cd(II, Hg(II, Co(II, Ni(II, Cu(II and Zn(II binary complexes of l-methionine in 1,2-propanediol-water mixtures

    Directory of Open Access Journals (Sweden)

    M. Padma Latha

    2007-04-01

    Full Text Available Chemical speciation of Pb(II, Cd(II, Hg(II, Co(II, Ni(II, Cu(II and Zn(II complexes of L-methionine in 0.0-60 % v/v 1,2-propanediol-water mixtures maintaining an ionic strength of 0.16 M at 303 K has been studied pH metrically. The active forms of ligand are LH2+, LH and L-. The predominant species detected are ML, MLH, ML2, ML2H, ML2H2 and MLOH. Models containing different numbers of species were refined by using the computer program MINIQUAD 75. The best-fit chemical models were arrived at based on statistical parameters. The trend in variation of complex stability constants with change in the dielectric constant of the medium is explained on the basis of electrostatic and non-electrostatic forces.

  10. Combination of electrospray ionization, atmospheric pressure photoionization and laser desorption ionization Fourier transform ion cyclotronic resonance mass spectrometry for the investigation of complex mixtures - Application to the petroleomic analysis of bio-oils.

    Science.gov (United States)

    Hertzog, Jasmine; Carré, Vincent; Le Brech, Yann; Mackay, Colin Logan; Dufour, Anthony; Mašek, Ondřej; Aubriet, Frédéric

    2017-05-29

    The comprehensive description of complex mixtures such as bio-oils is required to understand and improve the different processes involved during biological, environmental or industrial operation. In this context, we have to consider how different ionization sources can improve a non-targeted approach. Thus, the Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been coupled to electrospray ionization (ESI), laser desorption ionization (LDI) and atmospheric pressure photoionization (APPI) to characterize an oak pyrolysis bio-oil. Close to 90% of the all 4500 compound formulae has been attributed to C x H y O z with similar oxygen class compound distribution. Nevertheless, their relative abundance in respect with their double bound equivalent (DBE) value has evidenced significant differences depending on the ion source used. ESI has allowed compounds with low DBE but more oxygen atoms to be ionized. APPI has demonstrated the efficient ionization of less polar compounds (high DBE values and less oxygen atoms). The LDI behavior of bio-oils has been considered intermediate in terms of DBE and oxygen amounts but it has also been demonstrated that a significant part of the features are specifically detected by this ionization method. Thus, the complementarity of three different ionization sources has been successfully demonstrated for the exhaustive characterization by petroleomic approach of a complex mixture. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Phase equilibria at low temperature for light hydrocarbons-methanol-water-acid gases mixtures: measurements and modelling; Equilibres de phases a basse temperature de systemes complexes CO{sub 2} - hydrocarbures legers - methanol - eau: mesures et modelisation

    Energy Technology Data Exchange (ETDEWEB)

    Ruffine, L.

    2005-10-15

    The need to develop and improve natural gas treatment processes is real. The petroleum industry usually uses separation processes which require phase equilibrium phenomena. Yet, the complexity of the phase equilibria involved results in a lack of data, which in turn limits the development of thermodynamic models. The first part of this work is devoted to experimental investigations for systems containing light hydrocarbons, methanol, water and acid gases. We present a new apparatus that was developed to measure vapor-liquid and vapor-liquid-liquid equilibria. It allowed us to obtain new phase composition data for the methanol-ethane binary system and different mixtures, and also to determine a part of the three phases equilibrium envelope of the same systems. In the second part of this work, we have developed a thermodynamic model based on the CPA equation of state. This choice may be justified by the presence of associating components like methanol, hydrogen sulfide and water in the systems. Such model is necessary for the design of gas treatment plants. Our model provides good results for phase equilibrium calculations for binaries systems without binary interaction parameter in many cases, and describes correctly the vapour-liquid and vapor-liquid-liquid equilibria for complex mixtures. (author)

  12. Uncertainty estimation of predictions of peptides' chromatographic retention times in shotgun proteomics.

    Science.gov (United States)

    Maboudi Afkham, Heydar; Qiu, Xuanbin; The, Matthew; Käll, Lukas

    2017-02-15

    Liquid chromatography is frequently used as a means to reduce the complexity of peptide-mixtures in shotgun proteomics. For such systems, the time when a peptide is released from a chromatography column and registered in the mass spectrometer is referred to as the peptide's retention time . Using heuristics or machine learning techniques, previous studies have demonstrated that it is possible to predict the retention time of a peptide from its amino acid sequence. In this paper, we are applying Gaussian Process Regression to the feature representation of a previously described predictor E lude . Using this framework, we demonstrate that it is possible to estimate the uncertainty of the prediction made by the model. Here we show how this uncertainty relates to the actual error of the prediction. In our experiments, we observe a strong correlation between the estimated uncertainty provided by Gaussian Process Regression and the actual prediction error. This relation provides us with new means for assessment of the predictions. We demonstrate how a subset of the peptides can be selected with lower prediction error compared to the whole set. We also demonstrate how such predicted standard deviations can be used for designing adaptive windowing strategies. lukas.kall@scilifelab.se. Our software and the data used in our experiments is publicly available and can be downloaded from https://github.com/statisticalbiotechnology/GPTime . © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  13. A ternary-complex of a suicide gene, a RAGE-binding peptide, and polyethylenimine as a gene delivery system with anti-tumor and anti-angiogenic dual effects in glioblastoma.

    Science.gov (United States)

    Choi, Eunji; Oh, Jungju; Lee, Dahee; Lee, Jaewon; Tan, Xiaonan; Kim, Minkyung; Kim, Gyeungyun; Piao, Chunxian; Lee, Minhyung

    2018-04-13

    The receptor for advanced glycation end-products (RAGE) is involved in tumor angiogenesis. Inhibition of RAGE might be an effective anti-angiogenic therapy for cancer. In this study, a cationic RAGE-binding peptide (RBP) was produced as an antagonist of RAGE, and a ternary-complex consisting of RBP, polyethylenimine (2 kDa, PEI2k), and a suicide gene (pHSVtk) was developed as a gene delivery system with dual functions: the anti-tumor effect of pHSVtk and anti-angiogenic effect of RBP. As an antagonist of RAGE, RBP decreased the secretion of vascular-endothelial growth factor (VEGF) in activated macrophages and reduced the tube-formation of endothelial cells in vitro. In in vitro transfection assays, the RBP/PEI2k/plasmid DNA (pDNA) ternary-complex had higher transfection efficiency than the PEI2k/pDNA binary-complex. In an intracranial glioblastoma animal model, the RBP/PEI2k/pHSVtk ternary-complex reduced α-smooth muscle actin expression, suggesting that the complex has an anti-angiogenic effect. In addition, the ternary-complex had higher pHSVtk delivery efficiency than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes in an animal model. As a result, the ternary-complex induced apoptosis and reduced tumor volume more effectively than the PEI2k/pHSVtk and PEI25k/pHSVtk binary-complexes. In conclusion, due to its dual anti-tumor and anti-angiogenesis effects, the RBP/PEI2k/pHSVtk ternary-complex might be an efficient gene delivery system for the treatment of glioblastoma. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Dietary Intervention with β-Lactoglobulin-Derived Peptides and a Specific Mixture of Fructo-Oligosaccharides and Bifidobacterium breve M-16V Facilitates the Prevention of Whey-Induced Allergy in Mice by Supporting a Tolerance-Prone Immune Environment

    Directory of Open Access Journals (Sweden)

    Atanaska I. Kostadinova

    2017-10-01

    Full Text Available Cow’s milk allergy (CMA prevails in infants and brings increased risk of developing other allergic diseases. Oral administration of specific β-lactoglobulin (BLG-derived peptides (PepMix and a specific blend of short- and long-chain fructo-oligosaccharides and Bifidobacterium breve M-16V (FF/Bb was found to partially prevent CMA development in mice. In this study, we aimed to expand the knowledge on the preventive potential and the underlying mechanisms of this approach. Three-week-old female C3H/HeOuJ mice were orally exposed to PepMix±FF/Bb prior to a 5-week oral sensitization with whole whey and cholera toxin as an adjuvant. The acute allergic skin response was determined after an intradermal challenge with whole whey protein. Following an oral challenge with whey, regulatory T cells (Tregs in the small intestine lamina propria (SI-LP and mRNA expression of immune markers in the Peyer’s patches (PP were investigated. The early impact of PepMix and FF/Bb interventions on the immune system during the oral tolerance (OT induction phase was investigated after the last OT administration. Pre-exposing mice to PepMix+FF/Bb partially prevented the acute allergic skin response compared to PBS and increased Tregs and activated T cells in the SI-LP compared to sham-sensitized mice. It also increased the mRNA expression of Tbet over GATA3 in the PP of whey-sensitized mice. Directly upon the 6-day OT phase, FF/Bb intervention enhanced cecal content levels of propionic and butyric acid in PepMix-fed mice and the former was positively correlated with Foxp3+ cell numbers in the colon. In the PP of PepMix+FF/Bb-exposed mice, IL-22 mRNA expression increased and IL-10 followed the same tendency, while the Foxp3 expression was increased over GATA3 and RorγT. In the colon, the Tbet mRNA expression increased over GATA3, while IL-22 decreased. In addition, the Foxp3+/GATA3+ and regulatory/effector T cell ratios in the mesenteric lymph nodes and the CD11b

  15. ALF: a strategy for identification of unauthorized GMOs in complex mixtures by a GW-NGS method and dedicated bioinformatics analysis.

    Science.gov (United States)

    Košir, Alexandra Bogožalec; Arulandhu, Alfred J; Voorhuijzen, Marleen M; Xiao, Hongmei; Hagelaar, Rico; Staats, Martijn; Costessi, Adalberto; Žel, Jana; Kok, Esther J; Dijk, Jeroen P van

    2017-10-26

    The majority of feed products in industrialised countries contains materials derived from genetically modified organisms (GMOs). In parallel, the number of reports of unauthorised GMOs (UGMOs) is gradually increasing. There is a lack of specific detection methods for UGMOs, due to the absence of detailed sequence information and reference materials. In this research, an adapted genome walking approach was developed, called ALF: Amplification of Linearly-enriched Fragments. Coupling of ALF to NGS aims for simultaneous detection and identification of all GMOs, including UGMOs, in one sample, in a single analysis. The ALF approach was assessed on a mixture made of DNA extracts from four reference materials, in an uneven distribution, mimicking a real life situation. The complete insert and genomic flanking regions were known for three of the included GMO events, while for MON15985 only partial sequence information was available. Combined with a known organisation of elements, this GMO served as a model for a UGMO. We successfully identified sequences matching with this organisation of elements serving as proof of principle for ALF as new UGMO detection strategy. Additionally, this study provides a first outline of an automated, web-based analysis pipeline for identification of UGMOs containing known GM elements.

  16. Fast and Accurate Identification of Cross-Linked Peptides for the Structural Analysis of Large Protein Complexes and Elucidation of Interaction Networks. / Tahir, Salman; Bukowski-Wills, Jimi-Carlo; Rasmussen, Morten; Rappsilber, Juri

    DEFF Research Database (Denmark)

    Rasmussen, Morten

    to investigate protein structure and protein-protein interactions. When applied to single proteins or small purified protein complexes, this methodology works well. However certain challenges arise when applied to more complex samples. One of the main problems is the combinatorial increase in the search space...... simplify a spectrum because we remove all peaks that are accounted for by the fragmentation of peptide one. This approach is highly sensitive and scales well as revealed by searching our data of synthetic cross-links against a large sequence database. Currently, against a protein database of >1300 proteins...... a spectrum is searched in 0.35 seconds - a vast improvement when compared to the exhaustive search method of combining every potential cross-link for each spectrum(60 hours). In fact the search time is comparable, if not better, than existing linear search engines. Furthermore, we auto-validate the results...

  17. Development of a dedicated peptide tandem mass spectral library for conservation science.

    Science.gov (United States)

    Fremout, Wim; Dhaenens, Maarten; Saverwyns, Steven; Sanyova, Jana; Vandenabeele, Peter; Deforce, Dieter; Moens, Luc

    2012-05-30

    In recent years, the use of liquid chromatography tandem mass spectrometry (LC-MS/MS) on tryptic digests of cultural heritage objects has attracted much attention. It allows for unambiguous identification of peptides and proteins, and even in complex mixtures species-specific identification becomes feasible with minimal sample consumption. Determination of the peptides is commonly based on theoretical cleavage of known protein sequences and on comparison of the expected peptide fragments with those found in the MS/MS spectra. In this approach, complex computer programs, such as Mascot, perform well identifying known proteins, but fail when protein sequences are unknown or incomplete. Often, when trying to distinguish evolutionarily well preserved collagens of different species, Mascot lacks the required specificity. Complementary and often more accurate information on the proteins can be obtained using a reference library of MS/MS spectra of species-specific peptides. Therefore, a library dedicated to various sources of proteins in works of art was set up, with an initial focus on collagen rich materials. This paper discusses the construction and the advantages of this spectral library for conservation science, and its application on a number of samples from historical works of art. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Simultaneous determination of intestinal permeability and potential drug interactions of complex mixtures using Caco-2 cells and high-resolution mass spectrometry: Studies with Rauwolfia serpentina extract.

    Science.gov (United States)

    Flynn, Thomas J; Vohra, Sanah N

    2018-06-25

    Caco-2 cells are a commonly used model for estimating the intestinal bioavailability of single chemical entity pharmaceuticals. Caco-2 cells, when induced with calcitriol, also express other biological functions such as phase I (CYP) and phase II (glucuronosyltransferases) drug metabolizing enzymes which are relevant to drug-supplement interactions. Intestinal bioavailability is an important factor in the overall safety assessment of products consumed orally. Foods, including herbal dietary supplements, are complex substances with multiple chemical components. Because of potential interactions between components of complex mixtures, more reliable safety assessments can be obtained by studying the commercial products "as consumed" rather than by testing individual chemical components one at a time. The present study evaluated the apparent intestinal permeability (P app ) of a model herbal extract, Rauwolfia serpentina, using both whole plant extracts and the individual purified Rauwolfia alkaloids. All test compounds, endpoint substrates, and their metabolites were quantified using liquid chromatography and high-resolution mass spectrometry. The P app values for individual Rauwolfia alkaloids were comparable whether measured individually or as components of the complete extract. Both Rauwolfia extract and all individual Rauwolfia alkaloids except yohimbine inhibited CYP3A4 activity (midazolam 1'-hydroxylation). Both Rauwolfia extract and all individual Rauwolfia alkaloids except corynanthine and reserpic acid significantly increased glucuronosyltransferase activity (glucuronidation of 4-methylumbelliferone). The positive control, ketoconazole, significantly inhibited both CYP3A4 and glucuronosyltransferase activities. These findings suggest that the Caco-2 assay is capable of simultaneously identifying both bioavailability and potentially hazardous intestinal drug-supplement interactions in complex mixtures. Published by Elsevier B.V.

  19. Peptide release, side-chain deprotection, work-up, and isolation

    DEFF Research Database (Denmark)

    Pedersen, Søren Ljungberg; Jensen, Knud Jørgen

    2013-01-01

    After having successfully synthesized a peptide, it has to be released from the solid support, unless it is being used for on-resin display. The linker and, in some cases, the cleavage mixture determine the C-terminal functionality of the released peptide. In most cases, the peptide is released w...

  20. Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

    OpenAIRE

    Uteng, Marianne; Hauge, Håvard Hildeng; Brondz, Ilia; Nissen-Meyer, Jon; Fimland, Gunnar

    2002-01-01

    A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a lo...

  1. Affinity capture of biotinylated proteins at acidic conditions to facilitate hydrogen/deuterium exchange mass spectrometry analysis of multimeric protein complexes

    DEFF Research Database (Denmark)

    Jensen, Pernille Foged; Jørgensen, Thomas J. D.; Koefoed, Klaus

    2013-01-01

    Characterization of conformational and dynamic changes associated with protein interactions can be done by hydrogen/deuterium exchange mass spectrometry (HDX-MS) by comparing the deuterium uptake in the bound and unbound state of the proteins. Investigation of local hydrogen/deuterium exchange...... in heteromultimeric protein complexes poses a challenge for the method due to the increased complexity of the mixture of peptides originating from all interaction partners in the complex. Previously, interference of peptides from one interaction partner has been removed by immobilizing the intact protein on beads...... complexes without interference of peptides originating from other interaction partners in the complex. The biotin-streptavidin strategy has been successfully implemented in a model system with two recombinant monoclonal antibodies that target nonoverlapping epitopes on the human epidermal growth factor...

  2. Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

    DEFF Research Database (Denmark)

    Oleksiewicz, M.B.; Kristensen, B.; Ladekjaer-Mikkelsen, A.S.

    2002-01-01

    The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class 1) were cloned and sequenced for two haplotypes (114 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs...

  3. Associated equilibria with participatian of single and mixed silver, lead and cadmium halide complexes in mixtures of molten alkali and alkaline earth metal nitrates

    International Nuclear Information System (INIS)

    Gouk, Kh.S.; Gupta, R.K.; Vekma, K.V.

    1983-01-01

    Associated equilibria in the systems, which contain single and mixed silver, cadmium and lead halide complexes in the KNO 3 -Ba(N0 3 ) 2 (87.6:12.4 and 89:11 mol.%) and NaNO 3 -Ba(NO 3 ) 2 (94.2-5.8 mol%) melts in the temperature range from 568.2 up to 698.2 K are investigated. Applicability of equations derivated on the base of quasi-lattice model to description of temperature coefficients of association constants is analized

  4. Formation of molecular complexes of salicylic acid, acetylsalicylic acid, and methyl salicylate in a mixture of supercritical carbon dioxide with a polar cosolvent

    Science.gov (United States)

    Petrenko, V. E.; Antipova, M. L.; Gurina, D. L.; Odintsova, E. G.

    2015-08-01

    The solvate structures formed by salicylic acid, acetylsalicylic acid, and methyl salicylate in supercritical (SC) carbon dioxide with a polar cosolvent (methanol, 0.03 mole fractions) at a density of 0.7 g/cm3 and a temperature of 318 K were studied by the molecular dynamics method. Salicylic and acetylsalicylic acids were found to form highly stable hydrogen-bonded complexes with methanol via the hydrogen atom of the carboxyl group. For methyl salicylate in which the carboxyl hydrogen is substituted by a methyl radical, the formation of stable hydrogen bonds with methanol was not revealed. The contribution of other functional groups of the solute to the interactions with the cosolvent was much smaller. An analysis of correlations between the obtained data and the literature data on the cosolvent effect on the solubility of the compounds in SC CO2 showed that the dissolving ability of SC CO2 with respect to a polar organic substance in the presence of a cosolvent increased only when stable hydrogen-bonded complexes are formed between this substance and the cosolvent.

  5. Raman Spectroscopic Study of Tungsten(VI) Oxosulfato Complexes in WO3–K2S2O7–K2SO4 Molten Mixtures: Stoichiometry, Vibrational Properties and Molecular Structure

    DEFF Research Database (Denmark)

    Paulson, Andreas L.; Kalampounias, Angelos G.; Berg, Rolf W.

    2011-01-01

    The dissolution reaction of WO3 in pure molten K2S2O7 and in molten K2S2O7-K2SO4 mixtures is studied under static equilibrium conditions in the XWO3 0 = 0-0.33 mol fraction range at temperatures up to 860 C. High temperature Raman spectroscopy shows that the dissolution leads to formation of WVI...... configuration as a core unit within the oxosulfato complexes formed. A quantitative exploitation of the relative Raman intensities in the binary WO3-K2S2O7 system allows the determination of the stoichiometric coefficient, n, of the complex formation reaction WO3 þ nS2O7 2-fC2n-. It is found that n = 1......; therefore, the reaction WO3 þ S2O7 2- f WO2(SO4)2 2- with six-fold W coordination is proposed as fully consistent with the observed Raman features. The effects of the incremental dissolution and presence of K2SO4 inWO3-K2S2O7 melts point to aWO3 3 K2S2O7 3 K2SO4 stoichiometry and a corresponding complex...

  6. Combination of electrospray ionization, atmospheric pressure photoionization and laser desorption ionization Fourier transform ion cyclotronic resonance mass spectrometry for the investigation of complex mixtures – Application to the petroleomic analysis of bio-oils

    Energy Technology Data Exchange (ETDEWEB)

    Hertzog, Jasmine [LCP-A2MC, FR 2843 Institut Jean Barriol de Chimie et Physique Moléculaires et Biomoléculaires, FR 3624 Réseau National de Spectrométrie de Masse FT-ICR à très haut champ, Université de Lorraine, ICPM, 1 boulevard Arago, 57078 Metz Cedex 03 (France); Carré, Vincent, E-mail: vincent.carre@univ-lorraine.fr [LCP-A2MC, FR 2843 Institut Jean Barriol de Chimie et Physique Moléculaires et Biomoléculaires, FR 3624 Réseau National de Spectrométrie de Masse FT-ICR à très haut champ, Université de Lorraine, ICPM, 1 boulevard Arago, 57078 Metz Cedex 03 (France); Le Brech, Yann [LRGP, CNRS, Université de Lorraine, ENSIC, 1, Rue Grandville, 54000 Nancy (France); Mackay, Colin Logan [SIRCAMS, School of Chemistry, University of Edinburgh, Edinburgh, EH9 3FJ, Scotland (United Kingdom); Dufour, Anthony [LRGP, CNRS, Université de Lorraine, ENSIC, 1, Rue Grandville, 54000 Nancy (France); Mašek, Ondřej [UK Biochar Research Center, School of Geosciences, University of Edinburgh, Kings Buildings, Edinburgh, EH9 3JN (United Kingdom); and others

    2017-05-29

    The comprehensive description of complex mixtures such as bio-oils is required to understand and improve the different processes involved during biological, environmental or industrial operation. In this context, we have to consider how different ionization sources can improve a non-targeted approach. Thus, the Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has been coupled to electrospray ionization (ESI), laser desorption ionization (LDI) and atmospheric pressure photoionization (APPI) to characterize an oak pyrolysis bio-oil. Close to 90% of the all 4500 compound formulae has been attributed to C{sub x}H{sub y}O{sub z} with similar oxygen class compound distribution. Nevertheless, their relative abundance in respect with their double bound equivalent (DBE) value has evidenced significant differences depending on the ion source used. ESI has allowed compounds with low DBE but more oxygen atoms to be ionized. APPI has demonstrated the efficient ionization of less polar compounds (high DBE values and less oxygen atoms). The LDI behavior of bio-oils has been considered intermediate in terms of DBE and oxygen amounts but it has also been demonstrated that a significant part of the features are specifically detected by this ionization method. Thus, the complementarity of three different ionization sources has been successfully demonstrated for the exhaustive characterization by petroleomic approach of a complex mixture. - Highlights: • Non-targeted mass spectrometry by combining electrospray ionization, atmospheric pressure photoionization and laser/desorption ionization. • Exhaustive description of pyrolytic bio-oil components. • Distinction of sugaric derivatives, lignin derivatives and lipids contained in a woody-based pyrolytic bio-oil.

  7. GRAVITY PIPELINE TRANSPORT FOR HARDENING FILLING MIXTURES

    Directory of Open Access Journals (Sweden)

    Leonid KROUPNIK

    2015-12-01

    Full Text Available In underground mining of solid minerals becoming increasingly common development system with stowing hardening mixtures. In this case the natural ore array after it is replaced by an artificial excavation of solidified filling mixture consisting of binder, aggregates and water. Such a mixture is prepared on the surface on special stowing complexes and transported underground at special stowing pipelines. However, it is transported to the horizons of a few kilometers, which requires a sustainable mode of motion of such a mixture in the pipeline. Hardening stowing mixture changes its rheological characteristics over time, which complicates the calculation of the parameters of pipeline transportation. The article suggests a method of determining the initial parameters of such mixtures: the status coefficient, indicator of transportability, coefficient of hydrodynamic resistance to motion of the mixture. These indicators characterize the mixture in terms of the possibility to transport it through pipes. On the basis of these indicators is proposed methodology for calculating the parameters of pipeline transport hardening filling mixtures in drift mode when traffic on the horizontal part of the mixture under pressure column of the mixture in the vertical part of the backfill of the pipeline. This technique allows stable operation is guaranteed to provide pipeline transportation.

  8. Rapid Two-Step Procedure for Large-Scale Purification of Pediocin-Like Bacteriocins and Other Cationic Antimicrobial Peptides from Complex Culture Medium

    Science.gov (United States)

    Uteng, Marianne; Hauge, Håvard Hildeng; Brondz, Ilia; Nissen-Meyer, Jon; Fimland, Gunnar

    2002-01-01

    A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis. PMID:11823243

  9. Comparison of two association models (Elliott-Suresh-Donohue and simplified PC-SAFT) for complex phase equilibria of hydrocarbon-water and amine-containing mixtures

    DEFF Research Database (Denmark)

    Grenner, Andreas; Schmelzer, Jürgen; von Solms, Nicolas

    2006-01-01

    , and water. Furthermore, the predictive capabilities of the models are investigated for four ternary systems composed of these components, which exhibit complex liquid-liquid(-liquid) equilibria (LLLE). Various aspects of association models which have an influence in the results are studied for the PC......, both models perform overall similarly for the binary systems, although ESD shows a remarkably good behavior despite its simplicity and the use of only the two-site scheme for all associating compounds. The prediction of the LLE in the ternary systems water + octane + aniline and water + CHA + aniline......Two Wertheim-based association models, the simplified PC-SAFT and the Elliott-Suresh-Donohue (ESD) equation of state, are compared in this work for the description of vapor-liquid equilibria (VLE) and liquid-liquid equilibria (LLE) in binary systems of aniline, cyclohexylamine (CHA), hydrocarbons...

  10. Low-Molecular Weight Polyethylenimine Modified with Pluronic 123 and RGD- or Chimeric RGD-NLS Peptide: Characteristics and Transfection Efficacy of Their Complexes with Plasmid DNA

    Directory of Open Access Journals (Sweden)

    Jing Hu

    2016-05-01

    Full Text Available To solve the problem of transfection efficiency vs. cytotoxicity and tumor-targeting ability when polyethylenimine (PEI was used as a nonviral gene delivery vector, new degradable PEI polymers were synthesized via cross-linking low-molecular-weight PEI with Pluronic P123 and then further coupled with a targeting peptide R4 (RGD and a bifunctional R11 (RGD-NLS, which were termed as P123-PEI-R4 and P123-PEI-R11, respectively. Agarose gel electrophoresis showed that both P123-PEI-R4 and P123-PEI-R11 efficaciously condense plasmid DNA at a polymer-to-pDNA w/w ratio of 3.0 and 0.4, respectively. The polyplexes were stable in the presence of serum and could protect plasmid DNA against DNaseI. They had uniform spherical nanoparticles with appropriate sizes around 100–280 nm and zeta-potentials about +40 mV. Furthermore, in vitro experiments showed that these polyplexes had lower cytotoxicity at any concentration compared with PEI 25 kDa, thus giving promise to high transfection efficiency as compared with another P123-PEI derivate conjugated with trifunctional peptide RGD-TAT-NLS (P123-PEI-R18. More importantly, compared with the other polymers, P123-PEI-R11 showed the highest transfection efficiency with relatively lower cytotoxicity at any concentration, indicating that the new synthetic polymer P123-PEI-R11 could be used as a safe and efficient gene deliver vector.

  11. Bicyclic peptide inhibitor of urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Roodbeen, Renée; Jensen, Berit Paaske; Jiang, Longguang

    2013-01-01

    The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide-based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established...... investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X-ray structure of the bicyclic-peptide-uPA complex confirmed an interaction similar to that for the previous upain-1/upain-2-uPA complexes. These physical studies of the peptide...

  12. Optimal mixture experiments

    CERN Document Server

    Sinha, B K; Pal, Manisha; Das, P

    2014-01-01

    The book dwells mainly on the optimality aspects of mixture designs. As mixture models are a special case of regression models, a general discussion on regression designs has been presented, which includes topics like continuous designs, de la Garza phenomenon, Loewner order domination, Equivalence theorems for different optimality criteria and standard optimality results for single variable polynomial regression and multivariate linear and quadratic regression models. This is followed by a review of the available literature on estimation of parameters in mixture models. Based on recent research findings, the volume also introduces optimal mixture designs for estimation of optimum mixing proportions in different mixture models, which include Scheffé’s quadratic model, Darroch-Waller model, log- contrast model, mixture-amount models, random coefficient models and multi-response model.  Robust mixture designs and mixture designs in blocks have been also reviewed. Moreover, some applications of mixture desig...

  13. Separation of Am(III) from Eu(III) by mixtures of triazynylbipyridine and bis(dicarbollide) extractants. The composition of the metal complexes extracted

    International Nuclear Information System (INIS)

    Narbutt, J.; Krejzler, J.

    2006-01-01

    Separation of trivalent actinides, in particular americium and curium, from lanthanides is an important step in an advanced partitioning process for future reprocessing of spent nuclear fuels. The use of soft donor (N and S) ligands makes it possible to separate the two groups of elements, probably because of the more covalent character in the complexes with actinides compared to the lanthanides. The aim of present work was to study solvent extraction of Am(III) and Eu(III) in a similar system with diethylhemi-BTP and COSAN: protonated bis(chlorodicarbollido)cobalt(III) or commo-3,3-cobalta-bis(8,9,12-trichlora-1,2-dicarbaclosododecaborane)ic acid. The present research was focused on both the determination of conditions for the separation of 241 Am(III) from 152 Eu in aqueous nitrate solution by using a synergistic extraction system and on the modelling of the process by slope analysis. Obtained values of the separation factors supported by the computer modelling permitted drawing the conclusions on the mechanism of the process and on the structure of extracted species

  14. Novel phosphine-peptide hybrids as selective catalysts

    DEFF Research Database (Denmark)

    Nygaard, David

    (His(Trt), Gln, Gln(Trt), Cys(tBu), Thr(OtBu), azido- Dab, Asp(OtBu), Arg(Pmc))) yielding a range of novel modified peptides. Peptides containing one secondary amine were phosphinylated and captured as either phosphine-boranes or oxides. Both borane and oxide protection of phosphine-peptide hybrids...... was discovered and the compounds were structurally elucidated via NMR and mass spectroscopy. Two of these compounds were incorporated into peptides. An existing method of obtaining peptides containing secondary amines in the peptide backbone have been expanded for incorporation of functional amino acids as well...... palladium chloride dimer did not yield an observable phosphine-palladium complex. A peptide containing two secondary amine sites was synthesized, phosphinylated and complexed to respectively palladium and copper. The palladium complex was utilized successfully as a palladium catalyst in a model Sonogashira...

  15. Superior Antifouling Performance of a Zwitterionic Peptide Compared to an Amphiphilic, Non-Ionic Peptide.

    Science.gov (United States)

    Ye, Huijun; Wang, Libing; Huang, Renliang; Su, Rongxin; Liu, Boshi; Qi, Wei; He, Zhimin

    2015-10-14

    The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.

  16. Peptides and the new endocrinology

    Science.gov (United States)

    Schwyzer, Robert

    1982-01-01

    The discovery of regulatory peptides common to the nervous and the endocrine systems (brain, gut, and skin) has brought about a revolution in our concepts of endocrinology and neurology. We are beginning to understand some of the complex interrelationships between soma and psyche that might, someday, be important for an integrated treatment of diseases. Examples of the actions of certain peptides in the periphery and in the central nervous system are given, and their biosynthesis and molecular anatomy as carriers for information are discussed.

  17. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2, wh...

  18. Towards automated discrimination of lipids versus peptides from full scan mass spectra

    Directory of Open Access Journals (Sweden)

    Piotr Dittwald

    2014-09-01

    Full Text Available Although physicochemical fractionation techniques play a crucial role in the analysis of complex mixtures, they are not necessarily the best solution to separate specific molecular classes, such as lipids and peptides. Any physical fractionation step such as, for example, those based on liquid chromatography, will introduce its own variation and noise. In this paper we investigate to what extent the high sensitivity and resolution of contemporary mass spectrometers offers viable opportunities for computational separation of signals in full scan spectra. We introduce an automatic method that can discriminate peptide from lipid peaks in full scan mass spectra, based on their isotopic properties. We systematically evaluate which features maximally contribute to a peptide versus lipid classification. The selected features are subsequently used to build a random forest classifier that enables almost perfect separation between lipid and peptide signals without requiring ion fragmentation and classical tandem MS-based identification approaches. The classifier is trained on in silico data, but is also capable of discriminating signals in real world experiments. We evaluate the influence of typical data inaccuracies of common classes of mass spectrometry instruments on the optimal set of discriminant features. Finally, the method is successfully extended towards the classification of individual lipid classes from full scan mass spectral features, based on input data defined by the Lipid Maps Consortium.

  19. Low-molecular-weight color pI markers to monitor on-line the peptide focusing process in OFFGEL fractionation.

    Science.gov (United States)

    Michelland, Sylvie; Bourgoin-Voillard, Sandrine; Cunin, Valérie; Tollance, Axel; Bertolino, Pascal; Slais, Karel; Seve, Michel

    2017-08-01

    High-throughput mass spectrometry-based proteomic analysis requires peptide fractionation to simplify complex biological samples and increase proteome coverage. OFFGEL fractionation technology became a common method to separate peptides or proteins using isoelectric focusing in an immobilized pH gradient. However, the OFFGEL focusing process may be further optimized and controlled in terms of separation time and pI resolution. Here we evaluated OFFGEL technology to separate peptides from different samples in the presence of low-molecular-weight (LMW) color pI markers to visualize the focusing process. LMW color pI markers covering a large pH range were added to the peptide mixture before OFFGEL fractionation using a 24-wells device encompassing the pH range 3-10. We also explored the impact of LMW color pI markers on peptide fractionation labeled previously for iTRAQ. Then, fractionated peptides were separated by RP_HPLC prior to MS analysis using MALDI-TOF/TOF mass spectrometry in MS and MS/MS modes. Here we report the performance of the peptide focusing process in the presence of LMW color pI markers as on-line trackers during the OFFGEL process and the possibility to use them as pI controls for peptide focusing. This method improves the workflow for peptide fractionation in a bottom-up proteomic approach with or without iTRAQ labeling. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Quantification of amino acids and peptides in an ionic liquid based aqueous two-phase system by LC-MS analysis.

    Science.gov (United States)

    Oppermann, Sebastian; Oppermann, Christina; Böhm, Miriam; Kühl, Toni; Imhof, Diana; Kragl, Udo

    2018-04-25

    Aqueous two-phase systems (ATPS) occur by the mixture of two polymers or a polymer and an inorganic salt in water. It was shown that not only polymers but also ionic liquids in combination with inorganic cosmotrophic salts are able to build ATPS. Suitable for the formation of ionic liquid-based ATPS systems are hydrophilic water miscible ionic liquids. To understand the driving force for amino acid and peptide distribution in IL-ATPS at different pH values, the ionic liquid Ammoeng 110™ and K 2 HPO 4 have been chosen as a test system. To quantify the concentration of amino acids and peptides in the different phases, liquid chromatography and mass spectrometry (LC-MS) technologies were used. Therefore the peptides and amino acids have been processed with EZ:faast™-Kit from Phenomenex for an easy and reliable quantification method even in complex sample matrices. Partitioning is a surface-dependent phenomenon, investigations were focused on surface-related amino acid respectively peptide properties such as charge and hydrophobicity. Only a very low dependence between the amino acids or peptides hydrophobicity and the partition coefficient was found. Nevertheless, the presented results show that electrostatic respectively ionic interactions between the ionic liquid and the amino acids or peptides have a strong impact on their partitioning behavior.

  1. Liquids and liquid mixtures

    CERN Document Server

    Rowlinson, J S; Baldwin, J E; Buckingham, A D; Danishefsky, S

    2013-01-01

    Liquids and Liquid Mixtures, Third Edition explores the equilibrium properties of liquids and liquid mixtures and relates them to the properties of the constituent molecules using the methods of statistical thermodynamics. Topics covered include the critical state, fluid mixtures at high pressures, and the statistical thermodynamics of fluids and mixtures. This book consists of eight chapters and begins with an overview of the liquid state and the thermodynamic properties of liquids and liquid mixtures, including vapor pressure and heat capacities. The discussion then turns to the thermodynami

  2. Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides.

    Directory of Open Access Journals (Sweden)

    Jamie L Schafer

    2015-09-01

    Full Text Available Natural killer (NK cell responses in primates are regulated in part through interactions between two highly polymorphic molecules, the killer-cell immunoglobulin-like receptors (KIRs on NK cells and their major histocompatibility complex (MHC class I ligands on target cells. We previously reported that the binding of a common MHC class I molecule in the rhesus macaque, Mamu-A1*002, to the inhibitory receptor Mamu-KIR3DL05 is stabilized by certain simian immunodeficiency virus (SIV peptides, but not by others. Here we investigated the functional implications of these interactions by testing SIV peptides bound by Mamu-A1*002 for the ability to modulate Mamu-KIR3DL05+ NK cell responses. Twenty-eight of 75 SIV peptides bound by Mamu-A1*002 suppressed the cytolytic activity of primary Mamu-KIR3DL05+ NK cells, including three immunodominant CD8+ T cell epitopes previously shown to stabilize Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. Substitutions at C-terminal positions changed inhibitory peptides into disinhibitory peptides, and vice versa, without altering binding to Mamu-A1*002. The functional effects of these peptide variants on NK cell responses also corresponded to their effects on Mamu-A1*002 tetramer binding to Mamu-KIR3DL05. In assays with mixtures of inhibitory and disinhibitory peptides, low concentrations of inhibitory peptides dominated to suppress NK cell responses. Consistent with the inhibition of Mamu-KIR3DL05+ NK cells by viral epitopes presented by Mamu-A1*002, SIV replication was significantly higher in Mamu-A1*002+ CD4+ lymphocytes co-cultured with Mamu-KIR3DL05+ NK cells than with Mamu-KIR3DL05- NK cells. These results demonstrate that viral peptides can differentially affect NK cell responses by modulating MHC class I interactions with inhibitory KIRs, and provide a mechanism by which immunodeficiency viruses may evade NK cell responses.

  3. Toward the Rational Use of Exposure Information in Mixtures Toxicology

    Science.gov (United States)

    Of all the disciplines of toxicology, perhaps none is as dependent on exposure information as Mixtures Toxicology. Identifying real world mixtures and replicating them in the laboratory (or in silico) is critical to understanding their risks. Complex mixtures such as cigarett...

  4. Context-dependent protein folding of a virulence peptide in the bacterial and host environments: structure of an SycH–YopH chaperone–effector complex

    International Nuclear Information System (INIS)

    Vujanac, Milos; Stebbins, C. Erec

    2013-01-01

    The structure of a SycH–YopH chaperone–effector complex from Yersinia reveals the bacterial state of a protein that adopts different folds in the host and pathogen environments. Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH–YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called β-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the β-motif. Biochemical studies establish that the β-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed

  5. Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation

    Science.gov (United States)

    McKay, Craig S.; Finn, M.G.

    2014-01-01

    The selective chemical modification of biological molecules drives a good portion of modern drug development and fundamental biological research. While a few early examples of reactions that engage amine and thiol groups on proteins helped establish the value of such processes, the development of reactions that avoid most biological molecules so as to achieve selectivity in desired bond-forming events has revolutionized the field. We provide an update on recent developments in bioorthogonal chemistry that highlights key advances in reaction rates, biocompatibility, and applications. While not exhaustive, we hope this summary allows the reader to appreciate the rich continuing development of good chemistry that operates in the biological setting. PMID:25237856

  6. X-ray studies on crystalline complexes involving amino acids and peptides. XXXII. Effect of chirality on ionisation state, stoichiometry and aggregation in the complexes of oxalic acid with DL- and L-lysine.

    Science.gov (United States)

    Venkatraman, J; Prabu, M M; Vijayan, M

    1997-08-01

    Crystals of the oxalic acid complex of DL-lysine (triclinic P1; a = 5.540(1), b = 10.764(2), c = 12.056(2) A, alpha = 77.8(1), beta = 80.6(1), gamma = 75.6(1).; R = 4.7% for 2023 observed reflections) contain lysine and semioxalate ions in the 1:1 ratio, whereas the ratio of lysine and semioxalate/oxalate ions is 2:3 in the crystals of the L-lysine complex (monoclinic P2(1); alpha = 4.906(1), b = 20.145(4), c = 12.455(1) A, beta = 92.5(1).; R = 4.4% for 1494 observed reflections). The amino acid molecule in the L-lysine complex has an unusual ionisation state with positively charged alpha- and side-chain amino groups and a neutral carboxyl group. The unlike molecules aggregate into separate alternating layers in the DL-lysine complex in a manner similar to that observed in several of the amino acid complexes. The L-lysine complex exhibits a new aggregation pattern which cannot be easily explained in terms of planar features, thus emphasizing the fundamental dependence of aggregation on molecular characteristics. Despite the differences in stoichiometry, ionisation state and long-range aggregation patterns, the basic element of aggregation in the two complexes exhibits considerable similarity.

  7. Elemental ratios for characterization of quantum-dots populations in complex mixtures by asymmetrical flow field-flow fractionation on-line coupled to fluorescence and inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Menendez-Miranda, Mario; Fernandez-Arguelles, Maria T; Costa-Fernandez, Jose M; Encinar, Jorge Ruiz; Sanz-Medel, Alfredo

    2014-08-11

    Separation and identification of nanoparticles of different composition, with similar particle diameter, coexisting in heterogeneous suspensions of polymer-coated CdSe/ZnS quantum dots (QDs) have been thoroughly assessed by asymmetric flow field-flow fractionation (AF4) coupled on-line to fluorescence and inductively coupled plasma mass spectrometry (ICPMS) detectors. Chemical characterization of any previously on-line separated nanosized species was achieved by the measurement of the elemental molar ratios of every element involved in the synthesis of the QDs, using inorganic standards and external calibration by flow injection analysis (FIA). Such elemental molar ratios, strongly limited so far to pure single nanoparticles suspensions, have been achieved with adequate accuracy by coupling for the first time an ICP-QQQ instrument to an AF4 system. This hyphenation turned out to be instrumental to assess the chemical composition of the different populations of nanoparticles coexisting in the relatively complex mixtures, due to its capabilities to detect the hardly detectable elements involved in the synthesis. Interestingly such information, complementary to that obtained by fluorescence, was very valuable to detect and identify unexpected nanosized species, present at significant level, produced during QDs synthesis and hardly detectable by standard approaches. Copyright © 2014. Published by Elsevier B.V.

  8. Elemental ratios for characterization of quantum-dots populations in complex mixtures by asymmetrical flow field-flow fractionation on-line coupled to fluorescence and inductively coupled plasma mass spectrometry

    International Nuclear Information System (INIS)

    Menendez-Miranda, Mario; Fernandez-Arguelles, Maria T.; Costa-Fernandez, Jose M.; Encinar, Jorge Ruiz; Sanz-Medel, Alfredo

    2014-01-01

    Highlights: • The hyphenated system allows unequivocal identification of nanoparticle populations. • AF4 separation permitted detection of unexpected nanosized species in a sample. • ICP-QQQ provides elemental ratios with adequate accuracy in every nanoparticle. • Purity and chemical composition of different quantum dot samples were assessed. - Abstract: Separation and identification of nanoparticles of different composition, with similar particle diameter, coexisting in heterogeneous suspensions of polymer-coated CdSe/ZnS quantum dots (QDs) have been thoroughly assessed by asymmetric flow field-flow fractionation (AF4) coupled on-line to fluorescence and inductively coupled plasma mass spectrometry (ICPMS) detectors. Chemical characterization of any previously on-line separated nanosized species was achieved by the measurement of the elemental molar ratios of every element involved in the synthesis of the QDs, using inorganic standards and external calibration by flow injection analysis (FIA). Such elemental molar ratios, strongly limited so far to pure single nanoparticles suspensions, have been achieved with adequate accuracy by coupling for the first time an ICP-QQQ instrument to an AF4 system. This hyphenation turned out to be instrumental to assess the chemical composition of the different populations of nanoparticles coexisting in the relatively complex mixtures, due to its capabilities to detect the hardly detectable elements involved in the synthesis. Interestingly such information, complementary to that obtained by fluorescence, was very valuable to detect and identify unexpected nanosized species, present at significant level, produced during QDs synthesis and hardly detectable by standard approaches

  9. Development and prototypical application of analysis methods for complex anion mixtures in waters and heavy metal organyls in sediments; Entwicklung und prototypische Anwendung von Analysenverfahren fuer komplexe Anionengemische in Waessern und Schwermetallorganylen in Sedimenten. Schlussbericht

    Energy Technology Data Exchange (ETDEWEB)

    Anders, B.; Knoechel, A.; Potgeter, H.; Staub, S.; Stocker, M.

    2002-07-01

    When it comes to assessing the hazards emanating from heavy pollutants in waters mere elemental analysis provides too little information. Due to the great differences in toxicity and mobility it is important to know more about the exact species in question. This is particularly true of heavy metals that form stable organyls, specifically As, Pb, Sn and Hg, but also of alkylated arsenic acids, which need to be measured in complex anion mixtures. The purpose of the present project was to develop robust, powerful analysis methods and thus overcome the existing deficit in reliable analysis methods for these substances. An important approach in this connection is the use of coupled chromatography and detection systems for separation and analysis. [German] Hinsichtlich der von einer Schwermetallbelastung in Gewaessern ausgehenden Gefahren liefert die reine Elementanalytik nur unzureichende Aussagen. Aufgrund der grossen Unterschiede in Toxiditaet und Mobilitaet ist die Kenntnis der jeweils vorliegenden Spezies bedeutungsvoll. Dies gilt in besonderem Masse fuer die stabile Organyle bildenden Schwermetalle As, Pb, Sn und Hg sowie die alkylierten Arsensaeuren, die es innerhalb komplexer Anionengemische zu bestimmen gilt. Hinsichtlich ihrer sicheren Bestimmung bestehen methodische Defizite, die das vorliegende Projekt durch die Entwicklung robuster, nachweisstarker Analysenverfahren zu beseitigen versucht. Grosse Bedeutung kommt dabei gekoppelten Systemen aus Chromatographie und Detektion als Trenn- und Bestimmungsmethode zu. (orig.)

  10. Structure of the Ni(II) complex of Escherichia coli peptide deformylase and suggestions on deformylase activities depending on different metal(II) centres.

    Science.gov (United States)

    Yen, Ngo Thi Hai; Bogdanović, Xenia; Palm, Gottfried J; Kühl, Olaf; Hinrichs, Winfried

    2010-02-01

    Crystal structures of polypeptide deformylase (PDF) of Escherichia coli with nickel(II) replacing the native iron(II) have been solved with chloride and formate as metal ligands. The chloro complex is a model for the correct protonation state of the hydrolytic hydroxo ligand and the protonated status of the Glu133 side chain as part of the hydrolytic mechanism. The ambiguity that recently some PDFs have been identified with Zn(2+) ion as the active-site centre whereas others are only active with Fe(2+) (or Co(2+), Ni(2+) is discussed with respect to Lewis acid criteria of the metal ion and substrate activation by the CD loop.

  11. PeptideAtlas

    Data.gov (United States)

    U.S. Department of Health & Human Services — PeptideAtlas is a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments. Mass...

  12. Development of Surface Complexation Models of Cr(VI) Adsorption on Soils, Sediments and Model Mixtures of Kaolinite, Montmorillonite, γ-Alumina, Hydrous Manganese and Ferric Oxides and Goethite

    Energy Technology Data Exchange (ETDEWEB)

    Koretsky, Carla [Western Michigan University

    2013-11-29

    Hexavalent chromium is a highly toxic contaminant that has been introduced into aquifers and shallow sediments and soils via many anthropogenic activities. Hexavalent chromium contamination is a problem or potential problem in the shallow subsurface at several DOE sites, including Hanford, Idaho National Laboratory, Los Alamos National Laboratory and the Oak Ridge Reservation (DOE, 2008). To accurately quantify the fate and transport of hexavalent chromium at DOE and other contaminated sites, robust geochemical models, capable of correctly predicting changes in chromium chemical form resulting from chemical reactions occurring in subsurface environments are needed. One important chemical reaction that may greatly impact the bioavailability and mobility of hexavalent chromium in the subsurface is chemical binding to the surfaces of particulates, termed adsorption or surface complexation. Quantitative thermodynamic surface complexation models have been derived that can correctly calculate hexavalent chromium adsorption on well-characterized materials over ranges in subsurface conditions, such pH and salinity. However, models have not yet been developed for hexavalent chromium adsorption on many important constituents of natural soils and sediments, such as clay minerals. Furthermore, most of the existing thermodynamic models have been developed for relatively simple, single solid systems and have rarely been tested for the complex mixtures of solids present in real sediments and soils. In this study, the adsorption of hexavalent chromium was measured as a function of pH (3-10), salinity (0.001 to 0.1 M NaNO3), and partial pressure of carbon dioxide(0-5%) on a suite of naturally-occurring solids including goethite (FeOOH), hydrous manganese oxide (MnOOH), hydrous ferric oxide (Fe(OH)3), γ-alumina (Al2O3), kaolinite (Al2Si2O5(OH)4), and montmorillonite (Na3(Al, Mg)2Si4O10(OH)2-nH2O). The results show that all of these materials can bind substantial quantities of

  13. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  14. PH dependent adhesive peptides

    Science.gov (United States)

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  15. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  16. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  17. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  18. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  19. Antimicrobial Peptides in 2014

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2015-03-01

    Full Text Available This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

  20. Peptide and Peptide-Dependent Motions in MHC Proteins: Immunological Implications and Biophysical Underpinnings

    Directory of Open Access Journals (Sweden)

    Cory M. Ayres

    2017-08-01

    Full Text Available Structural biology of peptides presented by class I and class II MHC proteins has transformed immunology, impacting our understanding of fundamental immune mechanisms and allowing researchers to rationalize immunogenicity and design novel vaccines. However, proteins are not static structures as often inferred from crystallographic structures. Their components move and breathe individually and collectively over a range of timescales. Peptides bound within MHC peptide-binding grooves are no exception and their motions have been shown to impact recognition by T cell and other receptors in ways that influence function. Furthermore, peptides tune the motions of MHC proteins themselves, which impacts recognition of peptide/MHC complexes by other proteins. Here, we review the motional properties of peptides in MHC binding grooves and discuss how peptide properties can influence MHC motions. We briefly review theoretical concepts about protein motion and highlight key data that illustrate immunological consequences. We focus primarily on class I systems due to greater availability of data, but segue into class II systems as the concepts and consequences overlap. We suggest that characterization of the dynamic “energy landscapes” of peptide/MHC complexes and the resulting functional consequences is one of the next frontiers in structural immunology.

  1. Use of synthetic peptide libraries for the H-2Kd binding motif identification.

    Science.gov (United States)

    Quesnel, A; Casrouge, A; Kourilsky, P; Abastado, J P; Trudelle, Y

    1995-01-01

    To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

  2. Structure and tunneling dynamics in a model system of peptide co-solvents: Rotational spectroscopy of the 2,2,2-trifluoroethanol⋯water complex

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, Javix; Xu, Yunjie, E-mail: yunjie.xu@ualberta.ca [Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2 (Canada)

    2014-06-21

    The hydrogen-bonding topology and tunneling dynamics of the binary adduct, 2,2,2-trifluoroethanol (TFE)⋯water, were investigated using chirped pulse and cavity based Fourier transform microwave spectroscopy with the aid of high level ab initio calculations. Rotational spectra of the most stable binary TFE⋯water conformer and five of its deuterium isotopologues were assigned. A strong preference for the insertion binding topology where water is inserted into the existing intramolecular hydrogen-bonded ring of TFE was observed. Tunneling splittings were detected in all of the measured rotational transitions of TFE⋯water. Based on the relative intensity of the two tunneling components and additional isotopic data, the splitting can be unambiguously attributed to the tunneling motion of the water subunit, i.e., the interchange of the bonded and nonbonded hydrogen atoms of water. The absence of any other splitting in the rotational transitions of all isotopologues observed indicates that the tunneling between g+ and g− TFE is quenched in the TFE⋯H{sub 2}O complex.

  3. Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

    Science.gov (United States)

    Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

    2017-11-13

    A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

  4. O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro.

    Science.gov (United States)

    Liu, Y; Egyhazi, S; Hansson, J; Bhide, S V; Kulkarni, P S; Grafström, R C

    1997-10-01

    Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.

  5. Identification and quantification of seven fused aromatic rings C26H14 peri-condensed benzenoid polycyclic aromatic hydrocarbons in a complex mixture of polycyclic aromatic hydrocarbons from coal tar.

    Science.gov (United States)

    Oña-Ruales, Jorge O; Ruiz-Morales, Yosadara; Wise, Stephen A

    2016-04-15

    A methodology for the characterization of groups of polycyclic aromatic hydrocarbons (PAHs) using a combination of normal phase liquid chromatography with ultraviolet-visible spectroscopy (NPLC/UV-vis) and gas chromatography with mass spectrometry (GC/MS) was used for the identification and quantification of seven fused aromatic rings C26H14 peri-condensed benzenoid polycyclic aromatic hydrocarbons, PAHs, in standard reference material (SRM) 1597a, complex mixture of PAHs from coal tar. The NPLC/UV-vis isolated the fractions based on the number of aromatic carbons and the GC/MS allowed the identification and quantification of five of the nine C26H14 PAH isomers; naphtho[1,2,3,4-ghi]perylene, dibenzo[b,ghi]perylene, dibenzo[b,pqr]perylene, naphtho[8,1,2-bcd]perylene, and dibenzo[cd,lm]perylene using a retention time comparison with authentic reference standards. For the other four benzenoid isomers with no available reference standards the following two approaches were used. First, the annellation theory was used to achieve the potential identification of benzo[qr]naphtho[3,2,1,8-defg]chrysene, and second, the elution distribution in the GC fractions was used to support the potential identification of benzo[qr]naphtho[3,2,1,8-defg]chrysene and to reach the tentative identifications of dibenzo[a,ghi]perylene, naphtho[7,8,1,2,3-pqrst]pentaphene, and anthra[2,1,9,8-opqra]naphthacene. It is the first time that naphtho[1,2,3,4-ghi]perylene, dibenzo[b,ghi]perylene, dibenzo[b,pqr]perylene, naphtho[8,1,2-bcd]perylene, and dibenzo[cd,lm]perylene are quantified, and the first time that benzo[qr]naphtho[3,2,1,8-defg]chrysene is potentially identified, in any sample, in any context. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Acetone-Linked Peptides: A Convergent Approach for Peptide Macrocyclization and Labeling.

    Science.gov (United States)

    Assem, Naila; Ferreira, David J; Wolan, Dennis W; Dawson, Philip E

    2015-07-20

    Macrocyclization is a broadly applied approach for overcoming the intrinsically disordered nature of linear peptides. Herein, it is shown that dichloroacetone (DCA) enhances helical secondary structures when introduced between peptide nucleophiles, such as thiols, to yield an acetone-linked bridge (ACE). Aside from stabilizing helical structures, the ketone moiety embedded in the linker can be modified with diverse molecular tags by oxime ligation. Insights into the structure of the tether were obtained through co-crystallization of a constrained S-peptide in complex with RNAse S. The scope of the acetone-linked peptides was further explored through the generation of N-terminus to side chain macrocycles and a new approach for generating fused macrocycles (bicycles). Together, these studies suggest that acetone linking is generally applicable to peptide macrocycles with a specific utility in the synthesis of stabilized helices that incorporate functional tags. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. [Plant signaling peptides. Cysteine-rich peptides].

    Science.gov (United States)

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

    Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation.

  8. Perception of trigeminal mixtures.

    Science.gov (United States)

    Filiou, Renée-Pier; Lepore, Franco; Bryant, Bruce; Lundström, Johan N; Frasnelli, Johannes

    2015-01-01

    The trigeminal system is a chemical sense allowing for the perception of chemosensory information in our environment. However, contrary to smell and taste, we lack a thorough understanding of the trigeminal processing of mixtures. We, therefore, investigated trigeminal perception using mixtures of 3 relatively receptor-specific agonists together with one control odor in different proportions to determine basic perceptual dimensions of trigeminal perception. We found that 4 main dimensions were linked to trigeminal perception: sensations of intensity, warmth, coldness, and pain. We subsequently investigated perception of binary mixtures of trigeminal stimuli by means of these 4 perceptual dimensions using different concentrations of a cooling stimulus (eucalyptol) mixed with a stimulus that evokes warmth perception (cinnamaldehyde). To determine if sensory interactions are mainly of central or peripheral origin, we presented stimuli in a physical "mixture" or as a "combination" presented separately to individual nostrils. Results showed that mixtures generally yielded higher ratings than combinations on the trigeminal dimensions "intensity," "warm," and "painful," whereas combinations yielded higher ratings than mixtures on the trigeminal dimension "cold." These results suggest dimension-specific interactions in the perception of trigeminal mixtures, which may be explained by particular interactions that may take place on peripheral or central levels. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Acyl transfer from membrane lipids to peptides is a generic process.

    Science.gov (United States)

    Dods, Robert H; Bechinger, Burkhard; Mosely, Jackie A; Sanderson, John M

    2013-11-15

    The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins. © 2013. Published by Elsevier Ltd. All rights reserved.

  10. Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

    2012-11-15

    Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

  11. Structural basis of nonribosomal peptide macrocyclization in fungi.

    Science.gov (United States)

    Zhang, Jinru; Liu, Nicholas; Cacho, Ralph A; Gong, Zhou; Liu, Zhu; Qin, Wenming; Tang, Chun; Tang, Yi; Zhou, Jiahai

    2016-12-01

    Nonribosomal peptide synthetases (NRPSs) in fungi biosynthesize important pharmaceutical compounds, including penicillin, cyclosporine and echinocandin. To understand the fungal strategy of forging the macrocyclic peptide linkage, we determined the crystal structures of the terminal condensation-like (C T ) domain and the holo thiolation (T)-C T complex of Penicillium aethiopicum TqaA. The first, to our knowledge, structural depiction of the terminal module in a fungal NRPS provides a molecular blueprint for generating new macrocyclic peptide natural products.

  12. Lipopolysaccharide interactions of C-terminal peptides from human thrombin.

    Science.gov (United States)

    Singh, Shalini; Kalle, Martina; Papareddy, Praveen; Schmidtchen, Artur; Malmsten, Martin

    2013-05-13

    Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred.

  13. Iterative Mixture Component Pruning Algorithm for Gaussian Mixture PHD Filter

    Directory of Open Access Journals (Sweden)

    Xiaoxi Yan

    2014-01-01

    Full Text Available As far as the increasing number of mixture components in the Gaussian mixture PHD filter is concerned, an iterative mixture component pruning algorithm is proposed. The pruning algorithm is based on maximizing the posterior probability density of the mixture weights. The entropy distribution of the mixture weights is adopted as the prior distribution of mixture component parameters. The iterative update formulations of the mixture weights are derived by Lagrange multiplier and Lambert W function. Mixture components, whose weights become negative during iterative procedure, are pruned by setting corresponding mixture weights to zeros. In addition, multiple mixture components with similar parameters describing the same PHD peak can be merged into one mixture component in the algorithm. Simulation results show that the proposed iterative mixture component pruning algorithm is superior to the typical pruning algorithm based on thresholds.

  14. Interaction of antimicrobial peptides with lipid membranes

    Energy Technology Data Exchange (ETDEWEB)

    Hanulova, Maria

    2008-12-15

    This study aims to investigate the difference in the interaction of antimicrobial peptides with two classes of zwitterionic peptides, phosphatidylethanolamines (PE) and phosphatidylcholines (PC). Further experiments were performed on model membranes prepared from specific bacterial lipids, lipopolysaccharides (LPS) isolated from Salmonella minnesota. The structure of the lipid-peptide aqueous dispersions was studied by small-and wide-angle X-ray diffraction during heating and cooling from 5 to 85 C. The lipids and peptides were mixed at lipid-to-peptide ratios 10-10000 (POPE and POPC) or 2-50 (LPS). All experiments were performed at synchrotron soft condensed matter beamline A2 in Hasylab at Desy in Hamburg, Germany. The phases were identified and the lattice parameters were calculated. Alamethicin and melittin interact in similar ways with the lipids. Pure POPC forms only lamellar phases. POPE forms lamellar phases at low temperatures that upon heating transform into a highly curved inverse hexagonal phase. Insertion of the peptide induced inverse bicontinuous cubic phases which are an ideal compromise between the curvature stress and the packing frustration. Melittin usually induced a mixture of two cubic phases, Im3m and Pn3m, with a ratio of lattice parameters close to 1.279, related to the underlying minimal surfaces. They formed during the lamellar to hexagonal phase transition and persisted during cooling till the onset of the gel phase. The phases formed at different lipid-to-peptide ratios had very similar lattice parameters. Epitaxial relationships existed between coexisting cubic phases and hexagonal or lamellar phases due to confinement of all phases to an onion vesicle, a vesicle with several layers consisting of different lipid phases. Alamethicin induced the same cubic phases, although their formation and lattice parameters were dependent on the peptide concentration. The cubic phases formed during heating from the lamellar phase and their onset

  15. Interaction of antimicrobial peptides with lipid membranes

    International Nuclear Information System (INIS)

    Hanulova, Maria

    2008-12-01

    This study aims to investigate the difference in the interaction of antimicrobial peptides with two classes of zwitterionic peptides, phosphatidylethanolamines (PE) and phosphatidylcholines (PC). Further experiments were performed on model membranes prepared from specific bacterial lipids, lipopolysaccharides (LPS) isolated from Salmonella minnesota. The structure of the lipid-peptide aqueous dispersions was studied by small-and wide-angle X-ray diffraction during heating and cooling from 5 to 85 C. The lipids and peptides were mixed at lipid-to-peptide ratios 10-10000 (POPE and POPC) or 2-50 (LPS). All experiments were performed at synchrotron soft condensed matter beamline A2 in Hasylab at Desy in Hamburg, Germany. The phases were identified and the lattice parameters were calculated. Alamethicin and melittin interact in similar ways with the lipids. Pure POPC forms only lamellar phases. POPE forms lamellar phases at low temperatures that upon heating transform into a highly curved inverse hexagonal phase. Insertion of the peptide induced inverse bicontinuous cubic phases which are an ideal compromise between the curvature stress and the packing frustration. Melittin usually induced a mixture of two cubic phases, Im3m and Pn3m, with a ratio of lattice parameters close to 1.279, related to the underlying minimal surfaces. They formed during the lamellar to hexagonal phase transition and persisted during cooling till the onset of the gel phase. The phases formed at different lipid-to-peptide ratios had very similar lattice parameters. Epitaxial relationships existed between coexisting cubic phases and hexagonal or lamellar phases due to confinement of all phases to an onion vesicle, a vesicle with several layers consisting of different lipid phases. Alamethicin induced the same cubic phases, although their formation and lattice parameters were dependent on the peptide concentration. The cubic phases formed during heating from the lamellar phase and their onset

  16. A Fly-Through Mission Strategy Targeting Peptide as a Signature of Chemical Evolution and Possible Life in Enceladus Plumes

    Science.gov (United States)

    Fujishima, Kosuke; Dziomba, Szymon; Takahagi, Wataru; Shibuya, Takazo; Takano, Yoshinori; Guerrouache, Mohamed; Carbonnier, Benjamin; Takai, Ken; Rothschild, Lynn J.; Yano, Hajime

    2016-01-01

    In situ detection of organic molecules in the extraterrestrial environment provides a key step towards better understanding the variety and the distribution of building blocks of life and it may ultimately lead to finding extraterrestrial life within the Solar System. Here we present combined results of two separate experiments that enable us to realize such in situ life signature detection from the deep habitats of the "Ocean World": a hydrothermal reactor experiment simulating complex organic synthesis and a simulated fly-through capture experiment of organic-bearing microparticles using silica aerogels, followed by subsequent analysis. Both experiments employ peptide as a plausible organics existing in Encleadus plume particles produced in its subsurface ocean. Recent laboratory hydrothermal experiments and a theoretical model on silica saturation indicated an on going hydrothermal reactions in subsurface Enceladus ocean. Given the porous chondritic origin of the core, it is likely that organic compounds originated by radiation chemistry such as amino acid precursors could have been provided, leached, and altered through widespread water-rock interactions. By using the same laboratory experimental setup from the latest water-rock interaction study, we performed amino acid polymerization experiments for 144 days and monitored the organic complexity changing over time. So far over 3,000 peaks up to the size of greater than 600 MW were observed through the analysis of capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) with an indication of amino acid derivatives and short peptides. Generally abiotic polymerization of enantiomeric amino acids results in forming stereoisomeric peptides with identical molecular weight and formula as opposed to homochiral biopolymers. Assuming Enceladus plume particles may contain a mixture of stereoisomeric peptides, we were able to distinguish 16 of the 17 stereoisomeric tripeptides as a test sample using

  17. Peptides in melanoma therapy.

    Science.gov (United States)

    Mocellin, Simone

    2012-01-01

    Peptides derived from tumor associated antigens can be utilized to elicit a therapeutically effective immune response against melanoma in experimental models. However, patient vaccination with peptides - although it is often followed by the induction of melanoma- specific T lymphocytes - is rarely associated with tumor response of clinical relevance. In this review I summarize the principles of peptide design as well as the results so far obtained in the clinical setting while treating cutaneous melanoma by means of this active immunotherapy strategy. I also discuss some immunological and methodological issues that might be helpful for the successful development of peptide-based vaccines.

  18. NetMHCpan 4.0: Improved peptide-MHC class I interaction predictions integrating eluted ligand and peptide binding affinity data

    OpenAIRE

    Jurtz, Vanessa; Paul, Sinu; Andreatta, Massimo; Marcatili, Paolo; Peters, Bjoern; Nielsen, Morten

    2017-01-01

    Cytotoxic T cells are of central importance in the immune systems response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC (major histocompatibility complex) class I molecules. Peptide binding to MHC molecules is the single most selective step in the antigen presentation pathway. On the quest for T cell epitopes, the prediction of peptide binding to MHC molecules has therefore attracted large attention. In the past, predictors of peptide-...

  19. Design and initial characterization of the SC-200 proteomics standard mixture.

    Science.gov (United States)

    Bauman, Andrew; Higdon, Roger; Rapson, Sean; Loiue, Brenton; Hogan, Jason; Stacy, Robin; Napuli, Alberto; Guo, Wenjin; van Voorhis, Wesley; Roach, Jared; Lu, Vincent; Landorf, Elizabeth; Stewart, Elizabeth; Kolker, Natali; Collart, Frank; Myler, Peter; van Belle, Gerald; Kolker, Eugene

    2011-01-01

    High-throughput (HTP) proteomics studies generate large amounts of data. Interpretation of these data requires effective approaches to distinguish noise from biological signal, particularly as instrument and computational capacity increase and studies become more complex. Resolving this issue requires validated and reproducible methods and models, which in turn requires complex experimental and computational standards. The absence of appropriate standards and data sets for validating experimental and computational workflows hinders the development of HTP proteomics methods. Most protein standards are simple mixtures of proteins or peptides, or undercharacterized reference standards in which the identity and concentration of the constituent proteins is unknown. The Seattle Children's 200 (SC-200) proposed proteomics standard mixture is the next step toward developing realistic, fully characterized HTP proteomics standards. The SC-200 exhibits a unique modular design to extend its functionality, and consists of 200 proteins of known identities and molar concentrations from 6 microbial genomes, distributed into 10 molar concentration tiers spanning a 1,000-fold range. We describe the SC-200's design, potential uses, and initial characterization. We identified 84% of SC-200 proteins with an LTQ-Orbitrap and 65% with an LTQ-Velos (false discovery rate = 1% for both). There were obvious trends in success rate, sequence coverage, and spectral counts with protein concentration; however, protein identification, sequence coverage, and spectral counts vary greatly within concentration levels.

  20. Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

    Science.gov (United States)

    Zhang, Yun; Zhang, Hao; Cui, Weidong; Chen, Hao

    2011-09-01

    Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se-S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen ( m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se-S cleavage, analogous to the S-S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se-S of the tag to the S-S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se-S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.

  1. Metal Ion Controlled Polymorphism of a Peptide

    DEFF Research Database (Denmark)

    Hemmingsen, Lars Bo Stegeager; Jancso, Attila; Szunyogh, Daniel

    2011-01-01

    ions on fully or partially unstructured proteins, or the effect of metal ions on protein aggregation. Metal ions may be employed to fold (or misfold) individual peptides in a controlled manner depending on the potential metal ion coordinating amino acid side chains (Cys, His, Asp, Glu......In this work a metal ion binding model dodecapeptide was investigated in terms of its capacity to adopt different structures depending on the metal ion to peptide stoichiometry. The dodecapeptide is much simpler than real proteins, yet displays sufficient complexity to model the effect of metal......, …) in the peptide, and the ligand and structural preferences of the metal ion (in our studies Zn2+, Cd2+, Hg2+, Cu+/2+). Simultaneously, new species such as metal ion bridged ternary complexes or even oligomers may be formed. In recent previous studies we have observed similar polymorphism of zinc finger model...

  2. Rapid purification of radioiodinated peptides with Sep-Pak reversed phase cartridges and HPLC

    International Nuclear Information System (INIS)

    Miller, J.J.; Schultz, G.S.; Levy, R.S.

    1984-01-01

    A simple, rapid method is described for the purification of radioiodinated peptides for use in radioimmuno- and in radioreceptor assays. Iodinated reaction mixtures are applied directly onto Sep-Pak disposable, reversed phase cartridges equilibrated with phosphate buffer. Unreacted 125-iodide and other non-peptide reaction components are eluted with buffer. The peptide fraction is then eluted with 70% buffer:30% acetonitrile. The peptide fraction is further purified by reversed phase high pressure liquid chromatography to separate the native peptide and the mono- and diiodo-derivatives. In this study the method is used to prepare 125-iodide-labeled monoiodo-leucine enkephalin and monoiodo-angiotensin II, which are free of the parent peptides and diiodo-derivatives and are of maximum obtainable specific radioactivity. The usefulness of these labeled peptides in radioimmuno- and radioreceptor assays is demonstrated by their binding to specific antibodies and receptors, respectively. (author)

  3. Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

    Science.gov (United States)

    Kabytaev, Kuanysh; Durairaj, Anita; Shin, Dmitriy; Rohlfing, Curt L; Connolly, Shawn; Little, Randie R; Stoyanov, Alexander V

    2016-02-01

    A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Insulin C-peptide test

    Science.gov (United States)

    C-peptide ... the test depends on the reason for the C-peptide measurement. Ask your health care provider if ... C-peptide is measured to tell the difference between insulin the body produces and insulin someone injects ...

  5. Immunogenicity of peptides of measles virus origin and influence of adjuvants.

    Science.gov (United States)

    Halassy, Beata; Mateljak, Sanja; Bouche, Fabienne B; Pütz, Mike M; Muller, Claude P; Frkanec, Ruza; Habjanec, Lidija; Tomasić, Jelka

    2006-01-12

    Epitope-based peptide antigens have been under development for protection against measles virus. The immunogenicity of five peptides composed of the same B cell epitope (BCE) (H236-250 of the measles virus hemagglutinin), and different T cell epitopes of measles virus fusion protein (F421-435, F256-270, F288-302) and nucleoprotein (NP335-345) was studied in mice (subcutaneous immunisation). The adjuvant effects of peptidoglycan monomer (PGM), Montanide ISA 720 and 206 were also investigated. Results showed basic differences in peptide immunogenicity that were consistent with already described structural differences. PGM elevated peptide-specific IgG when applied together with four of five tested peptides. A strong synergistic effect was observed after co-immunisation of mice with a mixture containing all five chimeric peptides in small and equal amounts. Results revealed for the first time that immunisation with several peptides having the common BCE generated significantly higher levels of both anti-peptide and anti-BCE IgG in comparison to those obtained after immunisation with a single peptide in much higher quantity. Further improvement of immune response was obtained after incorporation of such a peptide mixture into oil-based adjuvants.

  6. Complexation behavior of oppositely charged polyelectrolytes: Effect of charge distribution

    International Nuclear Information System (INIS)

    Zhao, Mingtian; Li, Baohui; Zhou, Jihan; Su, Cuicui; Niu, Lin; Liang, Dehai

    2015-01-01

    Complexation behavior of oppositely charged polyelectrolytes in a solution is investigated using a combination of computer simulations and experiments, focusing on the influence of polyelectrolyte charge distributions along the chains on the structure of the polyelectrolyte complexes. The simulations are performed using Monte Carlo with the replica-exchange algorithm for three model systems where each system is composed of a mixture of two types of oppositely charged model polyelectrolyte chains (EGEG) 5 /(KGKG) 5 , (EEGG) 5 /(KKGG) 5 , and (EEGG) 5 /(KGKG) 5 , in a solution including explicit solvent molecules. Among the three model systems, only the charge distributions along the chains are not identical. Thermodynamic quantities are calculated as a function of temperature (or ionic strength), and the microscopic structures of complexes are examined. It is found that the three systems have different transition temperatures, and form complexes with different sizes, structures, and densities at a given temperature. Complex microscopic structures with an alternating arrangement of one monolayer of E/K monomers and one monolayer of G monomers, with one bilayer of E and K monomers and one bilayer of G monomers, and with a mixture of monolayer and bilayer of E/K monomers in a box shape and a trilayer of G monomers inside the box are obtained for the three mixture systems, respectively. The experiments are carried out for three systems where each is composed of a mixture of two types of oppositely charged peptide chains. Each peptide chain is composed of Lysine (K) and glycine (G) or glutamate (E) and G, in solution, and the chain length and amino acid sequences, and hence the charge distribution, are precisely controlled, and all of them are identical with those for the corresponding model chain. The complexation behavior and complex structures are characterized through laser light scattering and atomic force microscopy measurements. The order of the apparent weight

  7. Modelling interactions in grass-clover mixtures

    NARCIS (Netherlands)

    Nassiri Mahallati, M.

    1998-01-01

    The study described in this thesis focuses on a quantitative understanding of the complex interactions in binary mixtures of perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) under cutting. The first part of the study describes the dynamics of growth, production

  8. Modeling text with generalizable Gaussian mixtures

    DEFF Research Database (Denmark)

    Hansen, Lars Kai; Sigurdsson, Sigurdur; Kolenda, Thomas

    2000-01-01

    We apply and discuss generalizable Gaussian mixture (GGM) models for text mining. The model automatically adapts model complexity for a given text representation. We show that the generalizability of these models depends on the dimensionality of the representation and the sample size. We discuss...

  9. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

  10. Atrial natriuretic peptides in plasma

    DEFF Research Database (Denmark)

    Goetze, Jens Peter; Hansen, Lasse H; Terzic, Dijana

    2014-01-01

    Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone....... These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid...

  11. Atrial natriuretic peptides in plasma

    DEFF Research Database (Denmark)

    Goetze, Jens P; Holst Hansen, Lasse; Terzic, Dijana

    2015-01-01

    Measurement of cardiac natriuretic peptides in plasma has gained a diagnostic role in the assessment of heart failure. Plasma measurement is though hampered by the marked instability of the hormones, which has led to the development of analyses that target N-terminal fragments from the prohormone....... These fragments are stable in plasma and represent surrogate markers of the actual natriuretic hormone. Post-translational processing of the precursors, however, is revealing itself to be a complex event with new information still being reported on proteolysis, covalent modifications, and amino acid...

  12. Structural basis for precursor protein-directed ribosomal peptide macrocyclization

    Science.gov (United States)

    Li, Kunhua; Condurso, Heather L.; Li, Gengnan; Ding, Yousong; Bruner, Steven D.

    2016-01-01

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides whose members target proteases with potent reversible inhibition. The product structure is constructed by three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here, we describe the detailed structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases, MdnC and MdnB, interact with a conserved α-helix of the precursor peptide using a novel precursor peptide recognition mechanism. The results provide insight into the unique protein/protein interactions key to the chemistry, suggest an origin of the natural combinatorial synthesis of microviridin peptides and provide a framework for future engineering efforts to generate designed compounds. PMID:27669417

  13. Structural basis for precursor protein-directed ribosomal peptide macrocyclization.

    Science.gov (United States)

    Li, Kunhua; Condurso, Heather L; Li, Gengnan; Ding, Yousong; Bruner, Steven D

    2016-11-01

    Macrocyclization is a common feature of natural product biosynthetic pathways including the diverse family of ribosomal peptides. Microviridins are architecturally complex cyanobacterial ribosomal peptides that target proteases with potent reversible inhibition. The product structure is constructed via three macrocyclizations catalyzed sequentially by two members of the ATP-grasp family, a unique strategy for ribosomal peptide macrocyclization. Here we describe in detail the structural basis for the enzyme-catalyzed macrocyclizations in the microviridin J pathway of Microcystis aeruginosa. The macrocyclases MdnC and MdnB interact with a conserved α-helix of the precursor peptide using a novel precursor-peptide recognition mechanism. The results provide insight into the unique protein-protein interactions that are key to the chemistry, suggest an origin for the natural combinatorial synthesis of microviridin peptides, and provide a framework for future engineering efforts to generate designed compounds.

  14. Soluble elastin peptides in cardiovascular homeostasis: Foe or ally.

    Science.gov (United States)

    Qin, Zhenyu

    2015-05-01

    Elastin peptides, also known as elastin-derived peptides or elastokines, are soluble polypeptides in blood and tissue. The blood levels of elastin peptides are usually low but can increase during cardiovascular diseases, such as atherosclerosis, aortic aneurysm and diabetes with vascular complications. Generally, elastin peptides are derived from the degradation of insoluble elastic polymers. The biological activities of elastin peptides are bidirectional, e.g., a pro-inflammatory effect on monocyte migration induction vs. a protective effect on vasodilation promotion. However, recent in vivo studies have demonstrated that elastin peptides promote the formation of atherosclerotic plaques in hypercholesterolemic mice and induce hyperglycemia and elevations in plasma lipid levels in fasted mice. More important, the detrimental effects induced by elastin peptides can be largely inhibited by genetic or pharmacological blockade of the elastin receptor complex or by neutralization of an antibody against elastin peptides. These studies indicate new therapeutic strategies for the treatment of cardiovascular diseases by targeting elastin peptide metabolism. Therefore, the goal of this review is to summarize current knowledge about elastin peptides relevant to cardiovascular pathologies to further delineate their potential application in cardiovascular disease. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Peptide stabilized amphotericin B nanodisks

    Science.gov (United States)

    Tufteland, Megan; Pesavento, Joseph B.; Bermingham, Rachelle L.; Hoeprich, Paul D.; Ryan, Robert O.

    2007-01-01

    Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic α-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a ~7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv = 7.7 × 104. Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND. PMID:17293004

  16. pH dependence of MHC class I-restricted peptide presentation

    DEFF Research Database (Denmark)

    Stryhn, A; Pedersen, L O; Romme, T

    1996-01-01

    The function of MHC class I molecules is to bind and present antigenic peptides to cytotoxic T cells. Here, we report that class I-restricted peptide presentation is strongly pH dependent. The presentation of some peptides was enhanced at acidic pH, whereas the presentation of others was inhibited....... Biochemical peptide-MHC class I binding assays demonstrated that peptide-MHC class I complexes are more stable at neutral pH than at acidic pH. We suggest that acid-dependent peptide dissociation can generate empty class I molecules and that the resulting binding potential can be exploited by a subset...

  17. Complexity Plots

    KAUST Repository

    Thiyagalingam, Jeyarajan

    2013-06-01

    In this paper, we present a novel visualization technique for assisting the observation and analysis of algorithmic complexity. In comparison with conventional line graphs, this new technique is not sensitive to the units of measurement, allowing multivariate data series of different physical qualities (e.g., time, space and energy) to be juxtaposed together conveniently and consistently. It supports multivariate visualization as well as uncertainty visualization. It enables users to focus on algorithm categorization by complexity classes, while reducing visual impact caused by constants and algorithmic components that are insignificant to complexity analysis. It provides an effective means for observing the algorithmic complexity of programs with a mixture of algorithms and black-box software through visualization. Through two case studies, we demonstrate the effectiveness of complexity plots in complexity analysis in research, education and application. © 2013 The Author(s) Computer Graphics Forum © 2013 The Eurographics Association and Blackwell Publishing Ltd.

  18. Genotoxicity and cytotoxicity response to environmentally relevant complex metal mixture (Zn, Cu, Ni, Cr, Pb, Cd) accumulated in Atlantic salmon (Salmo salar). Part I: importance of exposure time and tissue dependence.

    Science.gov (United States)

    Stankevičiūtė, Milda; Sauliutė, Gintarė; Svecevičius, Gintaras; Kazlauskienė, Nijolė; Baršienė, Janina

    2017-10-01

    Health impact of metal mixture at environment realistic concentrations are difficult to predict especially for long-term effects where cause-and-effect relationships may not be directly obvious. This study was aimed to evaluate metal mixture (Zn-0.1, Cu-0.01, Ni-0.01, Cr-0.01, Pb-0.005 and Cd-0.005 mg/L, respectively for 1, 2, 4, 7, 14 and 28 days at concentrations accepted for the inland waters in EU) genotoxicity (micronuclei, nuclear buds, nuclear buds on filament), cytotoxicity (8-shaped nuclei, fragmented-apoptotic erythrocytes), bioaccumulation, steady-state and the reference level of geno-cytotoxicity in hatchery-reared Atlantic salmon tissues. Metals accumulated mostly in gills and kidneys, to the lesser extent in the muscle. Uptake of metals from an entire mixture in the fish for 14 days is sufficient to reach steady-state Cr, Pb concentrations in all tissues; Zn, Cu-in kidneys and muscle, Ni-in liver, kidneys, muscle and Cd-in muscle. Treatment with metal mixture significantly increased summed genotoxicity levels at 7 days of exposure in peripheral blood and liver erythrocytes, at 14 days of exposure in gills and kidney erythrocytes. Significant elevation of cytotoxicity was detected after 2 and 14 days of exposure in gills erythrocytes and after 28 days-in peripheral blood erythrocytes. The amount of Cu, Cr, Pb and Cd accumulated in tissues was dependent upon duration of exposure; nuclear buds, 8-shaped nuclei frequencies also were dependent upon duration of exposure. This study indicates that metals at low levels when existing in mixture causes significant geno-cytotoxicity responses and metals bioaccumulation in salmon.

  19. Mixtures Estimation and Applications

    CERN Document Server

    Mengersen, Kerrie; Titterington, Mike

    2011-01-01

    This book uses the EM (expectation maximization) algorithm to simultaneously estimate the missing data and unknown parameter(s) associated with a data set. The parameters describe the component distributions of the mixture; the distributions may be continuous or discrete. The editors provide a complete account of the applications, mathematical structure and statistical analysis of finite mixture distributions along with MCMC computational methods, together with a range of detailed discussions covering the applications of the methods and features chapters from the leading experts on the subject

  20. Nanotexture Optimization by Oxygen Plasma of Mesoporous Silica Thin Film for Enrichment of Low Molecular Weight Peptides Captured from Human Serum

    Science.gov (United States)

    Hu, Ye; Peng, Yang; Brousseau, Louis; Bouamrani, Ali; Liu, Xuewu; Ferrari, Mauro

    2010-01-01

    This study investigated the optimization of mesoporous silica thin films by nanotexturing using oxygen plasma versus thermal oxidation. Calcination in oxygen plasma provides superior control over pore formation with regard to the pore surface and higher fidelity to the structure of the polymer template. The resulting porous film offers an ideal substrate for the selective partitioning of peptides from complex mixtures. The improved chemico-physical characteristics of porous thin films (pore size distribution, nanostructure, surface properties and pore connectivity) were systematically characterized with XRD, Ellipsometry, FTIR, TEM and N2 adsorption/desorption. The enrichment of low molecular weight proteins captured from human serum on mesoporous silica thin films fabricated by both methodologies were investigated by comparison of their MALDI-TOF MS profiles. This novel on-chip fractionation technology offers advantages in recovering the low molecular weight peptides from human serum, which has been recognized as an informative resource for early diagnosis of cancer and other diseases. PMID:21179395

  1. Microwave heating in solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Pedersen, Søren Ljungberg; Shelton, Anne Pernille Tofteng; Malik, Leila

    2012-01-01

    synthesis, precise microwave irradiation to heat the reaction mixture during coupling and N(a)-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially...... relevant for sequences which might form ß-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating...... in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of ß-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references)....

  2. On modeling of structured multiphase mixtures

    International Nuclear Information System (INIS)

    Dobran, F.

    1987-01-01

    The usual modeling of multiphase mixtures involves a set of conservation and balance equations of mass, momentum, energy and entropy (the basic set) constructed by an averaging procedure or postulated. The averaged models are constructed by averaging, over space or time segments, the local macroscopic field equations of each phase, whereas the postulated models are usually motivated by the single phase multicomponent mixture models. In both situations, the resulting equations yield superimposed continua models and are closed by the constitutive equations which place restrictions on the possible material response during the motion and phase change. In modeling the structured multiphase mixtures, the modeling of intrinsic motion of grains or particles is accomplished by adjoining to the basic set of field equations the additional balance equations, thereby placing restrictions on the motion of phases only within the imposed extrinsic and intrinsic sources. The use of the additional balance equations has been primarily advocated in the postulatory theories of multiphase mixtures and are usually derived through very special assumptions of the material deformation. Nevertheless, the resulting mixture models can predict a wide variety of complex phenomena such as the Mohr-Coulomb yield criterion in granular media, Rayleigh bubble equation, wave dispersion and dilatancy. Fundamental to the construction of structured models of multiphase mixtures are the problems pertaining to the existence and number of additional balance equations to model the structural characteristics of a mixture. Utilizing a volume averaging procedure it is possible not only to derive the basic set of field equation discussed above, but also a very general set of additional balance equations for modeling of structural properties of the mixture

  3. Skin Delivery of EGCG and Silibinin: Potential of Peptide Dendrimers for Enhanced Skin Permeation and Deposition.

    Science.gov (United States)

    Shetty, Pallavi Krishna; Manikkath, Jyothsna; Tupally, Karnaker; Kokil, Ganesh; Hegde, Aswathi R; Raut, Sushil Y; Parekh, Harendra S; Mutalik, Srinivas

    2017-08-01

    The aim of the present study was to evaluate the ability of the peptide dendrimers to facilitate transdermal delivery of antioxidants, silibinin, and epigallocatechin-3-gallate (EGCG). Drug-peptide dendrimer complexes were prepared and evaluated for their ability to permeate across the skin. The data revealed the ready formation of complexes between drug and peptide dendrimer in a molar ratio of 1:1. In vitro permeation studies using excised rat skin and drug-peptide dendrimer complexes showed highest values for cumulative drug permeation at the end of 12 h (Q 12 ), with corresponding permeability coefficient (Kp) and enhancement ratio values also determined at this time point. With silibinin, 3.96-, 1.81-, and 1.06-fold increase in skin permeation was observed from silibinin-peptide dendrimer complex, simultaneous application of silibinin + peptide dendrimer, and pretreatment of skin with peptide dendrimer, respectively, in comparison with passive diffusion. With EGCG, 9.82-, 2.04-, and 1.72-fold increase in skin permeation was observed from EGCG-peptide dendrimer complex, simultaneous application of EGCG + peptide dendrimer, and pretreatment of skin with peptide dendrimer, respectively, in comparison with passive diffusion. The present study demonstrates the application of peptide dendrimers in effectively delivering antioxidants such as EGCG and silibinin into the skin, thus offering the potential to provide antioxidant effects when delivered via appropriately formulated topical preparations.

  4. High pH reversed-phase chromatography as a superior fractionation scheme compared to off-gel isoelectric focusing for complex proteome analysis.

    Science.gov (United States)

    Stein, Derek R; Hu, Xiaojie; McCorrister, Stuart J; Westmacott, Garrett R; Plummer, Francis A; Ball, Terry B; Carpenter, Michael S

    2013-10-01

    MS/MS is the technology of choice for analyzing complex protein mixtures. However, due to the intrinsic complexity and dynamic range present in higher eukaryotic proteomes, prefractionation is an important step to maximize the number of proteins identified. Off-gel IEF (OG-IEF) and high pH RP (Hp-RP) column chromatography have both been successfully utilized as a first-dimension peptide separation technique in shotgun proteomic experiments. Here, a direct comparison of the two methodologies was performed on ex vivo peripheral blood mononuclear cell lysate. In 12-fraction replicate analysis, Hp-RP resulted in more peptides and proteins identified than OG-IEF fractionation. Distributions of peptide pIs and hydropathy did not reveal any appreciable bias in either technique. Resolution, defined here as the ability to limit a specific peptide to one particular fraction, was significantly better for Hp-RP. This leads to a more uniform distribution of total and unique peptides for Hp-RP across all fractions collected. These results suggest that fractionation by Hp-RP over OG-IEF is the better choice for typical complex proteome analysis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. I-optimal mixture designs

    OpenAIRE

    GOOS, Peter; JONES, Bradley; SYAFITRI, Utami

    2013-01-01

    In mixture experiments, the factors under study are proportions of the ingredients of a mixture. The special nature of the factors in a mixture experiment necessitates specific types of regression models, and specific types of experimental designs. Although mixture experiments usually are intended to predict the response(s) for all possible formulations of the mixture and to identify optimal proportions for each of the ingredients, little research has been done concerning their I-optimal desi...

  6. Systematic Moiety Variations of Ultrashort Peptides Produce Profound Effects on Self-Assembly, Nanostructure Formation, Hydrogelation, and Phase Transition

    KAUST Repository

    Chan, Kiat Hwa; Xue, Bo; Robinson, Robert C.; Hauser, Charlotte

    2017-01-01

    Self-assembly of small biomolecules is a prevalent phenomenon that is increasingly being recognised to hold the key to building complex structures from simple monomeric units. Small peptides, in particular ultrashort peptides containing up to seven

  7. Radio peptide imaging and therapy

    International Nuclear Information System (INIS)

    Buscombe, Jonh

    1997-01-01

    Full text. The concept of the magic bullet retains its attraction to us. If only we could take a drug or radioisotope and inject this intravenously and then will attach to the target cancer. This may allow imaging if labelled with a radio pharmaceutical or possibly even effective therapy. Initially work was started using antibodies of mouse origin. These have shown some utility in targeting tumors but there are problems in that these are essentially non-human proteins, often derived from mice. This leads to the formation of antibodies against that antibody so that repeat administrations lead to reduced efficacy and possibly may carry a risk anaphylaxis for the patient. Two different methods have evolved to deal with this situation. Either make antibodies more human or use smaller fragments, so that they are less likely to cause allergic reactions. The second method is to try and use a synthetic peptide. This will contain a series of amino acids which recognize a certain cell receptor. For example the somatostatin analogue Octreotide is an 8 amino acid peptide which has the same biological actions as natural somatostatin but an increased plasma half life. To this is added a linker a good example being DTPA and then radioisotope for example In-111. There we can have the complex In-111-DTPA-Octreotide which can be used to image somatostatin receptors in vivo. The main advantage over antibodies is that the cost production is less and many different variation of peptides for a particular receptor can be manufactured and assessed to find which is the optimal agent tumour imaging at a fraction of the cost of antibody production. There are two main approaches. Firstly to take a natural peptide hormone such as insulin or VIP and label by a simple method such as iodination with I-123. A group in Vienna have done it and shown good uptake of I-123 Insulin in primary hepatomas and of I-123 VIP in pancreatic cancers. Many natural peptide hormones however have a short plasma half

  8. Diversity-oriented peptide stapling

    DEFF Research Database (Denmark)

    Tran, Thu Phuong; Larsen, Christian Ørnbøl; Røndbjerg, Tobias

    2017-01-01

    as a powerful method for peptide stapling. However, to date CuAAC stapling has not provided a simple method for obtaining peptides that are easily diversified further. In the present study, we report a new diversity-oriented peptide stapling (DOPS) methodology based on CuAAC chemistry. Stapling of peptides...

  9. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.

    1995-01-01

    Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

  10. Tumor penetrating peptides

    Directory of Open Access Journals (Sweden)

    Tambet eTeesalu

    2013-08-01

    Full Text Available Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC, contains the integrin-binding RGD motif. RGD mediates tumor homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular zip code of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is

  11. A Universal Method for Fishing Target Proteins from Mixtures of Biomolecules using Isothermal Titration Calorimetry

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, X.; Sun, Q; Kini, R; Sivaraman, J

    2008-01-01

    The most challenging tasks in biology include the identification of (1) the orphan receptor for a ligand, (2) the ligand for an orphan receptor protein, and (3) the target protein(s) for a given drug or a lead compound that are critical for the pharmacological or side effects. At present, several approaches are available, including cell- or animal-based assays, affinity labeling, solid-phase binding assays, surface plasmon resonance, and nuclear magnetic resonance. Most of these techniques are not easy to apply when the target protein is unknown and the compound is not amenable to labeling, chemical modification, or immobilization. Here we demonstrate a new universal method for fishing orphan target proteins from a complex mixture of biomolecules using isothermal titration calorimetry (ITC) as a tracking tool. We took snake venom, a crude mixture of several hundred proteins/peptides, as a model to demonstrate our proposed ITC method in tracking the isolation and purification of two distinct target proteins, a major component and a minor component. Identities of fished out target proteins were confirmed by amino acid sequencing and inhibition assays. This method has the potential to make a significant advancement in the area of identifying orphan target proteins and inhibitor screening in drug discovery and characterization.

  12. Perspective of Use of Antiviral Peptides against Influenza Virus

    Directory of Open Access Journals (Sweden)

    Sylvie Skalickova

    2015-10-01

    Full Text Available The threat of a worldwide influenza pandemic has greatly increased over the past decade with the emergence of highly virulent avian influenza strains. The increased frequency of drug-resistant influenza strains against currently available antiviral drugs requires urgent development of new strategies for antiviral therapy, too. The research in the field of therapeutic peptides began to develop extensively in the second half of the 20th century. Since then, the mechanisms of action for several peptides and their antiviral prospect received large attention due to the global threat posed by viruses. Here, we discussed the therapeutic properties of peptides used in influenza treatment. Peptides with antiviral activity against influenza can be divided into three main groups. First, entry blocker peptides such as a Flupep that interact with influenza hemagglutinin, block its binding to host cells and prevent viral fusion. Second, several peptides display virucidal activity, disrupting viral envelopes, e.g., Melittin. Finally, a third set of peptides interacts with the viral polymerase complex and act as viral replication inhibitors such as PB1 derived peptides. Here, we present a review of the current literature describing the antiviral activity, mechanism and future therapeutic potential of these influenza antiviral peptides.

  13. Maturation processes and structures of small secreted peptides in plants

    Directory of Open Access Journals (Sweden)

    Ryo eTabata

    2014-07-01

    Full Text Available In the past decade, small secreted peptides have proven to be essential for various aspects of plant growth and development, including the maintenance of certain stem cell populations. Most small secreted peptides identified in plants to date are recognised by membrane-localized receptor kinases, the largest family of receptor proteins in the plant genome. This peptide-receptor interaction is essential for initiating intracellular signalling cascades. Small secreted peptides often undergo post-translational modifications and proteolytic processing to generate the mature peptides. Recent studies suggest that, in contrast to the situation in mammals, the proteolytic processing of plant peptides involves a number of complex steps. Furthermore, NMR-based structural analysis demonstrated that post-translational modifications induce the conformational changes needed for full activity. In this mini review, we summarise recent advances in our understanding of how small secreted peptides are modified and processed into biologically active peptides and describe the mature structures of small secreted peptides in plants.

  14. Reactions of tritium atoms with amino acids, deuterated amino acids and mixtures of amino acids. Additivity property and isotope effect

    International Nuclear Information System (INIS)

    Badun, G.A.; Filatov, Eh.S.

    1988-01-01

    Interaction of tritium atoms with glycine (1) and leucine (2) amino acids, deuterated amino acids, their mixtures and glycylleucine (3) peptide in the 77-300 K temperature range is studied in isothermal and gradient regimes. Tagged amino acids were separated from targets after conducting the reaction. At T 150 K are associated with intermolecular transmission of free valence in the mixture of amino acids. Regularities of the reaction found for the mixture of amino acids are conserved for (3) as well, i.e. the peptide bond does not essentially affect the reaction of isotopic exchange conditioned by atomic tritium

  15. EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond

    Science.gov (United States)

    Samgina, Tatiana Yu; Kovalev, Sergey V.; Tolpina, Miriam D.; Trebse, Polonca; Torkar, Gregor; Lebedev, Albert T.

    2018-05-01

    Our scientific interests involve de novo sequencing of non-tryptic natural amphibian skin peptides including those with intramolecular S-S bond by means of exclusively mass spectrometry. Reliable discrimination of the isomeric leucine/isoleucine residues during peptide sequencing by means of mass spectrometry represents a bottleneck in the workflow for complete automation of the primary structure elucidation of these compounds. MS3 is capable of solving the problem. Earlier we demonstrated the advanced efficiency of ETD-HCD method to discriminate Leu/Ile in individual peptides by consecutive application of ETD to the polyprotonated peptides followed by HCD applied to the manually selected primary z-ions with the targeted isomeric residues at their N-termini and registration of the characteristic w-ions. Later this approach was extended to deal with several (4-7) broad band mass ranges, without special isolation of the primary z-ions. The present paper demonstrates an advanced version of this method when EThcD is applied in the whole mass range to a complex mixture of natural non-tryptic peptides without their separation and intermediate isolation of the targeted z-ions. The proposed EThcD method showed over 81% efficiency for the large natural peptides with intact disulfide ring, while the interfering process of radical site migration is suppressed. Due to higher speed and sensitivity, the proposed EThcD approach facilitates the analytical procedure and allows for the automation of the entire experiment and data processing. Moreover, in some cases it gives a chance to establish the nature of the residues in the intact intramolecular disulfide loops. [Figure not available: see fulltext.

  16. Mixture based outlier filtration

    Czech Academy of Sciences Publication Activity Database

    Pecherková, Pavla; Nagy, Ivan

    2006-01-01

    Roč. 46, č. 2 (2006), s. 30-35 ISSN 1210-2709 R&D Projects: GA MŠk 1M0572; GA MDS 1F43A/003/120 Institutional research plan: CEZ:AV0Z10750506 Keywords : data filtration * system modelling * mixture models Subject RIV: BD - Theory of Information http://library.utia.cas.cz/prace/20060165.pdf

  17. Extracting Markov Models of Peptide Conformational Dynamics from Simulation Data.

    Science.gov (United States)

    Schultheis, Verena; Hirschberger, Thomas; Carstens, Heiko; Tavan, Paul

    2005-07-01

    A high-dimensional time series obtained by simulating a complex and stochastic dynamical system (like a peptide in solution) may code an underlying multiple-state Markov process. We present a computational approach to most plausibly identify and reconstruct this process from the simulated trajectory. Using a mixture of normal distributions we first construct a maximum likelihood estimate of the point density associated with this time series and thus obtain a density-oriented partition of the data space. This discretization allows us to estimate the transfer operator as a matrix of moderate dimension at sufficient statistics. A nonlinear dynamics involving that matrix and, alternatively, a deterministic coarse-graining procedure are employed to construct respective hierarchies of Markov models, from which the model most plausibly mapping the generating stochastic process is selected by consideration of certain observables. Within both procedures the data are classified in terms of prototypical points, the conformations, marking the various Markov states. As a typical example, the approach is applied to analyze the conformational dynamics of a tripeptide in solution. The corresponding high-dimensional time series has been obtained from an extended molecular dynamics simulation.

  18. Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses.

    Science.gov (United States)

    Kinumi, Tomoya; Goto, Mari; Eyama, Sakae; Kato, Megumi; Kasama, Takeshi; Takatsu, Akiko

    2012-07-01

    A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of β-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography-mass spectrometry and hydrophilic chromatography-mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.

  19. Mixture for plugging absorption zones

    Energy Technology Data Exchange (ETDEWEB)

    Sitinkov, G V; Kovalenko, N G; Makarov, L V; Zinnatulchin, Ts Kh

    1981-01-17

    A mixture is proposed for plugging absorption zones. The mixture contains synthetic polymer and a solvent. So as to increase the penetrability of the mixture through a reduction in its viscosity and an increase in insulation properties, the compound contains either Capron or Neilon as the synthetic polyamide resin polmyer, and concentrated chloride as the solvent. The mixture is prepared in a special AzINMASh-30 unit (acid cart). After the mixture has been produced, it is injected into the borehole by means of an acid cart pump. So as to prevent coaggulation at the point when the mixture in injected into the stratum through tubes, the mixture is placed betwen chemically inert fluids, for example, a clay mortar. The inert and compressed fluids are injected by means of a cementing unit. The entire process of production and application of the mixture is simple and fully automated through the use of well-known equipment.

  20. Bayesian mixture analysis for metagenomic community profiling.

    Science.gov (United States)

    Morfopoulou, Sofia; Plagnol, Vincent

    2015-09-15

    Deep sequencing of clinical samples is now an established tool for the detection of infectious pathogens, with direct medical applications. The large amount of data generated produces an opportunity to detect species even at very low levels, provided that computational tools can effectively profile the relevant metagenomic communities. Data interpretation is complicated by the fact that short sequencing reads can match multiple organisms and by the lack of completeness of existing databases, in particular for viral pathogens. Here we present metaMix, a Bayesian mixture model framework for resolving complex metagenomic mixtures. We show that the use of parallel Monte Carlo Markov chains for the exploration of the species space enables the identification of the set of species most likely to contribute to the mixture. We demonstrate the greater accuracy of metaMix compared with relevant methods, particularly for profiling complex communities consisting of several related species. We designed metaMix specifically for the analysis of deep transcriptome sequencing datasets, with a focus on viral pathogen detection; however, the principles are generally applicable to all types of metagenomic mixtures. metaMix is implemented as a user friendly R package, freely available on CRAN: http://cran.r-project.org/web/packages/metaMix sofia.morfopoulou.10@ucl.ac.uk Supplementary data are available at Bionformatics online. © The Author 2015. Published by Oxford University Press.

  1. Environmentally relevant chemical mixtures of concern in waters of United States tributaries to the Great Lakes

    Science.gov (United States)

    Elliott, Sarah M.; Brigham, Mark E.; Kiesling, Richard L.; Schoenfuss, Heiko L.; Jorgenson, Zachary G.

    2018-01-01

    The North American Great Lakes are a vital natural resource that provide fish and wildlife habitat, as well as drinking water and waste assimilation services for millions of people. Tributaries to the Great Lakes receive chemical inputs from various point and nonpoint sources, and thus are expected to have complex mixtures of chemicals. However, our understanding of the co‐occurrence of specific chemicals in complex mixtures is limited. To better understand the occurrence of specific chemical mixtures in the US Great Lakes Basin, surface water from 24 US tributaries to the Laurentian Great Lakes was collected and analyzed for diverse suites of organic chemicals, primarily focused on chemicals of concern (e.g., pharmaceuticals, personal care products, fragrances). A total of 181 samples and 21 chemical classes were assessed for mixture compositions. Basin wide, 1664 mixtures occurred in at least 25% of sites. The most complex mixtures identified comprised 9 chemical classes and occurred in 58% of sampled tributaries. Pharmaceuticals typically occurred in complex mixtures, reflecting pharmaceutical‐use patterns and wastewater facility outfall influences. Fewer mixtures were identified at lake or lake‐influenced sites than at riverine sites. As mixture complexity increased, the probability of a specific mixture occurring more often than by chance greatly increased, highlighting the importance of understanding source contributions to the environment. This empirically based analysis of mixture composition and occurrence may be used to focus future sampling efforts or mixture toxicity assessments. 

  2. Plasma glucagon-like peptide 1 and peptide YY levels are not altered in symptomatic fructose-sorbitol malabsorption

    DEFF Research Database (Denmark)

    Valeur, Jørgen; Øines, Eliann; Morken, Mette Helvik

    2008-01-01

    consecutive patients with functional abdominal complaints, referred to our clinic for investigation of self-reported food hypersensitivity, were included in the study and compared with 15 healthy volunteers. All subjects ingested a mixture of 25 g fructose and 5 g sorbitol. Pulmonary hydrogen and methane...... excretion and plasma glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) levels were measured during the next 3 h. Both habitual and post-test symptoms were assessed. RESULTS: Malabsorption of fructose and sorbitol was present in 61% of the patients and 73% of the controls. Nevertheless, the patients...

  3. Separating Underdetermined Convolutive Speech Mixtures

    DEFF Research Database (Denmark)

    Pedersen, Michael Syskind; Wang, DeLiang; Larsen, Jan

    2006-01-01

    a method for underdetermined blind source separation of convolutive mixtures. The proposed framework is applicable for separation of instantaneous as well as convolutive speech mixtures. It is possible to iteratively extract each speech signal from the mixture by combining blind source separation...

  4. Mixtures of truncated basis functions

    DEFF Research Database (Denmark)

    Langseth, Helge; Nielsen, Thomas Dyhre; Rumí, Rafael

    2012-01-01

    In this paper we propose a framework, called mixtures of truncated basis functions (MoTBFs), for representing general hybrid Bayesian networks. The proposed framework generalizes both the mixture of truncated exponentials (MTEs) framework and the mixture of polynomials (MoPs) framework. Similar t...

  5. Nitrogen Heterocycles in Miller-Urey Spark-Discharge Mixtures: Using Chemical Trends to Elucidate Plausible Pre-RNAs on the Early Earth

    Science.gov (United States)

    Rodriguez, L. E.; House, C. H.; Callahan, M. P.

    2017-07-01

    We incubated 53 nitrogen heterocycles with spark-discharge mixtures and found that they react with only a handful of nitriles to yield adducts that may polymerize. Whether these adducts can form a monomer of Peptide Nucleic Acid was investigated.

  6. Artificial neural network study on organ-targeting peptides

    Science.gov (United States)

    Jung, Eunkyoung; Kim, Junhyoung; Choi, Seung-Hoon; Kim, Minkyoung; Rhee, Hokyoung; Shin, Jae-Min; Choi, Kihang; Kang, Sang-Kee; Lee, Nam Kyung; Choi, Yun-Jaie; Jung, Dong Hyun

    2010-01-01

    We report a new approach to studying organ targeting of peptides on the basis of peptide sequence information. The positive control data sets consist of organ-targeting peptide sequences identified by the peroral phage-display technique for four organs, and the negative control data are prepared from random sequences. The capacity of our models to make appropriate predictions is validated by statistical indicators including sensitivity, specificity, enrichment curve, and the area under the receiver operating characteristic (ROC) curve (the ROC score). VHSE descriptor produces statistically significant training models and the models with simple neural network architectures show slightly greater predictive power than those with complex ones. The training and test set statistics indicate that our models could discriminate between organ-targeting and random sequences. We anticipate that our models will be applicable to the selection of organ-targeting peptides for generating peptide drugs or peptidomimetics.

  7. Bioprinting synthetic self-assembling peptide hydrogels for biomedical applications

    International Nuclear Information System (INIS)

    Loo, Yihua; Hauser, Charlotte A E

    2016-01-01

    Three-dimensional (3D) bioprinting is a disruptive technology for creating organotypic constructs for high-throughput screening and regenerative medicine. One major challenge is the lack of suitable bioinks. Short synthetic self-assembling peptides are ideal candidates. Several classes of peptides self-assemble into nanofibrous hydrogels resembling the native extracellular matrix. This is a conducive microenvironment for maintaining cell survival and physiological function. Many peptides also demonstrate stimuli-responsive gelation and tuneable mechanical properties, which facilitates extrusion before dispensing and maintains the shape fidelity of the printed construct in aqueous media. The inherent biocompatibility and biodegradability bodes well for in vivo applications as implantable tissues and drug delivery matrices, while their short length and ease of functionalization facilitates synthesis and customization. By applying self-assembling peptide inks to bioprinting, the dynamic complexity of biological tissue can be recreated, thereby advancing current biomedical applications of peptide hydrogel scaffolds. (paper)

  8. Peptide Integrated Optics.

    Science.gov (United States)

    Handelman, Amir; Lapshina, Nadezda; Apter, Boris; Rosenman, Gil

    2018-02-01

    Bio-nanophotonics is a wide field in which advanced optical materials, biomedicine, fundamental optics, and nanotechnology are combined and result in the development of biomedical optical chips. Silk fibers or synthetic bioabsorbable polymers are the main light-guiding components. In this work, an advanced concept of integrated bio-optics is proposed, which is based on bioinspired peptide optical materials exhibiting wide optical transparency, nonlinear and electrooptical properties, and effective passive and active waveguiding. Developed new technology combining bottom-up controlled deposition of peptide planar wafers of a large area and top-down focus ion beam lithography provides direct fabrication of peptide optical integrated circuits. Finding a deep modification of peptide optical properties by reconformation of biological secondary structure from native phase to β-sheet architecture is followed by the appearance of visible fluorescence and unexpected transition from a native passive optical waveguiding to an active one. Original biocompatibility, switchable regimes of waveguiding, and multifunctional nonlinear optical properties make these new peptide planar optical materials attractive for application in emerging technology of lab-on-biochips, combining biomedical photonic and electronic circuits toward medical diagnosis, light-activated therapy, and health monitoring. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Antimicrobial Peptides from Plants

    Directory of Open Access Journals (Sweden)

    James P. Tam

    2015-11-01

    Full Text Available Plant antimicrobial peptides (AMPs have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic, lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.

  10. Prevalence Incidence Mixture Models

    Science.gov (United States)

    The R package and webtool fits Prevalence Incidence Mixture models to left-censored and irregularly interval-censored time to event data that is commonly found in screening cohorts assembled from electronic health records. Absolute and relative risk can be estimated for simple random sampling, and stratified sampling (the two approaches of superpopulation and a finite population are supported for target populations). Non-parametric (absolute risks only), semi-parametric, weakly-parametric (using B-splines), and some fully parametric (such as the logistic-Weibull) models are supported.

  11. LESSONS IN DE NOVO PEPTIDE SEQUENCING BY TANDEM MASS SPECTROMETRY

    Science.gov (United States)

    Medzihradszky, Katalin F.; Chalkley, Robert J.

    2015-01-01

    Mass spectrometry has become the method of choice for the qualitative and quantitative characterization of protein mixtures isolated from all kinds of living organisms. The raw data in these studies are MS/MS spectra, usually of peptides produced by proteolytic digestion of a protein. These spectra are “translated” into peptide sequences, normally with the help of various search engines. Data acquisition and interpretation have both been automated, and most researchers look only at the summary of the identifications without ever viewing the underlying raw data used for assignments. Automated analysis of data is essential due to the volume produced. However, being familiar with the finer intricacies of peptide fragmentation processes, and experiencing the difficulties of manual data interpretation allow a researcher to be able to more critically evaluate key results, particularly because there are many known rules of peptide fragmentation that are not incorporated into search engine scoring. Since the most commonly used MS/MS activation method is collision-induced dissociation (CID), in this article we present a brief review of the history of peptide CID analysis. Next, we provide a detailed tutorial on how to determine peptide sequences from CID data. Although the focus of the tutorial is de novo sequencing, the lessons learned and resources supplied are useful for data interpretation in general. PMID:25667941

  12. AnchorDock for Blind Flexible Docking of Peptides to Proteins.

    Science.gov (United States)

    Slutzki, Michal; Ben-Shimon, Avraham; Niv, Masha Y

    2017-01-01

    Due to increasing interest in peptides as signaling modulators and drug candidates, several methods for peptide docking to their target proteins are under active development. The "blind" docking problem, where the peptide-binding site on the protein surface is unknown, presents one of the current challenges in the field. AnchorDock protocol was developed by Ben-Shimon and Niv to address this challenge.This protocol narrows the docking search to the most relevant parts of the conformational space. This is achieved by pre-folding the free peptide and by computationally detecting anchoring spots on the surface of the unbound protein. Multiple flexible simulated annealing molecular dynamics (SAMD) simulations are subsequently carried out, starting from pre-folded peptide conformations, constrained to the various precomputed anchoring spots.Here, AnchorDock is demonstrated using two known protein-peptide complexes. A PDZ-peptide complex provides a relatively easy case due to the relatively small size of the protein, and a typical peptide conformation and binding region; a more challenging example is a complex between USP7 N-term and a p53-derived peptide, where the protein is larger, and the peptide conformation and a binding site are generally assumed to be unknown. AnchorDock returned native-like solutions ranked first and third for the PDZ and USP7 complexes, respectively. We describe the procedure step by step and discuss possible modifications where applicable.

  13. Acylation of Therapeutic Peptides

    DEFF Research Database (Denmark)

    Trier, Sofie; Henriksen, Jonas Rosager; Jensen, Simon Bjerregaard

    ) , which promotes intestinal growth and is used to treat bowel disorders such as inflammatory bowel diseases and short bowel syndrome, and the 32 amino acid salmon calcitonin (sCT), which lowers blood calcium and is employed in the treatment of post-menopausal osteoporosis and hypercalcemia. The two...... peptides are similar in size and structure, but oppositely charged at physiological pH. Both peptides were acylated with linear acyl chains of systematically increasing length, where sCT was furthermore acylated at two different positions on the peptide backbone. For GLP-2, we found that increasing acyl...... remained optimal overall. The results indicate that rational acylation of GLP-2 can increase its in vitro intestinal absorption, alone or in combination with permeation enhancers, and are consistent with the initial project hypothesis. For sCT, an unpredicted effect of acylation largely superseded...

  14. 111In and 90Y labelled peptide radiopharmaceuticals for diagnosis and therapy

    International Nuclear Information System (INIS)

    Obenaus, E.; Crudo, J.L.; Castiglia, S.G. de

    2004-01-01

    Full text: The application of 111In-labelled octreotide to target somatostatin receptors on tumour cells has gained acceptance as a diagnostic procedure for demonstrating neuroendocrine and other SSTR-positive tumors. A further advance in the field of somatostatin analogues is the development of macrocyclic chelators that also bind beta particle emitters like 90Y and 177Lu. [90Y-DOTA, Tyr3] octreotide and [177Lu-DOTA, Tyr3] octreotate are currently being tried as the therapeutic agents. The aim of this work was to label two somatostatin analogues (Tyr3 Octreotide and Lanreotide) with 111In and 90Y using DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as a chelating agent. DOTA in the form of the tri-t-butyl ester was coupled to the Lys5 (BOC) protected Tyr3octreotide and Lys5 (BOC) protected lanreotide in N,N-dimethylformamide, in a three-step reaction involving conjugation, using HATU and diisopropylethylamine as coupling reagents, de-protection with trifluoroacetic and HPLC purification of the conjugates. The DOTALAN and DOTATOC were labelled with 111In and 90Y and assayed by HPLC. In both cases solutions of gentisic acid in sodium acetate 0.4M pH=5.0 and conjugated peptides were added to the 111In or 90Y chloride. The final solution was heated at 90 deg. C for 25 minutes. We tried different amounts of the two peptide complexes per mCi of 111In/90Y and quality control of the labelled products was performed with the following HPLC system: reverse phase column, flow rate 1ml-min, UV/radiometric detection and a gradient solvent: solvent A: acetonitrile, solvent B: water TFA 0.1%, gradient: 0-3min 100% B, linear increase of eluent A to 50% from 3-13 min, 13-18 min 50% A, 18-20 min linear increase of eluent A to 70%. Serum stability was determined after 4 and 24 hr. incubation of the labeled peptide in human serum at 37 deg. C. After precipitation of proteins with acetonitrile the incubation mixture was analyzed by HPLC. Normal Wistar rats were

  15. Separation of gas mixtures

    International Nuclear Information System (INIS)

    1981-01-01

    Apparatus is described for the separation of a gaseous plasma mixture into components in some of which the original concentration of a specific ion has been greatly increased or decreased, comprising: a source for converting the gaseous mixture into a train of plasma packets; an open-ended vessel with a main section and at least one branch section, adapted to enclose along predetermined tracks the original plasma packets in the main section, and the separated plasma components in the branch sections; drive means for generating travelling magnetic waves along the predetermined tracks with the magnetic flux vector of the waves transverse to each of the tracks; and means for maintaining phase coherence between the plasma packets and the magnetic waves at a value needed for accelerating the components of the packets to different velocities and in such different directions that the plasma of each packet is divided into distinctly separate packets in some of which the original concentration of a specific ion has been greatly increased or decreased, and which plasma packets are collected from the branch sections of the vessels. (author)

  16. Use of the nonsteady monotonic heating method for complex determination of thermophysical properties of chemically reacting mixture in the case of non-equilibrium proceeding of the chemical reaction

    International Nuclear Information System (INIS)

    Serebryanyj, G.Z.

    1984-01-01

    Theoretical analysis is made for the monotonic heating method as applied for complex determination of thermophysical properties of chemically reacting gases. The possibility is shown of simultaneous determination of frozen and equilibrium heat capacity, frozen and equilibrium heat conduction provided non-equilibrium occuring of the reaction in the wide range of temperatures and pressures. The monotonic heating method can be used for complex determination of thermophysical properties of chemically reacting systems in case of non-equilibrium proceeding of the chemical reaction

  17. A highly scalable peptide-based assay system for proteomics.

    Directory of Open Access Journals (Sweden)

    Igor A Kozlov

    Full Text Available We report a scalable and cost-effective technology for generating and screening high-complexity customizable peptide sets. The peptides are made as peptide-cDNA fusions by in vitro transcription/translation from pools of DNA templates generated by microarray-based synthesis. This approach enables large custom sets of peptides to be designed in silico, manufactured cost-effectively in parallel, and assayed efficiently in a multiplexed fashion. The utility of our peptide-cDNA fusion pools was demonstrated in two activity-based assays designed to discover protease and kinase substrates. In the protease assay, cleaved peptide substrates were separated from uncleaved and identified by digital sequencing of their cognate cDNAs. We screened the 3,011 amino acid HCV proteome for susceptibility to cleavage by the HCV NS3/4A protease and identified all 3 known trans cleavage sites with high specificity. In the kinase assay, peptide substrates phosphorylated by tyrosine kinases were captured and identified by sequencing of their cDNAs. We screened a pool of 3,243 peptides against Abl kinase and showed that phosphorylation events detected were specific and consistent with the known substrate preferences of Abl kinase. Our approach is scalable and adaptable to other protein-based assays.

  18. Therapeutic HIV Peptide Vaccine

    DEFF Research Database (Denmark)

    Fomsgaard, Anders

    2015-01-01

    Therapeutic vaccines aim to control chronic HIV infection and eliminate the need for lifelong antiretroviral therapy (ART). Therapeutic HIV vaccine is being pursued as part of a functional cure for HIV/AIDS. We have outlined a basic protocol for inducing new T cell immunity during chronic HIV-1...... infection directed to subdominant conserved HIV-1 epitopes restricted to frequent HLA supertypes. The rationale for selecting HIV peptides and adjuvants are provided. Peptide subunit vaccines are regarded as safe due to the simplicity, quality, purity, and low toxicity. The caveat is reduced immunogenicity...

  19. Descriptors for antimicrobial peptides

    DEFF Research Database (Denmark)

    Jenssen, Håvard

    2011-01-01

    of these are currently being used in quantitative structure--activity relationship (QSAR) studies for AMP optimization. Additionally, some key commercial computational tools are discussed, and both successful and less successful studies are referenced, illustrating some of the challenges facing AMP scientists. Through...... examples of different peptide QSAR studies, this review highlights some of the missing links and illuminates some of the questions that would be interesting to challenge in a more systematic fashion. Expert opinion: Computer-aided peptide QSAR using molecular descriptors may provide the necessary edge...

  20. Combustion of powdery tungsten in pyrotechnic mixtures

    International Nuclear Information System (INIS)

    Ivanov, G.V.; Reshetov, A.A.; Viktorenko, A.M.; Surkov, V.G.; Karmadonov, L.N.

    1982-01-01

    The basic regularities of tungsten burning (powder 2-5 μm) with oxidizers most typical for pyrotechnics: nitrates, lead and barium peroxides (powder, 2-8 μm) and potassium perchlorate (powder, 2-8 μm) are studied. Dependences of burning rate as a function of pressure and ratio of components are established. It is supposed that tungsten burning in mixtures with the mentioned nitrates is a complex and multistage process the rate of which is determined by tungsten dissolution in nitrate melts. Analysis of burning products using available methods is complex

  1. Antimicrobial peptides interact with peptidoglycan

    Science.gov (United States)

    Neelay, Om P.; Peterson, Christian A.; Snavely, Mary E.; Brown, Taylor C.; TecleMariam, Ariam F.; Campbell, Jennifer A.; Blake, Allison M.; Schneider, Sydney C.; Cremeens, Matthew E.

    2017-10-01

    Traditional therapeutics are losing effectiveness as bacterial resistance increases, and antimicrobial peptides (AMPs) can serve as an alternative source for antimicrobial agents. Their mode of action is commonly hypothesized to involve pore formation in the lipid membrane, thereby leading to cell death. However, bacterial cell walls are much more complex than just the lipid membrane. A large portion of the wall is comprised of peptidoglycan, yet we did not find any report of AMP-peptidoglycan interactions. Consequently, this work evaluated AMP-peptidoglycan and AMP-phospholipid (multilamellar vesicles) interactions through tryptophan fluorescence. Given that peptidoglycan is insoluble and vesicles are large particles, we took advantage of the unique properties of Trp-fluorescence to use one technique for two very different systems. Interestingly, melittin and cecropin A interacted with peptidoglycan to a degree similar to vancomycin, a positive control. Whether these AMP-peptidoglycan interactions relate to a killing mode of action requires further study.

  2. DOTA-TATE peptides labelling with Lutetium 177: Preliminary study

    International Nuclear Information System (INIS)

    Aliaga, Eleazar; Robles, Anita; Ramos, Bertha; Martinez, Flor

    2014-01-01

    he peptide DOTA-TATE was labeled with lutetium 177 according to the methodology provided under the regional project RLA/6/074, sponsored by the IAEA. The labeling was done in 0.26 M gentisic acid solution in 0.8 M sodium acetate buffer, pH 5, at 100 °C for 30 minutes in a dry heating block. The radiochemical purity was assessed by thin layer chromatography, using ITLC SG strips and a mixture of 0.15 M ammonium acetate - methanol (1:1) as solvent. The radiolabeled peptide 177 Lu-DOTA-TATE reached a radiochemical purity of 98 % with a specific activity of 2,8 mCi/µg of peptide. (authors).

  3. Construction of tunable peptide nucleic acid junctions.

    Science.gov (United States)

    Duan, Tanghui; He, Liu; Tokura, Yu; Liu, Xin; Wu, Yuzhou; Shi, Zhengshuang

    2018-03-15

    We report here the construction of 3-way and 4-way peptide nucleic acid (PNA) junctions as basic structural units for PNA nanostructuring. The incorporation of amino acid residues into PNA chains makes PNA nanostructures with more structural complexity and architectural flexibility possible, as exemplified by building 3-way PNA junctions with tunable nanopores. Given that PNA nanostructures have good thermal and enzymatic stabilities, they are expected to have broad potential applications in biosensing, drug delivery and bioengineering.

  4. Interpretation of Tandem Mass Spectrometry (MSMS) Spectra for Peptide Analysis

    DEFF Research Database (Denmark)

    Hjernø, Karin; Højrup, Peter

    2015-01-01

    The aim of this chapter is to give a short introduction to peptide analysis by mass spectrometry (MS) and interpretation of fragment mass spectra. Through examples and guidelines we demonstrate how to understand and validate search results and how to perform de novo sequencing based on the often...... very complex fragmentation pattern obtained by tandem mass spectrometry (also referred to as MSMS). The focus is on simple rules for interpretation of MSMS spectra of tryptic as well as non-tryptic peptides....

  5. Research of Deformation of Clay Soil Mixtures Mixtures

    OpenAIRE

    Romas Girkontas; Tadas Tamošiūnas; Andrius Savickas

    2014-01-01

    The aim of this article is to determine clay soils and clay soils mixtures deformations during drying. Experiments consisted from: a) clay and clay mixtures bridges (height ~ 0,30 m, span ~ 1,00 m); b) tiles of clay and clay, sand and straw (height, length, wide); c) cylinders of clay; clay and straw; clay, straw and sand (diameter; height). According to the findings recommendations for clay and clay mixtures drying technology application were presented. During the experiment clay bridge bear...

  6. Antimicrobial Peptides: An Introduction.

    Science.gov (United States)

    Haney, Evan F; Mansour, Sarah C; Hancock, Robert E W

    2017-01-01

    The "golden era" of antibiotic discovery has long passed, but the need for new antibiotics has never been greater due to the emerging threat of antibiotic resistance. This urgency to develop new antibiotics has motivated researchers to find new methods to combat pathogenic microorganisms resulting in a surge of research focused around antimicrobial peptides (AMPs; also termed host defense peptides) and their potential as therapeutics. During the past few decades, more than 2000 AMPs have been identified from a diverse range of organisms (animals, fungi, plants, and bacteria). While these AMPs share a number of common features and a limited number of structural motifs; their sequences, activities, and targets differ considerably. In addition to their antimicrobial effects, AMPs can also exhibit immunomodulatory, anti-biofilm, and anticancer activities. These diverse functions have spurred tremendous interest in research aimed at understanding the activity of AMPs, and various protocols have been described to assess different aspects of AMP function including screening and evaluating the activities of natural and synthetic AMPs, measuring interactions with membranes, optimizing peptide function, and scaling up peptide production. Here, we provide a general overview of AMPs and introduce some of the methodologies that have been used to advance AMP research.

  7. Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry.

    Science.gov (United States)

    Molloy, M P; Andrews, P C

    2001-11-15

    The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site o