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Sample records for reverse phase protein

  1. Mixed-mode reversed phase/positively charged repulsion chromatography for intact protein separation.

    Science.gov (United States)

    Ding, Ling; Guo, Zhimou; Hu, Zhuo; Liang, Xinmiao

    2017-05-10

    A mixed-mode reversed phase/positively charged repulsion stationary phase C8PN composed of octyl and amino group has been developed for separation of intact protein. Before the separation of proteins, a set of probe compounds were employed to evaluate the chromatographic properties of C8PN, demonstrating typical reversed phase/positively charged repulsion interaction on this stationary phase as estimated. Then the new C8PN stationary phase was used to separate a standard protein mixture on the reversed phase mode. Compared with a commercial C4 stationary phase, it showed different selectivity for some proteins. In order to better understand the properties of C8PN, the effect of acetonitrile content was investigated based on retention equation. Higher values of the equation parameters on C8PN demonstrated that the protein retentions were more sensitive to the change of acetonitrile content. Besides, the influences of buffer salt additives on the protein retentions were also studied. The retention factors of the proteins got larger with the increase of buffer salt concentration, which confirmed the positively charged repulsion interaction on the column. Finally, the C8PN was further applied to separate oxidized- and reduced- forms of Recombinant Human Growth Hormone. Our study indicated the advantages and application potential of mixed-mode reversed phase/positively charged repulsion stationary phase for intact protein separation. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Reverse Phase Protein Arrays for High-throughput Toxicity Screening

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    High-throughput screening is extensively applied for identification of drug targets and drug discovery and recently it found entry into toxicity testing. Reverse phase protein arrays (RPPAs) are used widespread for quantification of protein markers. We reasoned that RPPAs also can be utilized...... beneficially in automated high-throughput toxicity testing. An advantage of using RPPAs is that, in addition to the baseline toxicity readout, they allow testing of multiple markers of toxicity, such as inflammatory responses, which do not necessarily cumulate in cell death. We used transfection of si......RNAs with known killing effects as a model system to demonstrate that RPPA-based protein quantification can serve as substitute readout of cell viability, hereby reliably reflecting toxicity. In terms of automation, cell exposure, protein harvest, serial dilution and sample reformatting were performed using...

  3. Solid-phase synthesis of protein-polymers on reversible immobilization supports.

    Science.gov (United States)

    Murata, Hironobu; Carmali, Sheiliza; Baker, Stefanie L; Matyjaszewski, Krzysztof; Russell, Alan J

    2018-02-27

    Facile automated biomacromolecule synthesis is at the heart of blending synthetic and biologic worlds. Full access to abiotic/biotic synthetic diversity first occurred when chemistry was developed to grow nucleic acids and peptides from reversibly immobilized precursors. Protein-polymer conjugates, however, have always been synthesized in solution in multi-step, multi-day processes that couple innovative chemistry with challenging purification. Here we report the generation of protein-polymer hybrids synthesized by protein-ATRP on reversible immobilization supports (PARIS). We utilized modified agarose beads to covalently and reversibly couple to proteins in amino-specific reactions. We then modified reversibly immobilized proteins with protein-reactive ATRP initiators and, after ATRP, we released and analyzed the protein polymers. The activity and stability of PARIS-synthesized and solution-synthesized conjugates demonstrated that PARIS was an effective, rapid, and simple method to generate protein-polymer conjugates. Automation of PARIS significantly reduced synthesis/purification timelines, thereby opening a path to changing how to generate protein-polymer conjugates.

  4. RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

    OpenAIRE

    Stanislaus, Romesh; Carey, Mark; Deus, Helena F; Coombes, Kevin; Hennessy, Bryan T; Mills, Gordon B; Almeida, Jonas S

    2008-01-01

    Abstract Background Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of inter...

  5. PURIFICATION OF Cd-BINDING PROTEIN FROM MAIZE ROOTS BY REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

    Institute of Scientific and Technical Information of China (English)

    何笃修; 罗建沅; 全胜

    1991-01-01

    An efficient procedure of purification of Cd-binding protein in roots of maize has been established. Young seedlings of maize were exposed to a medium containing CdCl2 to induce the production of Cd-binding protein in their roots. The protein was purified after heat treatment by ion-exchange chromatography and reverse-phase HPLC. The resulting protein was identified as a purified product by N-terminal amino acid with the dansyl method. Its molecular weight was 3200 dalton, the cysteine content was 29.5%, about 3 Cd atoms were bound to one molecule of the protein and the Cd : cystine ratio was 1 : 2.3. According to its character, this protein could be a kind of plant metallothionein-like protein.

  6. Reverse phase protein microarray technology in traumatic brain injury.

    Science.gov (United States)

    Gyorgy, Andrea B; Walker, John; Wingo, Dan; Eidelman, Ofer; Pollard, Harvey B; Molnar, Andras; Agoston, Denes V

    2010-09-30

    Antibody based, high throughput proteomics technology represents an exciting new approach in understanding the pathobiologies of complex disorders such as cancer, stroke and traumatic brain injury. Reverse phase protein microarray (RPPA) can complement the classical methods based on mass spectrometry as a high throughput validation and quantification method. RPPA technology can address problematic issues, such as sample complexity, sensitivity, quantification, reproducibility and throughput, which are currently associated with mass spectrometry-based approaches. However, there are technical challenges, predominantly associated with the selection and use of antibodies, preparation and representation of samples and with analyzing and quantifying primary RPPA data. Here we present ways to identify and overcome some of the current issues associated with RPPA. We believe that using stringent quality controls, improved bioinformatics analysis and interpretation of primary RPPA data, this method will significantly contribute in generating new level of understanding about complex disorders at the level of systems biology. Published by Elsevier B.V.

  7. RPPAML/RIMS: a metadata format and an information management system for reverse phase protein arrays.

    Science.gov (United States)

    Stanislaus, Romesh; Carey, Mark; Deus, Helena F; Coombes, Kevin; Hennessy, Bryan T; Mills, Gordon B; Almeida, Jonas S

    2008-12-22

    Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1,000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest. In this report an RPPA Information Management System (RIMS) is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML). RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape. The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis.

  8. RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

    Directory of Open Access Journals (Sweden)

    Hennessy Bryan T

    2008-12-01

    Full Text Available Abstract Background Reverse Phase Protein Arrays (RPPA are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest. Results In this report an RPPA Information Management System (RIMS is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML. RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape. Conclusion The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis.

  9. Enrichment and identification of integral membrane proteins from barley aleurone layers by reversed-phase chromatography, SDS-PAGE and LC-MS/MS

    DEFF Research Database (Denmark)

    Hynek, Radovan; Svensson, Birte; Nørregaard Jensen, Ole

    2006-01-01

    was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral...

  10. Three-dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation

    OpenAIRE

    Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W. Andy

    2016-01-01

    Glycoproteins have vast structural diversity which plays an important role in many biological processes and have great potential as disease biomarkers. Here we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase GlycoProtein Array (polyGPA), to specifically capture and profile glycoproteomes, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture pre-oxidized glycan...

  11. Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

    Science.gov (United States)

    Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

    2013-01-01

    A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O

  12. A reverse-phase protein microarray-based screen identifies host signaling dynamics upon Burkholderia spp. infection

    Directory of Open Access Journals (Sweden)

    Chih-Yuan eChiang

    2015-07-01

    Full Text Available Burkholderia is a diverse genus of Gram-negative bacteria that cause high mortality rate in humans and cattle. The lack of effective therapeutic treatments poses serious public health threats. Insights toward host-Burkholderia spp. interaction are critical in understanding the pathogenesis of the infection as well as identifying therapeutic targets for drug development. Reverse-phase protein microarray (RPMA technology was previously proven to characterize novel biomarkers and molecular signatures associated with infectious diseases and cancers. In the present study, this technology was utilized to interrogate changes in host protein expression and post-translational phosphorylation events in macrophages infected with a collection of geographically diverse strains of Burkholderia spp. The expression or phosphorylation state of 25 proteins was altered during Burkholderia spp. infections and of which eight proteins were selected for further validation by immunoblotting. Kinetic expression patterns of phosphorylated AMPK-α1, Src, and GSK3β suggested the importance of their roles in regulating Burkholderia spp. mediated innate immune responses. Modulating inflammatory responses by perturbing AMPK-α1, Src, and GSK3β activities may provide novel therapeutic targets for future treatments.

  13. Online combination of reversed-phase/reversed-phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples.

    Science.gov (United States)

    Lam, Maggie P Y; Lau, Edward; Siu, S O; Ng, Dominic C M; Kong, Ricky P W; Chiu, Philip C N; Yeung, William S B; Lo, Clive; Chu, Ivan K

    2011-11-01

    In this paper, we describe an online combination of reversed-phase/reversed-phase (RP-RP) and porous graphitic carbon (PGC) liquid chromatography (LC) for multicomponent analysis of proteomics and glycoproteomics samples. The online RP-RP portion of this system provides comprehensive 2-D peptide separation based on sequence hydrophobicity at pH 2 and 10. Hydrophilic components (e.g. glycans, glycopeptides) that are not retained by RP are automatically diverted downstream to a PGC column for further trapping and separation. Furthermore, the RP-RP/PGC system can provide simultaneous extension of the hydropathy range and peak capacity for analysis. Using an 11-protein mixture, we found that the system could efficiently separate native peptides and released N-glycans from a single sample. We evaluated the applicability of the system to the analysis of complex biological samples using 25 μg of the lysate of a human choriocarcinoma cell line (BeWo), confidently identifying a total of 1449 proteins from a single experiment and up to 1909 distinct proteins from technical triplicates. The PGC fraction increased the sequence coverage through the inclusion of additional hydrophilic sequences that accounted for up to 6.9% of the total identified peptides from the BeWo lysate, with apparent preference for the detection of hydrophilic motifs and proteins. In addition, RP-RP/PGC is applicable to the analysis of complex glycomics samples, as demonstrated by our analysis of a concanavalin A-extracted glycoproteome from human serum; in total, 134 potentially N-glycosylated serum proteins, 151 possible N-glycosylation sites, and more than 40 possible N-glycan structures recognized by concanavalin A were simultaneously detected. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation.

    Science.gov (United States)

    Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W Andy

    2016-11-30

    Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.

  15. Analysis of Reverse Phase Protein Array Data: From Experimental Design towards Targeted Biomarker Discovery

    Directory of Open Access Journals (Sweden)

    Astrid Wachter

    2015-11-01

    Full Text Available Mastering the systematic analysis of tumor tissues on a large scale has long been a technical challenge for proteomics. In 2001, reverse phase protein arrays (RPPA were added to the repertoire of existing immunoassays, which, for the first time, allowed a profiling of minute amounts of tumor lysates even after microdissection. A characteristic feature of RPPA is its outstanding sample capacity permitting the analysis of thousands of samples in parallel as a routine task. Until today, the RPPA approach has matured to a robust and highly sensitive high-throughput platform, which is ideally suited for biomarker discovery. Concomitant with technical advancements, new bioinformatic tools were developed for data normalization and data analysis as outlined in detail in this review. Furthermore, biomarker signatures obtained by different RPPA screens were compared with another or with that obtained by other proteomic formats, if possible. Options for overcoming the downside of RPPA, which is the need to steadily validate new antibody batches, will be discussed. Finally, a debate on using RPPA to advance personalized medicine will conclude this article.

  16. NormaCurve: a SuperCurve-based method that simultaneously quantifies and normalizes reverse phase protein array data.

    Directory of Open Access Journals (Sweden)

    Sylvie Troncale

    Full Text Available MOTIVATION: Reverse phase protein array (RPPA is a powerful dot-blot technology that allows studying protein expression levels as well as post-translational modifications in a large number of samples simultaneously. Yet, correct interpretation of RPPA data has remained a major challenge for its broad-scale application and its translation into clinical research. Satisfying quantification tools are available to assess a relative protein expression level from a serial dilution curve. However, appropriate tools allowing the normalization of the data for external sources of variation are currently missing. RESULTS: Here we propose a new method, called NormaCurve, that allows simultaneous quantification and normalization of RPPA data. For this, we modified the quantification method SuperCurve in order to include normalization for (i background fluorescence, (ii variation in the total amount of spotted protein and (iii spatial bias on the arrays. Using a spike-in design with a purified protein, we test the capacity of different models to properly estimate normalized relative expression levels. The best performing model, NormaCurve, takes into account a negative control array without primary antibody, an array stained with a total protein stain and spatial covariates. We show that this normalization is reproducible and we discuss the number of serial dilutions and the number of replicates that are required to obtain robust data. We thus provide a ready-to-use method for reliable and reproducible normalization of RPPA data, which should facilitate the interpretation and the development of this promising technology. AVAILABILITY: The raw data, the scripts and the normacurve package are available at the following web site: http://microarrays.curie.fr.

  17. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

    Science.gov (United States)

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes. PMID:21960964

  18. Late protein synthesis-dependent phases in CTA long-term memory: BDNF requirement

    Directory of Open Access Journals (Sweden)

    Araceli eMartínez-Moreno

    2011-09-01

    Full Text Available It has been proposed that long-term memory persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related long-term memory when protein synthesis was inhibited. Our previous studies on the insular cortex (IC, a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA, have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis dependent in different time-windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 hours after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes.

  19. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement.

    Science.gov (United States)

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F; Escobar, Martha L

    2011-01-01

    It has been proposed that long-term memory (LTM) persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related LTM when protein synthesis was inhibited. Our previous studies on the insular cortex (IC), a region of the temporal cortex implicated in the acquisition and storage of conditioned taste aversion (CTA), have demonstrated that intracortical delivery of BDNF reverses the deficit in CTA memory caused by the inhibition of IC protein synthesis due to anisomycin administration during early acquisition. In this work, we first analyze whether CTA memory storage is protein synthesis-dependent in different time windows. We observed that CTA memory become sensible to protein synthesis inhibition 5 and 7 h after acquisition. Then, we explore the effect of BDNF delivery (2 μg/2 μl per side) in the IC during those late protein synthesis-dependent phases. Our results show that BDNF reverses the CTA memory deficit produced by protein synthesis inhibition in both phases. These findings support the notion that recurrent rounds of consolidation-like events take place in the neocortex for maintenance of CTA memory trace and that BDNF is an essential component of these processes.

  20. Highly sensitive reversed-phase high-performance liquid chromatography assay for the detection of Tamm-Horsfall protein in human urine.

    Science.gov (United States)

    Akimoto, Masaru; Hokazono, Eisaku; Ota, Eri; Tateishi, Takiko; Kayamori, Yuzo

    2016-01-01

    Tamm-Horsfall protein (also known as uromodulin) is the most abundant urinary protein in healthy individuals. Since initially characterized by Tamm and Horsfall, the amount of urinary excretion and structural mutations of Tamm-Horsfall protein is associated with kidney diseases. However, currently available assays for Tamm-Horsfall protein, which are mainly enzyme-linked immunosorbent assay-based, suffer from poor reproducibility and might give false negative results. We developed a novel, quantitative assay for Tamm-Horsfall protein using reversed-phase high-performance liquid chromatography. A precipitation pretreatment avoided urine matrix interference and excessive sample dilution. High-performance liquid chromatography optimization based on polarity allowed excellent separation of Tamm-Horsfall protein from other major urine components. Our method exhibited high precision (based on the relative standard deviations of intraday [≤2.77%] and interday [≤5.35%] repetitions). The Tamm-Horsfall protein recovery rate was 100.0-104.2%. The mean Tamm-Horsfall protein concentration in 25 healthy individuals was 31.6 ± 18.8 mg/g creatinine. There was a strong correlation between data obtained by high-performance liquid chromatography and enzyme-linked immunosorbent assay (r = 0.906), but enzyme-linked immunosorbent assay values tended to be lower than high-performance liquid chromatography values at low Tamm-Horsfall protein concentrations. The high sensitivity and reproducibility of our Tamm-Horsfall protein assay will reduce the number of false negative results of the sample compared with enzyme-linked immunosorbent assay. Moreover, our method is superior to other high-performance liquid chromatography methods, and a simple protocol will facilitate further research on the physiological role of Tamm-Horsfall protein. © The Author(s) 2015.

  1. Doping reversed-phase media for improved peptide purification.

    Science.gov (United States)

    Khalaf, Rushd; Forrer, Nicola; Buffolino, Gianluca; Gétaz, David; Bernardi, Susanna; Butté, Alessandro; Morbidelli, Massimo

    2015-06-05

    The purification of therapeutic peptides is most often performed using one or more reversed phase chromatography steps. This ensures high purities while keeping the costs of purification under control. In this paper, a doped reversed phase chromatographic material is tested and compared to traditional reversed phase materials. The doping consists of adding limited amounts of ion exchange ligands to the surface of the material to achieve orthogonal separation and increase the non-hydrophobic interactions with the surface. These ionic groups can either be attractive (opposite charge), or repulsive (same charge) to the peptide. The benefit of this new doped reversed phase material is shown through increases in selectivity in diluted conditions and yield and productivity in overloaded (i.e. industrial) conditions. It is the conjectured that all performance characteristics should increase using repulsive doping groups, whereas these characteristics should decrease when using attractive doping groups. This conjecture is shown to be true through several examples, including purifications of industrially relevant peptide crudes, in industrially relevant conditions. Moreover, the effect of ionic strength and organic modifier concentration was explored and shown to be in line with the expected behavior. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Strong and Reversible Monovalent Supramolecular Protein Immobilization

    NARCIS (Netherlands)

    Young, Jacqui F.; Nguyen, Hoang D.; Yang, Lanti; Huskens, Jurriaan; Jonkheijm, Pascal; Brunsveld, Luc

    2010-01-01

    Proteins with an iron clasp: Site-selective incorporation of a ferrocene molecule into a protein allows for easy, strong, and reversible supramolecular protein immobilization through a selective monovalent interaction of the ferrocene with a cucurbit[7]uril immobilized on a gold surface. The

  3. Role of Reversible Phase Transformation for Strong Piezoelectric Performance at the Morphotropic Phase Boundary

    Science.gov (United States)

    Liu, Hui; Chen, Jun; Huang, Houbing; Fan, Longlong; Ren, Yang; Pan, Zhao; Deng, Jinxia; Chen, Long-Qing; Xing, Xianran

    2018-01-01

    A functional material with coexisting energetically equivalent phases often exhibits extraordinary properties such as piezoelectricity, ferromagnetism, and ferroelasticity, which is simultaneously accompanied by field-driven reversible phase transformation. The study on the interplay between such phase transformation and the performance is of great importance. Here, we have experimentally revealed the important role of field-driven reversible phase transformation in achieving enhanced electromechanical properties using in situ high-energy synchrotron x-ray diffraction combined with 2D geometry scattering technology, which can establish a comprehensive picture of piezoelectric-related microstructural evolution. High-throughput experiments on various Pb /Bi -based perovskite piezoelectric systems suggest that reversible phase transformation can be triggered by an electric field at the morphotropic phase boundary and the piezoelectric performance is highly related to the tendency of electric-field-driven phase transformation. A strong tendency of phase transformation driven by an electric field generates peak piezoelectric response. Further, phase-field modeling reveals that the polarization alignment and the piezoelectric response can be much enhanced by the electric-field-driven phase transformation. The proposed mechanism will be helpful to design and optimize the new piezoelectrics, ferromagnetics, or other related functional materials.

  4. Reversals and collisions optimize protein exchange in bacterial swarms

    Energy Technology Data Exchange (ETDEWEB)

    Amiri, Aboutaleb; Harvey, Cameron; Buchmann, Amy; Christley, Scott; Shrout, Joshua D.; Aranson, Igor S.; Alber, Mark

    2017-03-01

    Swarming groups of bacteria coordinate their behavior by self-organizing as a population to move over surfaces in search of nutrients and optimal niches for colonization. Many open questions remain about the cues used by swarming bacteria to achieve this self-organization. While chemical cue signaling known as quorum sensing is well-described, swarming bacteria often act and coordinate on time scales that could not be achieved via these extracellular quorum sensing cues. Here, cell-cell contact-dependent protein exchange is explored as amechanism of intercellular signaling for the bacterium Myxococcus xanthus. A detailed biologically calibrated computational model is used to study how M. xanthus optimizes the connection rate between cells and maximizes the spread of an extracellular protein within the population. The maximum rate of protein spreading is observed for cells that reverse direction optimally for swarming. Cells that reverse too slowly or too fast fail to spread extracellular protein efficiently. In particular, a specific range of cell reversal frequencies was observed to maximize the cell-cell connection rate and minimize the time of protein spreading. Furthermore, our findings suggest that predesigned motion reversal can be employed to enhance the collective behavior of biological synthetic active systems.

  5. Reversible, on-demand generation of aqueous two-phase microdroplets

    Science.gov (United States)

    Collier, Charles Patrick; Retterer, Scott Thomas; Boreyko, Jonathan Barton; Mruetusatorn, Prachya

    2017-08-15

    The present invention provides methods of on-demand, reversible generation of aqueous two-phase microdroplets core-shell microbeads, microparticle preparations comprising the core-shell microbeads, and drug delivery formulation comprising the microparticle preparations. Because these aqueous microdroplets have volumes comparable to those of cells, they provide an approach to mimicking the dynamic microcompartmentation of biomaterial that naturally occurs within the cytoplasm of cells. Hence, the present methods generate femtoliter aqueous two-phase droplets within a microfluidic oil channel using gated pressure pulses to generate individual, stationary two-phase microdroplets with a well-defined time zero for carrying out controlled and sequential phase transformations over time. Reversible phase transitions between single-phase, two-phase, and core-shell microbead states are obtained via evaporation-induced dehydration and water rehydration.

  6. Polarity-based fractionation in proteomics: hydrophilic interaction vs reversed-phase liquid chromatography.

    Science.gov (United States)

    Jafari, M; Mirzaie, M; Khodabandeh, M; Rezadoost, H; Ghassempour, A; Aboul-Enein, H Y

    2016-07-01

    During recent decades, hydrophilic interaction liquid chromatography (HILIC) ahs been introduced to fractionate or purify especially polar solutes such as peptides and proteins while reversed-phase liquid chromatography (RPLC) is also a common strategy. RPLC is also a common dimension in multidimensional chromatography. In this study, the potential of HILIC vs RPLC chromatography was compared for proteome mapping of human peripheral blood mononuclear cell extract. In HILIC a silica-based stationary phase and for RPLC a C18 column were applied. Then separated proteins were eluted to an ion trap mass spectrometry system. Our results showed that the HILIC leads to more proteins being identified in comparison to RPLC. Among the total 181 identified proteins, 56 and 38 proteins were fractionated specifically by HILIC and RPLC, respectively. In order to demonstrate this, the physicochemical properties of identified proteins such as polarity and hydrophobicity were considered. This analysis indicated that polarity may play a major role in the HILIC separation of proteins vs RPLC. Using gene ontology enrichment analysis, it was also observed that differences in physicochemical properties conform to the cellular compartment and biological features. Finally, this study highlighted the potential of HILIC and the great orthogonality of RPLC in gel-free proteomic studies. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  7. Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin☆

    OpenAIRE

    Roche, Camille J.; Dantsker, David; Heller, Elizabeth R.; Sabat, Joseph E.; Friedman, Joel M.

    2013-01-01

    Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to mani...

  8. Stainless austenitic steels strengthened due to reversible phase transformations and by ageing

    International Nuclear Information System (INIS)

    Sagaradze, V.V.; Kositsyna, I.I.; Ozhiganov, A.V.

    1981-01-01

    The effect of the reversible phase transformations, consisting in the conduction of the direct and reverse martensite transformations and aging, during which the intermetallide γ'-phase of the composition Ni 3 Ti is formed, on the streng-thening of alloys in the Fe-Cr-Ni-Ti system is considered. Stainless austenitic steels Kh12N12T3 and Kh12N14T3, which acquire high mechanical properties: σsub(0.2)=685-785 MPa, σsub(B)=1275 MPa, delta >= 20%, as a result of reversible phase transformations and aging, are suggested. After the reversible phase transformations and ageing the steels possess a high resistance to γ-α-transformation during cold treatment [ru

  9. Temperature induced reversible polymorphic phase transformations in a bis-hydrazone compound

    Science.gov (United States)

    Jayant, Vikrant; Das, Dinabandhu

    2018-03-01

    Two reversible polymorphic phase transformation of 2,3-butanedione, 2,3- bis[4,4‧-bis(diethylamino)benzophenone hydrazone] (DEBH) have been identified in DSC experiment. Topotactic phase transformation of three polymorphs has been observed in variable temperature Single Crystal X-ray Diffraction experiment. The reversible phase transformation of bulk material has been confirmed by Powder X-ray diffraction study.

  10. Martensite shear phase reversion-induced nanograined/ultrafine-grained Fe-16Cr-10Ni alloy: The effect of interstitial alloying elements and degree of austenite stability on phase reversion

    Energy Technology Data Exchange (ETDEWEB)

    Misra, R.D.K., E-mail: dmisra@louisiana.edu [Center for Structural and Functional Materials, University of Louisiana at Lafayette, Madison Hall Room 217, P.O. Box 44130, Lafayette, LA 70504-1430 (United States); Zhang, Z.; Venkatasurya, P.K.C. [Center for Structural and Functional Materials, University of Louisiana at Lafayette, Madison Hall Room 217, P.O. Box 44130, Lafayette, LA 70504-1430 (United States); Somani, M.C.; Karjalainen, L.P. [Department of Mechanical Engineering, University of Oulu, P.O. Box 4200, Oulu 90014 (Finland)

    2010-11-15

    Research highlights: {yields} Development of a novel process involving phase-reversion annealing process. {yields} Austensite stability strongly influences development of nanograined structure. {yields} Interstitial elements influence microstructural evolution during annealing. - Abstract: We describe here an electron microscopy study of microstructural evolution associated with martensitic shear phase reversion-induced nanograined/ultrafine-grained (NG/UFG) structure in an experimental Fe-16Cr-10Ni alloy with very low interstitial content. The primary objective is to understand and obtain fundamental insights on the influence of degree of austenite stability (Fe-16Cr-10Ni, 301LN, and 301 have different austenite stability index) and interstitial elements (carbon and nitrogen) in terms of phase reversion process, microstructural evolution during reversion annealing, and temperature-time annealing sequence. A relative comparison of Fe-16Cr-10Ni alloy with 301LN and 301 austenitic stainless steels indicated that phase reversion in Fe-16Cr-10Ni occurred by shear mechanism, which is similar to that observed for 301, but is different from the diffusional mechanism in 301LN steel. While the phase reversion in the experimental Fe-16Cr-10Ni alloy and 301 austenitic stainless steel occurred by shear mechanism, there were fundamental differences between these two alloys. The reversed strain-free austenite grains in Fe-16Cr-10Ni alloy were characterized by nearly same crystallographic orientation, where as in 301 steel there was evidence of break-up of martensite laths during reversion annealing resulting in several regions of misoriented austenite grains in 301 steel. Furthermore, a higher phase reversion annealing temperature range (800-900 deg. C) was required to obtain a fully NG/UFG structure of grain size 200-600 nm. The difference in the phase reversion and the temperature-time sequence in the three stages is explained in terms of Gibbs free energy change that

  11. Reclaimable Thermally Reversible Polymers for AM Feedstock, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — CRG proposes to continue efforts from the 2016 NASA SBIR Phase I topic H5.04 Reclaimable Thermally Reversible Polymers for AM Feedstock. In Phase II, CRG will refine...

  12. Benefits and Limitations of Lab-on-a-Chip Method over Reversed-Phase High-Performance Liquid Chromatography Method in Gluten Proteins Evaluation

    Directory of Open Access Journals (Sweden)

    Dragan Živančev

    2015-01-01

    Full Text Available RP-HPLC (reversed-phase high-performance liquid chromatography is widely used to determine the amounts of the different gluten protein types. However, this method is time-consuming, especially at early stages of wheat breeding, when large number of samples needs to be analyzed. On the other hand, LoaC (Lab-on-a-Chip technique has the potential for a fast, reliable, and automatable analysis of proteins. In the present study, benefits and limitations of Lab-on-a-Chip method over RP-HPLC method in gluten proteins evaluation were explored in order to determine in which way LoaC method should be improved in order to make its results more compliant with the results of RP-HPLC method. Strong correlation (P≤0.001 was found between numbers of HMW glutenin peaks determined by LoaC and RP-HPLC methods. Significant correlations (P≤0.05 were obtained between percentages of HMW and LMW glutenin subunits calculated with regard to total HMW + LMW area. Even more significant correlation (P≤0.001 was found when percentages of individual HMW areas were calculated with regard to total HMW. RP-HPLC method showed superiority in determination of gliadins since larger number and better resolution of gliadin peaks were obtained by this method.

  13. Small scale affinity purification and high sensitivity reversed phase nanoLC-MS N-glycan characterization of mAbs and fusion proteins.

    Science.gov (United States)

    Higel, Fabian; Seidl, Andreas; Demelbauer, Uwe; Sörgel, Fritz; Frieß, Wolfgang

    2014-01-01

    N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.

  14. Graphene/phase change material nanocomposites: light-driven, reversible electrical resistivity regulation via form-stable phase transitions.

    Science.gov (United States)

    Wang, Yunming; Mi, Hongyi; Zheng, Qifeng; Ma, Zhenqiang; Gong, Shaoqin

    2015-02-04

    Innovative photoresponsive materials are needed to address the complexity of optical control systems. Here, we report a new type of photoresponsive nanomaterial composed of graphene and a form-stable phase change material (PCM) that exhibited a 3 orders of magnitude change in electrical resistivity upon light illumination while retaining its overall original solid form at the macroscopic level. This dramatic change in electrical resistivity also occurred reversibly through the on/off control of light illumination. This was attributed to the reversible phase transition (i.e., melting/recrystallization) behavior of the microscopic crystalline domains present in the form-stable PCM. The reversible phase transition observed in the graphene/PCM nanocomposite was induced by a reversible temperature change through the on/off control of light illumination because graphene can effectively absorb light energy and convert it to thermal energy. In addition, this graphene/PCM nanocomposite also possessed excellent mechanical properties. Such photoresponsive materials have many potential applications, including flexible electronics.

  15. Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

    Science.gov (United States)

    Roche, Camille J.; Dantsker, David; Heller, Elizabeth R.; Sabat, Joseph E.; Friedman, Joel M.

    2013-08-01

    Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to manipulate water content. Hydration properties are probed using the water-sensitive fluorescence from Hb bound pyranine and covalently attached Badan. Protein dynamics are probed through ligand recombination traces derived from photodissociated carbonmonoxy hemoglobin on a log scale that exposes the potential role of both α and β solvent fluctuations in modulating protein dynamics. The results open the possibility of probing hydration level phenomena in this system using a combination of NMR and optical probes.

  16. Studying protein assembly with reversible Brownian dynamics of patchy particles

    International Nuclear Information System (INIS)

    Klein, Heinrich C. R.; Schwarz, Ulrich S.

    2014-01-01

    Assembly of protein complexes like virus shells, the centriole, the nuclear pore complex, or the actin cytoskeleton is strongly determined by their spatial structure. Moreover, it is becoming increasingly clear that the reversible nature of protein assembly is also an essential element for their biological function. Here we introduce a computational approach for the Brownian dynamics of patchy particles with anisotropic assemblies and fully reversible reactions. Different particles stochastically associate and dissociate with microscopic reaction rates depending on their relative spatial positions. The translational and rotational diffusive properties of all protein complexes are evaluated on-the-fly. Because we focus on reversible assembly, we introduce a scheme which ensures detailed balance for patchy particles. We then show how the macroscopic rates follow from the microscopic ones. As an instructive example, we study the assembly of a pentameric ring structure, for which we find excellent agreement between simulation results and a macroscopic kinetic description without any adjustable parameters. This demonstrates that our approach correctly accounts for both the diffusive and reactive processes involved in protein assembly

  17. Studying protein assembly with reversible Brownian dynamics of patchy particles

    Energy Technology Data Exchange (ETDEWEB)

    Klein, Heinrich C. R. [Institute for Theoretical Physics, Heidelberg University, 69120 Heidelberg (Germany); Schwarz, Ulrich S., E-mail: ulrich.schwarz@bioquant.uni-heidelberg.de [Institute for Theoretical Physics, Heidelberg University, 69120 Heidelberg (Germany); BioQuant, Heidelberg University, 69120 Heidelberg (Germany)

    2014-05-14

    Assembly of protein complexes like virus shells, the centriole, the nuclear pore complex, or the actin cytoskeleton is strongly determined by their spatial structure. Moreover, it is becoming increasingly clear that the reversible nature of protein assembly is also an essential element for their biological function. Here we introduce a computational approach for the Brownian dynamics of patchy particles with anisotropic assemblies and fully reversible reactions. Different particles stochastically associate and dissociate with microscopic reaction rates depending on their relative spatial positions. The translational and rotational diffusive properties of all protein complexes are evaluated on-the-fly. Because we focus on reversible assembly, we introduce a scheme which ensures detailed balance for patchy particles. We then show how the macroscopic rates follow from the microscopic ones. As an instructive example, we study the assembly of a pentameric ring structure, for which we find excellent agreement between simulation results and a macroscopic kinetic description without any adjustable parameters. This demonstrates that our approach correctly accounts for both the diffusive and reactive processes involved in protein assembly.

  18. Characterization of retentivity of reversed phase liquid chromatography columns.

    Science.gov (United States)

    Ying, P T; Dorsey, J G

    1991-03-01

    There are dozens of commercially available reversed phase columns, most marketed as C-8 or C-18 materials, but with no useful way of classifying their retentivity. A useful way of ranking these columns in terms of column "strength" or retentivity is presented. The method utilizes a value for ln k'(w), the estimated retention of a solute from a mobile phase of 100% water, and the slope of the plot of ln k' vsE(T)(30), the solvent polarity. The method is validated with 26 solutes varying in ln k'(w) from about 2 to over 20, on 14 different reversed phase columns. In agreement with previous work, it is found that the phase volume ratio of the column is the most important parameter in determining retentivity. It is strongly suggested that manufacturers adopt a uniform method of calculating this value and that it be made available in advertising, rather than the uninterpretable "% carbon".

  19. A time-reversal invariant topological phase at the surface of a 3D topological insulator

    International Nuclear Information System (INIS)

    Bonderson, Parsa; Nayak, Chetan; Qi, Xiao-Liang

    2013-01-01

    A 3D fermionic topological insulator has a gapless Dirac surface state protected by time-reversal symmetry and charge conservation symmetry. The surface state can be gapped by introducing ferromagnetism to break time-reversal symmetry, introducing superconductivity to break charge conservation, or entering a topological phase. In this paper, we construct a minimal gapped topological phase that preserves both time-reversal and charge conservation symmetries and supports Ising-type non-Abelian anyons. This phase can be understood heuristically as emerging from a surface s-wave superconducting state via the condensation of eight-vortex composites. The topological phase inherits vortices supporting Majorana zero modes from the surface superconducting state. However, since it is time-reversal invariant, the surface topological phase is a distinct phase from the Ising topological phase, which can be viewed as a quantum-disordered spin-polarized p x + ip y superconductor. We discuss the anyon model of this topological phase and the manner in which time-reversal symmetry is realized in it. We also study the interfaces between the topological state and other surface gapped phases. (paper)

  20. Development of Reverse-Phase HPLC Method for Simultaneous ...

    African Journals Online (AJOL)

    Erah

    Purpose: To develop a simple, sensitive and rapid reverse phase HPLC method for the simultaneous analysis of metoprolol succinate and hydrochlorothiazide in a solid dosage form. Methods: The .... Extraction was carried out three times with.

  1. Reverse Phase Protein Arrays for High-Throughput Protein Measurements in Mammospheres

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    Protein Array (RPPA)-based readout format integrated into robotic siRNA screening. This technique would allow post-screening high-throughput quantification of protein changes. Recently, breast cancer stem cells (BCSCs) have attracted much attention, as a tumor- and metastasis-driving subpopulation...

  2. Separation of Chloroplast Pigments Using Reverse Phase Chromatography.

    Science.gov (United States)

    Reese, R. Neil

    1997-01-01

    Presents a protocol that uses reverse phase chromatography for the separation of chloroplast pigments. Provides a simple and relatively safe procedure for use in teaching laboratories. Discusses pigment extraction, chromatography, results, and advantages of the process. (JRH)

  3. A reversed-phase compatible thin-layer chromatography autography for the detection of acetylcholinesterase inhibitors.

    Science.gov (United States)

    Ramallo, I Ayelen; García, Paula; Furlan, Ricardo L E

    2015-11-01

    A dual readout autographic assay to detect acetylcholinesterase inhibitors present in complex matrices adsorbed on reversed-phase or normal-phase thin-layer chromatography plates is described. Enzyme gel entrapment with an amphiphilic copolymer was used for assay development. The effects of substrate and enzyme concentrations, pH, incubation time, and incubation temperature on the sensitivity and the detection limit of the assay were evaluated. Experimental design and response surface methodology were used to optimize conditions with a minimum number of experiments. The assay allowed the detection of 0.01% w/w of physostigmine in both a spiked Sonchus oleraceus L. extract chromatographed on normal phase and a spiked Pimenta racemosa (Mill.) J.W. Moore leaf essential oil chromatographed on reversed phase. Finally, the reversed-phase thin-layer chromatography assay was applied to reveal the presence of an inhibitor in the Cymbopogon citratus (DC.) Stapf essential oil. The developed assay is able to detect acetylcholinesterase inhibitors present in complex matrixes that were chromatographed in normal phase or reversed-phase thin-layer chromatography. The detection limit for physostigmine on both normal and reversed phase was of 1×10(-4) μg. The results can be read by a change in color and/or a change in fluorescence. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. C18, C8, and perfluoro reversed phases on diamond for solid-phase extraction.

    Science.gov (United States)

    Saini, Gaurav; Wiest, Landon A; Herbert, David; Biggs, Katherine N; Dadson, Andrew; Vail, Michael A; Linford, Matthew R

    2009-04-17

    In spite of advances in solid-phase extraction (SPE) technology there are certain disadvantages to current SPE silica-based, column packings. The pH range over which extraction can occur is limited and each column is generally only used once. New diamond-based reversed SPE phases (C(18), C(8), and perfluorinated) were developed in our laboratories. Studies were done which show that these phases do not have the same limitations as traditional silica-based stationary phases. The synthesis and properties of these diamond-based phases are presented, and the stability, percent recovery, and column capacity are given for the C(18) phase.

  5. Simultaneous determination of caffeine, theophylline and theobromine in human plasma by on-line solid-phase extraction coupled to reversed-phase chromatography.

    Science.gov (United States)

    Emara, Samy

    2004-10-01

    A reversed-phase liquid chromatographic column switching system was described for the determination of caffeine (CF), theophylline (TH) and theobromine (TB) in human plasma with a direct injection procedure. A short protein-coated mu Bondapak CN silica pre-column (20 x 3 mm, i.d.) was used for enrichment of the drugs and clean up from weakly retained plasma components using phosphate buffer saline pH 7.4. After washing step, the retained drugs were flushed into a reversed-phase column (5 microm TSK gel ODS-80 TM, 150 x 4.6 mm i.d.) with a mobile phase of methanol-0.01 M phosphate buffer, pH 3.5 (30:70, v/v) for the final separation. The eluent was monitored with a UV detector at 275 nm. The resulting chromatograms showed no interference from endogenous plasma components. A linear relationship between the concentration of drug and peak height was confirmed in the range of 0.5-20 microg/mL for all drugs. High extraction recoveries from plasma ranging from 96.12 to 100.32% were achieved. Validation of the method was examined performing intra- and inter-day accuracy and precision and was found to be satisfactory. The coefficients of variation of the three drugs were less than 3% for intra-day and less than 4% for inter-day run assays.

  6. Gradient Scouting in Reversed-Phase HPLC Revisited

    Science.gov (United States)

    Alcazar, A.; Jurado, J. M.; Gonzalez, A. G.

    2011-01-01

    Gradient scouting is the best way to decide the most suitable elution mode in reversed-phase high-performance liquid chromatography (RP-HPLC). A simple rule for this decision involves the evaluation of the ratio [delta]t/t[subscript G] (where [delta]t is the difference in the retention time between the last and the first peak and t[subscript G] is…

  7. The use of Fourier reverse transforms in crystallographic phase refinement

    Energy Technology Data Exchange (ETDEWEB)

    Ringrose, Sharon [Iowa State Univ., Ames, IA (United States)

    1997-10-08

    Often a crystallographer obtains an electron density map which shows only part of the structure. In such cases, the phasing of the trial model is poor enough that the electron density map may show peaks in some of the atomic positions, but other atomic positions are not visible. There may also be extraneous peaks present which are not due to atomic positions. A method for determination of crystal structures that have resisted solution through normal crystallographic methods has been developed. PHASER is a series of FORTRAN programs which aids in the structure solution of poorly phased electron density maps by refining the crystallographic phases. It facilitates the refinement of such poorly phased electron density maps for difficult structures which might otherwise not be solvable. The trial model, which serves as the starting point for the phase refinement, may be acquired by several routes such as direct methods or Patterson methods. Modifications are made to the reverse transform process based on several assumptions. First, the starting electron density map is modified based on the fact that physically the electron density map must be non-negative at all points. In practice a small positive cutoff is used. A reverse Fourier transform is computed based on the modified electron density map. Secondly, the authors assume that a better electron density map will result by using the observed magnitudes of the structure factors combined with the phases calculated in the reverse transform. After convergence has been reached, more atomic positions and less extraneous peaks are observed in the refined electron density map. The starting model need not be very large to achieve success with PHASER; successful phase refinement has been achieved with a starting model that consists of only 5% of the total scattering power of the full molecule. The second part of the thesis discusses three crystal structure determinations.

  8. Phase retrieval with the reverse projection method in the presence of object's scattering

    International Nuclear Information System (INIS)

    Wang, Zhili; Gao, Kun; Wang, Dajiang

    2017-01-01

    X-ray grating interferometry can provide substantially increased contrast over traditional attenuation-based techniques in biomedical applications, and therefore novel and complementary information. Recently, special attention has been paid to quantitative phase retrieval in X-ray grating interferometry, which is mandatory to perform phase tomography, to achieve material identification, etc. An innovative approach, dubbed “Reverse Projection” (RP), has been developed for quantitative phase retrieval. The RP method abandons grating scanning completely, and is thus advantageous in terms of higher efficiency and reduced radiation damage. Therefore, it is expected that this novel method would find its potential in preclinical and clinical implementations. Strictly speaking, the reverse projection method is applicable for objects exhibiting only absorption and refraction. In this contribution, we discuss the phase retrieval with the reverse projection method for general objects with absorption, refraction and scattering simultaneously. Especially, we investigate the influence of the object's scattering on the retrieved refraction signal. Both theoretical analysis and numerical experiments are performed. The results show that the retrieved refraction signal is the product of object's refraction and scattering signals for small values. In the case of a strong scattering, the reverse projection method cannot provide reliable phase retrieval. Those presented results will guide the use of the reverse projection method for future practical applications, and help to explain some possible artifacts in the retrieved images and/or reconstructed slices. - Highlights: • Accurate phase retrieval by the reverse projection method without object's scattering. • Retrieved refraction signal contaminated by the object's scattering. • Refraction signal underestimated by the reverse projection method. • Guide the use of the reverse projection method for

  9. Attractive interactions between reverse aggregates and phase separation in concentrated malonamide extractant solutions

    International Nuclear Information System (INIS)

    Erlinger, C.; Belloni, L.; Zemb, T.; Madic, C.

    1999-01-01

    Using small angle X-ray scattering, conductivity, and phase behavior determination, the authors show that concentrated solutions of malonamide extractants, dimethyldibutyltetradecylmalonamide (DMDBTDMA), are organized in reverse oligomeric aggregates which have many features in common with reverse micelles. The aggregation numbers of these reverse globular aggregates as well as their interaction potential are determined from absolute scattering curves. An attractive interaction is responsible for the demixing of the oil phase when in equilibrium with excess oil. Prediction of conductivity as well as the formation conditions for the third phase is possible using standard liquid theory applied to the extractant aggregates. The interactions, modeled with the sticky sphere model proposed by Baster, are shown to be due to steric interactions resulting from the hydrophobic tails of the extractant molecule and van der Waals forces between the highly polarizable water core of the reverse micelles. The attractive interaction in the oil phase, equilibrated with water, is determined as a function of temperature, extractant molecule concentration, and proton and neodynium(III) cation concentration. It is shown that van der Waals interactions, with an effective Hamaker constant of 3kT, quantitatively explain the behavior of DMDBTDMA in n-dodecane in terms of scattering as well as phase stability limits

  10. Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry

    Science.gov (United States)

    Choi, Sam B.; Lombard-Banek, Camille; Muñoz-LLancao, Pablo; Manzini, M. Chiara; Nemes, Peter

    2018-05-01

    The ability to detect peptides and proteins in single cells is vital for understanding cell heterogeneity in the nervous system. Capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) provides high-resolution mass spectrometry (HRMS) with trace-level sensitivity, but compressed separation during CE challenges protein identification by tandem HRMS with limited MS/MS duty cycle. Here, we supplemented ultrasensitive CE-nanoESI-HRMS with reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons. An 1 to 20 μg neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custom-built CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates 274,000 versus 412,000 maximum, resp.), which enhanced detection sensitivity (2.5-fold higher signal-to-noise ratio), minimized co-isolation spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to 57%. From 1 ng of protein digest (organization. [Figure not available: see fulltext.

  11. ANALYSIS OF BASIC PSYCHOTROPIC DRUGS IN BIOLOGICAL FLUIDS AND TISSUES BY REVERSED-PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY.

    Science.gov (United States)

    Petruczynik, Anna; Waksmundzka-Hajnos, Monika

    2017-03-01

    The review of the RP HPLC analysis of basic psychotropic drugs is presented. It contains sample preparation methods with centrifugation, protein precipitation, liquid-liquid extraction (LLE), dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), solid-phase microextraction (SPME), microwave-assisted extraction (MAE) and RP-HPLC analysis. Chromatographic behavior of basic drugs in aqueous media - eluents used in reversed phase systems is discussed. Methods of blocking of residue surface silanols' interaction are mentioned. Analytical methods used for the analysis are divided into parts according with the above methods: the use of low-pH eluents, the use of high-pH eluents, the use of silanol blockers, special stationary phases for basic analytes. Literature connected with the sample preparation methods and analytical systems for the drug analysis are cited in details and presented also in Table 1.

  12. Reverse screening methods to search for the protein targets of chemopreventive compounds

    Science.gov (United States)

    Huang, Hongbin; Zhang, Guigui; Zhou, Yuquan; Lin, Chenru; Chen, Suling; Lin, Yutong; Mai, Shangkang; Huang, Zunnan

    2018-05-01

    This article is a systematic review of reverse screening methods used to search for the protein targets of chemopreventive compounds or drugs. Typical chemopreventive compounds include components of traditional Chinese medicine, natural compounds and Food and Drug Administration (FDA)-approved drugs. Such compounds are somewhat selective but are predisposed to bind multiple protein targets distributed throughout diverse signaling pathways in human cells. In contrast to conventional virtual screening, which identifies the ligands of a targeted protein from a compound database, reverse screening is used to identify the potential targets or unintended targets of a given compound from a large number of receptors by examining their known ligands or crystal structures. This method, also known as in silico or computational target fishing, is highly valuable for discovering the target receptors of query molecules from terrestrial or marine natural products, exploring the molecular mechanisms of chemopreventive compounds, finding alternative indications of existing drugs by drug repositioning, and detecting adverse drug reactions and drug toxicity. Reverse screening can be divided into three major groups: shape screening, pharmacophore screening and reverse docking. Several large software packages, such as Schrödinger and Discovery Studio; typical software/network services such as ChemMapper, PharmMapper, idTarget and INVDOCK; and practical databases of known target ligands and receptor crystal structures, such as ChEMBL, BindingDB and the Protein Data Bank (PDB), are available for use in these computational methods. Different programs, online services and databases have different applications and constraints. Here, we conducted a systematic analysis and multilevel classification of the computational programs, online services and compound libraries available for shape screening, pharmacophore screening and reverse docking to enable non-specialist users to quickly learn and

  13. The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

    Directory of Open Access Journals (Sweden)

    Gronenborn Angela M

    2010-05-01

    Full Text Available Abstract Background Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H domain of HIV-1 reverse transcriptase (RT and influences the efficiency of viral reverse transcription (Lemay et al., 2008, Retrovirology 5:47. HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs that bind AU-rich elements (AREs. To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s by which HuR influences HIV-1 reverse transcription activity in vitro, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only. Results All four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with 15N labeled HuR variants or the 15N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription in vitro, even on RNA templates that contain AREs. Conclusions Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.

  14. Application of Ionic Liquids in High Performance Reversed-Phase Chromatography

    Directory of Open Access Journals (Sweden)

    Wentao Bi

    2009-06-01

    Full Text Available Ionic liquids, considered “green” chemicals, are widely used in many areas of analytical chemistry due to their unique properties. Recently, ionic liquids have been used as a kind of novel additive in separation and combined with silica to synthesize new stationary phase as separation media. This review will focus on the properties and mechanisms of ionic liquids and their potential applications as mobile phase modifier and surface-bonded stationary phase in reversed-phase high performance liquid chromatography (RP-HPLC. Ionic liquids demonstrate advantages and potential in chromatographic field.

  15. Reverse-phase HPLC analysis of human alpha crystallin.

    Science.gov (United States)

    Swamy, M S; Abraham, E C

    1991-03-01

    A rapid and highly sensitive reverse-phase HPLC (RP-HPLC) method was used to separate crystallin subunits from human alpha crystallin. Three distinct peaks were separated; by electrophoretic and immunological analyses the first and second peaks were identified as alpha B and alpha A respectively. On the other hand, peak 3 appeared to be a modified form of alpha crystallin. The ratio of alpha A and alpha B proteins was 3:1 in 1 day old lenses which gradually changed to 2:1 in 17 year old lenses and to 1:1 in the 50 and 82 year old whole lenses and 82 year old lens cortex, with a concomitant increase in the modified alpha, suggesting that alpha A subunits are relatively more involved in aggregation. Analysis of the 82 year old lens nucleus also supported this conclusion. The RP-HPLC analysis of the HMW aggregate fraction showed substantial enrichment of the modified alpha. The alpha A and alpha B subunits independently reassociated to form polymeric alpha crystallin whereas the modified alpha reassociated to form HMW aggregates as shown by molecular sieve HPLC. Hence it appears that the HMW aggregate peak was constituted by modified alpha crystallin. Only in the peak 3 material the 280 nm absorbance was about 2-fold higher than what was expected from the actual protein content. The data suggest that the changes induced by post-translational modifications may have some role in the formation of modified alpha. The present RP-HPLC method is useful in separating these modified alpha from the unmodified alpha A and alpha B subunits.

  16. Ion-pairing reversed-phased chromatography/mass spectrometry of heparin

    DEFF Research Database (Denmark)

    Henriksen, Jens; Roepstorff, Peter; Ringborg, Lene H.

    2006-01-01

    not well characterised. In order to further characterise such mixtures, two on-line ion-pairing reverse-phased chromatography electrospray ionisation (ESI) mass spectrometry methods have been developed. One of the systems allows the determination of more than 200 components in a medium molecular weight...

  17. Applications of the solvation parameter model in reversed-phase liquid chromatography.

    Science.gov (United States)

    Poole, Colin F; Lenca, Nicole

    2017-02-24

    The solvation parameter model is widely used to provide insight into the retention mechanism in reversed-phase liquid chromatography, for column characterization, and in the development of surrogate chromatographic models for biopartitioning processes. The properties of the separation system are described by five system constants representing all possible intermolecular interactions for neutral molecules. The general model can be extended to include ions and enantiomers by adding new descriptors to encode the specific properties of these compounds. System maps provide a comprehensive overview of the separation system as a function of mobile phase composition and/or temperature for method development. The solvation parameter model has been applied to gradient elution separations but here theory and practice suggest a cautious approach since the interpretation of system and compound properties derived from its use are approximate. A growing application of the solvation parameter model in reversed-phase liquid chromatography is the screening of surrogate chromatographic systems for estimating biopartitioning properties. Throughout the discussion of the above topics success as well as known and likely deficiencies of the solvation parameter model are described with an emphasis on the role of the heterogeneous properties of the interphase region on the interpretation and understanding of the general retention mechanism in reversed-phase liquid chromatography for porous chemically bonded sorbents. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Reverse triiodothyronine in protein energy malnutrition

    International Nuclear Information System (INIS)

    Hafiez, A.A.; Abdel-Salam, E.; Abbas, E.Z.; Halawa, F.A.; El-Hefnawy, N.

    1984-01-01

    Serum levels of thyroxine (T 4 ), triiodothyronine (T 3 ), reverse triiodothyronine (rT 3 ) and thyrotropin (TSH) were determined in cases of kwashiorkor and marasmus. Decreased levels of T 4 and T 3 , and increased levels of rT 3 with no change in TSH were obtained. Thus in infants suffering from protein energy malnutrition there is a state of thyroid dysfunction as well as a shift in the peripheral T 4 metabolism being converted to the inert rT 3 rather than to the physiologically active T 3 . (author)

  19. Development and validation of a reversed phase High Performance ...

    African Journals Online (AJOL)

    A simple, rapid, accurate and economical isocratic Reversed Phase High Performance Liquid Chromatography (RPHPLC) method was developed, validated and used for the evaluation of content of different brands of paracetamol tablets. The method was validated according to ICH guidelines and may be adopted for the ...

  20. Monitoring aged reversed-phase high performance liquid chromatography columns

    NARCIS (Netherlands)

    Bolck, A; Smilde, AK; Bruins, CHP

    1999-01-01

    In this paper, a new approach for the quality assessment of routinely used reversed-phase high performance liquid chromatography columns is presented. A used column is not directly considered deteriorated when changes in retention occur. If attention is paid to the type and magnitude of the changes,

  1. A Technical Assessment of the Utility of Reverse Phase Protein Arrays for the Study of the Functional Proteome in Non-microdissected Human Breast Cancers.

    LENUS (Irish Health Repository)

    Hennessy, Bryan T

    2010-12-01

    INTRODUCTION: The lack of large panels of validated antibodies, tissue handling variability, and intratumoral heterogeneity potentially hamper comprehensive study of the functional proteome in non-microdissected solid tumors. The purpose of this study was to address these concerns and to demonstrate clinical utility for the functional analysis of proteins in non-microdissected breast tumors using reverse phase protein arrays (RPPA). METHODS: Herein, 82 antibodies that recognize kinase and steroid signaling proteins and effectors were validated for RPPA. Intraslide and interslide coefficients of variability were <15%. Multiple sites in non-microdissected breast tumors were analyzed using RPPA after intervals of up to 24 h on the benchtop at room temperature following surgical resection. RESULTS: Twenty-one of 82 total and phosphoproteins demonstrated time-dependent instability at room temperature with most variability occurring at later time points between 6 and 24 h. However, the 82-protein functional proteomic "fingerprint" was robust in most tumors even when maintained at room temperature for 24 h before freezing. In repeat samples from each tumor, intratumoral protein levels were markedly less variable than intertumoral levels. Indeed, an independent analysis of prognostic biomarkers in tissue from multiple tumor sites accurately and reproducibly predicted patient outcomes. Significant correlations were observed between RPPA and immunohistochemistry. However, RPPA demonstrated a superior dynamic range. Classification of 128 breast cancers using RPPA identified six subgroups with markedly different patient outcomes that demonstrated a significant correlation with breast cancer subtypes identified by transcriptional profiling. CONCLUSION: Thus, the robustness of RPPA and stability of the functional proteomic "fingerprint" facilitate the study of the functional proteome in non-microdissected breast tumors.

  2. A systems biology approach to the pathogenesis of obesity-related nonalcoholic fatty liver disease using reverse phase protein microarrays for multiplexed cell signaling analysis.

    Science.gov (United States)

    Calvert, Valerie S; Collantes, Rochelle; Elariny, Hazem; Afendy, Arian; Baranova, Ancha; Mendoza, Michael; Goodman, Zachary; Liotta, Lance A; Petricoin, Emanuel F; Younossi, Zobair M

    2007-07-01

    Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. Omental adipose tissue, a biologically active organ secreting adipokines and cytokines, may play a role in the development of NAFLD. We tested this hypothesis with reverse-phase protein microarrays (RPA) for multiplexed cell signaling analysis of adipose tissue from patients with NAFLD. Omental adipose tissue was obtained from 99 obese patients. Liver biopsies obtained at the time of surgery were all read by the same hepatopathologist. Adipose tissue was exposed to rapid pressure cycles to extract protein lysates. RPA was used to investigate intracellular signaling. Analysis of 54 different kinase substrates and cell signaling endpoints showed that an insulin signaling pathway is deranged in different locations in NAFLD patients. Furthermore, components of insulin receptor-mediated signaling differentiate most of the conditions on the NAFLD spectrum. For example, PKA (protein kinase A) and AKT/mTOR (protein kinase B/mammalian target of rapamycin) pathway derangement accurately discriminates patients with NASH from those with the non-progressive forms of NAFLD. PKC (protein kinase C) delta, AKT, and SHC phosphorylation changes occur in patients with simple steatosis. Amounts of the FKHR (forkhead factor Foxo1)phosphorylated at S256 residue were significantly correlated with AST/ALT ratio in all morbidly obese patients. Furthermore, amounts of cleaved caspase 9 and pp90RSK S380 were positively correlated in patients with NASH. Specific insulin pathway signaling events are altered in the adipose tissue of patients with NASH compared with patients with nonprogressive forms of NAFLD. These findings provide evidence for the role of omental fat in the pathogenesis, and potentially, the progression of NAFLD.

  3. Protein-surface interactions on stimuli-responsive polymeric biomaterials.

    Science.gov (United States)

    Cross, Michael C; Toomey, Ryan G; Gallant, Nathan D

    2016-03-04

    Responsive surfaces: a review of the dependence of protein adsorption on the reversible volume phase transition in stimuli-responsive polymers. Specifically addressed are a widely studied subset: thermoresponsive polymers. Findings are also generalizable to other materials which undergo a similarly reversible volume phase transition. As of 2015, over 100,000 articles have been published on stimuli-responsive polymers and many more on protein-biomaterial interactions. Significantly, fewer than 100 of these have focused specifically on protein interactions with stimuli-responsive polymers. These report a clear trend of increased protein adsorption in the collapsed state compared to the swollen state. This control over protein interactions makes stimuli-responsive polymers highly useful in biomedical applications such as wound repair scaffolds, on-demand drug delivery, and antifouling surfaces. Outstanding questions are whether the protein adsorption is reversible with the volume phase transition and whether there is a time-dependence. A clear understanding of protein interactions with stimuli-responsive polymers will advance theoretical models, experimental results, and biomedical applications.

  4. Flooding and flow reversal of two-phase annular flow

    International Nuclear Information System (INIS)

    Asahi, Y.

    1978-01-01

    The flooding and flow reversal conditions of two-phase annular flow are mathematically defined in terms of a characteristic function representing a force balance. Sufficiently below the flooding point in counter-current flow, the interface is smooth and the characteristic equation reduces to the Nusselt relationship. Just below flooding point and above the flow reversal point in cocurrent flow, the interface is 'wavy', so that the interfacial shear effect plays an important role. The theoretical analysis is compared with experimental results by others. It is suggested that the various length effects which have been experimentally observed may be accounted for by the spatial variation of the droplet entrainment. (Auth.)

  5. Reversed-phase thin-layer chromatography of homologs of Antimycin-A and related derivatives

    Science.gov (United States)

    Abidi, Sharon L.

    1989-01-01

    Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, Ala, Alb, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins Al, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifiers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpretated based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.

  6. Reverse triiodothyronine in protein energy malnutrition

    Energy Technology Data Exchange (ETDEWEB)

    Hafiez, A A; Abdel-Salam, E; Abbas, E Z; Halawa, F A; El-Hefnawy, N [Cairo Univ. (Egypt)

    1984-08-01

    Serum levels of thyroxine (T/sub 4/), triiodothyronine (T/sub 3/), reverse triiodothyronine (rT/sub 3/) and thyrotropin (TSH) were determined in cases of kwashiorkor and marasmus. Decreased levels of T/sub 4/ and T/sub 3/, and increased levels of rT/sub 3/ with no change in TSH were obtained. Thus in infants suffering from protein energy malnutrition there is a state of thyroid dysfunction as well as a shift in the peripheral T/sub 4/ metabolism being converted to the inert rT/sub 3/ rather than to the physiologically active T/sub 3/.

  7. Quantification of whey proteins by reversed phase-HPLC and effectiveness of mid-infrared spectroscopy for their rapid prediction in sweet whey.

    Science.gov (United States)

    Sturaro, Alba; De Marchi, Massimo; Masi, Antonio; Cassandro, Martino

    2016-01-01

    In the dairy industry, membrane filtration is used to reduce the amount of whey waste and, simultaneously, to recover whey proteins (WP). The composition of WP can strongly affect the filtration treatment of whey, and rapid determination of WP fractions would be of interest for dairy producers to monitor WP recovery. This study aimed to develop mid-infrared spectroscopy (MIRS) prediction models for the rapid quantification of protein in sweet whey, using a validated rapid reversed phase (RP)-HPLC as a reference method. Quantified WP included α-lactalbumin (α-LA), β-lactoglobulin (β-LG) A and B, bovine serum albumin, caseinomacropeptides, and proteose peptone. Validation of RP-HPLC was performed by calculating the relative standard deviation (RSD) in repeatability and reproducibility tests for WP retention time and peak areas. Samples of liquid whey (n=187) were analyzed by RP-HPLC and scanned through MIRS to collect spectral information (900 to 4,000 cm(-1)); statistical analysis was carried out through partial least squares regression and random cross-validation procedure. Retention times in RP-HPLC method were stable (RSD between 0.03 and 0.80%), whereas the RSD of peak area (from 0.25 to 8.48%) was affected by WP relative abundance. Higher coefficients of determination in validation for MIRS model were obtained for protein fractions present in whey in large amounts, such as β-LG (0.58), total identified WP (0.58), and α-LA (0.56). Results of this study suggest that MIRS is an easy method for rapid quantification of detail protein in sweet whey, even if better resolution was achieved with the method based on RP-HPLC. The prediction of WP in sweet whey by MIRS might be used for screening and for classifying sweet whey according to its total and individual WP contents. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  8. Two proteins with reverse transcriptase activities associated with hepatitis B virus-like particles

    International Nuclear Information System (INIS)

    Bavand, M.R.; Laub, O.

    1988-01-01

    Recent studies suggest that hepatitis B virus (HBV), despite being a DNA virus, replicates via an RNA intermediate. The HBV life cycle is therefore a permuted version of the RNA retroviral life cycle. Sequence homology between retroviral reverse transcriptase and the putative HBV polymerase gene product suggests the presence of an HBV reverse transcriptase. As yet, there has been no direct evidence that reverse transcriptase activity is present in the viral particle. The authors used activity gel analysis to detect the in situ catalytic activities of DNA polymerases after sodium dodecyl sulfate-polyacrylamide gel electrophorsis. These studies demonstrated that HBV-like particles secreted by a differentiated human hepatoma cell line tranfected with genomic HBV DNA contain two major polymerase activities which migrate as ∼90- and ∼70-kilodalton (kDa) proteins. This demonstrated, for the first time, that HBV-like particles contain a novel DNA polymerase-reverse transcriptase activity. Furthermore, they propose that the 70-kDa reverse transcriptase may be produced by proteolytic self-cleavage of the 90-kDa precursor protein

  9. Targeting protein kinases to reverse multidrug resistance in sarcoma.

    Science.gov (United States)

    Chen, Hua; Shen, Jacson; Choy, Edwin; Hornicek, Francis J; Duan, Zhenfeng

    2016-02-01

    Sarcomas are a group of cancers that arise from transformed cells of mesenchymal origin. They can be classified into over 50 subtypes, accounting for approximately 1% of adult and 15% of pediatric cancers. Wide surgical resection, radiotherapy, and chemotherapy are the most common treatments for the majority of sarcomas. Among these therapies, chemotherapy can palliate symptoms and prolong life for some sarcoma patients. However, sarcoma cells can have intrinsic or acquired resistance after treatment with chemotherapeutics drugs, leading to the development of multidrug resistance (MDR). MDR attenuates the efficacy of anticancer drugs and results in treatment failure for sarcomas. Therefore, overcoming MDR is an unmet need for sarcoma therapy. Certain protein kinases demonstrate aberrant expression and/or activity in sarcoma cells, which have been found to be involved in the regulation of sarcoma cell progression, such as cell cycle, apoptosis, and survival. Inhibiting these protein kinases may not only decrease the proliferation and growth of sarcoma cells, but also reverse their resistance to chemotherapeutic drugs to subsequently reduce the doses of anticancer drugs and decrease drug side-effects. The discovery of novel strategies targeting protein kinases opens a door to a new area of sarcoma research and provides insight into the mechanisms of MDR in chemotherapy. This review will focus on the recent studies in targeting protein kinase to reverse chemotherapeutic drug resistance in sarcoma. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Auditory sensitivity to spectral modulation phase reversal as a function of modulation depth.

    Science.gov (United States)

    Buss, Emily; Grose, John

    2018-01-01

    The present study evaluated auditory sensitivity to spectral modulation by determining the modulation depth required to detect modulation phase reversal. This approach may be preferable to spectral modulation detection with a spectrally flat standard, since listeners appear unable to perform the task based on the detection of temporal modulation. While phase reversal thresholds are often evaluated by holding modulation depth constant and adjusting modulation rate, holding rate constant and adjusting modulation depth supports rate-specific assessment of modulation processing. Stimuli were pink noise samples, filtered into seven octave-wide bands (0.125-8 kHz) and spectrally modulated in dB. Experiment 1 measured performance as a function of modulation depth to determine appropriate units for adaptive threshold estimation. Experiment 2 compared thresholds in dB for modulation detection with a flat standard and modulation phase reversal; results supported the idea that temporal cues were available at high rates for the former but not the latter. Experiment 3 evaluated spectral modulation phase reversal thresholds for modulation that was restricted to either one or two neighboring bands. Flanking bands of unmodulated noise had a larger detrimental effect on one-band than two-band targets. Thresholds for high-rate modulation improved with increasing carrier frequency up to 2 kHz, whereas low-rate modulation appeared more consistent across frequency, particularly in the two-band condition. Experiment 4 measured spectral weights for spectral modulation phase reversal detection and found higher weights for bands in the spectral center of the stimulus than for the lowest (0.125 kHz) or highest (8 kHz) band. Experiment 5 compared performance for highly practiced and relatively naïve listeners, and found weak evidence of a larger practice effect at high than low spectral modulation rates. These results provide preliminary data for a task that may provide a better estimate of

  11. 3D printed titanium micro-bore columns containing polymer monoliths for reversed-phase liquid chromatography.

    Science.gov (United States)

    Gupta, Vipul; Talebi, Mohammad; Deverell, Jeremy; Sandron, Sara; Nesterenko, Pavel N; Heery, Brendan; Thompson, Fletcher; Beirne, Stephen; Wallace, Gordon G; Paull, Brett

    2016-03-03

    The potential of 3D selective laser melting (SLM) technology to produce compact, temperature and pressure stable titanium alloy chromatographic columns is explored. A micro bore channel (0.9 mm I.D. × 600 mm long) was produced within a 5 × 30 × 30 mm titanium alloy (Ti-6Al-4V) cuboid, in form of a double handed spiral. A poly(butyl methacrylate-co-ethyleneglycoldimethacrylate) (BuMA-co-EDMA) monolithic stationary phase was thermally polymerised within the channel for application in reversed-phase high-performance liquid chromatography. The prepared monolithic column was applied to the liquid chromatographic separation of intact proteins and peptides. Peak capacities of 69-76 (for 6-8 proteins respectively) were observed during isothermal separation of proteins at 44 °C which were further increased to 73-77 using a thermal step gradient with programmed temperature from 60 °C to 35 °C using an in-house built direct-contact heater/cooler platform based upon matching sized Peltier thermoelectric modules. Rapid temperature gradients were possible due to direct-contact between the planar metal column and the Peltier module, and the high thermal conductivity of the titanium column as compared to a similar stainless steel printed column. The separation of peptides released from a digestion of E.coli was also achieved in less than 35 min with ca. 40 distinguishable peaks at 210 nm. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Study of plasma equilibrium during the AC current reversal phase in STOR-M

    International Nuclear Information System (INIS)

    Xiao, C.

    2002-01-01

    Alternating current (AC) tokamak operation and equilibrium studies have been performed on the STOR-M tokamak. The recent experiments have achieved consistent smooth current reversal through the implementation of a hybrid digital-analog position controller and by careful density control. In order to study the plasma equilibrium during the current reversal phase with negligible rotational transform, a segmented limiter with four isolated conducting plates has been installed. The plates can be connected outside the vacuum vessel, which allows measurements of currents flowing between limiter plates. When the current reversal is smooth with zero dwell time, the hydrogen line emission level and electron density remain finite, indicating a finite particle confinement. The current from the top to the bottom limiter plate is also finite and its direction is consistent with that of the grad-B drift. The observation suggests that the limiter and other conducting structures surrounding the plasmas plays the role, during the current reversal phase of AC tokamak operation, to short out the charge separation arising from the grad-B drift and to maintain a finite particle confinement. (author)

  13. Nanoscale Phase Stability Reversal During the Nucleation and Growth of Titanium Oxide Minerals

    Science.gov (United States)

    Hummmer, D. R.; Heaney, P. J.; Kubicki, J. D.; Kent, P. R.; Post, J. E.

    2008-12-01

    Fine-grained titanium oxide minerals are important in soils, where they affect a variety of geochemical processes. They are also industrially important as catalysts, pigments, food additives, and dielectrics. Recent research has indicated an apparent reversal of thermodynamic stability between TiO2 phases at the nanoscale thought to be caused by an increased contribution of surface energy to the total free energy. Time-resolved X-ray diffraction (XRD) experiments in which titanium oxides crystallize from aqueous TiCl4 solutions confirm that anatase, a metastable phase, is always the first phase to nucleate under our range of initial conditions. Rutile peaks are observed only minutes after the first appearance of anatase, after which anatase abundance slowly decreases while rutile continues to form. Whole pattern refinement of diffraction data reveals that lattice constants of both phases increase throughout the crystallization process. In addition, transmission electron microscope (TEM) observations and kinetic modeling indicate that anatase does not undergo a solid-state transformation to the rutile structure as once thought. Instead, anatase appears to re-dissolve and then feed the growth of already nucleated rutile nanocrystals. Density functional theory (DFT) calculations were employed to model 1, 2, and 3 nm particles of both mineral phases. The total surface energies calculated from these models did yield lower values for anatase than for rutile by 8-13 kJ/mol depending on particle size, indicating that surface free energy is sufficient to account for stability reversal. However, these whole-particle surface energies were much higher than the sum of energies of each particle's constituent crystallographic surfaces. We attribute the excess energy to defects associated with the edges and corners of nanoparticles, which are not present on a 2-D periodic surface. This previously unreported edge and corner energy may play a dominant role in the stability reversal

  14. The reversal phase of the bone-remodeling cycle

    DEFF Research Database (Denmark)

    Delaisse, Jean-Marie

    2014-01-01

    coincides with decreased osteoblast recruitment and impaired initiation of bone formation, that is, uncoupling. Overall, this review stresses that coupling does not only depend on molecules able to activate osteogenesis, but that it also demands the presence of osteoprogenitors and ordered cell......The reversal phase couples bone resorption to bone formation by generating an osteogenic environment at remodeling sites. The coupling mechanism remains poorly understood, despite the identification of a number of 'coupling' osteogenic molecules. A possible reason is the poor attention...

  15. Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.

    Science.gov (United States)

    Romero-Romero, Sergio; Costas, Miguel; Rodríguez-Romero, Adela; Alejandro Fernández-Velasco, D

    2015-08-28

    Temperature is one of the main variables that modulate protein function and stability. Thermodynamic studies of oligomeric proteins, the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. There are no reports on the reversible equilibrium thermal unfolding of proteins composed of (β/α)8 barrel subunits, albeit this "TIM barrel" topology is one of the most abundant and versatile in nature. We studied the eponymous TIM barrel, triosephosphate isomerase (TIM), belonging to five species of different bacterial taxa. All of them were found to be catalytically efficient dimers. The three-dimensional structure of four enzymes was solved at high/medium resolution. Irreversibility and kinetic control were observed in the thermal unfolding of two TIMs, while for the other three the thermal unfolding was found to follow a two-state equilibrium reversible process. Shifts in the global stability curves of these three proteins are related to the organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. Reversibility appears to correlate with the low isoelectric point, the absence of a residual structure in the unfolded state, small cavity volume in the native state, low conformational stability and a low melting temperature. Furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce aggregation and favour reversibility. It is therefore very thought-provoking to find that a common topological ensemble, such as the TIM barrel, can unfold/refold in the Anfinsen way, i.e. without the help of the cellular machinery.

  16. Acute-phase proteins: As diagnostic tool

    Directory of Open Access Journals (Sweden)

    Sachin Jain

    2011-01-01

    Full Text Available The varied reactions of the host to infection, inflammation, or trauma are collectively known as the acute-phase response and encompass a wide range of pathophysiological responses such as pyrexia, leukocytosis, hormone alterations, and muscle protein depletion combining to minimize tissue damage while enhancing the repair process. The mechanism for stimulation of hepatic production of acute-phase proteins is by proinflammatory cytokines. The functions of positive acute-phase proteins (APP are regarded as important in optimization and trapping of microorganism and their products, in activating the complement system, in binding cellular remnants like nuclear fractions, in neutralizing enzymes, scavenging free hemoglobin and radicals, and in modulating the host′s immune response. APP can be used as diagnostic tool in many diseases like bovine respiratory syncytial virus, prostate cancer, bronchopneumonia, multiple myeloma, mastitis, Streptococcus suis infection, starvation, or lymphatic neoplasia. Thus, acute-phase proteins may provide an alternative means of monitoring animal health.

  17. Murine leukemia virus pol gene products: analysis with antisera generated against reverse transcriptase and endonuclease fusion proteins expressed in Escherichia coli

    International Nuclear Information System (INIS)

    Hu, S.C.; Court, D.L.; Zweig, M.; Levin, J.G.

    1986-01-01

    The organization of the murine leukemia virus (MuLV) pol gene was investigated by expressing molecular clones containing AKR MuLV reverse transcriptase or endonuclease or both gene segments in Escherichia coli and generating specific antisera against the expressed bacterial proteins. Reaction of these antisera with detergent-disrupted virus precipitated and 80-kilodalton (kDa) protein, the MuLV reverse transcriptase, and a 46-kDa protein which we believe is the viral endonuclease. A third (50-kDa) protein, related to reverse transcriptase, was also precipitated. Bacterial extracts of clones expressing reverse transcriptase and endonuclease sequences competed with the viral 80- and 46-kDa proteins, respectively. These results demonstrate that the antisera are specific for viral reverse transcriptase and endonuclease. Immunoprecipitation of AKR MuLV with antisera prepared against a bacterial protein containing only endonuclease sequences led to the observation that reverse transcriptase and endonuclease can be associated as a complex involving a disulfide bond(s)

  18. Reversed phase column extraction studies to recovery of uranium using a modified perlite

    International Nuclear Information System (INIS)

    Akcay, H.

    2006-01-01

    Reversed phase column (RPC) extraction chromatography is an useful technique and has been carried out successfully to the separation of various metals and organic compounds. Its application has received considerable attention because it combines the selectivity of LLE with the advantage of chromatography. The efficiency of the separation by RPC depends on both the chemical and physical properties of the solid support and the stationary phase. This work describes the preparation of an improved adsorbent from natural perlite and its properties to uptake of uranyl ion. Perlite is a volcanic glassy rock produced in commercially workable quantities from mines of Aegean region of Turkey and contains 70-75% CO 2 .The CO 2 was converted to soluble silicates with NaOH modification then its acidification to form hydrogen which turned into xerogels upon drying. Fundamental parameters (particle size, specific surface area, pore size and volume, surface hydroxyl group density) were determined for modified perlite and it was silanized then loaded with 20% (w/w) TBP before being used as reversed phase column chromatography solid support. Finally the sorption of UO 2 + 2 from aqueous solutions by the modified perlite was investigated using Batch techniques. The use of TBP-loaded perlite as a reversed phase column (RPC) extraction chromatography support seems to be useful to uptake of UO 2 + 2 from aqueous solutions and to separate from various cations

  19. Experimental hydrophobicity parameters of perfluorinated alkylated substances from reversed-phase high performance liquid chromatography

    NARCIS (Netherlands)

    de Voogt, P.; Zurano, L.; Serné, P.; Haftka, J.J.H.

    2012-01-01

    Capacity factors of perfluorinated alkylated substances were obtained from isocratic reversed-phase high-performance liquid chromatography-mass spectrometry experiments at different organic modifier strengths of the mobile phase. The resulting capacity factor v. modifier strengths plots were

  20. Coexistence of Phases in a Protein Heterodimer

    Science.gov (United States)

    Krokhotin, Andrey; Liwo, Adam; Niemi, Antti J.; Scheraga, Harold A.

    2012-07-01

    A heterodimer consisting of two or more different kinds of proteins can display an enormous number of distinct molecular architectures. The conformational entropy is an essential ingredient in the Helmholtz free energy and, consequently, these heterodimers can have a very complex phase structure. Here, it is proposed that there is a state of proteins, in which the different components of a heterodimer exist in different phases. For this purpose, the structures in the protein data bank (PDB) have been analyzed, with radius of gyration as the order parameter. Two major classes of heterodimers with their protein components coexisting in different phases have been identified. An example is the PDB structure 3DXC. This is a transcriptionally active dimer. One of the components is an isoform of the intra-cellular domain of the Alzheimer-disease related amyloid precursor protein (AICD), and the other is a nuclear multidomain adaptor protein in the Fe65 family. It is concluded from the radius of gyration that neither of the two components in this dimer is in its own collapsed phase, corresponding to a biologically active protein. The UNRES energy function has been utilized to confirm that, if the two components are separated from each other, each of them collapses. The results presented in this work show that heterodimers whose protein components coexist in different phases, can have intriguing physical properties with potentially important biological consequences.

  1. Reversed-phase thin-layer chromatography behavior of aldopentose derivatives

    Directory of Open Access Journals (Sweden)

    Malbaša Radomir V.

    2012-01-01

    Full Text Available Quantitative structure-retention relationships (QSRR have been used to study the chromatographic behavior of some aldopentose. The behavior of aldopentose derivatives was investigated by means of the reversed-phase thin-layer chromatography (RP TLC on the silica gel impregnated with paraffin oil stationary phases. Binary mixtures of methanol-water, acetone-water and dioxane-water were used as mobile phases. Retention factors, RM0, corresponding to zero percent organic modifier in the aqueous mobile phase was determined. Lipophilicity C0 was calculated as the ratio of the intercept and slope values. There was satisfactory correlation between them and log P values calculated using different theoretical procedures. Some of these correlations offer very good predicting models, which are important for a better understanding of the relationships between chemical structure and retention. The study showed that the hydrophobic parameters RM0 and C0 can be used as a measures of lipophilicity of investigated compounds.

  2. Origin of Reversible Photoinduced Phase Separation in Hybrid Perovskites.

    Science.gov (United States)

    Bischak, Connor G; Hetherington, Craig L; Wu, Hao; Aloni, Shaul; Ogletree, D Frank; Limmer, David T; Ginsberg, Naomi S

    2017-02-08

    The distinct physical properties of hybrid organic-inorganic materials can lead to unexpected nonequilibrium phenomena that are difficult to characterize due to the broad range of length and time scales involved. For instance, mixed halide hybrid perovskites are promising materials for optoelectronics, yet bulk measurements suggest the halides reversibly phase separate upon photoexcitation. By combining nanoscale imaging and multiscale modeling, we find that the nature of halide demixing in these materials is distinct from macroscopic phase separation. We propose that the localized strain induced by a single photoexcited charge interacting with the soft, ionic lattice is sufficient to promote halide phase separation and nucleate a light-stabilized, low-bandgap, ∼8 nm iodide-rich cluster. The limited extent of this polaron is essential to promote demixing because by contrast bulk strain would simply be relaxed. Photoinduced phase separation is therefore a consequence of the unique electromechanical properties of this hybrid class of materials. Exploiting photoinduced phase separation and other nonequilibrium phenomena in hybrid materials more generally could expand applications in sensing, switching, memory, and energy storage.

  3. Implications of protein polymorphism on protein phase behaviour

    NARCIS (Netherlands)

    Stegen, J.; Schoot, van der P.P.A.M.

    2015-01-01

    The phase behaviour of small globular proteins is often modeled by approximating them as spherical particles with fixed internal structure. However, changes in the local environment of a protein can lead to changes in its conformation rendering this approximation invalid. We present a simple

  4. Origin of Reversible Photoinduced Phase Separation in Hybrid Perovskites

    Science.gov (United States)

    Bischak, Connor G.; Hetherington, Craig L.; Wu, Hao; Aloni, Shaul; Ogletree, D. Frank; Limmer, David T.; Ginsberg, Naomi S.

    2017-02-01

    Nonequilibrium processes occurring in functional materials can significantly impact device efficiencies and are often difficult to characterize due to the broad range of length and time scales involved. In particular, mixed halide hybrid perovskites are promising for optoelectronics, yet the halides reversibly phase separate when photo-excited, significantly altering device performance. By combining nanoscale imaging and multiscale modeling, we elucidate the mechanism underlying this phenomenon, demonstrating that local strain induced by photo-generated polarons promotes halide phase separation and leads to nucleation of light-stabilized iodide-rich clusters. This effect relies on the unique electromechanical properties of hybrid materials, characteristic of neither their organic nor inorganic constituents alone. Exploiting photo-induced phase separation and other nonequilibrium phenomena in hybrid materials, generally, could enable new opportunities for expanding the functional applications in sensing, photoswitching, optical memory, and energy storage.

  5. Reversed phase parallel artificial membrane permeation assay for log P measurement

    Directory of Open Access Journals (Sweden)

    Zihao Song

    2016-03-01

    Full Text Available A reversed phase parallel artificial membrane permeation assay (RP-PAMPA was newly invented for log P measurement. An oil/water/oil sandwich was constructed using a conventional PAMPA instrument. 1 % agarose was used to improve the physical stability of the water phase. A linear correlation between log P and the apparent permeability was observed in the -0.24 < log P < 2.85 region (R2 = 0.98. RP-PAMPA was also applied to pKa measurement.

  6. Rapid separation of lanthanides and actinides on small particle based reverse phase supports

    Energy Technology Data Exchange (ETDEWEB)

    Datta, A.; Sivaraman, N.; Srinivasan, T.G.; Vasudeva Rao, P.R. [Chemistry Group, Indira Gandhi Centre for Atomic Research, Kalpakkam (India)

    2010-07-01

    This paper presents the results on the use of short columns (3-5 cm long) with small particle size (1.8 {mu}m) for high performance liquid chromatographic separation of individual lanthanides and uranium from plutonium as well as uranium from thorium to achieve rapid separations i.e. separation time as short as 3.6 min for individual lanthanides, 1 min for thorium-uranium and 4.2 min for uranium from plutonium. These advantages can be exploited to significantly reduce analysis time, liquid waste generation as well as dose to operator when radioactive samples are analysed e.g. burn-up determination. In the present work, a dynamic ion-exchange chromatographic separation technique was employed using camphor-10-sulfonic acid (CSA) as the ion-pairing reagent and {alpha}-hydroxy isobutyric acid ({alpha}-HIBA) as the complexing reagent for the isolation of individual lanthanides as well as the separation of uranium from thorium. Uranium was separated from Pu(III) as well as Pu(IV) by reverse phase HPLC technique. The reverse phase HPLC was also investigated for the isolation and quantitative determination of uranium from thorium as well as lanthanide group from uranium. The dynamic ion-exchange technique using small particle support was demonstrated for measuring the concentrations of lanthanide fission products such as La, Ce, Pr, Nd and Sm in the dissolver solution of fast reactor fuel. Similarly, the assay of uranium in the dissolver solution of fast reactor was carried out using reverse phase HPLC technique. The rapid separation technique using reverse phase HPLC was also demonstrated for separation of lanthanides as a group from uranium matrix; samples of LiCl-KCl eutectic salt containing chlorides of lanthanides in uranium matrix (typically 1: 2000) were analysed. (orig.)

  7. Local wettability reversal during steady-state two-phase flow in porous media.

    Science.gov (United States)

    Sinha, Santanu; Grøva, Morten; Ødegården, Torgeir Bryge; Skjetne, Erik; Hansen, Alex

    2011-09-01

    We study the effect of local wettability reversal on remobilizing immobile fluid clusters in steady-state two-phase flow in porous media. We consider a two-dimensional network model for a porous medium and introduce a wettability alteration mechanism. A qualitative change in the steady-state flow patterns, destabilizing the percolating and trapped clusters, is observed as the system wettability is varied. When capillary forces are strong, a finite wettability alteration is necessary to move the system from a single-phase to a two-phase flow regime. When both phases are mobile, we find a linear relationship between fractional flow and wettability alteration.

  8. Critical indices for reversible gamma-alpha phase transformation in metallic cerium

    Science.gov (United States)

    Soldatova, E. D.; Tkachenko, T. B.

    1980-08-01

    Critical indices for cerium have been determined within the framework of the pseudobinary solution theory along the phase equilibrium curve, the critical isotherm, and the critical isobar. The results obtained verify the validity of relationships proposed by Rushbrook (1963), Griffiths (1965), and Coopersmith (1968). It is concluded that reversible gamma-alpha transformation in metallic cerium is a critical-type transformation, and cerium has a critical point on the phase diagram similar to the critical point of the liquid-vapor system.

  9. Sensitive determination of nitrophenol isomers by reverse-phase high-performance liquid chromatography in conjunction with liquid-liquid extraction

    Science.gov (United States)

    A method for the highly sensitive determination of 2-, 3- and 4- nitrophenols was developed using reverse-phase high-performance liquid chromatography (RP-HPLC) with a UV photodiode array detector. Using a reverse-phase column and 40% aqueous acetonitrile as an eluent (i.e. isocratic elution), the i...

  10. High-performance liquid chromatography using reversed-phase stationary phases dynamically modified with organophosphorus compound for the separation and determination of lanthanoid elements

    International Nuclear Information System (INIS)

    Tsuyoshi, Akira; Akiba, Kenichi

    2000-01-01

    An acidic organophosphorus compound, 2-ethylhexylphosphonic acid mono-2-ethlhexyl ester (EHPA), has been applied to reversed-phase high-performance liquid chromatography (RP-HPLC). The reversed-phase stationary phase was dynamically modified with EHPA by flowing the mobile phase of an acetone-water mixture containing the extracting regent. The retention of lanthanoid elements was widely varied by changing the conditions of the mobile phase, i.e., the pH, the EHPA concentration and the acetone content. The selectivity of EHPA is well reflected to the chromatographic systems, and a precise separation of lanthanoid elements was achieved with sufficient resolution. The determination of Sm was examined in the presence of a large amount of Nd. A linear calibration graph was obtained for Sm at the level of 10 -7 mol dm -3 , in the presence of 1x10 -4 mol dm -3 of Nd. (author)

  11. Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors.

    Science.gov (United States)

    Pardon, Els; Betti, Cecilia; Laeremans, Toon; Chevillard, Florent; Guillemyn, Karel; Kolb, Peter; Ballet, Steven; Steyaert, Jan

    2018-05-04

    The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β 2 -adrenoreceptor-nanobody fusion locked in its active-state conformation by a G-protein-mimicking nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody-enabled reverse pharmacology. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Preparation and characterization of a new microwave immobilized poly(2-phenylpropyl)methylsiloxane stationary phase for reversed phase high-performance liquid chromatography.

    Science.gov (United States)

    Begnini, Fernanda R; Jardim, Isabel C S F

    2013-07-05

    A new reversed phase high-performance liquid chromatography (RP-HPLC) stationary phase was prepared and its chromatographic and physical-chemical properties were evaluated. The new stationary phase was prepared with a silica support and poly(2-phenylpropyl)methylsiloxane (PPPMS), a phenyl type polysiloxane copolymer. Since this is a new copolymer and there is little information in the literature, it was submitted to physical-chemical characterization by infrared spectroscopy and thermogravimetry. The chromatographic phase was prepared through sorption and microwave immobilization of the copolymer onto a silica support. The chromatographic performance was evaluated by employing test procedures suggested by Engelhardt and Jungheim, Tanaka and co-workers, Neue, and Szabó and Csató. These test mixtures provide information about the hydrophobic selectivity, silanophilic activity, ion-exchange capacity, shape selectivity and interaction with polar analytes of the new Si-PPPMS reversed phase. Stability tests were developed using accelerated aging tests under both basic and acidic conditions to provide information about the lifetime of the packed columns. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Reversibility of magnetic field driven transition from electronic phase separation state to single-phase state in manganites: A microscopic view

    Science.gov (United States)

    Liu, Hao; Lin, Lingfang; Yu, Yang; Lin, Hanxuan; Zhu, Yinyan; Miao, Tian; Bai, Yu; Shi, Qian; Cai, Peng; Kou, Yunfang; Lan, Fanli; Wang, Wenbin; Zhou, Xiaodong; Dong, Shuai; Yin, Lifeng; Shen, Jian

    2017-11-01

    Electronic phase separation (EPS) is a common phenomenon in strongly correlated oxides. For colossal magnetoresistive (CMR) manganites, the EPS is so pronounced that not only does it govern the CMR behavior, but also raises a question whether EPS exists as a ground state for systems or a metastable state. While it has been well known that a magnetic field can drive the transition of the EPS state into a single-phase state in manganites, the reversibility of this transition is not well studied. In this work we use magnetic force microscopy (MFM) to directly visualize the reversibility of the field driven transition between the EPS state and the single-phase state at different temperatures. The MFM images correspond well with the global magnetic and transport property measurements, uncovering the underlying mechanism of the field driven transition between the EPS state and the single-phase state. We argue that EPS state is a consequence of system quenching whose response to an external magnetic field is governed by a local energy landscape.

  14. Molecular differences between deuterated and protonated polystyrenes using reversed-phase high-performance liquid chromatography.

    Science.gov (United States)

    Kayillo, Sindy; Gray, Michael J; Shalliker, R Andrew; Dennis, Gary R

    2005-05-06

    Isotopic substitution is a technique used to highlight particular bonds within a molecule for kinetic, spectroscopic and structure analysis. It is presumed that although some properties such as stretching frequencies will not be the same for substituted analogues, the chemical interactions will not vary appreciably as a function of labelling. Reversed-phase liquid chromatography has been used to demonstrate that there are significant differences between the chromatographic behaviour of a sequence of deuterated and protonated oligomeric polystyrenes. Two-dimensional reversed-phase liquid chromatography was used to show that even the diasteromers of the oligomers (n = 5) have retention mechanisms that are dependent on the subtle changes to the molecular conformation and electronic structure, which are a consequence of deuteration.

  15. Recovery of Picloram and 2,4-Dichlorophenoxyacetic Acid from Aqueous Samples by Reversed-Phase Solid-Phase Extraction

    Science.gov (United States)

    Martha J.M. Wells; Jerry L. Michael

    1987-01-01

    Extensive preparation of samples before chromatographic analysis is usually the most time-consuming process in the determination of many organic compounds in environmental matrices. In the past, removal of some organic from aqueous solution was commonly done by liquid/liquid extraction. However, the introduction of stable, covalently bonded reversed-phase sorbents now...

  16. Uniform magnetization reversal in dual main-phase (Ce,Nd)2Fe14B sintered magnets with inhomogeneous microstructure

    International Nuclear Information System (INIS)

    Zhang, Le-le; Li, Zhu-bai; Zhang, Xue-feng; Ma, Qiang; Liu, Yan-li; Li, Yong-feng; Zhao, Qian

    2017-01-01

    The element distribution and the magnetic properties were investigated in (Ce,Nd)–Fe–B sintered magnets prepared by mixing Nd 13.5 Fe 80 B 6.5 and Ce 9 Nd 4.5 Fe 80 B 6.5 powders with different mass ratios. Two main phases exist, but element diffusion is evident, and the chemical composition of the main phase is widely different from that of the master alloy. The Ce element tends to be expelled from the Ce-rich Re 2 Fe 14 B phase. Compared with the Ce-rich main phase, the Nd-rich Re 2 Fe 14 B phase is more stable in structure. Although the microstructure is inhomogeneous and the magnetocrystalline anisotropy is variable, the magnetization reversal is uniform in these dual main-phase magnets, which should ascribe to the existence of the exchange coupling, and magnetization reversal undergoes the nucleation of the reversed domain in irreversible magnetization. It is expected to further improve the coercivity by optimizing the distribution of the Nd-rich main phase in preparing the resource-saving (Ce,Nd) 2 Fe 14 B sintered magnets. (paper)

  17. Validated Reverse Phase HPLC Method for the Determination of Impurities in Etoricoxib

    Directory of Open Access Journals (Sweden)

    S. Venugopal

    2011-01-01

    Full Text Available This paper describes the development of reverse phase HPLC method for etoricoxib in the presence of impurities and degradation products generated from the forced degradation studies. The drug substance was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. The degradation of etoricoxib was observed under base and oxidation environment. The drug was found stable in other stress conditions studied. Successful separation of the drug from the process related impurities and degradation products were achieved on zorbax SB CN (250 x 4.6 mm 5 μm particle size column using reverse phase HPLC method. The isocratic method employed with a mixture of buffer and acetonitrile in a ratio of 60:40 respectively. Disodium hydrogen orthophosphate (0.02 M is used as buffer and pH adjusted to 7.20 with 1 N sodium hydroxide solution. The HPLC method was developed and validated with respect to linearity, accuracy, precision, specificity and ruggedness.

  18. The Effect of Pitching Phase on the Vortex Circulation for a Flapping Wing During Stroke Reversal

    Science.gov (United States)

    Burge, Matthew; Ringuette, Matthew

    2017-11-01

    We study the effect of pitching-phase on the circulation behavior for the 3D flow structures produced during stroke reversal for a 2-degree-of-freedom flapping wing executing hovering kinematics. Previous research has related the choice in pitching-phase with respect to the wing rotation during stroke reversal (advanced vs. symmetric pitch-timing) to a lift peak preceding stroke reversal. However, results from experiments on the time-varying circulation contributions from the 3D vortex structures across the span produced by both rotation and pitching are lacking. The objective of this research is to quantitatively examine how the spanwise circulation of these structures is affected by the pitching-phase for several reduced pitching frequencies. We employ a scaled wing model in a glycerin-water mixture and measure the time-varying velocity using multiple planes of stereo digital particle image velocimetry. Data-plane positions along the wing span are informed by the unsteady behavior of the 3D vortex structures found in our prior flow visualization movies. Individual vortices are identified to calculate their circulation. This work is aimed at understanding how the behavior of the vortex structures created during stroke reversal vary with key motion parameters. This work is supported by the National Science Foundation, Award Number 1336548, supervised by Dr. Ronald Joslin.

  19. The use of dihexyldithiocarbamate in reverse-phase HPLC of metal chelates

    Science.gov (United States)

    Fatimah, S. S.; Bahti, H. H.; Hastiawan, I.; Permanasari, A.

    2018-05-01

    Dialkyldithiocarbamates have long been used as chelating agents in reverse-phase HPLC of transition metals. In the previous study, an alkyl homolog of this type of ligand, namely dihexyldithiocarbamate (DHDTC), was synthesized and characterized. The use of this particular ligand in the revese-phase HPLC of some selected transition metal ions is now reported for the first time. The mobile phase comprising of the flow rate and of the detection, in the separation of the metal chelates of Cd (II), Fe (III), Cu (II), and Co (III), were investigated on a C-18 column. The results showed that dihexylditiocarbamate could be used for separating Cd (II), Fe(III), Cu(II), and Co(III). Therefore, it could be used in simultaneous analysis.

  20. [Determination of acyclovir in mouse plasma and tissues by reversed-phase high performance liquid chromatography].

    Science.gov (United States)

    Xu, Y; Zhou, S W; Tang, J L; Huang, L Q

    2001-11-01

    The aim of this study was to establish an high performance liquid chromatographic method for determining acyclovir (ACV) concentration in mouse plasma and tissues. A solution of 0.25 mL 60 g/L perchloric acid and 0.25 mL acetonitrile was added into 0.2 mL plasma or 0.2 g tissues to precipitate proteins. Following centrifugation, the supernatant obtained was injected into a reversed-phase column. Operating conditions were Hypersil ODS column(250 mm x 4.6 mm i.d., 5 microns), methanol-water-acetic acid(1:99:0.5, volume ratio) solution as mobile phase at a flow rate of 1.5 mL/min, UV detection at 252 nm. The detection limit of ACV concentration in plasma was 20 micrograms/L and that in tissues was 50 ng/g. The standard curves for ACV were linear in plasma and homogenate of tissues (r > 0.99). The precision of the method was good and the recoveries of ACV were higher than 97.5%. So this method is rapid, accurate and convenient for determination of ACV concentrations in plasma and tissues.

  1. The porcine acute phase response to infection with Actinobacillus pleuropneumoniae. Haptoglobin, C-reactive protein, major acute phase protein and serum amyloid a protein are sensitive indicators of infection

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Klausen, Joan; Nielsen, J.P.

    1998-01-01

    response peaking at around 2 days after infection. Haptoglobin, C-reactive protein (CRP), and major acute phase protein (MAP) responded with large increases in serum levels, preceding the development of specific antibodies by 4-5 days. Serum amyloid A protein (SAA) was also strongly induced. The increase......, kinetics of induction and normalization were different between these proteins. It is concluded that experimental Ap-infection by the aerosol route induces a typical acute phase reaction in the pig, and that pig Hp, CRP, MAP, and SAA are major acute phase reactants. These findings indicate the possibility...

  2. Purification of nattokinase by reverse micelles extraction from fermentation broth: effect of temperature and phase volume ratio.

    Science.gov (United States)

    Liu, Jun-Guo; Xing, Jian-Min; Chang, Tian-Shi; Liu, Hui-Zhou

    2006-03-01

    Nattokinase is a novel fibrinolytic enzyme that is considered to be a promising agent for thrombosis therapy. In this study, reverse micelles extraction was applied to purify and concentrate nattokinase from fermentation broth. The effects of temperature and phase volume ratio used for the forward and backward extraction on the extraction process were examined. The optimal temperature for forward and backward extraction were 25 degrees C and 35 degrees C respectively. Nattokinase became more thermosensitive during reverse micelles extraction. And it could be enriched in the stripping phase eight times during backward extraction. It was found that nattokinase could be purified by AOT reverse micelles with up to 80% activity recovery and with a purification factor of 3.9.

  3. Supercritical fluid reverse micelle separation

    Science.gov (United States)

    Fulton, J.L.; Smith, R.D.

    1993-11-30

    A method of separating solute material from a polar fluid in a first polar fluid phase is provided. The method comprises combining a polar fluid, a second fluid that is a gas at standard temperature and pressure and has a critical density, and a surfactant. The solute material is dissolved in the polar fluid to define the first polar fluid phase. The combined polar and second fluids, surfactant, and solute material dissolved in the polar fluid is maintained under near critical or supercritical temperature and pressure conditions such that the density of the second fluid exceeds the critical density thereof. In this way, a reverse micelle system defining a reverse micelle solvent is formed which comprises a continuous phase in the second fluid and a plurality of reverse micelles dispersed in the continuous phase. The solute material is dissolved in the polar fluid and is in chemical equilibrium with the reverse micelles. The first polar fluid phase and the continuous phase are immiscible. The reverse micelles each comprise a dynamic aggregate of surfactant molecules surrounding a core of the polar fluid. The reverse micelle solvent has a polar fluid-to-surfactant molar ratio W, which can vary over a range having a maximum ratio W[sub o] that determines the maximum size of the reverse micelles. The maximum ratio W[sub o] of the reverse micelle solvent is then varied, and the solute material from the first polar fluid phase is transported into the reverse micelles in the continuous phase at an extraction efficiency determined by the critical or supercritical conditions. 27 figures.

  4. Purification of flavonoids from licorice using an off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method.

    Science.gov (United States)

    Fan, Yunpeng; Fu, Yanhui; Fu, Qing; Cai, Jianfeng; Xin, Huaxia; Dai, Mei; Jin, Yu

    2016-07-01

    An orthogonal (71.9%) off-line preparative two-dimensional normal-phase liquid chromatography/reversed-phase liquid chromatography method coupled with effective sample pretreatment was developed for separation and purification of flavonoids from licorice. Most of the nonflavonoids were firstly removed using a self-made Click TE-Cys (60 μm) solid-phase extraction. In the first dimension, an industrial grade preparative chromatography was employed to purify the crude flavonoids. Click TE-Cys (10 μm) was selected as the stationary phase that provided an excellent separation with high reproducibility. Ethyl acetate/ethanol was selected as the mobile phase owing to their excellent solubility for flavonoids. Flavonoids co-eluted in the first dimension were selected for further purification using reversed-phase liquid chromatography. Multiple compounds could be isolated from one normal-phase fraction and some compounds with bad resolution in one-dimensional liquid chromatography could be prepared in this two-dimensional system owing to the orthogonal separation. Moreover, this two-dimensional liquid chromatography method was beneficial for the preparation of relatively trace flavonoid compounds, which were enriched in the first dimension and further purified in the second dimension. Totally, 24 flavonoid compounds with high purity were obtained. The results demonstrated that the off-line two-dimensional liquid chromatography method was effective for the preparative separation and purification of flavonoids from licorice. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Liquid-liquid extraction by reversed micelles in biotechnological processes

    Directory of Open Access Journals (Sweden)

    Kilikian B. V.

    2000-01-01

    Full Text Available In biotechnology there is a need for new purification and concentration processes for biologically active compounds such as proteins, enzymes, nucleic acids, or cells that combine a high selectivity and biocompatibility with an easy scale-up. A liquid-liquid extraction with a reversed micellar phase might serve these purposes owing to its capacity to solubilize specific biomolecules from dilute aqueous solutions such as fermentation and cell culture media. Reversed micelles are aggregates of surfactant molecules containing an inner core of water molecules, dispersed in a continuous organic solvent medium. These reversed micelles are capable of selectively solubilizing polar compounds in an apolar solvent. This review gives an overview of liquid-liquid extraction by reversed micelles for a better understanding of this process.

  6. 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)

  7. In-situ characterization of highly reversible phase transformation by synchrotron X-ray Laue microdiffraction

    International Nuclear Information System (INIS)

    Chen, Xian; Tamura, Nobumichi; MacDowell, Alastair; James, Richard D.

    2016-01-01

    The alloy Cu_2_5Au_3_0Zn_4_5 undergoes a huge first-order phase transformation (6% strain) and shows a high reversibility under thermal cycling and an unusual martensitc microstructure in sharp contrast to its nearby compositions. This alloy was discovered by systematically tuning the composition so that its lattice parameters satisfy the cofactor conditions (i.e., the kinematic conditions of compatibility between phases). It was conjectured that satisfaction of these conditions is responsible for the enhanced reversibility as well as the observed unusual fluid-like microstructure during transformation, but so far, there has been no direct evidence confirming that these observed microstructures are those predicted by the cofactor conditions. To verify this hypothesis, we use synchrotron X-ray Laue microdiffraction to measure the orientations and structural parameters of variants and phases near the austenite/martensite interface. The areas consisting of both austenite and multi-variants of martensite are scanned by microLaue diffraction. The cofactor conditions have been examined from the kinematic relation of lattice vectors across the interface. The continuity condition of the interface is precisely verified from the correspondent lattice vectors between two phases.

  8. Reversible Copolymer Materials for FDM 3-D Printing of Non-Standard Plastics, Phase II

    Data.gov (United States)

    National Aeronautics and Space Administration — Cornerstone Research Group Inc. (CRG) proposes to continue efforts from the 2015 NASA SBIR Phase I topic H14.03 ?Reversible Copolymer Materials for FDM 3D Printing...

  9. Reverse Algols

    Science.gov (United States)

    Leung, K. C.

    1989-01-01

    Reverse Algols, binary systems with a semidetached configuration in which the more massive component is in contact with the critical equipotential surface, are examined. Observational evidence for reverse Algols is presented and the parameters of seven reverse Algols are listed. The evolution of Algols and reverse Algols is discussed. It is suggested that, because reverse Algols represent the premass-reversal semidetached phase of close binary evolution, the evolutionary time scale between regular and reverse Algols is the ratio of the number of confirmed systems of these two Algol types.

  10. Simultaneous determination of ascorbic acid and caffeine in commercial soft drinks using reversed-phase ultraperformance liquid chromatography.

    Science.gov (United States)

    Turak, Fatma; Güzel, Remziye; Dinç, Erdal

    2017-04-01

    A new reversed-phase ultraperformance liquid chromatography method with a photodiode array detector was developed for the quantification of ascorbic acid (AA) and caffeine (CAF) in 11 different commercial drinks consisting of one energy drink and 10 ice tea drinks. Separation of the analyzed AA and CAF with an internal standard, caffeic acid, was performed on a Waters BEH C 18 column (100 mm × 2.1 mm, 1.7 μm i.d.), using a mobile phase consisting of acetonitrile and 0.2M H 3 PO 4 (11:89, v/v) with a flow rate of 0.25 mL/min and an injection volume of 1.0 μL. Calibration graphs for AA and CAF were computed from the peak area ratio of AA/internal standard and CAF/internal standard detected at 244.0 nm and 273.6 nm, respectively. The developed reversed-phase ultraperformance liquid chromatography method was validated by analyzing standard addition samples. The proposed reversed-phase ultraperformance liquid chromatography method gave us successful results for the quantitative analysis of commercial drinks containing AA and CAF substances. Copyright © 2016. Published by Elsevier B.V.

  11. Simultaneous determination of ascorbic acid and caffeine in commercial soft drinks using reversed-phase ultraperformance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Fatma Turak

    2017-04-01

    Full Text Available A new reversed-phase ultraperformance liquid chromatography method with a photodiode array detector was developed for the quantification of ascorbic acid (AA and caffeine (CAF in 11 different commercial drinks consisting of one energy drink and 10 ice tea drinks. Separation of the analyzed AA and CAF with an internal standard, caffeic acid, was performed on a Waters BEH C18 column (100 mm × 2.1 mm, 1.7 μm i.d., using a mobile phase consisting of acetonitrile and 0.2M H3PO4 (11:89, v/v with a flow rate of 0.25 mL/min and an injection volume of 1.0 μL. Calibration graphs for AA and CAF were computed from the peak area ratio of AA/internal standard and CAF/internal standard detected at 244.0 nm and 273.6 nm, respectively. The developed reversed-phase ultraperformance liquid chromatography method was validated by analyzing standard addition samples. The proposed reversed-phase ultraperformance liquid chromatography method gave us successful results for the quantitative analysis of commercial drinks containing AA and CAF substances.

  12. Prediction of mutational tolerance in HIV-1 protease and reverse transcriptase using flexible backbone protein design.

    Directory of Open Access Journals (Sweden)

    Elisabeth Humphris-Narayanan

    Full Text Available Predicting which mutations proteins tolerate while maintaining their structure and function has important applications for modeling fundamental properties of proteins and their evolution; it also drives progress in protein design. Here we develop a computational model to predict the tolerated sequence space of HIV-1 protease reachable by single mutations. We assess the model by comparison to the observed variability in more than 50,000 HIV-1 protease sequences, one of the most comprehensive datasets on tolerated sequence space. We then extend the model to a second protein, reverse transcriptase. The model integrates multiple structural and functional constraints acting on a protein and uses ensembles of protein conformations. We find the model correctly captures a considerable fraction of protease and reverse-transcriptase mutational tolerance and shows comparable accuracy using either experimentally determined or computationally generated structural ensembles. Predictions of tolerated sequence space afforded by the model provide insights into stability-function tradeoffs in the emergence of resistance mutations and into strengths and limitations of the computational model.

  13. Dissection of membrane protein degradation mechanisms by reversible inhibitors

    International Nuclear Information System (INIS)

    Hare, J.F.

    1988-01-01

    The degradation of slowly turning over 125I-lactoperoxidase-labeled plasma membrane polypeptides in response to reversible temperature and lysosomotropic inhibitors was studied in rat hepatoma cultures. Cells were radiolabeled and left for 24 h to allow the removal of rapidly degraded proteins. Remaining trichloroacetic acid-precipitable protein was degraded (t 1/2 = 40-68 h) by an apparent first order process 60-86% sensitive to 10 mM NH4Cl or 5 mM methylamine and greater than 95% inhibited by temperature reduction to 18 degrees C. Thus, membrane proteins are selected for degradation in a time-dependent manner by a system which is sensitive to both 18 degrees C and to lysosomotropic amines. When inhibitory conditions were removed after 40-48 h, degradation of 125I-labeled protein resumed at the same rate as that seen in their absence. Since membrane proteins do not exhibit accelerated degradation after removal of inhibitory conditions, there can be no marking or sorting of those proteins destined for degradation during the 40-h exposure to inhibitory conditions. Exposure to amines or 18 degrees C did not affect the position of two-dimensionally resolved labeled polypeptides. Fractionation of labeled cells on Percoll gradients after 40 h of exposure to low temperature or amines showed that labeled protein remained in the plasma membrane fractions of the gradient although shifted to a slightly lower buoyant density in the presence of amines. These results support the notion that selection of plasma membrane proteins for degradation requires their internalization into acidic vesicles. Lysosomotropic amines and reduced temperature interfere with the selection process by preventing membrane fusion events

  14. On the relevance of kinking to reversible hysteresis in MAX phases

    International Nuclear Information System (INIS)

    Jones, N.G.; Humphrey, C.; Connor, L.D.; Wilhelmsson, O.; Hultman, L.; Stone, H.J.; Giuliani, F.; Clegg, W.J.

    2014-01-01

    This paper examines the idea that reversible hysteresis in MAX phases is caused by the formation, growth and collapse of unstable, or incipient, kink bands. In situ X-ray diffraction of polycrystalline Ti 3 SiC 2 in compression showed that residual elastic lattice strains developed during the first loading cycle and remained approximately constant afterwards. These residual strains were compressive in grains with a low Schmid factor and tensile in grains with a high Schmid factor, consistent with previous observations of plastically deformed hexagonal metals. In contrast, incipient kink bands would be expected to collapse completely, without any residual strain. Elastoplastic self-consistent simulations showed that reversible hysteresis is predicted if some grains yield by slip on the basal plane, while others remain predominantly elastic, giving both the experimentally observed magnitude of the work dissipated and its dependence on the maximum applied stress. The reversible hysteresis in single crystals was studied by cyclically indenting thin films of Ti 3 SiC 2 and Ti 3 SiC 2 /TiC multilayers on Al 2 O 3 substrates. The work dissipated in the multilayer films was greater than in Ti 3 SiC 2 alone, despite the reduction in volume fraction of Ti 3 SiC 2 . Reversible hysteresis was also observed during indentation of single-crystal cubic MgO, demonstrating that this behaviour can occur if there are insufficient slip systems to accommodate the strain around the indentation. These results show that reversible hysteresis is associated with conventional dislocation flow, without the need for unstable kinking

  15. Polarity-dependent reversible resistance switching in Ge-Sb-Te phase-change thin films

    NARCIS (Netherlands)

    Pandian, Ramanathaswamy; Kooi, Bart J.; Palasantzas, George; De Hosson, Jeff T. M.; Pauza, Andrew

    2007-01-01

    In this paper, we demonstrate reversible resistance switching in a capacitorlike cell using a Ge-Sb-Te film that does not rely on amorphous-crystalline phase change. The polarity of the applied electric field switches the cell resistance between lower- and higher-resistance states, as was observed

  16. On-line comprehensive two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography for preparative isolation of Peucedanum praeruptorum.

    Science.gov (United States)

    Wang, Xin-Yuan; Li, Jia-Fu; Jian, Ya-Mei; Wu, Zhen; Fang, Mei-Juan; Qiu, Ying-Kun

    2015-03-27

    A new on-line comprehensive preparative two-dimensional normal-phase liquid chromatography × reversed-phase liquid chromatography (2D NPLC × RPLC) system was developed for the separation of complicated natural products. It was based on the use of a silica gel packed medium-pressure column as the first dimension and an ODS preparative HPLC column as the second dimension. The two dimensions were connected with normal-phase (NP) and reversed-phase (RP) enrichment units, involving a newly developed airflow assisted adsorption (AAA) technique. The instrument operation and the performance of this NPLC × RPLC separation method were illustrated by gram-scale isolation of ethanol extract from the roots of Peucedanum praeruptorum. In total, 19 compounds with high purity were obtained via automated multi-step preparative separation in a short period of time using this system, and their structures were comprehensively characterized by ESI-MS, (1)H NMR, and (13)C NMR. Including two new compounds, five isomers in two groups with identical HPLC and TLC retention values were also obtained and identified by 1D NMR and 2D NMR. This is the first report of an NPLC × RPLC system successfully applied in an on-line preparative process. This system not only solved the interfacing problem of mobile-phase immiscibility caused by NP and RP separation, it also exhibited apparent advantages in separation efficiency and sample treatment capacity compared with conventional methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Determination of Picloram in Soil and Water by Reversed-Phase Liquid Chromatography

    Science.gov (United States)

    M.J.M. Wells; J.L. Michael; D.G. Neary

    1984-01-01

    A reversed-phase liquid chromatographic method is presneted for the determination of picloram in the parts per billion (ppb) range in soil, soil solution, and stream samples. Quanitification is effected by UV absorpation at 254 nm. Derivatization is not necessary. The method permits 92% ± 7.1 recovery from water samples and 61.8% ± 11.1 recovery from soil samples....

  18. Molecular-level characterization of crude oil compounds combining reversed-phase high-performance liquid chromatography with off-line high-resolution mass spectrometry

    Science.gov (United States)

    Sim, Arum; Cho, Yunju; Kim, Daae; Witt, Matthias; Birdwell, Justin E.; Kim, Byung Ju; Kim, Sunghwan

    2014-01-01

    A reversed-phase separation technique was developed in a previous study (Loegel et al., 2012) and successfully applied to the de-asphalted fraction of crude oil. However, to the best of our knowledge, the molecular-level characterization of oil fractions obtained by reversed-phase high-performance liquid chromatography (HPLC) coupled with high-resolution mass spectrometry (MS) has not yet been reported. A detailed characterization of the oil fractions prepared by reversed-phase HPLC was performed in this study. HPLC fractionation was carried out on conventional crude oil and an oil shale pyrolysate. The analyses of the fractions showed that the carbon number of alkyl chains and the double bond equivalent (DBE) value were the major factors determining elution order. The compounds with larger DBE (presumably more condensed aromatic structures) and smaller carbon number (presumably compounds with short side chains) were eluted earlier but those compounds with lower DBE values (presumably less aromatic structures) and higher carbon number (presumably compounds with longer alkyl chains) eluted later in the chromatograms. This separation behavior is in good agreement with that expected from the principles of reversed-phase separation. The data presented in this study show that reversed-phase chromatography is effective in separating crude oil compounds and can be combined with ultrahigh-resolution MS data to better understand natural oils and oil shale pyrolysates.

  19. Phase lag control of tidally reversing mega-ripple geometry and bed stress in tidal inlets

    Science.gov (United States)

    Traykovski, P.

    2016-02-01

    Recent observations in the Columbia River Mouth, New River Inlet, and Wasque Shoals have shown that tidally reversing mega-ripples are an ubiquitous bedform morphology in energetic tidal inlets. As the name implies, these bedforms reverse asymmetry and migration direction in each half tidal cycle. With wavelengths of 2 to 5 m and heights of 0.2 to 0.5 m, these bedforms are larger than current formed ripples, but smaller than dunes. Unlike dunes which have a depth dependent geometry, observations indicate the tidally reversing mega-ripples geometry is related to the time dependent tidal flow and independent of depth. Previous empirical relations for predicting the geometry of ripples or dunes do not successfully predict the geometry of these features. A time dependent geometric model was developed that accounts for the reversal of migration and asymmetry to successfully predict bedform geometry. The model requires sufficient sediment transport in each half tidal cycle to reverse the asymmetry before the bedforms begin to grow. Both the observations and model indicate that the complete reversal of asymmetry and development of a steep lee face occurs near or after maximum flow in each half tidal cycle. This phase lag in bedform response to tidal forcing also has important implications for bed stress in tidal inlets. Observations of frictional drag in the Columbia River mouth based on a tidal momentum balance of surface slope over 10 km regressed against quadratic near bed velocity show drag coefficients that fall off as CD U-1.4. Reynolds stress measurements performed using the dual ADV differencing technique show similar relations. The Reynolds stress measurements also show a dramatic asymmetry between accelerating flows and decelerating flows with a factor of 5 increase during deceleration. Pulse coherent Doppler profiles of near bed turbulence indicate that the turbulence is dominated by energetic fluctuations in separation zones downstream of steep lee faces. The

  20. Analysis of electrophoretic soil humic acids fractions by reversed-phase high performance liquid chromatography with on-line absorbance and fluorescence detection.

    Science.gov (United States)

    Trubetskoj, Oleg A; Richard, Claire; Guyot, Ghislain; Voyard, Guillaume; Trubetskaya, Olga E

    2012-06-22

    A combination of reversed-phase high performance liquid chromatography (RP HPLC) with on-line absorbance and fluorescence detection was used for analysis of chernozem soil humic acids (HAs) and their fractions A, B and C+D with different electrophoretic mobility (EM) and molecular size (MS). Samples were injected onto the column at the identical volume and absorbance. All chromatograms exhibit the resolution of seven peaks. The estimation of relative recovery of HAs and fractions from the reverse-phase column has been done. High MS fraction A, which possesses the low EM, is essentially more hydrophobic (73% of the fraction amount remained adsorbed on the column) and aliphatic than medium MS and EM fraction B (33% of the fraction amount remained adsorbed on the column). The most hydrophilic and aromatic properties belong to low MS fraction C+D, which possess the highest EM and practically was not adsorbed on the column. The hydrophobicity of the bulk HAs lies within the range of fractions hydrophobicity. The absorption spectra of bulk HAs, electrophoretic fractions A, B, C+D and corresponding RP HPLC peaks were featureless but had differences in the values of absorbance ratio at 300 and 400 nm (A3/A4). For fractions A and B this ratio gradually decreased from peak 1 to 7 (from 3.05 to 2.80 and 3.00 to 2.40, respectively). This trend was less pronounced in HAs and practically absent in fraction C+D, where ratio A3/A4 varied within a small range. The strong relationship between fluorescence properties, EM, MS, polarity and aliphaticity/aromaticity of HAs fractions was found. Humic and protein-like fluorescence had different polarity nature. The protein-like fluorescence is located in humic material which irreversibly adsorbed on the reverse-phase column and not subjected to RP HPLC characterization. The humic-like fluorescence at Ex/Em 270/450 nm is mostly located in the hydrophilic peak of low MS fraction C+D. Taking into account that high MS fraction A consisted

  1. Additional phase information from UV damage of selenomethionine labelled proteins

    Energy Technology Data Exchange (ETDEWEB)

    Sanctis, Daniele de [ESRF, Structural Biology Group, 6 rue Jules Horowitz, 38043 Grenoble Cedex (France); Tucker, Paul A.; Panjikar, Santosh, E-mail: panjikar@embl-hamburg.de [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany)

    2011-05-01

    Successful examples of ultraviolet radiation-damage-induced phasing with anomalous scattering from selenomethionine protein crystals have been demonstrated. Currently, selenium is the most widely used phasing vehicle for experimental phasing, either by single anomalous scattering or multiple-wavelength anomalous dispersion (MAD) procedures. The use of the single isomorphous replacement anomalous scattering (SIRAS) phasing procedure with selenomethionine containing proteins is not so commonly used, as it requires isomorphous native data. Here it is demonstrated that isomorphous differences can be measured from intensity changes measured from a selenium labelled protein crystal before and after UV exposure. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment. The phasing procedure for two selenomethionine proteins, the feruloyl esterase module of xylanase 10B from Clostridium thermocellum and the Mycobacterium tuberculosis chorismate synthase, have been investigated using datasets collected near the absorption edge of selenium before and after UV radiation. The utility of UV radiation in measuring radiation damage data for isomorphous differences is highlighted and it is shown that, after such measurements, the UV-RIPAS procedure yields comparable phase sets with those obtained from the conventional MAD procedure. The results presented are encouraging for the development of alternative phasing approaches for selenomethionine proteins in difficult cases.

  2. Menin-MLL inhibitors reverse oncogenic activity of MLL fusion proteins in leukemia.

    Science.gov (United States)

    Grembecka, Jolanta; He, Shihan; Shi, Aibin; Purohit, Trupta; Muntean, Andrew G; Sorenson, Roderick J; Showalter, Hollis D; Murai, Marcelo J; Belcher, Amalia M; Hartley, Thomas; Hess, Jay L; Cierpicki, Tomasz

    2012-01-29

    Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.

  3. Controlled VLF phase reversal experiment in the magnetosphere

    International Nuclear Information System (INIS)

    Koons, H.C.; Dazey, M.H.; Dowden, R.L.; Amon, L.E.S.

    1976-01-01

    During the 1973 operations of the transportable very low frequency transmitter near Anchorage, Alaska (Lapprox.4), an experiment was performed to determine the effect of controlled phase change of the transmitted wave on the magnetospherically propagated signal received in the conjugate region. At periodic intervals the phase of the driving voltage was changed (essentially instantaneously) by 180degree. The amplitude of the 6.6-kHz signal detected in the conjugate region went to zero and recovered with a characteristic time constant of 33 ms. This is 10 times longer than the antenna current response time and is in fact comparable with characteristic electron interaction times with whistler mode waves. Between the times at which the phase reversals occurred the received signal was amplitude modulated. The period of the modulation was approx.26 ms. An upper side band was present in the spectrum while these pulsations were occurring. These characteristic times are in general agreement with theoretical predictions of bandwidths, growth rates, and particle-trapping frequencies for whistler instabilities in the magnetosphere. Data obtained from the controlled transmissions and from lightning-generated whistlers propagating in the same duct were combined to determine the plasma and wave parameters at the geomagnetic equator. Of particular interest is the level at which the magnetic field of the wave saturated. During the time period for which the data were analyzed this was found to be 3.5 pT (mγ)

  4. The liquid protein phase in crystallization: a case study—intact immunoglobulins

    Science.gov (United States)

    Kuznetsov, Yurii G.; Malkin, Alexander J.; McPherson, Alexander

    2001-11-01

    A common observation by protein chemists has been the appearance, for many proteins in aqueous solutions, of oil like droplets, or in more extreme cases the formation of a second oil like phase. These may accompany the formation of precipitate in "salting out" or "salting in' procedures, but more commonly appear in place of any precipitate. Such phase separations also occur, with even greater frequency, in the presence of polymeric precipitants such as polyethyleneglycol (PEG). In general the appearance of a second liquid phase has been taken as indicative of protein aggregation, though an aggregate state distinctly different from that characteristic of amorphous precipitate. While the latter is thought to be composed of linear and branched assemblies, polymers of a sort, the oil phase suggests a more compact, three-dimensional, but fluid state. An important property of an alternate, fluid phase is that it can mediate transitions between other states, for example, between protein molecules free in solution and protein molecules immobilized in amorphous precipitate or crystals. The "liquid protein" phase can be readily observed in many crystallization experiments either prior to the appearance of visible crystals, or directly participating in the crystal growth process. In some cases the relationship between the liquid phase and developing crystals is intimate. Crystals grow directly from the liquid phase, or appear only after the visible formation of the liquid phase. We describe here our experience with a class of macromolecules, immunoglobulins, and particularly IDEC-151, an IgG specific for CD4 on human lymphocytes. This protein has been crystallized from a Jeffamine-LiSO 4 mother liquor and, its crystallization illustrates many of the features associated with the liquid protein, or protein rich phase.

  5. Histone fractionation by high-performance liquid chromatography on cyanoalkylsilane (CN) reverse-phase columns

    International Nuclear Information System (INIS)

    Gurley, L.R.; Prentice, D.A.; Valdez, J.G.; Spall, W.D.

    1983-01-01

    Previous work described conditions for the rapid fractionation of histones by high-performance liquid chromatography (HPLC) using a reverse-phase μBondapak C 18 column. That procedure resolved the major classes of histones with one exception: the more hydrophobic H2A variant, (MHP)H2A, was not resolved from the H4 histone class. This report extends that work describing experiments using a μBondapak CN column which better resolves the classes of histones from each other including the resolution of (MHP)H2A from the H4. In addition, the less hydrophobic H2A variant, (LHP)H2A, is partially resolved from the (MHP)H2A, and the less hydrophobic H3 variant, (LHP)H3, is resolved from the more hydrophobic H3 variant, (MHP)H3. Lower trifluoroacetic acid (TFA) concentrations (0.1%) in the eluting water/acetonitrile solvent were used with the CN column than were used with the C 18 column which increased the sensitivity of histone detection by ultraviolet absorption at 206 nm. Greater than 95% of the total [ 3 H]lysine-labeled protein applied to the CN column was eluted from the column. Contaminating nonhistone proteins were found to chromatograph in the region of histone elution. These were greatly reduced by isolating nuclei prior to histone preparation. The fractionation of the histones appears to be based on the hydrophobic properties of the proteins. The histone fractions (identified by their electrophoretic mobilities) were eluted from the CN column in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3. Phosphorylated and acetylated histone species were not resolved from their unmodified parental species

  6. Leukotriene B4 catabolism: quantitation of leukotriene B4 and its omega-oxidation products by reversed-phase high-performance liquid chromatography.

    Science.gov (United States)

    Shak, S

    1987-01-01

    LTB4 and its omega-oxidation products may be rapidly, sensitively, and specifically quantitated by the methods of solid-phase extraction and reversed-phase high-performance liquid chromatography (HPLC), which are described in this chapter. Although other techniques, such as radioimmunoassay or gas chromatography-mass spectrometry, may be utilized for quantitative analysis of the lipoxygenase products of arachidonic acid, only the technique of reversed-phase HPLC can quantitate as many as 10 metabolites in a single analysis, without prior derivatization. In this chapter, we also reviewed the chromatographic theory which we utilized in order to optimize reversed-phase HPLC analysis of LTB4 and its omega-oxidation products. With this information and a gradient HPLC system, it is possible for any investigator to develop a powerful assay for the potent inflammatory mediator, LTB4, or for any other lipoxygenase product of arachidonic acid.

  7. Integrated Automation of High-Throughput Screening and Reverse Phase Protein Array Sample Preparation

    DEFF Research Database (Denmark)

    Pedersen, Marlene Lemvig; Block, Ines; List, Markus

    into automated robotic high-throughput screens, which allows subsequent protein quantification. In this integrated solution, samples are directly forwarded to automated cell lysate preparation and preparation of dilution series, including reformatting to a protein spotter-compatible format after the high......-throughput screening. Tracking of huge sample numbers and data analysis from a high-content screen to RPPAs is accomplished via MIRACLE, a custom made software suite developed by us. To this end, we demonstrate that the RPPAs generated in this manner deliver reliable protein readouts and that GAPDH and TFR levels can...

  8. Enantiomeric separation of type I and type II pyrethroid insecticides with different chiral stationary phases by reversed-phase high-performance liquid chromatography.

    Science.gov (United States)

    Zhang, Ping; Yu, Qian; He, Xiulong; Qian, Kun; Xiao, Wei; Xu, Zhifeng; Li, Tian; He, Lin

    2018-04-01

    The enantiomeric separation of type I (bifenthrin, BF) and type II (lambda-cyhalothrin, LCT) pyrethroid insecticides on Lux Cellulose-1, Lux Cellulose-3, and Chiralpak IC chiral columns was investigated by reversed-phase high-performance liquid chromatography. Methanol/water or acetonitrile/water was used as mobile phase at a flow rate of 0.8 mL/min. The effects of chiral stationary phase, mobile phase composition, column temperature, and thermodynamic parameters on enantiomer separation were carefully studied. Bifenthrin got a partial separation on Lux Cellulose-1 column and baseline separation on Lux Cellulose-3 column, while LCT enantiomers could be completely separated on both Lux Cellulose-1 and Lux Cellulose-3 columns. Chiralpak IC provided no separation ability for both BF and LCT. Retention factor (k) and selectivity factor (α) decreased with the column temperature increasing from 10°C to 40°C for both BF and LCT enantiomers. Thermodynamic parameters including ∆H and ∆S were also calculated, and the maximum R s were not always obtained at lowest temperature. Furthermore, the quantitative analysis methods for BF and LCT enantiomers in soil and water were also established. Such results provide a new approach for pyrethroid separation under reversed-phase condition and contribute to environmental risk assessment of pyrethroids at enantiomer level. © 2017 Wiley Periodicals, Inc.

  9. MODELS OF PROJECT REVERSE ENGINEERING

    Directory of Open Access Journals (Sweden)

    Віктор Володимирович ІВАНОВ

    2017-03-01

    Full Text Available Reverse engineering decided important scientific and technical problems of increasing the cost of the existing technical product by transforming it into a product with other features or design. Search ideas of the new application of existing products on the base of heuristic analysis were created. The concept of reverse engineering and its division into three types: conceptual, aggregate and complete was expanded. The use of heuristic methods for reverse engineering concept was showed. The modification model of Reverse engineering based on the model of РМВОК was developed. Our model includes two new phases: identification and transformation. At the identification phase, technical control is made. At the transformation phase, search heuristic idea of the new applied existing technical product was made. The model of execution phase that included heuristic methods, metrological equipment, and CAD/CAM/CAE program complex was created. The model that connected economic indicators of reverse engineering project was developed.

  10. Acute Phase Proteins and Variables of Protein Metabolism in Dairy Cows during the Pre- and Postpartal Period

    Directory of Open Access Journals (Sweden)

    Cs. Tóthová

    2008-01-01

    Full Text Available The objective of the present study was to compare the concentrations of acute phase proteins and selected variables of protein metabolism in dairy cows of the Slovak Spotted breed from 4 weeks before parturition to 10 weeks after parturition. Acute phase proteins - haptoglobin (Hp and serum amyloid A (SAA - and variables of protein metabolism - total proteins, albumin, urea, creatinine, total immunoglobulins - were evaluated in blood serum. Significant differences were found in average values of the Hp and SAA concentrations in several groups during the monitored period (P P P P P P P P P < 0.001. The above mentioned results indicate that in the time around parturition there are significant changes in concentrations of acute phase proteins, as well as in the whole protein metabolism of dairy cows. These facts suggest that the postparturient period is a critical biological phase, throughout which there is the highest incidence of metabolic disorders.

  11. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    Energy Technology Data Exchange (ETDEWEB)

    Albariño, César G., E-mail: calbarino@cdc.gov; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-10-15

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies.

  12. Development of a reverse genetics system to generate a recombinant Ebola virus Makona expressing a green fluorescent protein

    International Nuclear Information System (INIS)

    Albariño, César G.; Wiggleton Guerrero, Lisa; Lo, Michael K.; Nichol, Stuart T.; Towner, Jonathan S.

    2015-01-01

    Previous studies have demonstrated the potential application of reverse genetics technology in studying a broad range of aspects of viral biology, including gene regulation, protein function, cell entry, and pathogenesis. Here, we describe a highly efficient reverse genetics system used to generate recombinant Ebola virus (EBOV) based on a recent isolate from a human patient infected during the 2014–2015 outbreak in Western Africa. We also rescued a recombinant EBOV expressing a fluorescent reporter protein from a cleaved VP40 protein fusion. Using this virus and an inexpensive method to quantitate the expression of the foreign gene, we demonstrate its potential usefulness as a tool for screening antiviral compounds and measuring neutralizing antibodies. - Highlights: • Recombinant Ebola virus (EBOV) derived from Makona variant was rescued. • New protocol for viral rescue allows 100% efficiency. • Modified EBOV expresses a green fluorescent protein from a VP40-fused protein. • Modified EBOV was tested as tool to screen antiviral compounds and measure neutralizing antibodies

  13. Reversible temperature regulation of electrical and thermal conductivity using liquid–solid phase transitions

    Science.gov (United States)

    Zheng, Ruiting; Gao, Jinwei; Wang, Jianjian; Chen, Gang

    2011-01-01

    Reversible temperature tuning of electrical and thermal conductivities of materials is of interest for many applications, including seasonal regulation of building temperature, thermal storage and sensors. Here we introduce a general strategy to achieve large contrasts in electrical and thermal conductivities using first-order phase transitions in percolated composite materials. Internal stress generated during a phase transition modulates the electrical and thermal contact resistances, leading to large contrasts in the electrical and thermal conductivities at the phase transition temperature. With graphite/hexadecane suspensions, the electrical conductivity changes 2 orders of magnitude and the thermal conductivity varies up to 3.2 times near 18 °C. The generality of the approach is also demonstrated in other materials such as graphite/water and carbon nanotube/hexadecane suspensions. PMID:21505445

  14. Reversible temperature regulation of electrical and thermal conductivity using liquid-solid phase transitions.

    Science.gov (United States)

    Zheng, Ruiting; Gao, Jinwei; Wang, Jianjian; Chen, Gang

    2011-01-01

    Reversible temperature tuning of electrical and thermal conductivities of materials is of interest for many applications, including seasonal regulation of building temperature, thermal storage and sensors. Here we introduce a general strategy to achieve large contrasts in electrical and thermal conductivities using first-order phase transitions in percolated composite materials. Internal stress generated during a phase transition modulates the electrical and thermal contact resistances, leading to large contrasts in the electrical and thermal conductivities at the phase transition temperature. With graphite/hexadecane suspensions, the electrical conductivity changes 2 orders of magnitude and the thermal conductivity varies up to 3.2 times near 18 °C. The generality of the approach is also demonstrated in other materials such as graphite/water and carbon nanotube/hexadecane suspensions.

  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. Topological phases in a three-dimensional topological insulator with a time-reversal invariant external field

    International Nuclear Information System (INIS)

    Guo, Xiaoyong; Ren, Xiaobin; Wang, Gangzhi; Peng, Jie

    2014-01-01

    We investigate the impact of a time-reversal invariant external field on the topological phases of a three-dimensional (3D) topological insulator. By taking the momentum k z as a parameter, we calculate the spin-Chern number analytically. It is shown that both the quantum spin Hall phase and the integer quantum Hall phase can be realized in our system. When the strength of the external field is varied, a series of topological phase transitions occurs with the closing of the energy gap or the spin-spectrum gap. In a tight-binding form, the surface modes are discussed numerically to confirm the analytically results. (paper)

  17. Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

    Science.gov (United States)

    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.

    2014-01-01

    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

  18. Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.

    Directory of Open Access Journals (Sweden)

    Daniel Bello-Gil

    Full Text Available We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

  19. Determination of Fluorescent Whitening Agents in Paper Materials by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

    International Nuclear Information System (INIS)

    Kim, Jeong Soo; Kim, Keon; Kim, Do Hwan

    2012-01-01

    A simple method was developed for the analysis of seven stilbene-type fluorescent whitening agents (FWAs) in paper materials by ion-pair reversed-phase high-performance liquid chromatography with fluorescence detection. These stilbene-type FWAs included two disulfonate, two tetrasulfonate, and three hexasulfonate compounds. After optimization of chromatographic conditions, the FWAs were satisfactorily separated using a reversed-phase column (RP-18) with the following isocratic mobile phase: methanol-water (60:40) containing 17.5 mM TBABr and 10 mM citrate buffer (pH = 7.0). The calibration plot was linear in the range from 5 to 500 ng/mL for two disulfo-FWAs and from 1 to 500 ng/mL for the other five FWAs. Precision levels of the calibration curve as indicated by RSD of response factors were 1.2 and 8.1%. Limits of quantitation (LOQ) ranged from 1.2 to 11 ng/mL

  20. Determination of Fluorescent Whitening Agents in Paper Materials by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jeong Soo; Kim, Keon [Korea Univ., Seoul (Korea, Republic of); Kim, Do Hwan [Daegu Univ., Gyeongsan (Korea, Republic of)

    2012-12-15

    A simple method was developed for the analysis of seven stilbene-type fluorescent whitening agents (FWAs) in paper materials by ion-pair reversed-phase high-performance liquid chromatography with fluorescence detection. These stilbene-type FWAs included two disulfonate, two tetrasulfonate, and three hexasulfonate compounds. After optimization of chromatographic conditions, the FWAs were satisfactorily separated using a reversed-phase column (RP-18) with the following isocratic mobile phase: methanol-water (60:40) containing 17.5 mM TBABr and 10 mM citrate buffer (pH = 7.0). The calibration plot was linear in the range from 5 to 500 ng/mL for two disulfo-FWAs and from 1 to 500 ng/mL for the other five FWAs. Precision levels of the calibration curve as indicated by RSD of response factors were 1.2 and 8.1%. Limits of quantitation (LOQ) ranged from 1.2 to 11 ng/mL.

  1. Reversed-phase thin-layer chromatography of the rare earth elements

    International Nuclear Information System (INIS)

    Kuroda, R.; Adachi, M.; Oguma, K.

    1988-01-01

    Partition chromatographic behaviour of the rare earth elements on C 18 bonded silica reversed-phase material has been investigated by thin-layer chromatography in methanol - lactate media. The rare earth lactato complexes are distributed and fractionated on bonded silica layers without ion-interaction reagents. The concentration and pH of lactate solution, methanol concentration and temperature have effects on the migration and resolution of the rare earth elements. The partition system is particularly suited to separate adjacent rare earths of middle atomic weight groups, allowing the separation of gadolinium, terbium, dysprosium, holmium, erbium and thulium to be achieved by development to 18 cm distance. (orig.)

  2. Theoretical aspects of gradient reversed-phase high performance liquid chromatography of styrene-butylacrylate block copolymers

    NARCIS (Netherlands)

    Kolarova, L.; Jandera, P.; Vonk, E.C.; Claessens, H.A.

    2004-01-01

    Butylacrylate – styrene co-polymers prepared by atom transfer radical polymeratization were separated on an octadecyl silica column by gradient elution with tetrahydrofuran in water, up to the molar masses 10,000. In reversed-phase high performance liquid chromatography (RP-HPLC), the retention of

  3. Carrier effect on separation efficiency of the column in reversed-phase partition chromatography

    International Nuclear Information System (INIS)

    Pszonicka, M.; Siekierski, S.

    1972-01-01

    Chromatographic columns were filled with carriers of diatomaceous earth type (Hyflo Super Cell, and Celite 545) of large pores, and two microporous silica gels respectively. These columns were used for the separation of europium and gadolinium by reversed-phase partition chromatography in the system: stationary phase-diethylhexylphosphoric acid (HDEHP) mobile phase-0.4 N nitric acid. In each case the separation of the above mentioned elements was achieved. The plate height that characterizes separation efficiency of the column, decreases with the decrease of particle diameter of the carrier. Best columns were obtained from carriers of large pores (Hyflo Super Cell and Celite 545) for which plate hights below 0.1 mm could be achieved. Columns filled with microporous silica gels showed plate heights of 0.2-0.3 mm. (author)

  4. Comparison of Separation of Seed Oil Triglycerides Containing Isomeric Conjugated Octadecatrienoic Acid Moieties by Reversed-Phase HPLC

    OpenAIRE

    Anh Van Nguyen; Victor Deineka; Lumila Deineka; Anh Vu Thi Ngoc

    2017-01-01

    Relative retention analysis and increment approach were applied for the comparison of triglycerides (TGs) retention of a broad set of plant seed oils with isomeric conjugated octadecatrienoic acids (CLnA) by reversed-phase HPLC for “propanol-2-acetonitrile” mobile phases and Kromasil 100-5C18 stationary phase with diode array detection (DAD) and mass spectrometric (MS) detection. The subjects of investigation were TGs of seed oils: Calendula officinalis, Catalpa ovata, Jacaranda mimosifolia, ...

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

    Science.gov (United States)

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

    2017-06-02

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

  6. Effect of grain boundary phase on the magnetization reversal process of nanocrystalline magnet using large-scale micromagnetic simulation

    Directory of Open Access Journals (Sweden)

    Hiroshi Tsukahara

    2018-05-01

    Full Text Available We investigated the effects of grain boundary phases on magnetization reversal in permanent magnets by performing large-scale micromagnetic simulations based on Landau–Lifshitz–Gilbert equation under a periodic boundary. We considered planar grain boundary phases parallel and perpendicular to an easy axis of the permanent magnet and assumed the saturation magnetization and exchange stiffness constant of the grain boundary phase to be 10% and 1%, respectively, for Nd2Fe14B grains. The grain boundary phase parallel to the easy axis effectively inhibits propagation of magnetization reversal. In contrast, the domain wall moves across the grain boundary perpendicular to the easy axis. These properties of the domain wall motion are explained by dipole interaction, which stabilizes the antiparallel magnetic configuration in the direction perpendicular to the magnetization orientation. On the other hand, the magnetization is aligned in the same direction by the dipole interaction parallel to the magnetization orientation. This anisotropy of the effect of the grain boundary phase shows that improvement of the grain boundary phase perpendicular to the easy axis effectively enhances the coercivity of permanent magnets.

  7. A Simple and Validated Reverse Phase HPLC Methodfor the Determination of Rabeprazole inPharmaceutical Dosage Forms

    Directory of Open Access Journals (Sweden)

    Uma Mahesh Karra

    2010-01-01

    Full Text Available A simple and rapid reverse phase high performance liquid chromatography (RP-HPLC method was developed and validated for quantitative determination of rabeprazole in bulk drug samples and formulations. Rabeprazole was analyzed by using reverse phase LC-GC column (Inertsil ODS, 4.6 mm x 25 cm, 5 microns, with mobile phase consisting of methanol: water (78:22 v/v. The flow rate was set 1.0 mL/min and analysis was performed at wavelength 288 nm using Photo Diode Array (PDA detector at ambient temperature. The method was validated and stability studies were conducted under different conditions. The retention time for rabeprazole was around 4.12 minutes. The calibration curves were linear (r≥0.9998 over a concentration range from 20.0 to 80.0 μg/mL. Limit of detection (LOD and Limit of quantitation (LOQ were 8 ng/mL and 24 ng/mL respectively. The developed method was successfully applied to estimate the amount of rabeprazole in tablet formulations.

  8. Differential screening and mass mapping of proteins from premalignant and cancer cell lines using nonporous reversed-phase HPLC coupled with mass spectrometric analysis.

    Science.gov (United States)

    Chong, B E; Hamler, R L; Lubman, D M; Ethier, S P; Rosenspire, A J; Miller, F R

    2001-03-15

    Nonporous (NPS) RP-HPLC has been used to rapidly separate proteins from whole cell lysates of human breast cell lines. The nonporous separation involves the use of hard-sphere silica beads of 1.5-microm diameter coated with C18, which can be used to separate proteins ranging from 5 to 90 kDa. Using only 30-40 microg of total protein, the protein molecular weights are detectable on-line using an ESI-oaTOF MS. Of hundreds of proteins detected in this mass range, approxinately 75-80 are more highly expressed. The molecular weight profiles can be displayed as a mass map analogous to a virtual "1-D gel" and differentially expressed proteins can be compared by image analysis. The separated proteins can also be detected by UV absorption and differentially expressed proteins quantified. The eluting proteins can be collected in the liquid phase and the molecular weight and peptide maps determined by MALDI-TOF MS for identification. It is demonstrated that the expressed protein profiles change during neoplastic progression and that many oncoproteins are readily detected. It is also shown that the response of premalignant cancer cells to estradiol can be rapidly screened by this method, demonstrating significant changes in response to an external agent. Ultimately, the proteins can be studied by peptide mapping to search for posttranslational modifications of the oncoproteins accompanying progression.

  9. Optimization of protein extraction process from jackfruit seed flour by reverse micelle system

    Directory of Open Access Journals (Sweden)

    Maycon Fagundes Teixeira Reis

    2016-06-01

    Full Text Available The extraction of protein from flour of jackfruit seeds by reverse micelles was evaluated. Reverse micelle system was composed of sodium dodecyl sulfate (SDS as surfactant, butanol as solvent, and water. The effects of stirring time, temperature, molar ratio H2O SDS-1, concentration of butanol (mass percentage and flour mass were tested in batch systems. Based on the adjusted linear regression model, only butanol concentration provided optimum extraction conditions (41.16%. Based on the analysis of surface response, the best extraction yield could be obtained at 25°C, stirring time of 120 min, mass of flour of 100 mg, and a ratio H2O SDS-1 of 50. Experimental results showed that a 79.00% extraction yield could be obtained.

  10. Combined effects of potassium chloride and ethanol as mobile phase modulators on hydrophobic interaction and reversed-phase chromatography of three insulin variants.

    Science.gov (United States)

    Johansson, Karolina; Frederiksen, Søren S; Degerman, Marcus; Breil, Martin P; Mollerup, Jørgen M; Nilsson, Bernt

    2015-02-13

    The two main chromatographic modes based on hydrophobicity, hydrophobic interaction chromatography (HIC) and reversed-phase chromatography (RPC), are widely used for both analytical and preparative chromatography of proteins in the pharmaceutical industry. Despite the extensive application of these separation methods, and the vast amount of studies performed on HIC and RPC over the decades, the underlying phenomena remain elusive. As part of a systematic study of the influence of mobile phase modulators in hydrophobicity-based chromatography, we have investigated the effects of both KCl and ethanol on the retention of three insulin variants on two HIC adsorbents and two RPC adsorbents. The focus was on the linear adsorption range, separating the modulator effects from the capacity effects, but some complementary experiments at higher load were included to further investigate observed phenomena. The results show that the modulators have the same effect on the two RPC adsorbents in the linear range, indicating that the modulator concentration only affects the activity of the solute in the mobile phase, and not that of the solute-ligand complex, or that of the ligand. Unfortunately, the HIC adsorbents did not show the same behavior. However, the insulin variants displayed a strong tendency toward self-association on both HIC adsorbents; on one in particular. Since this causes peak fronting, the retention is affected, and this could probably explain the lack of congruity. This conclusion was supported by the results from the non-linear range experiments which were indicative of double-layer adsorption on the HIC adsorbents, while the RPC adsorbents gave the anticipated increased tailing at higher load. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. ''reverse'' solid-phase radioimmunoasssay for IgM-antibodies to hepatitis A virus

    Energy Technology Data Exchange (ETDEWEB)

    Meurman, O H; Matter, L; Krishna, R V; Krech, U H [Institute of Medical Microbiology, St. Gallen, Switzerland

    1981-01-01

    A ''reverse'' solid-phase radio-immuno-assay for IgM antibodies to hepatitis A virus (HAV) was developed. Anti-human IgM immunoglobulins were bound on the wells of polyvinylchloride microtiter plates. Serum specimens were incubated in the anti-human IgM coated wells and bound IgM antibodies were then assayed for antigen specificity by subsequent incubations with HAV antigen and /sup 125/I-labelled human anti-HAV IgG. The test showed a high sensitivity and specificity for anti-HAV IgM antibodies. No false-positive reactions were observed either in the sera from patients with hepatobiliary disorders other than HAV infection or in the sera containing both rheumatoid factor and anti-HAV IgG antibodies. In acute HAV infections specific IgM antibodies were present already in the first specimens taken within a few days after the onset of jaundice. The persistence of the IgM antibodies was from 4 to 6 months. IgM antibody titers up to 1,000,000 were observed in the acute phase of HAV infection. In routine diagnostic work the titration of the sera was not necessary, since a reliable qualitative result was obtained by testing the sera in a single dilution of 1:100. A similar reverse immuno-assay principle may be adaptable for the diagnostic determination of IgM antibodies to different viral and microbial antigens.

  12. Inhibition of protein kinase A and GIRK channel reverses fentanyl-induced respiratory depression.

    Science.gov (United States)

    Liang, Xiaonan; Yong, Zheng; Su, Ruibin

    2018-06-11

    Opioid-induced respiratory depression is a major obstacle to improving the clinical management of moderate to severe chronic pain. Opioids inhibit neuronal activity via various pathways, including calcium channels, adenylyl cyclase, and potassium channels. Currently, the underlying molecular pathway of opioid-induced respiratory depression is only partially understood. This study aimed to investigate the mechanisms of opioid-induced respiratory depression in vivo by examining the effects of different pharmacological agents on fentanyl-induced respiratory depression. Respiratory parameters were detected using whole body plethysmography in conscious rats. We show that pre-treatment with the protein kinase A (PKA) inhibitor H89 reversed the fentanyl-related effects on respiratory rate, inspiratory time, and expiratory time. Pre-treatment with the G protein-gated inwardly rectifying potassium (GIRK) channel blocker Tertiapin-Q dose-dependently reversed the fentanyl-related effects on respiratory rate and inspiratory time. A phosphodiesterase 4 (PDE4) inhibitor and cyclic adenosine monophosphate (cAMP) analogs did not affect fentanyl-induced respiratory depression. These findings suggest that PKA and GIRK may be involved in fentanyl-induced respiratory depression and could represent useful therapeutic targets for the treatment of fentanyl-induced ventilatory depression. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Quality control of Photosystem II: reversible and irreversible protein aggregation decides the fate of Photosystem II under excessive illumination

    Directory of Open Access Journals (Sweden)

    Yasusi eYamamoto

    2013-10-01

    Full Text Available In response to excessive light, the thylakoid membranes of higher plant chloroplasts show dynamic changes including the degradation and reassembly of proteins, a change in the distribution of proteins, and large-scale structural changes such as unstacking of the grana. Here, we examined the aggregation of light-harvesting chlorophyll-protein complexes and Photosystem II core subunits of spinach thylakoid membranes under light stress with 77K chlorophyll fluorescence; aggregation of these proteins was found to proceed with increasing light intensity. Measurement of changes in the fluidity of thylakoid membranes with fluorescence polarization of diphenylhexatriene showed that membrane fluidity increased at a light intensity of 500–1,000 µmol photons m-2 s-1, and decreased at very high light intensity (1,500 µmol photons m-2 s-1. The aggregation of light-harvesting complexes at moderately high light intensity is known to be reversible, while that of Photosystem II core subunits at extremely high light intensity is irreversible. It is likely that the reversibility of protein aggregation is closely related to membrane fluidity: increases in fluidity should stimulate reversible protein aggregation, whereas irreversible protein aggregation might decrease membrane fluidity. When spinach leaves were pre-illuminated with moderately high light intensity, the qE component of non-photochemical quenching and the optimum quantum yield of Photosystem II increased, indicating that Photosystem II/ light-harvesting complexes rearranged in the thylakoid membranes to optimize Photosystem II activity. Transmission electron microscopy revealed that the thylakoids underwent partial unstacking under these light stress conditions. Thus, protein aggregation is involved in thylakoid dynamics and regulates photochemical reactions, thereby deciding the fate of Photosystem II.

  14. 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.

  15. Analysis of the free-energy surface of proteins from reversible folding simulations.

    Directory of Open Access Journals (Sweden)

    Lucy R Allen

    2009-07-01

    Full Text Available Computer generated trajectories can, in principle, reveal the folding pathways of a protein at atomic resolution and possibly suggest general and simple rules for predicting the folded structure of a given sequence. While such reversible folding trajectories can only be determined ab initio using all-atom transferable force-fields for a few small proteins, they can be determined for a large number of proteins using coarse-grained and structure-based force-fields, in which a known folded structure is by construction the absolute energy and free-energy minimum. Here we use a model of the fast folding helical lambda-repressor protein to generate trajectories in which native and non-native states are in equilibrium and transitions are accurately sampled. Yet, representation of the free-energy surface, which underlies the thermodynamic and dynamic properties of the protein model, from such a trajectory remains a challenge. Projections over one or a small number of arbitrarily chosen progress variables often hide the most important features of such surfaces. The results unequivocally show that an unprojected representation of the free-energy surface provides important and unbiased information and allows a simple and meaningful description of many-dimensional, heterogeneous trajectories, providing new insight into the possible mechanisms of fast-folding proteins.

  16. An analysis of dissolved organic matter from freshwater Karelian Lakes using reversed-phase high-performance liquid chromatography with online absorbance and fluorescence analysis

    Science.gov (United States)

    Khundzhua, D. A.; Patsaeva, S. V.; Trubetskoj, O. A.; Trubetskaya, O. E.

    2017-01-01

    The spectral and optical properties of the fractionated components of dissolved organic matter (DOM) of three freshwater lakes in Karelia were studied using reversed-phase high-performance liquid chromatography (RP-HPLC) with online detection of fluorescence and absorption spectra. It is shown that the DOM fractions are qualitatively similar, but differ quantitatively in the ratio of components and consist of at least three types of fluorophores: (1) hydrophilic "humic-like" fluorophore(s) with the emission maximum in the region of 420 nm and an absorption band at 260-270 nm; (2) hydrophobic "humic-like" fluorophore(s) with the emission maximum at approximately 450 nm that has no characteristic absorption maxima in the region from 220 to 400 nm; and (3) a "protein-like" fluorophore with the emission maximum in the region of 340-350 nm, which is typical of proteins and peptides containing tryptophan.

  17. Development of a perfusion reversed-phase high performance liquid chromatography method for the characterisation of maize products using multivariate analysis.

    Science.gov (United States)

    Rodriguez-Nogales, J M; Garcia, M C; Marina, M L

    2006-02-03

    A perfusion reversed-phase high performance liquid chromatography (RP-HPLC) method has been designed to allow rapid (3.4 min) separations of maize proteins with high resolution. Several factors, such as extraction conditions, temperature, detection wavelength and type and concentration of ion-pairing agent were optimised. A fine optimisation of the gradient elution was also performed by applying experimental design. Commercial maize products for human consumption (flours, precocked flours, fried snacks and extruded snacks) were characterised for the first time by perfusion RP-HPLC and their chromatographic profiles allowed a differentiation among products relating the different technological process used for their preparation. Furthermore, applying discriminant analysis makes it possible to group the samples according with the technological process suffered by maize products, obtaining a good prediction in 92% of the samples.

  18. [Separation of p-aminobenzenearsonic acid and its oxide by ion-pair reversed-phase high performance liquid chromatography].

    Science.gov (United States)

    Kang, J; Ma, X; Meng, L; Ma, D

    1999-05-01

    To study the separation of p-aminobenzenearsonic acid (PABAA) and its oxide, p-aminophenylarsine oxide (PAPAO), both the absorption spectra were scanned at the wavelengths from 200 nm to 380 nm. PABAA had absorption maximum at 254 nm and PAPAO 258 nm. The effects of salt concentration, column temperature, methanol and ion-pair agent concentrations on the capacity factor were investigated. Compounds of high polarity showed almost no retention on reversed-phase column; as the volume fraction of the methanol decreased from 90% to 10%, the retention time of PABAA gradually increased with broad peak, and partially eluted when methanol volume fraction being below 20%. With temperature rising, the retention time of PABAA was decreased. But PABAA capacity factor can be increased by selecting an appropriate salt concentration for the mobile phase. The cetyltrimethyl and tetrabutyl ammonium ions were separately added as ion-pair agents to the mobile phase containing methanol in phosphate buffer of 10 mmol/L, the changes of retention time were observed. The mechanism of retention based on reversed phase ion-pair model is proposed. Besides, the retention behaviour is also influenced by size exclusion in stationary phase as well as polar interactions with residual silanol group on the silica surface.

  19. Diagnostical meaning acute phase proteins in cerebrospinal liquid in children with neuroinfections

    OpenAIRE

    L. A. Alekseeva; N. V. Skripchenko; T. V. Bessonova

    2010-01-01

    In the article presented results of the examination of acute phase proteins in cerebrospinal liquid in 237 children with meningitis and encephalitis viral and bacterial etiology. The dependence between the level of acute phase proteins in cerebrospinal liquid and etiology of neuroinfectional process, the severity of brain damage and the process stage was determined. Diagnostic and prognostic efficiency of the acute phase proteins (C-reactive protein, albumin, alpha-1-antitripsin, alpha-2-macr...

  20. Thermodynamics of the sorption of water-soluble vitamins in reverse-phase high performance liquid chromatography

    Science.gov (United States)

    Chirkin, V. A.; Karpov, S. I.; Selemenev, V. F.

    2012-12-01

    The thermodynamics of the sorption of certain water-soluble vitamins on a C18 reverse phase from water-acetonitrile solutions of different compositions is studied. The thermodynamic characteristics of the investigated chromatographic systems are calculated. The dependences of standard molar enthalpy and changes in entropy when the sorbate transfers from the bulk solution to the surface layer on the concentration of the organic component in the mobile phase are analyzed. The boundaries for applying the main retention models describing the sorption of the investigated compounds are discussed.

  1. Contributions to reversed-phase column selectivity: III. Column hydrogen-bond basicity.

    Science.gov (United States)

    Carr, P W; Dolan, J W; Dorsey, J G; Snyder, L R; Kirkland, J J

    2015-05-22

    Column selectivity in reversed-phase chromatography (RPC) can be described in terms of the hydrophobic-subtraction model, which recognizes five solute-column interactions that together determine solute retention and column selectivity: hydrophobic, steric, hydrogen bonding of an acceptor solute (i.e., a hydrogen-bond base) by a stationary-phase donor group (i.e., a silanol), hydrogen bonding of a donor solute (e.g., a carboxylic acid) by a stationary-phase acceptor group, and ionic. Of these five interactions, hydrogen bonding between donor solutes (acids) and stationary-phase acceptor groups is the least well understood; the present study aims at resolving this uncertainty, so far as possible. Previous work suggests that there are three distinct stationary-phase sites for hydrogen-bond interaction with carboxylic acids, which we will refer to as column basicity I, II, and III. All RPC columns exhibit a selective retention of carboxylic acids (column basicity I) in varying degree. This now appears to involve an interaction of the solute with a pair of vicinal silanols in the stationary phase. For some type-A columns, an additional basic site (column basicity II) is similar to that for column basicity I in primarily affecting the retention of carboxylic acids. The latter site appears to be associated with metal contamination of the silica. Finally, for embedded-polar-group (EPG) columns, the polar group can serve as a proton acceptor (column basicity III) for acids, phenols, and other donor solutes. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Nature of metastable amorphous-to-crystalline reversible phase transformations in GaSb

    Energy Technology Data Exchange (ETDEWEB)

    Kalkan, B. [Advanced Light Source, Lawrence Berkeley Laboratory, Berkeley, California 20015 (United States); Edwards, T. G.; Sen, S. [Department of Chemical Engineering and Materials Science, University of California, Davis, California 95616 (United States); Raoux, S. [IBM T. J. Watson Research Center, Yorktown Heights, New York 10598 (United States)

    2013-08-28

    The structural, thermodynamic, and kinetic aspects of the transformations between the metastable amorphous and crystalline phases of GaSb are investigated as a function of pressure at ambient temperature using synchrotron x-ray diffraction experiments in a diamond anvil cell. The results are consistent with the hypothesis that the pressure induced crystallization of amorphous GaSb into the β-Sn crystal structure near ∼5 GPa is possibly a manifestation of an underlying polyamorphic phase transition between a semiconducting, low density and a metallic, high density amorphous (LDA and HDA, respectively) phases. In this scenario, the large differences in the thermal crystallization kinetics between amorphous GaSb deposited in thin film form by sputtering and that prepared by laser melt quenching may be related to the relative location of the glass transition temperature of the latter in the pressure-temperature (P-T) space with respect to the location of the critical point that terminate the LDA ↔ HDA transition. The amorphous →β-Sn phase transition is found to be hysteretically reversible as the β-Sn phase undergoes decompressive amorphization near ∼2 GPa due to the lattice instabilities that give rise to density fluctuations in the crystal upon decompression.

  3. Nature of metastable amorphous-to-crystalline reversible phase transformations in GaSb

    Science.gov (United States)

    Kalkan, B.; Edwards, T. G.; Raoux, S.; Sen, S.

    2013-08-01

    The structural, thermodynamic, and kinetic aspects of the transformations between the metastable amorphous and crystalline phases of GaSb are investigated as a function of pressure at ambient temperature using synchrotron x-ray diffraction experiments in a diamond anvil cell. The results are consistent with the hypothesis that the pressure induced crystallization of amorphous GaSb into the β-Sn crystal structure near ˜5 GPa is possibly a manifestation of an underlying polyamorphic phase transition between a semiconducting, low density and a metallic, high density amorphous (LDA and HDA, respectively) phases. In this scenario, the large differences in the thermal crystallization kinetics between amorphous GaSb deposited in thin film form by sputtering and that prepared by laser melt quenching may be related to the relative location of the glass transition temperature of the latter in the pressure-temperature (P-T) space with respect to the location of the critical point that terminate the LDA ↔ HDA transition. The amorphous → β-Sn phase transition is found to be hysteretically reversible as the β-Sn phase undergoes decompressive amorphization near ˜2 GPa due to the lattice instabilities that give rise to density fluctuations in the crystal upon decompression.

  4. Nature of metastable amorphous-to-crystalline reversible phase transformations in GaSb

    International Nuclear Information System (INIS)

    Kalkan, B.; Edwards, T. G.; Sen, S.; Raoux, S.

    2013-01-01

    The structural, thermodynamic, and kinetic aspects of the transformations between the metastable amorphous and crystalline phases of GaSb are investigated as a function of pressure at ambient temperature using synchrotron x-ray diffraction experiments in a diamond anvil cell. The results are consistent with the hypothesis that the pressure induced crystallization of amorphous GaSb into the β-Sn crystal structure near ∼5 GPa is possibly a manifestation of an underlying polyamorphic phase transition between a semiconducting, low density and a metallic, high density amorphous (LDA and HDA, respectively) phases. In this scenario, the large differences in the thermal crystallization kinetics between amorphous GaSb deposited in thin film form by sputtering and that prepared by laser melt quenching may be related to the relative location of the glass transition temperature of the latter in the pressure-temperature (P-T) space with respect to the location of the critical point that terminate the LDA ↔ HDA transition. The amorphous →β-Sn phase transition is found to be hysteretically reversible as the β-Sn phase undergoes decompressive amorphization near ∼2 GPa due to the lattice instabilities that give rise to density fluctuations in the crystal upon decompression

  5. Peptide-tagged proteins in aqueous two-phase systems

    OpenAIRE

    Nilsson, Anna

    2002-01-01

    This thesis deals with proteins containing peptide tags for improved partitioning in aqueous two-phase systems. Qualitatively the peptide-tagged protein partitioning could be predicted from peptide data, i.e. partitioning trends found for peptides were also found for the peptide-tagged proteins. However, full effect of the tag as expected from peptide partitioning was not found in the tagged protein. When alkyl-ethylene oxide surfactant was included in a two-polymer system, almost full effect...

  6. Systematic Comparison of Reverse Phase and Hydrophilic Interaction Liquid Chromatography Platforms for the Analysis of N-linked Glycans

    Science.gov (United States)

    Walker, S. Hunter; Carlisle, Brandon C.; Muddiman, David C.

    2013-01-01

    Due to the hydrophilic nature of glycans, reverse phase chromatography has not been widely used as a glycomic separation technique coupled to mass spectrometry. Other approaches such as hydrophilic interaction chromatography and porous graphitized carbon chromatography are often employed, though these strategies frequently suffer from decreased chromatographic resolution, long equilibration times, indefinite retention, and column bleed. Herein, it is shown that through an efficient hydrazone formation derivatization of N-linked glycans (∼4 hr of additional sample preparation time which is carried out in parallel), numerous experimental and practical advantages are gained when analyzing the glycans by online reverse phase chromatography. These benefits include an increased number of glycans detected, increased peak capacity of the separation, and the ability to analyze glycans on the identical liquid chromatography-mass spectrometry platform commonly used for proteomic analyses. The data presented show that separation of derivatized N-linked glycans by reverse phase chromatography significantly out-performs traditional separation of native or derivatized glycans by hydrophilic interaction chromatography. Furthermore, the movement to a more ubiquitous separation technique will afford numerous research groups the opportunity to analyze both proteomic and glycomic samples on the same platform with minimal time and physical change between experiments, increasing the efficiency of ‘multi-omic’ biological approaches. PMID:22954204

  7. Reversible amorphous-crystalline phase changes in a wide range of Se1-xTex alloys studied using ultrafast differential scanning calorimetry

    NARCIS (Netherlands)

    Vermeulen, Paul. A.; Momand, Jamo; Kooi, Bart J.

    The reversible amorphous-crystalline phase change in a chalcogenide material, specifically the Se1-xTex alloy, has been investigated for the first time using ultrafast differential scanning calorimetry. Heating rates and cooling rates up to 5000 K/s were used. Repeated reversible

  8. Pharmaceutical Perspective on Opalescence and Liquid-Liquid Phase Separation in Protein Solutions.

    Science.gov (United States)

    Raut, Ashlesha S; Kalonia, Devendra S

    2016-05-02

    Opalescence in protein solutions reduces aesthetic appeal of a formulation and can be an indicator of the presence of aggregates or precursor to phase separation in solution signifying reduced product stability. Liquid-liquid phase separation of a protein solution into a protein-rich and a protein-poor phase has been well-documented for globular proteins and recently observed for monoclonal antibody solutions, resulting in physical instability of the formulation. The present review discusses opalescence and liquid-liquid phase separation (LLPS) for therapeutic protein formulations. A brief discussion on theoretical concepts based on thermodynamics, kinetics, and light scattering is presented. This review also discusses theoretical concepts behind intense light scattering in the vicinity of the critical point termed as "critical opalescence". Both opalescence and LLPS are affected by the formulation factors including pH, ionic strength, protein concentration, temperature, and excipients. Literature reports for the effect of these formulation factors on attractive protein-protein interactions in solution as assessed by the second virial coefficient (B2) and the cloud-point temperature (Tcloud) measurements are also presented. The review also highlights pharmaceutical implications of LLPS in protein solutions.

  9. Estimation of Ziprasidone Hydrochloride Monohydrate in Bulk and Capsules by Reverse Phase HPLC

    Directory of Open Access Journals (Sweden)

    B. Sudha Rani

    2006-01-01

    Full Text Available A reverse phase HPLC method is described for the determination of Ziprasidone HCl mono hydrate in bulk and pharmaceutical dosage forms. Chromatography was carried out on an ODS C18 column using a mixture of methanol and phosphate buffer (55:45v/v as the mobile phase at a flow rate of 1mL/min. Detection was carried out at 314nm. The retension time of the drug was 4.522 min. The method produced linear responses in the concentration range of 0.5-30 μg /mL of Ziprasidone HCl mono hydrate.The method was found to be applicable for determination of the drug in capsules.

  10. Lactobacillus salivarius reverse diabetes-induced intestinal defense impairment in mice through non-defensin protein.

    Science.gov (United States)

    Chung, Pei-Hsuan; Wu, Ying-Ying; Chen, Pei-Hsuan; Fung, Chang-Phone; Hsu, Ching-Mei; Chen, Lee-Wei

    2016-09-01

    Altered intestinal microbiota and subsequent endotoxemia play pathogenic roles in diabetes. We aimed to study the mechanisms of intestinal defense impairment in type 1 diabetes and the effects of Lactobacillus salivarius as well as fructooligosaccharides (FOS) supplementation on diabetes-induced bacterial translocation. Alterations in the enteric microbiome, expression of mucosal antibacterial proteins and bacteria-killing activity of the intestinal mucosa in streptozotocin (STZ)-induced diabetic mice and Ins2(Akita) mice were investigated. The effects of dead L. salivarius (2×10(8)CFU/ml) and FOS (250 mg per day) supplementation for 1 week on endotoxin levels and Klebsiella pneumoniae translocation were also examined. Finally, germ-free mice were cohoused with wild-type or Ins2(Akita) mice for 2 weeks to examine the contribution of microbiota on the antibacterial protein expression. STZ-induced diabetic mice developed intestinal defense impairment as demonstrated by decreased mucosal bacteria-killing activity; reduction of non-defensin family proteins, such as Reg3β, Reg3γ, CRP-ductin and RELMβ, but not the defensin family proteins; and increased bacterial translocation. Intestinal bacteria overgrowth, enteric dysbiosis and increased intestinal bacterial translocation, particularly pathogenic K. pneumoniae in STZ-induced diabetic mice and Ins2(Akita) mice, were noted. Treating diabetic mice with dead L. salivarius or FOS reversed enteric dysbiosis, restored mucosal antibacterial protein and lessened endotoxin levels as well as K. pneumoniae translocation. Moreover, germ-free mice cohoused with wild-type mice demonstrated more intestinal Reg3β and RELMβ expression than those cohoused with Ins2(Akita) mice. These results indicate that hyperglycemia induces enteric dysbiosis, reduction of non-defensin proteins as well as bacteria-killing activity of the intestinal mucosa and intestinal defense impairment. Reversal of enteric dysbiosis with dead L. salivarius or

  11. Determination of the Trans-resveratrol content of Champagne wines by reversed-phase HPLC

    Directory of Open Access Journals (Sweden)

    Philippe Jeandet

    2006-06-01

    Full Text Available Levels of trans-resveratrol in Champagne wines were determined by the use of reversed-phase HPLC with UV and fluorometric detection after liquid-liquid extraction with ethyl acetate. Resveratrol concentrations in Champagne wines range from 20 to 77 μg/L except for the Champagne rosé in which resveratrol reaches several hundred micrograms per litre. The resveratrol content of Champagne wines was also shown to decrease with aging on lees.

  12. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population.

    Science.gov (United States)

    Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine; Kjelgaard-Hansen, Mads; Christensen, Michelle; Hesta, Myriam; Tugirimana, Pierrot; Budd, Jane; Dermauw, Veronique; Janssens, Geert P J

    2014-09-01

    Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type. Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations within reference ranges for healthy domestic cats. In contrast, unhealthy cheetahs had higher (P cheetahs suffering from chronic kidney disease were significantly greater compared to the reportedly healthy cheetahs. Our study indicates that serum proteins in the cheetah can be analyzed by routine capillary electrophoresis, whereas acute-phase proteins can be measured using available immunoassays or non-species-specific techniques, which are also likely to be applicable in other exotic felids. Moreover, results suggest that serum amyloid A and haptoglobin are important acute-phase proteins in the diseased cheetah and highlight the need to evaluate their role as early-onset markers for disease.

  13. On thermodynamic and microscopic reversibility

    International Nuclear Information System (INIS)

    Crooks, Gavin E

    2011-01-01

    The word 'reversible' has two (apparently) distinct applications in statistical thermodynamics. A thermodynamically reversible process indicates an experimental protocol for which the entropy change is zero, whereas the principle of microscopic reversibility asserts that the probability of any trajectory of a system through phase space equals that of the time reversed trajectory. However, these two terms are actually synonymous: a thermodynamically reversible process is microscopically reversible, and vice versa

  14. A robust quantitative solid phase immunoassay for the acute phase protein C-reactive protein (CRP) based on cytidine 5 '-diphosphocholine coupled dendrimers

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Pedersen, H. G.; Jensen, A. L.

    2009-01-01

    C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP for phosp......C-reactive protein (CRP) is an important acute phase protein, being used as a sensitive indicator of inflammation and infection and is also associated with the risk of cardiovascular problems. The present paper describes a robust and sensitive ELISA for CRP, based on the affinity of CRP...... was applied to determination of pig and human CRP using commercially available antibodies against human CRP. The assay was shown to be more sensitive than previously published immunoassays employing albumin-coupled cytidine diphosphocholine. The coating was stable for at least 30 days at room temperature...

  15. Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.

    Directory of Open Access Journals (Sweden)

    Ellen Wallace

    Full Text Available The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive

  16. Cytokine and acute phase protein gene expression in liver biopsies from dairy cows with a lipopolysaccharide - induced mastitis

    DEFF Research Database (Denmark)

    Vels, J; Røntved, Christine M.; Bjerring, Martin

    2009-01-01

    A minimally invasive liver biopsy technique was tested for its applicability to study the hepatic acute phase response (APR) in dairy cows with Escherichia coli lipopolysaccharide (LPS)-induced mastitis. The hepatic mRNA expression profiles of the inflammatory cytokines, tumor necrosis factor (TNF......, a minimally invasive liver biopsy technique can be used for studying the hepatic APR in diseased cattle. Lipopolysaccharide-induced mastitis resulted in a time-dependent production of inflammatory cytokines and SAA and Hp in the liver of dairy cows.......- ), IL-1β, IL-6, and IL-10, and the acute phase proteins serum amyloid A isoform 3 (SAA3), haptoglobin (Hp), and 1-acid glycoprotein (AGP) were determined by real-time reverse transcription-PCR. Fourteen primiparous cows in mid lactation were challenged with 200 µg of LPS (n = 8) or NaCl solution (n = 6...

  17. Structural study of the AOT reverse micellar system. Influence of attractive interactions induced by the solubilisation of native and modified proteins

    International Nuclear Information System (INIS)

    Cassin, Guillaume

    1994-01-01

    This research thesis reports the study of the influence of intra-micellar attractions on the thermodynamic behaviour of reverse micellar systems, as well as of the effects induced by the solubilisation of natives or modified proteins. The author proposes a model to explain the decrease of attractions between droplets when the volume fraction occupied by reverse micelles increases. This model which highlights the importance of depletion forces between reverse micelles, allows the building up of a theoretical relationship between the bonding parameter and the volume fraction of reverse micelles. In order to understand the appearance of an attractive term related to the solubilisation of native cytochrome-c in these systems, this protein has been chemically modified. The author highlights the role of the charge born by a micellar probe on the thermodynamic behaviour of micro-emulsions. Then, the author applies the model of dimerizing adhesive spheres to reverse micellar systems containing native cytochrome-c. He shows that theoretical predictions of this model are in agreement with obtained experimental results [fr

  18. Two complementary reversed-phase separations for comprehensive coverage of the semipolar and nonpolar metabolome.

    Science.gov (United States)

    Naser, Fuad J; Mahieu, Nathaniel G; Wang, Lingjue; Spalding, Jonathan L; Johnson, Stephen L; Patti, Gary J

    2018-02-01

    Although it is common in untargeted metabolomics to apply reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) methods that have been systematically optimized for lipids and central carbon metabolites, here we show that these established protocols provide poor coverage of semipolar metabolites because of inadequate retention. Our objective was to develop an RPLC approach that improved detection of these metabolites without sacrificing lipid coverage. We initially evaluated columns recently released by Waters under the CORTECS line by analyzing 47 small-molecule standards that evenly span the nonpolar and semipolar ranges. An RPLC method commonly used in untargeted metabolomics was considered a benchmarking reference. We found that highly nonpolar and semipolar metabolites cannot be reliably profiled with any single method because of retention and solubility limitations of the injection solvent. Instead, we optimized a multiplexed approach using the CORTECS T3 column to analyze semipolar compounds and the CORTECS C 8 column to analyze lipids. Strikingly, we determined that combining these methods allowed detection of 41 of the total 47 standards, whereas our reference RPLC method detected only 10 of the 47 standards. We then applied credentialing to compare method performance at the comprehensive scale. The tandem method showed more than a fivefold increase in credentialing coverage relative to our RPLC benchmark. Our results demonstrate that comprehensive coverage of metabolites amenable to reversed-phase separation necessitates two reconstitution solvents and chromatographic methods. Thus, we suggest complementing HILIC methods with a dual T3 and C 8 RPLC approach to increase coverage of semipolar metabolites and lipids for untargeted metabolomics. Graphical abstract Analysis of semipolar and nonpolar metabolites necessitates two reversed-phase chromatography (RPLC) methods, which extend metabolome coverage more

  19. Preparation of organic monolithic columns in polytetrafluoroethylene tubes for reversed-phase liquid chromatography

    International Nuclear Information System (INIS)

    Catalá-Icardo, M.; Torres-Cartas, S.; Meseguer-Lloret, S.; Gómez-Benito, C.; Carrasco-Correa, E.; Simó-Alfonso, E.F.; Ramis-Ramos, G.; Herrero-Martínez, J.M.

    2017-01-01

    In this work, a method for the preparation and anchoring of polymeric monoliths in a polytetrafluoroethylene (PTFE) tubing as a column housing for microbore HPLC is described. In order to assure a covalent attachment of the monolith to the inner wall of the PTFE tube, a two-step procedure was developed. Two surface etching reagents, a commercial sodium naphthalene solution (Fluoroetch"®), or mixtures of H_2O_2 and H_2SO_4, were tried and compared. Then, the obtained hydroxyl groups on the PTFE surface were modified by methacryloylation. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and scanning electron microscopy (SEM) confirmed the successful modification of the tubing wall and the stable anchorage of monolith to the wall, respectively. Special emphasis was also put on the reduction of the unwanted effects of shrinking of monolith during polymerization, by using an external proper mold and by selecting the adequate monomers in order to increase the flexibility of the polymer. Poly(glycidyl methacrylate-co-divinylbenzene) monoliths were in situ synthesized by thermal polymerization within the confines of surface-vinylized PTFE tubes. The modified PTFE tubing tightly held the monolith, and the monolithic column exhibited good pressure resistance up to 20 MPa. The column performance was also evaluated via the isocratic separation of a series of alkylbenzenes in the reversed-phase mode. The optimized monolithic columns gave plate heights ranged between 70 and 80 μm. The resulting monoliths were also satisfactorily applied to the separation of proteins. - Highlights: • Successful surface etching of PTFE inner wall tubing was done. • The modified PTFE support was next methacryloylated with GMA. • Organic polymeric monolith was in situ prepared in the functionalized PTFE tube. • The monolithic columns gave suitable pressure resistance and separation of proteins.

  20. Preparation of organic monolithic columns in polytetrafluoroethylene tubes for reversed-phase liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Catalá-Icardo, M., E-mail: mocaic@qim.upv.es [Research Institute for Integrated Management of Coastal Areas, Universitat Politècnica de València, Paranimf 1, 46730, Grao de Gandía, Valencia (Spain); Torres-Cartas, S.; Meseguer-Lloret, S.; Gómez-Benito, C. [Research Institute for Integrated Management of Coastal Areas, Universitat Politècnica de València, Paranimf 1, 46730, Grao de Gandía, Valencia (Spain); Carrasco-Correa, E.; Simó-Alfonso, E.F.; Ramis-Ramos, G. [Department of Analytical Chemistry, Universitat de València, Dr. Moliner 50, 46100, Burjassot, Valencia (Spain); Herrero-Martínez, J.M., E-mail: jmherrer@uv.es [Department of Analytical Chemistry, Universitat de València, Dr. Moliner 50, 46100, Burjassot, Valencia (Spain)

    2017-04-01

    In this work, a method for the preparation and anchoring of polymeric monoliths in a polytetrafluoroethylene (PTFE) tubing as a column housing for microbore HPLC is described. In order to assure a covalent attachment of the monolith to the inner wall of the PTFE tube, a two-step procedure was developed. Two surface etching reagents, a commercial sodium naphthalene solution (Fluoroetch{sup ®}), or mixtures of H{sub 2}O{sub 2} and H{sub 2}SO{sub 4}, were tried and compared. Then, the obtained hydroxyl groups on the PTFE surface were modified by methacryloylation. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and scanning electron microscopy (SEM) confirmed the successful modification of the tubing wall and the stable anchorage of monolith to the wall, respectively. Special emphasis was also put on the reduction of the unwanted effects of shrinking of monolith during polymerization, by using an external proper mold and by selecting the adequate monomers in order to increase the flexibility of the polymer. Poly(glycidyl methacrylate-co-divinylbenzene) monoliths were in situ synthesized by thermal polymerization within the confines of surface-vinylized PTFE tubes. The modified PTFE tubing tightly held the monolith, and the monolithic column exhibited good pressure resistance up to 20 MPa. The column performance was also evaluated via the isocratic separation of a series of alkylbenzenes in the reversed-phase mode. The optimized monolithic columns gave plate heights ranged between 70 and 80 μm. The resulting monoliths were also satisfactorily applied to the separation of proteins. - Highlights: • Successful surface etching of PTFE inner wall tubing was done. • The modified PTFE support was next methacryloylated with GMA. • Organic polymeric monolith was in situ prepared in the functionalized PTFE tube. • The monolithic columns gave suitable pressure resistance and separation of proteins.

  1. Characterizing the selectivity of stationary phases and organic modifiers in reversed-phase high-performance liquid chromatographic systems by a general solvation equation using gradient elution.

    Science.gov (United States)

    Du, C M; Valko, K; Bevan, C; Reynolds, D; Abraham, M H

    2000-11-01

    Retention data for a set of 69 compounds using rapid gradient elution are obtained on a wide range of reversed-phase stationary phases and organic modifiers. The chromatographic stationary phases studied are Inertsil (IN)-ODS, pentafluorophenyl, fluoro-octyl, n-propylcyano, Polymer (PLRP-S 100), and hexylphenyl. The organic solvent modifiers are 2,2,2-trifluoroethanol (TFE); 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP); isopropanol; methanol (MeOH); acetonitrile (AcN); tetrahydrofuran; 1,4-dioxane; N,N-dimethylformamide; and mixed solvents of dimethylsulfoxide (DMSO) with AcN and DMSO with MeOH (1:1). A total of 25 chromatographic systems are analyzed using a solvation equation. In general, most of the systems give reasonable statistics. The selectivity of the reversed phase-high-performance liquid chromatographic (HPLC) systems with respect to the solute's dipolarity-polarity, hydrogen-bond acidity, and basicity are reflected in correspondingly large coefficients in the solvation equation. We wanted to find the most orthogonal HPLC systems, showing the highest possible selectivity difference in order to derive molecular descriptors using the gradient retention times of a compound. We selected eight chromatographic systems that have a large range of coefficients of interest (s, a, and b) similar to those found in water-solvent partitions used previously to derive molecular descriptors. The systems selected are IN-ODS phases with AcN, MeOH, TFE, and HFIP as mobile phase, PLRP-S 100 phase with AcN, propylcyano phase with AcN and MeOH, and fluorooctyl phase with TFE. Using the retention data obtained for a compound in the selected chromatographic systems, we can estimate the molecular descriptors with the faster and simpler gradient elution method.

  2. Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC.

    Science.gov (United States)

    Tarasova, Irina A; Goloborodko, Anton A; Perlova, Tatyana Y; Pridatchenko, Marina L; Gorshkov, Alexander V; Evreinov, Victor V; Ivanov, Alexander R; Gorshkov, Mikhail V

    2015-07-07

    The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC.

  3. [Determination of glycyrrhizinic acid in biotransformation system by reversed-phase high performance liquid chromatography].

    Science.gov (United States)

    Li, Hui; Lu, Dingqiang; Liu, Weimin

    2004-05-01

    A method for determining glycyrrhizinic acid in the biotransformation system by reversed-phase high performance liquid chromatography (RP-HPLC) was developed. The HPLC conditions were as follows: Hypersil C18 column (4.6 mm i.d. x 250 mm, 5 microm) with a mixture of methanol-water-acetic acid (70:30:1, v/v) as the mobile phase; flow rate at 1.0 mL/min; and UV detection at 254 nm. The linear range of glycyrrhizinic acid was 0.2-20 microg. The recoveries were 98%-103% with relative standard deviations between 0.16% and 1.58% (n = 3). The method is simple, rapid and accurate for determining glycyrrhizinic acid.

  4. Get phases from arsenic anomalous scattering: de novo SAD phasing of two protein structures crystallized in cacodylate buffer.

    Directory of Open Access Journals (Sweden)

    Xiang Liu

    Full Text Available The crystal structures of two proteins, a putative pyrazinamidase/nicotinamidase from the dental pathogen Streptococcus mutans (SmPncA and the human caspase-6 (Casp6, were solved by de novo arsenic single-wavelength anomalous diffraction (As-SAD phasing method. Arsenic (As, an uncommonly used element in SAD phasing, was covalently introduced into proteins by cacodylic acid, the buffering agent in the crystallization reservoirs. In SmPncA, the only cysteine was bound to dimethylarsinoyl, which is a pentavalent arsenic group (As (V. This arsenic atom and a protein-bound zinc atom both generated anomalous signals. The predominant contribution, however, was from the As anomalous signals, which were sufficient to phase the SmPncA structure alone. In Casp6, four cysteines were found to bind cacodyl, a trivalent arsenic group (As (III, in the presence of the reducing agent, dithiothreitol (DTT, and arsenic atoms were the only anomalous scatterers for SAD phasing. Analyses and discussion of these two As-SAD phasing examples and comparison of As with other traditional heavy atoms that generate anomalous signals, together with a few arsenic-based de novo phasing cases reported previously strongly suggest that As is an ideal anomalous scatterer for SAD phasing in protein crystallography.

  5. Automated de novo phasing and model building of coiled-coil proteins.

    Science.gov (United States)

    Rämisch, Sebastian; Lizatović, Robert; André, Ingemar

    2015-03-01

    Models generated by de novo structure prediction can be very useful starting points for molecular replacement for systems where suitable structural homologues cannot be readily identified. Protein-protein complexes and de novo-designed proteins are examples of systems that can be challenging to phase. In this study, the potential of de novo models of protein complexes for use as starting points for molecular replacement is investigated. The approach is demonstrated using homomeric coiled-coil proteins, which are excellent model systems for oligomeric systems. Despite the stereotypical fold of coiled coils, initial phase estimation can be difficult and many structures have to be solved with experimental phasing. A method was developed for automatic structure determination of homomeric coiled coils from X-ray diffraction data. In a benchmark set of 24 coiled coils, ranging from dimers to pentamers with resolutions down to 2.5 Å, 22 systems were automatically solved, 11 of which had previously been solved by experimental phasing. The generated models contained 71-103% of the residues present in the deposited structures, had the correct sequence and had free R values that deviated on average by 0.01 from those of the respective reference structures. The electron-density maps were of sufficient quality that only minor manual editing was necessary to produce final structures. The method, named CCsolve, combines methods for de novo structure prediction, initial phase estimation and automated model building into one pipeline. CCsolve is robust against errors in the initial models and can readily be modified to make use of alternative crystallographic software. The results demonstrate the feasibility of de novo phasing of protein-protein complexes, an approach that could also be employed for other small systems beyond coiled coils.

  6. Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

    Science.gov (United States)

    Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

    2017-11-01

    The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

  7. Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients

    NARCIS (Netherlands)

    Bischoff, Rainer; Roecklin, D.; Roitsch, C.

    1992-01-01

    Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase

  8. Localization of Reversion-Induced LIM Protein (RIL) in the Rat Central Nervous System

    International Nuclear Information System (INIS)

    Iida, Yuko; Matsuzaki, Toshiyuki; Morishima, Tetsuro; Sasano, Hiroshi; Asai, Kiyofumi; Sobue, Kazuya; Takata, Kuniaki

    2009-01-01

    Reversion-induced LIM protein (RIL) is a member of the ALP (actinin-associated LIM protein) subfamily of the PDZ/LIM protein family. RIL serves as an adaptor protein and seems to regulate cytoskeletons. Immunoblotting suggested that RIL is concentrated in the astrocytes in the central nervous system. We then examined the expression and localization of RIL in the rat central nervous system and compared it with that of water channel aquaporin 4 (AQP4). RIL was concentrated in the cells of ependyma lining the ventricles in the brain and the central canal in the spinal cord. In most parts of the central nervous system, RIL was expressed in the astrocytes that expressed AQP4. Double-labeling studies showed that RIL was concentrated in the cytoplasm of astrocytes where glial fibrillary acidic protein was enriched as well as in the AQP4-enriched regions such as the endfeet or glia limitans. RIL was also present in some neurons such as Purkinje cells in the cerebellum and some neurons in the brain stem. Differential expression of RIL suggests that it may be involved in the regulation of the central nervous system

  9. X-ray phase radiography and tomography with grating interferometry and the reverse projection technique

    International Nuclear Information System (INIS)

    Wang, Zhili; Gao, Kun; Ge, Xin; Wu, Zhao; Chen, Heng; Wang, Shenghao; Wu, Ziyu; Zhu, Peiping; Yuan, Qingxi; Huang, Wanxia; Zhang, Kai

    2013-01-01

    X-ray grating interferometry provides substantially increased contrast over conventional absorption-based imaging methods, and therefore new and complementary information. Compared with other phase-contrast imaging techniques, x-ray grating interferometry can overcome some of the problems that have impaired the applications of x-ray phase-contrast radiography and phase tomography. Recently, special attention has been paid to the development of quantitative phase retrieval methods, which is mandatory to perform x-ray phase tomography, to achieve material identification, to differentiate distinct tissues, etc. Typically, the phase-stepping approach has been utilized for phase retrieval in grating interferometry. This method requires a grating scanning and acquisition of multiple radiographic projections, and therefore is disadvantageous in terms of imaging speed and radiation damage. Here we present an innovative, highly sensitive approach, dubbed ‘reverse projection’ (RP), for quantitative phase retrieval. Compared with the phase-stepping approach, the present RP method abandons grating scanning completely, and thus is advantageous due to its much higher efficiency and the reduced radiation dose, without the degradation of reconstruction quality. This review presents a detailed explanation of the principle of the RP method. Both radiography and phase tomography experiments are performed to validate the RP method. We believe that this new technique will find widespread applications in biomedical imaging and in vivo studies. (paper)

  10. Strain-assisted current-induced magnetization reversal in magnetic tunnel junctions: A micromagnetic study with phase-field microelasticity

    International Nuclear Information System (INIS)

    Huang, H. B.; Hu, J. M.; Yang, T. N.; Chen, L. Q.; Ma, X. Q.

    2014-01-01

    Effect of substrate misfit strain on current-induced in-plane magnetization reversal in CoFeB-MgO based magnetic tunnel junctions is investigated by combining micromagnetic simulations with phase-field microelasticity theory. It is found that the critical current density for in-plane magnetization reversal decreases dramatically with an increasing substrate strain, since the effective elastic field can drag the magnetization to one of the four in-plane diagonal directions. A potential strain-assisted multilevel bit spin transfer magnetization switching device using substrate misfit strain is also proposed.

  11. Prediction of Molar Extinction Coefficients of Proteins and Peptides Using UV Absorption of the Constituent Amino Acids at 214 nm To Enable Quantitative Reverse Phase High-Performance Liquid Chromatography-Mass Spectrometry Analysis

    NARCIS (Netherlands)

    Kuipers, B.J.H.; Gruppen, H.

    2007-01-01

    The molar extinction coefficients of 20 amino acids and the peptide bond were measured at 214 nm in the presence of acetonitrile and formic acid to enable quantitative comparison of peptides eluting from reversed-phase high-performance liquid chromatography, once identified with mass spectrometry

  12. Simultaneous quantification of porcine myocardial adenine nucleotides and creatine phosphate by ion-pair reverse-phase high-performance liquid chromatography

    International Nuclear Information System (INIS)

    Cordis, G.A.; Das, D.K.

    1987-01-01

    In order to follow the energy metabolism and the levels of high-energy phosphate compounds in porcine myocardium subjected to ischemic insult, it was necessary to develop a high-performance liquid chromatography (HPLC) method where creatine phosphate (CP) and the adenine nucleotides could be measured simultaneously in a single run. Currently available ion-pair reverse-phase HPLC methods require a separate injection with a change in wavelength and mobile phase in order to measure the creatine phosphate, while baseline separation of AMP is lacking. The ion-exchange HPLC method includes a simultaneous determination, but the baseline drifts due to the gradient and baseline separation of AMP is not achieved. In the following ion-pair reverse-phase HPLC method, simultaneous measurements of porcine myocardial adenine nucleotides and creatine phosphate were achieved along with a stable baseline and homogeneous baseline separation of each measured compound, allowing accurate quantification

  13. A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation

    International Nuclear Information System (INIS)

    Wiegand, Kimberly C; Lu, Yiling; Zhang, Fan; Anglesio, Michael S; Gilks, Blake; Mills, Gordon B; Huntsman, David G; Carey, Mark S; Hennessy, Bryan T; Leung, Samuel; Wang, Yemin; Ju, Zhenlin; McGahren, Mollianne; Kalloger, Steve E; Finlayson, Sarah; Stemke-Hale, Katherine

    2014-01-01

    Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC. Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A. Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr 308 and AKT-Ser 473 phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in

  14. NONLINEAR-REGRESSION METHODS FOR MODELING OF HETEROSCEDASTIC RETENTION DATA IN REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

    NARCIS (Netherlands)

    HENDRIKS, MMWB; COENEGRACHT, PMJ; DOORNBOS, DA

    1994-01-01

    New models have been developed that accurately describe the response surfaces of capacity factors that are a function of changes in the pH and the fraction of organic modifier in reversed-phase high-performance liquid chromatography (RP-HPLC). The purpose of this article is to illustrate one of the

  15. Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

    Science.gov (United States)

    Dhandhukia, Jugal P; Brill, Dab A; Kouhi, Aida; Pastuszka, Martha K; MacKay, J Andrew

    2017-09-01

    Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent K d of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates. © 2017 The Protein Society.

  16. The TITAN Reversed-Field Pinch fusion reactor study: Scoping phase report

    International Nuclear Information System (INIS)

    1987-01-01

    The TITAN research program is a multi-institutional effort to determine the potential of the Reversed-Field Pinch (RFP) magnetic fusion concept as a compact, high-power-density, and ''attractive'' fusion energy system from economic (cost of electricity, COE), environmental, and operational viewpoints. In particular, a high neutron wall loading design (18 MW/m 2 ) has been chosen as the reference case in order to quantify the issue of engineering practicality, to determine the physics requirements and plasma operating mode, to assess significant benefits of compact systems, and to illuminate the main drawbacks. The program has been divided into two phases, each roughly one year in length: the Scoping Phase and the Design Phase. During the scoping phase, the TITAN design team has defined the parameter space for a high mass power density (MPD) RFP reactor, and explored a variety of approaches to the design of major subsystems. Two major design approaches consistent with high MPD and low COE, the lithium-vanadium blanket design and aqueous loop-in-pool design, have been selected for more detailed engineering evaluation in the design phase. The program has retained a balance in its approach to investigating high MPD systems. On the one hand, parametric investigations of both subsystems and overall system performance are carried out. On the other hand, more detailed analysis and engineering design and integration are performed, appropriate to determining the technical feasibility of the high MPD approach to RFP fusion reactors. This report describes the work of the scoping phase activities of the TITAN program. A synopsis of the principal technical findings and a brief description of the TITAN multiple-design approach is given. The individual chapters on Plasma Physics and Engineering, Parameter Systems Studies, Divertor, Reactor Engineering, and Fusion Power Core Engineering have been cataloged separately

  17. The TITAN Reversed-Field Pinch fusion reactor study: Scoping phase report

    Energy Technology Data Exchange (ETDEWEB)

    1987-01-01

    The TITAN research program is a multi-institutional effort to determine the potential of the Reversed-Field Pinch (RFP) magnetic fusion concept as a compact, high-power-density, and ''attractive'' fusion energy system from economic (cost of electricity, COE), environmental, and operational viewpoints. In particular, a high neutron wall loading design (18 MW/m/sup 2/) has been chosen as the reference case in order to quantify the issue of engineering practicality, to determine the physics requirements and plasma operating mode, to assess significant benefits of compact systems, and to illuminate the main drawbacks. The program has been divided into two phases, each roughly one year in length: the Scoping Phase and the Design Phase. During the scoping phase, the TITAN design team has defined the parameter space for a high mass power density (MPD) RFP reactor, and explored a variety of approaches to the design of major subsystems. Two major design approaches consistent with high MPD and low COE, the lithium-vanadium blanket design and aqueous loop-in-pool design, have been selected for more detailed engineering evaluation in the design phase. The program has retained a balance in its approach to investigating high MPD systems. On the one hand, parametric investigations of both subsystems and overall system performance are carried out. On the other hand, more detailed analysis and engineering design and integration are performed, appropriate to determining the technical feasibility of the high MPD approach to RFP fusion reactors. This report describes the work of the scoping phase activities of the TITAN program. A synopsis of the principal technical findings and a brief description of the TITAN multiple-design approach is given. The individual chapters on Plasma Physics and Engineering, Parameter Systems Studies, Divertor, Reactor Engineering, and Fusion Power Core Engineering have been cataloged separately.

  18. Echocardiographic phase imaging to predict reverse remodeling after cardiac resynchronization therapy.

    Science.gov (United States)

    Buss, Sebastian J; Humpert, Per M; Bekeredjian, Raffi; Hardt, Stefan E; Zugck, Christian; Schellberg, Dieter; Bauer, Alexander; Filusch, Arthur; Kuecherer, Helmut; Katus, Hugo A; Korosoglou, Grigorios

    2009-05-01

    The aim of our study was to investigate whether echocardiographic phase imaging (EPI) can predict response in patients who are considered for cardiac resynchronization therapy (CRT). CRT improves quality of life, exercise capacity, and outcome in patients with bundle-branch block and advanced heart failure. Previous studies used QRS duration to select patients for CRT; the accuracy of this parameter to predict functional recovery, however, is controversial. We examined 42 patients with advanced heart failure (New York Heart Association [NYHA] functional class III to IV, QRS duration >130 ms, and ejection fraction or=15% at 6 to 8 months of follow-up were defined as responders. All others were classified as nonresponders. The Ts-SD and the mean EPI-Index were related to Delta ESV (r = 0.43 for Ts-SD and r = 0.67 for mean EPI-Index, p < 0.01 for both), and both parameters yielded similar accuracy for the prediction of LV remodeling (area under the curve of 0.87 for TDI vs. 0.90 for EPI, difference between areas = 0.03, p = NS) and ejection fraction (EF) improvement (area under the curve of 0.87 for TDI vs. 0.93 for EPI, difference between areas = 0.06, p = NS). Furthermore, patients classified as responders by EPI (mean EPI-Index phase imaging can predict functional recovery, reverse LV remodeling, and clinical outcomes in patients who undergo CRT. EPI is a method that objectively and accurately quantifies LV dyssynchrony and seems to be noninferior to TDI for the prediction of reverse LV remodeling and functional recovery.

  19. A Switchable Molecular Dielectric with Two Sequential Reversible Phase Transitions: [(CH3)4P]4[Mn(SCN)6].

    Science.gov (United States)

    Li, Qiang; Shi, Ping-Ping; Ye, Qiong; Wang, Hui-Ting; Wu, De-Hong; Ye, Heng-Yun; Fu, Da-Wei; Zhang, Yi

    2015-11-16

    A new organic-inorganic hybrid switchable and tunable dielectric compound, [(CH3)4P]4[Mn(SCN)6] (1), exhibits three distinct dielectric states above room temperature and undergoes two reversible solid-state phase transitions, including a structural phase transition at 330 K and a ferroelastic phase transition with the Aizu notation of mmmF2/m at 352 K. The variable-temperature structural analyses disclose that the origin of the phase transitions and dielectric anomalies can be ascribed to the reorientation or motion of both the [(CH3)4P](+) cations and [Mn(SCN)6](4-) anions in solid-state crystals.

  20. Optimisation and characterisation of silica-based reversed-phase liquid chromatographic systems for the analysis of basic pharmaceuticals

    NARCIS (Netherlands)

    Vervoort, R.J.M.; Debets, A.J.J.; Claessens, H.A.; Cramers, C.A.M.G.; Jong, de G.J.

    2000-01-01

    Reversed-phase liquid chromatography using silica-based columns is successfully applied in many separations. However, also some drawbacks exist, i.e. the analysis of basic compounds is often hampered by ionic interaction of the basic analytes with residual silanols present on the silica surface,

  1. Serum protein capillary electrophoresis and measurement of acute phase proteins in a captive cheetah (Acinonyx jubatus) population

    DEFF Research Database (Denmark)

    Depauw, Sarah; Delanghe, Joris; Whitehouse-Tedd, Katherine

    2014-01-01

    Renal and gastrointestinal pathologies are widespread in the captive cheetah (Acinonyx jubatus) population but are often diagnosed at a late stage, because diagnostic tools are limited to the evaluation of clinical signs or general blood examination. Presently, no data are available on serum...... proteins and acute-phase proteins in cheetahs during health or disease, although they might be important to improve health monitoring. This study aimed to quantify serum proteins by capillary electrophoresis in 80 serum samples from captive cheetahs, categorized according to health status and disease type....... Moreover, serum amyloid A concentrations were measured via a turbidimetric immunoassay validated in domestic cats, whereas haptoglobin and C-reactive protein were determined by non-species-specific functional tests. Cheetahs classified as healthy had serum protein and acute phase protein concentrations...

  2. Differential Regulation of Telomerase Reverse Transcriptase Promoter Activation and Protein Degradation by Histone Deacetylase Inhibition.

    Science.gov (United States)

    Qing, Hua; Aono, Jun; Findeisen, Hannes M; Jones, Karrie L; Heywood, Elizabeth B; Bruemmer, Dennis

    2016-06-01

    Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti-proliferative efficacy of HDAC inhibitors. © 2015 Wiley Periodicals, Inc.

  3. Reversible Resistance Switching Effect in Amorphous Ge1Sb4Te7 Thin Films without Phase Transformation

    International Nuclear Information System (INIS)

    Hua-Jun, Sun; Li-Song, Hou; Yi-Qun, Wu; Xiao-Dong, Tang

    2009-01-01

    We demonstrate a reversible resistance switching effect that does not rely on amorphous-crystalline phase transformation in a nanoscale capacitor-like cell using Ge 1 Sb 4 Te 7 films as the working material. The polarity and amplitude of the applied electric voltage switches the cell resistance between low- and high-resistance states, as revealed in the current-voltage characteristics of the film by conductive atomic force microscopy (CAFM). This reversible SET/RESET switching effect is induced by voltage pulses and their polarity. The change of electrical resistance due to the switching effect is approximately two orders of magnitude

  4. A Typology of Reverse Innovation

    DEFF Research Database (Denmark)

    von Zedtwitz, Max; Corsi, Simone; Søberg, Peder Veng

    2015-01-01

    secondary market introduction, this study expands the espoused definition of reverse innovation beyond its market-introduction focus with reversals in the flow of innovation in the ideation and product development phases. Recognizing that each phase can take place in different geographical locations...... taking place in an emerging country. This analytical framework allows recasting of current research at the intersection between innovation and international business. Of the 10 reverse innovation flows, six are new and have not been covered in the literature to date. The study addresses questions......’s portfolio of global innovation competence and capability. The implications for management are concerned with internal and external resistance to reverse innovation. Most significantly, while greater recognition and power of innovation in formerly subordinate organizational units is inconvenient to some...

  5. Early postmenopausal phase is associated with reduced prostacyclin-induced vasodilation that is reversed by exercise training

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Egelund, Jon; Mandrup Jensen, Camilla Maria

    2016-01-01

    The postmenopausal phase is associated with an accelerated rate of rise in the prevalence of vascular dysfunction and hypertension; however, the mechanisms underlying these adverse vascular changes and whether exercise training can reverse the decline in vascular function remains unclear. We...... increased (Pphase is associated with a marked reduction in vascular function. Despite of a reduced sensitivity...

  6. Diagnostical meaning acute phase proteins in cerebrospinal liquid in children with neuroinfections

    Directory of Open Access Journals (Sweden)

    L. A. Alekseeva

    2010-01-01

    Full Text Available In the article presented results of the examination of acute phase proteins in cerebrospinal liquid in 237 children with meningitis and encephalitis viral and bacterial etiology. The dependence between the level of acute phase proteins in cerebrospinal liquid and etiology of neuroinfectional process, the severity of brain damage and the process stage was determined. Diagnostic and prognostic efficiency of the acute phase proteins (C-reactive protein, albumin, alpha-1-antitripsin, alpha-2-macroglobulin, gaptoglobin examination in children with neuroinfections was specified. Developed method of express diagnostics of the severity of inflammatory damage of the brain in bacterial meningitis in children by determination in cerebrospinal liquid alpha-2-macroglobulin is described.

  7. Type 2 diabetes mellitus with early phase acute inflammatory protein on serum protein electrophoresis

    Directory of Open Access Journals (Sweden)

    ET Tuladhar

    2012-03-01

    Full Text Available Background: The onset of Type 2 diabetes has been associated with low grade systemic inflammation. The inflammatory status has been studied by measuring acute phase reactant proteins like hsCRP, α1- antitrypsin, α1-acid glycoprotein, ceruloplasmin, fibrinogen. Most of these acute phase reactants form α1 and α2 bands on electropherogram of serum proteins. The aim of this study was to evaluate inflammatory status in controlled and uncontrolled type 2 diabetes using cellulose acetate electrophoresis and to find the impact of glycemic status as indicated by HbA1c on inflammation process. Materials and Methods: Serum protein electrophoresis was done on serum samples of 60 cases of Diabetes [controlled and uncontrolled] using cellulose acetate paper technique. The electropherogram obtained was stained with Ponseu S and then quantitated using densitometer. Glycemic status was studied by HbA1c analysis. The density of α1and α2 bands in electropherogram were correlated with HbA1c level. Result: A significant increase in the percentage of α1 and α2 band proteins (0.765 and 0.716, p<0.001 were found with the increasing level of HbA1c. With cutoff of HbA1c 7% (American Diabetic Association recommended, the α1 and α2 serum proteins concentration are significantly higher (p<0.001 in uncontrolled diabetes mellitus compared to controlled diabetes mellitus Conclusion: Cellulose acetate electrophoresis of serum proteins show early phase acute inflammatory status in uncontrolled type 2 diabetes mellitus. The process of systemic inflammation worsens with uncontrolled glycemia as indicated by HbA1c. Inflammatory status should be studied adjunct to glycemic status. DOI: http://dx.doi.org/10.3126/jpn.v2i3.6024 JPN 2012; 2(3: 211-214

  8. Structure-retention and mobile phase-retention relationships for reversed-phase high-performance liquid chromatography of several hydroxythioxanthone derivatives in binary acetonitrile-water mixtures

    International Nuclear Information System (INIS)

    Amiri, Ali Asghar; Hemmateenejad, Bahram; Safavi, Afsaneh; Sharghi, Hashem; Beni, Ali Reza Salimi; Shamsipur, Mojtaba

    2007-01-01

    The reversed-phase high-performance liquid chromatographic (RP-HPLC) behavior of some newly synthesized hydroxythioxanthone derivatives using binary acetonitrile-water mixtures as mobile phase has been examined. First, the variation in the retention time of each molecule as a function of mobile phase properties was studied by Kamlet-Taft solvatochromic equations. Then, the influences of molecular structure of the hydroxythioxanthone derivatives on their retention time in various mobile phase mixtures were investigated by quantitative structure-property relationship (QSPR) analysis. Finally, a unified model containing both the molecular structure parameters and mobile phase properties was developed to describe the chromatographic behavior of the systems studied. Among the solvent properties, polarity/polarizability parameter (π * ) and hydrogen-bond basicity (β), and among the solute properties, the most positive local charge (MPC), the sum of positive charges on hydrogen atoms contributing in hydrogen bonding (SPCH) and lipophilicity index (log P) were identified as controlling factors in the RP-HPLC behavior of hydroxythioxanthone derivatives in actonitrile-water binary solvents

  9. Solid-Phase Extraction and Reverse-Phase HPLC: Application to Study the Urinary Excretion Pattern of Benzophenone-3 and its Metabolite 2,4-Dihydroxybenzophenone in Human Urine

    Directory of Open Access Journals (Sweden)

    Helena Gonzalez

    2008-01-01

    Full Text Available Background Benzophenone-3 (BZ-3 is a common ultraviolet (UV absorbing compound in sunscreens. It is the most bioavailable species of all UV-absorbing compounds after topical application and can be found in plasma and urine. Objectives The aim of this study was to develop a reverse-phase high performance liquid chromatography (HPLC method for determining the amounts BZ-3 and its metabolite 2,4-dihydroxybenzophenone (DHB in human urine. The method had to be suitable for handling a large number of samples. It also had to be rapid and simple, but still sensitive, accurate and reproducible. The assay was applied to study the urinary excretion pattern after repeated whole-body applications of a commercial sunscreen, containing 4% BZ-3, to 25 healthy volunteers. Methods Each sample was analyzed with regard to both conjugated/non-conjugated BZ-3 and conjugated/non-conjugated DHB, since both BZ-3 and DHB are extensively conjugated in the body. Solid-phase extraction (SPE with C8 columns was followed by reverse-phase HPLC. For separation a Genesis C18 column was used with an acethonitrile-water mobile phase and the UV-detector was set at 287 nm. Results The assay was linear r 2 > 0.99, with detection limits for BZ-3 and DHB of 0.01 µmol L -1 and 0.16 µmol L -1 respectively. Relative standard deviation (RSD was less than 10% for BZ-3 and less than 13% for DHB. The excretion pattern varied among the human volunteers; we discerned different patterns among the individuals. Conclusions The reverse-phase HPLC assay and extraction procedures developed are suitable for use when a large number of samples need to be analyzed and the method fulfilled our objectives. The differences in excretion pattern may be due to differences in enzyme activity but further studies, especially about genetic polymorphism, need to be performed to verify this finding.

  10. Synthesis of a mixed-model stationary phase derived from glutamine for HPLC separation of structurally different biologically active compounds: HILIC and reversed-phase applications.

    Science.gov (United States)

    Aral, Tarık; Aral, Hayriye; Ziyadanoğulları, Berrin; Ziyadanoğulları, Recep

    2015-01-01

    A novel mixed-mode stationary phase was synthesised starting from N-Boc-glutamine, aniline and spherical silica gel (4 µm, 60 Å). The prepared stationary phase was characterized by IR and elemental analysis. The new stationary phase bears an embedded amide group into phenyl ring, highly polar a terminal amide group and non-polar groups (phenyl and alkyl groups). At first, this new mixed-mode stationary phase was used for HILIC separation of four nucleotides and five nucleosides. The effects of different separation conditions, such as pH value, mobile phase and temperature, on the separation process were investigated. The optimum separation for nucleotides was achieved using HILIC isocratic elution with aqueous mobile phase and acetonitrile with 20°C column temperature. Under these conditions, the four nucleotides could be separated and detected at 265 nm within 14 min. Five nucleosides were separated under HILIC isocratic elution with aqueous mobile phase containing pH=3.25 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 265 nm within 14 min. Chromatographic parameters as retention factor, selectivity, theoretical plate number and peak asymmetry factor were calculated for the effect of temperature and water content in mobile phase on the separation process. The new column was also tested for nucleotides and nucleosides mixture and six analytes were separated in 10min. The chromatographic behaviours of these polar analytes on the new mixed-model stationary phase were compared with those of HILIC columns under similar conditions. Further, phytohormones and phenolic compounds were separated in order to see influence of the new stationary phase in reverse phase conditions. Eleven plant phytohormones were separated within 13 min using RP-HPLC gradient elution with aqueous mobile phase containing pH=2.5 phosphate buffer (10mM) and acetonitrile with 20°C column temperature and detected at 230 or 278 nm. The best separation

  11. Preparation, characterization and application of a reversed phase liquid chromatography/hydrophilic interaction chromatography mixed-mode C18-DTT stationary phase.

    Science.gov (United States)

    Wang, Qing; Long, Yao; Yao, Lin; Xu, Li; Shi, Zhi-Guo; Xu, Lanying

    2016-01-01

    A mixed-mode chromatographic stationary phase, C18-DTT (dithiothreitol) silica (SiO2) was prepared through "thiol-ene" click chemistry. The obtained material was characterized by fourier transform infrared spectroscope, nitrogen adsorption analysis and contact angle analysis. Chromatographic performance of the C18-DTT was systemically evaluated by studying the effect of acetonitrile content, pH, buffer concentration of the mobile phase and column temperature. It was demonstrated that the novel stationary phase possessed reversed phase liquid chromatography (RPLC)/hydrophilic interaction liquid chromatography (HILIC) mixed-mode property. The stop-flow test revealed that C18-DTT exhibited excellent compatibility with 100% aqueous mobile phase. Additionally, the stability and column-to-column reproducibility of the C18-DTT material were satisfactory, with relative standard deviations of retention factor of the tested analytes (verapamil, fenbufen, guanine, tetrandrine and nicotinic acid) in the range of 1.82-3.72% and 0.85-1.93%, respectively. Finally, the application of C18-DTT column was demonstrated in the separation of non-steroidal anti-inflammatory drugs, aromatic carboxylic acids, alkaloids, nucleo-analytes and polycyclic aromatic hydrocarbons. It had great resolving power in the analysis of various compounds in HILIC and RPLC chromatographic conditions and was a promising RPLC/HILIC mixed-mode stationary phase. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Effect of molecular interactions on retention and selectivity in reversed-phase liquid chromatography.

    Science.gov (United States)

    Szepesy, László

    2002-06-25

    The linear solvation energy relationships (LSERs) have been applied in the last years for description and prediction of retention and selectivity in reversed-phase liquid chromatography with good results. Widely different stationary phases have been compared and characterized by LSERs. In recent publications the influence of the type of the organic moderator and the composition of the mobile phase have also been described. However, the influence of the molecular properties of the solutes to be separated has never been discussed. According to the LSER model variation in retention factors (log k) with solute structure can be related to their potential for various intermolecular interactions. The retention factor is given as the sum of the terms of the LSER equation representing various types of molecular interactions. For this reason the influence of the structure and molecular properties of the solutes to be separated can also be investigated using the LSER equation. In this study we shall demonstrate how the specific molecular interactions influence chromatographic retention and selectivity. We intend to show that retention and selectivity depend on all participants of the system. In addition to the structure and properties of the stationary phase and the type and composition of the mobile phase the molecular properties of the solutes, characterized by the solvation parameters, will also influence the type and extent of the various molecular interactions governing retention and selectivity.

  13. Rapid Quantitative Analysis of Naringenin in the Fruit Bodies of Inonotus vaninii by Two-phase Acid Hydrolysis Followed by Reversed Phase-high Performance Liquid Chromatography-ultra Violet.

    Science.gov (United States)

    Guohua, Xia; Pan, Ruirong; Bao, Rui; Ge, Yanru; Zhou, Cunshan; Shen, Yuping

    2017-01-01

    Sanghuang is one of mystical traditional Chinese medicines recorded earliest 2000 years ago, that included various fungi of Inonotus genus and was well-known for antitumor effect in modern medicine. Inonotus vaninii is grown in natural forest of Northeastern China merely and used as Sanghuang commercially, but it has no quality control specification until now. This study was to establish a rapid method of two-phase acid hydrolysis followed by reversed phase-high performance liquid chromatography-ultra violet (RP-HPLC-UV) to quantify naringenin in the fruit body of I. vaninii . Sample solution was prepared by pretreatment of raw material in two-phase acid hydrolysis and the hydrolysis technology was optimized. After reconstitution, analysis was performed using RP-HPLC-UV. The method validation was investigated and the naringenin content of sample and comparison were determined. The naringenin was obtained by two-phase acid hydrolysis method, namely, 10.0 g of raw material was hydrolyzed in 200 mL of 1% sulfuric acid aqueous solution (v/v) and 400 mL of chloroform in oil bath at 110°C for 2 h. Good linearity ( r = 0.9992) was achieved between concentration of analyte and peak area. The relative standard deviation (RSD) of precision was 2.47% and the RSD of naringenin contents for repeatability was 3.13%. The accuracy was supported with recoveries at 96.37%, 97.30%, and 99.31%. The sample solution prepared using the proposed method contained higher content of naringenin than conventional method and was stable for 8 h. Due to the high efficiency of sample preparation and high reliability of the HPLC method, it is feasible to use this method for routine analysis of naringenin in the fungus. A convenient two-phase acid hydrolysis was employed to produce naringenin from raw material, and then an efficient and reliable reversed phase-high performance liquid chromatography-ultra violet method was established to monitor naringenin in the fruit bodies of Inonotus vaninii

  14. Identification of regioisomers and enantiomers of triacylglycerols in different yeasts using reversed- and chiral-phase LC–MS

    Czech Academy of Sciences Publication Activity Database

    Řezanka, Tomáš; Kolouchová, I.; Čejková, A.; Cajthaml, Tomáš; Sigler, Karel

    2013-01-01

    Roč. 36, č. 20 (2013), s. 3310-3320 ISSN 1615-9306 R&D Projects: GA ČR(CZ) GAP503/11/0215 Institutional support: RVO:61388971 Keywords : Atmospheric pressure chemical ionization mass spectrometry * Chiral LC * Reversed phase LC Subject RIV: EE - Microbiology, Virology Impact factor: 2.594, year: 2013

  15. Concentrado proteico de folhas de mandioca na alimentação de tilápias-do-nilo na fase de reversão sexual Cassava leaf protein concentrate in Nile tilapia diets during sex reversal phase

    Directory of Open Access Journals (Sweden)

    Leandro Bohnenberger

    2010-06-01

    Full Text Available Este trabalho foi realizado com o objetivo de avaliar a inclusão de concentrado proteico de folhas de mandioca na alimentação de tilápias-do-nilo na fase de reversão sexual. Durante o período experimental, que durou 28 dias, foram utilizadas 300 larvas de tilápia com 7 dias de idade, distribuídas em 20 aquários com capacidade para 30 L, em um delineamento inteiramente casualisado, com cinco níveis do concentrado proteico de folhas de mandioca (0; 5; 10; 15 e 20%, cada um com quatro repetições, considerando unidade experimental um aquário com 15 larvas. As cinco rações foram isoproteicas e isoenergéticas, com 38,6% de proteína digestível e 3.300 kcal de ED/kg de ração. Os valores médios de temperatura, pH, oxigênio dissolvido e condutividade elétrica da água dos aquários durante o período experimental foram de 23,90 ± 1,35ºC; 7,23 ± 0,30; 5,15 ± 0,84 mg/L; e 105,91 ± 4,73 μS/cm, respectivamente. Ao final do período experimental, não foram observadas diferenças significativas no peso, no comprimento final, no ganho de peso nem na taxa de sobrevivência das larvas alimentadas com o concentrado proteico de folhas de mandioca. A utilização de concentrado proteico de folhas de mandioca em níveis de até 20% em rações para tilápia-do-nilo na fase de reversão sexual não prejudica o desempenho nem a sobrevivência dos animais.This paper aimed to evaluate the effect of inclusion of cassava leaf protein concentrate (CLPC in Nile tilapia diets during sex reversal phase. During the experiment, which lasted for 28 days, 300 tilapia larvae at 7 days of age were distributed into 20 aquarium tanks with 30 L of water capacity in a complete randomized design, with 5 levels of cassava leaf protein concentrate (0; 5; 10; 15; and 20%, each one with four replicates. One aquarium with 15 larvae was considered the experimental unit. The five rations were iso-proteic and iso-energetic containing 38.6% of digestible protein and 3

  16. Artificial Self-Sufficient P450 in Reversed Micelles

    Directory of Open Access Journals (Sweden)

    Teruyuki Nagamune

    2010-04-01

    Full Text Available Cytochrome P450s are heme-containing monooxygenases that require electron transfer proteins for their catalytic activities. They prefer hydrophobic compounds as substrates and it is, therefore, desirable to perform their reactions in non-aqueous media. Reversed micelles can stably encapsulate proteins in nano-scaled water pools in organic solvents. However, in the reversed micellar system, when multiple proteins are involved in a reaction they can be separated into different micelles and it is then difficult to transfer electrons between proteins. We show here that an artificial self-sufficient cytochrome P450, which is an enzymatically crosslinked fusion protein composed of P450 and electron transfer proteins, showed micelle-size dependent catalytic activity in a reversed micellar system. Furthermore, the presence of thermostable alcohol dehydrogenase promoted the P450-catalyzed reaction due to cofactor regeneration.

  17. Thermodynamic and kinetic characterization of polycyclic aromatic hydrocarbons in reversed-phase liquid chromatography.

    Science.gov (United States)

    Howerton, Samuel B; McGuffin, Victoria L

    2003-07-15

    The retention of six polycyclic aromatic hydrocarbons (PAHs) was characterized by reversed-phase liquid chromatography. The PAHs were detected by laser-induced fluorescence at four points along an optically transparent capillary column. The profiles were characterized in space and time using an exponentially modified Gaussian equation. The resulting parameters were used to calculate the retention factors, as well as the concomitant changes in molar enthalpy and molar volume, for each PAH on monomeric (2.7 micromol/m2) and polymeric (5.4 micromol/m2) octadecylsilica. The changes in molar enthalpy become more exothermic as ring number increases and as annelation structure becomes less condensed. The changes in molar volume become more negative as ring number increases for the planar PAHs, but are positive for the nonplanar solutes. In addition, the rate constants, as well as the concomitant activation enthalpy and activation volume, are calculated for the first time. The kinetic data demonstrate that many of the PAHs exhibit very fast transitions between the mobile and stationary phases. The transition state is very high in energy, and the activation enthalpies and volumes become greater as ring number increases and as annelation structure becomes less condensed. The changes in thermodynamic and kinetic behavior are much more pronounced for the polymeric phase than for the monomeric phase.

  18. Course 12: Proteins: Structural, Thermodynamic and Kinetic Aspects

    Science.gov (United States)

    Finkelstein, A. V.

    1 Introduction 2 Overview of protein architectures and discussion of physical background of their natural selection 2.1 Protein structures 2.2 Physical selection of protein structures 3 Thermodynamic aspects of protein folding 3.1 Reversible denaturation of protein structures 3.2 What do denatured proteins look like? 3.3 Why denaturation of a globular protein is the first-order phase transition 3.4 "Gap" in energy spectrum: The main characteristic that distinguishes protein chains from random polymers 4 Kinetic aspects of protein folding 4.1 Protein folding in vivo 4.2 Protein folding in vitro (in the test-tube) 4.3 Theory of protein folding rates and solution of the Levinthal paradox

  19. Separation and determination of synthetic impurities of difloxacin by reversed-phase high-performance liquid chromatography.

    Science.gov (United States)

    Rao, R Nageswara; Nagaraju, V

    2004-11-19

    A simple and rapid reversed-phase high-performance liquid chromatographic method for separation and determination of process-related impurities of difloxacin (DFL) was developed. The separation was achieved on a reversed-phase C(18) column using methanol-water-acetic acid (78:21.9:0.1, v/v/v) as a mobile solvent at a flow rate of 1.0 ml/min at 28 degrees C using UV detection at 230 nm. It was linear over a range of 0.03 x 10(-6) to 1.60 x 10(-6)g for process related impurities and 0.05 x 10(-6) to 2.40 x 10(-6)g for difloxacin. The detection limits were 0.009 x 10(-6) to 0.024 x 10(-6)g for all the compounds examined. The recoveries were found to be in the range of 97.6-102.0% for impurities as well as difloxacin. The precision and robustness of the method were evaluated. It was used for not only quality assurance, but also monitoring the synthetic reactions involved in the process development work of difloxacin. The method was found to be specific, precise and reliable for the determination of unreacted levels of raw materials, intermediates in the reaction mixtures and the finished products of difloxacin.

  20. 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

  1. Study of Stationary Phase Metabolism Via Isotopomer Analysis of Amino Acids from an Isolated Protein

    Energy Technology Data Exchange (ETDEWEB)

    Shaikh, AfshanS.; Tang, YinjieJ.; Mukhopadhyay, Aindrila; Martin, Hector Garcia; Gin, Jennifer; Benke, Peter; Keasling, Jay D.

    2009-09-14

    Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C-labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase.

  2. Chitosan-coated magnetic nanoparticles prepared in one-step by precipitation in a high-aqueous phase content reverse microemulsion.

    Science.gov (United States)

    Pineda, María Guadalupe; Torres, Silvia; López, Luis Valencia; Enríquez-Medrano, Francisco Javier; de León, Ramón Díaz; Fernández, Salvador; Saade, Hened; López, Raúl Guillermo

    2014-07-02

    Chitosan-coated magnetic nanoparticles (CMNP) were prepared in one-step by precipitation in a high-aqueous phase content reverse microemulsion in the presence of chitosan. The high-aqueous phase concentration led to productivities close to 0.49 g CMNP/100 g microemulsion; much higher than those characteristic of precipitation in reverse microemulsions for preparing magnetic nanoparticles. The obtained nanoparticles present a narrow particle size distribution with an average diameter of 4.5 nm; appearing to be formed of a single crystallite; furthermore they present superparamagnetism and high magnetization values; close to 49 emu/g. Characterization of CMNP suggests that chitosan is present as a non-homogeneous very thin layer; which explains the slight reduction in the magnetization value of CMNP in comparison with that of uncoated magnetic nanoparticles. The prepared nanoparticles show high heavy ion removal capability; as demonstrated by their use in the treatment of Pb2+ aqueous solutions; from which lead ions were completely removed within 10 min.

  3. Chitosan-Coated Magnetic Nanoparticles Prepared in One-Step by Precipitation in a High-Aqueous Phase Content Reverse Microemulsion

    Directory of Open Access Journals (Sweden)

    María Guadalupe Pineda

    2014-07-01

    Full Text Available Chitosan-coated magnetic nanoparticles (CMNP were prepared in one-step by precipitation in a high-aqueous phase content reverse microemulsion in the presence of chitosan. The high-aqueous phase concentration led to productivities close to 0.49 g CMNP/100 g microemulsion; much higher than those characteristic of precipitation in reverse microemulsions for preparing magnetic nanoparticles. The obtained nanoparticles present a narrow particle size distribution with an average diameter of 4.5 nm; appearing to be formed of a single crystallite; furthermore they present superparamagnetism and high magnetization values; close to 49 emu/g. Characterization of CMNP suggests that chitosan is present as a non-homogeneous very thin layer; which explains the slight reduction in the magnetization value of CMNP in comparison with that of uncoated magnetic nanoparticles. The prepared nanoparticles show high heavy ion removal capability; as demonstrated by their use in the treatment of Pb2+ aqueous solutions; from which lead ions were completely removed within 10 min.

  4. Rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs

    DEFF Research Database (Denmark)

    Skovgaard, Kerstin; Mortensen, Shila; Boye, Mette

    2009-01-01

    The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other...... parts of innate host defence reactions remain somewhat elusive. In order to gain new insight into this early host defence response in the context of bacterial infection we studied gene expression changes in peripheral lymphoid tissues as compared to hepatic expression changes, 14-18 h after lung...... with measurements of interleukin-6 and selected acute phase proteins in serum. C-reactive protein and serum amyloid A were clearly induced 14-18 h after infection. Extrahepatic expression of acute phase proteins was found to be dramatically altered as a result of the lung infection with an extrahepatic acute phase...

  5. Analysis of proinsulin and its conversion products by reversed-phase high-performance liquid chromatography

    DEFF Research Database (Denmark)

    Linde, S; Welinder, B S; Nielsen, Jens Høiriis

    1993-01-01

    . Most mammals produce a single insulin, but in rodents two non-allelic insulin genes are expressed. There is an inverse ratio between the two insulins in rats and mice, the reason for this being unknown. It has been suggested that differences in transcription, translation (biosynthesis) and...... PIM (intact proinsulin or its intermediates) has been incompletely determined. Studies of the biosynthesis of proinsulins and their conversion with the purpose of revealing some of these points depend on accessible reversed-phase high-performance liquid chromatographic (RP-HPLC) analyses capable...

  6. Programmable self-assembly of carbon nanotubes assisted by reversible denaturation of a protein

    International Nuclear Information System (INIS)

    Nithiyasri, P; Parthasarathy, M; Balaji, K; Brindha, P

    2012-01-01

    Self-assembly of pristine multi-walled carbon nanotubes (CNTs) in aqueous dispersion using a protein, bovine serum albumin (BSA), has been demonstrated. Step-wise conformational changes in BSA as a function of temperature have been deployed to direct the assembly of nanotubes. More specifically, CNTs distributed randomly in native BSA at 35 °C as well as completely denatured BSA solution at 80 °C self-assemble in the intermediate temperature range of 45–65 °C, as evident from scanning and transmission electron microscopy. Fourier transform infrared (FTIR) and fluorescence studies indicate significant changes in the α-helical content of the protein with respect to the amide I and II bands and tryptophan emission intensity, respectively. The stability of CNT dispersion in BSA solution has been attributed to the hydrophobic interaction between nanotubes and the protein molecule by adding sodium cholate to the dispersion. Moreover, a mechanism based on electrostatic repulsion between BSA-bound CNTs has been proposed for the thermally reversible assembly of CNTs in BSA solution based on evidence from zeta potential measurements and FTIR spectroscopy. Thus the present report demonstrates bio-mimetic self-assembly of as-synthesized CNTs using changes in surface charge and conformation of an unfolding protein for biomedical applications and nanobiotechnology. (paper)

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

    Science.gov (United States)

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

    2005-04-15

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

  8. [Reversed-phase high-performance liquid chromatograph--application to serum aluminium monitoring].

    Science.gov (United States)

    Hoshino, H; Kaneko, E

    1996-01-01

    High-Performance Liquid Chromatography (HPLC) with the reversed-phase partition mode separation (including ion-pair one) towards metal chelate compounds prepared in an off-line fashion (precolumn chelation) is most versatile in terms of high sensitivity with base-line flatness, unique selectivity and cost effectiveness. The extraordinary toughness to the complicated matrices encountered in clinical testing is exemplified by the successful application to the aluminium monitoring of human serum samples. The A1 chelate with 2,2'-dihydroxyazobenzene is efficiently chromatographed on a LiChroCART RP-18 column using an aqueous methanol eluent (63.6 wt%) containing tetrabutylammonium bromide as an ion-pair agent. The serum concentration level of A1 down to 6 micrograms dm-3 is readily monitored without influences from iron, chyle and haemolysis.

  9. Retention prediction and hydrophobicity estimation of weak acidic compounds by reversed-phase liquid chromatography using acetic and perchloric acids as ion suppressors.

    Science.gov (United States)

    Han, Shu-ying; Ming, Xin; Qi, Zheng-chun; Sheng, Dong; Lian, Hong-zhen

    2010-11-01

    Simple acids are usually applied to suppress the ionization of weakly ionizable acidic analytes in reversed-phase liquid chromatography. The purpose of this study is to investigate the retention behavior of various weak acidic compounds (monoprotic, diprotic, triprotic, and tetraprotic acids) using acetic or perchloric acid as ion suppressor in a binary hydroorganic mobile phase. The apparent n-octanol-water partition coefficient (K(ow)") was proposed to calibrate the n-octanol-water partition coefficient (K(ow)) of weak acidic compound. LogK(ow)" was found to have a better linear correlation with logk(w), the logarithm of the retention factor obtained by extrapolating to neat aqueous fraction of the mobile phase, for all weakly ionizable acidic compounds. This straightforward relationship offers a potential medium for direct measurement of K(ow) data of weak acidic analytes and can be used to predict retention behavior of these compounds in the ion suppression reversed-phase liquid chromatographic mode.

  10. Reversible assembly of protein-DNA nanostructures triggered by mediated electron transfer

    International Nuclear Information System (INIS)

    Vogt, Stephan; Wenderhold-Reeb, Sabine; Nöll, Gilbert

    2017-01-01

    Stable protein-DNA nanostructures have been assembled by reconstitution of the multi-ligand binding flavoprotein dodecin on top of flavin-terminated dsDNA monolayers on gold electrodes. These structures could be disassembled by electrochemical flavin reduction via mediated electron transfer. For this purpose a negative potential was applied at the Au working electrode in the presence of the redox mediator bis-(ammoniumethyl)-4,4′-bipyridinium tetrabromide. The stepwise formation of the flavin-terminated dsDNA monolayers as well as the binding and electrochemically triggered release of apododecin were monitored by surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) measurements. The assembly and disassembly of the protein-DNA nanostructures were fully reversible processes, which could be carried out multiple times at the same flavin-dsDNA modified surface. When a negative potential was applied in the absence of a redox mediator apododecin could not be released, i.e. direct electron transfer was not possible. As alternative redox mediators also methylene blue and phenosafranine were studied, but in the presence of these molecules apododecin was released without applying a potential, probably because the tricyclic aromatic compounds are able to replace the flavins at the binding sites.

  11. A new imidazolium-embedded C{sub 18} stationary phase with enhanced performance in reversed-phase liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Qiu Hongdeng [Department of Applied Chemistry and Biochemistry, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan); Lanzhou Institute of Chemical Physics, Chinese Academy of Science, Lanzhou 730000 (China); Mallik, Abul K. [Department of Applied Chemistry and Biochemistry, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan); Takafuji, Makoto [Department of Applied Chemistry and Biochemistry, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan); Kumamoto Institute for Photo-Electro Organics (Phoenics), Kumamoto 862-0901 (Japan); Liu Xia; Jiang Shengxiang [Lanzhou Institute of Chemical Physics, Chinese Academy of Science, Lanzhou 730000 (China); Ihara, Hirotaka, E-mail: ihara@kumamoto-u.ac.jp [Department of Applied Chemistry and Biochemistry, Kumamoto University, 2-39-1 Kurokami, Kumamoto 860-8555 (Japan); Kumamoto Institute for Photo-Electro Organics (Phoenics), Kumamoto 862-0901 (Japan)

    2012-08-13

    Highlights: Black-Right-Pointing-Pointer Imidazolium-embedded C{sub 18} stationary phase was prepared and characterized. Black-Right-Pointing-Pointer Enhanced chromatographic selectivity was observed in SiImC{sub 18} column. Black-Right-Pointing-Pointer Seven nucleosides and bases were separated using only water as eluent within 8 min. Black-Right-Pointing-Pointer Multiple-interactions induced by embedded polar imidazolium was investigated. - Abstract: In this paper, a new imidazolium-embedded C{sub 18} stationary phase (SiImC{sub 18}) for reversed-phase high-performance liquid chromatography is described. 1-Allyl-3-octadecylimidazolium bromide ionic liquid compound having a long alkyl chain and reactive groups was newly prepared and grafted onto 3-mercaptopropyltrimethoxysilane-modified silica via a surface-initiated radical-chain transfer addition reaction. The SiImC{sub 18} obtained was characterized by elemental analysis, infrared spectroscopy, thermogravimetric analysis, diffuse reflectance infrared Fourier transform, and solid-state {sup 13}C and {sup 29}Si cross-polarization/magic angle spinning nuclear magnetic resonance spectroscopy. The selectivity toward polycyclic aromatic hydrocarbons relative to that toward alkylbenzenes exhibited by SiImC{sub 18} was higher than the corresponding selectivity exhibited by a conventional octadecyl silica (ODS) column, which could be explained by electrostatic {pi}-{pi} interaction cationic imidazolium and electron-rich aromatic rings. On the other hand, SiImC{sub 18} also showed high selectivity for polar compounds, which was based on the multiple interaction and retention mechanisms of this phase with different analytes. 1,6-Dinitropyrene and 1,8-dinitropyrene, which form a positional isomer pair of dipolar compounds, were separated successfully with the SiImC{sub 18} phase. Seven nucleosides and bases (i.e. cytidine, uracil, uridine, thymine, guanosine, xanthosine, and adenosine) were separated using only water as

  12. HDAC inhibition induces HIV-1 protein and enables immune-based clearance following latency reversal

    DEFF Research Database (Denmark)

    Wu, Guoxin; Swanson, Michael; Talla, Aarthi

    2017-01-01

    Promising therapeutic approaches for eradicating HIV include transcriptional activation of provirus from latently infected cells using latency-reversing agents (LRAs) and immune-mediated clearance to purge reservoirs. Accurate detection of cells capable of producing viral antigens and virions......, and the measurement of clearance of infected cells, is essential to assessing therapeutic efficacy. Here, we apply enhanced methodology extending the sensitivity limits for the rapid detection of subfemtomolar HIV gag p24 capsid protein in CD4+ T cells from ART-suppressed HIV+ individuals, and we show viral protein...... induction following treatment with LRAs. Importantly, we demonstrate that clinical administration of histone deacetylase inhibitors (HDACis; vorinostat and panobinostat) induced HIV gag p24, and ex vivo stimulation produced sufficient viral antigen to elicit immune-mediated cell killing using anti-gp120/CD3...

  13. Arc is a flexible modular protein capable of reversible self-oligomerization

    Science.gov (United States)

    Myrum, Craig; Baumann, Anne; Bustad, Helene J.; Flydal, Marte Innselset; Mariaule, Vincent; Alvira, Sara; Cuéllar, Jorge; Haavik, Jan; Soulé, Jonathan; Valpuesta, José Maria; Márquez, José Antonio; Martinez, Aurora; Bramham, Clive R.

    2015-01-01

    The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes. PMID:25748042

  14. FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit.

    Science.gov (United States)

    Bibert, Stéphanie; Liu, Chia-Chi; Figtree, Gemma A; Garcia, Alvaro; Hamilton, Elisha J; Marassi, Francesca M; Sweadner, Kathleen J; Cornelius, Flemming; Geering, Käthi; Rasmussen, Helge H

    2011-05-27

    The seven members of the FXYD protein family associate with the Na(+)-K(+) pump and modulate its activity. We investigated whether conserved cysteines in FXYD proteins are susceptible to glutathionylation and whether such reactivity affects Na(+)-K(+) pump function in cardiac myocytes and Xenopus oocytes. Glutathionylation was detected by immunoblotting streptavidin precipitate from biotin-GSH loaded cells or by a GSH antibody. Incubation of myocytes with recombinant FXYD proteins resulted in competitive displacement of native FXYD1. Myocyte and Xenopus oocyte pump currents were measured with whole-cell and two-electrode voltage clamp techniques, respectively. Native FXYD1 in myocytes and FXYD1 expressed in oocytes were susceptible to glutathionylation. Mutagenesis identified the specific cysteine in the cytoplasmic terminal that was reactive. Its reactivity was dependent on flanking basic amino acids. We have reported that Na(+)-K(+) pump β(1) subunit glutathionylation induced by oxidative signals causes pump inhibition in a previous study. In the present study, we found that β(1) subunit glutathionylation and pump inhibition could be reversed by exposing myocytes to exogenous wild-type FXYD3. A cysteine-free FXYD3 derivative had no effect. Similar results were obtained with wild-type and mutant FXYD proteins expressed in oocytes. Glutathionylation of the β(1) subunit was increased in myocardium from FXYD1(-/-) mice. In conclusion, there is a dependence of Na(+)-K(+) pump regulation on reactivity of two specifically identified cysteines on separate components of the multimeric Na(+)-K(+) pump complex. By facilitating deglutathionylation of the β(1) subunit, FXYD proteins reverse oxidative inhibition of the Na(+)-K(+) pump and play a dynamic role in its regulation.

  15. Serum acute phase protein concentrations in female dogs with mammary tumors.

    Science.gov (United States)

    Tecles, Fernando; Caldín, Marco; Zanella, Anna; Membiela, Francisco; Tvarijonaviciute, Asta; Subiela, Silvia Martínez; Cerón, José Joaquín

    2009-03-01

    Acute phase proteins (APPs) are proteins whose concentrations in serum change after any inflammatory stimulus or tissue damage. The aim of the current study was to evaluate 3 positive APPs (C-reactive protein, serum amyloid A, and haptoglobin) and 1 negative APP (albumin) in female dogs with mammary neoplasia. Acute phase proteins were studied in 70 female dogs aged 8-12 years in the following groups: healthy (n = 10); mammary tumors in stages I (n = 19), II (n = 5), III (n = 6), IV (n = 5), and V (n = 7); and with mammary neoplasia plus a concomitant disease (n = 18). In animals with mammary neoplasia, significant increases of positive APPs were only detected in those that had metastasis or a neoplasm with a diameter greater than 5 cm and ulceration. Dogs with mammary neoplasia and a concomitant disease also had high C-reactive protein concentrations. Albumin concentration was decreased in animals with metastasis and with a concomitant disease. The results of the present study indicate that the acute phase response could be stimulated in female dogs with mammary gland tumors because of different factors, such as metastasis, large size of the primary mass, and ulceration or secondary inflammation of the neoplasm.

  16. Optimal combinations of acute phase proteins for detecting infectious disease in pigs

    DEFF Research Database (Denmark)

    Heegaard, Peter M. H.; Stockmarr, Anders; Piñeiro, Matilde

    2011-01-01

    The acute phase protein (APP) response is an early systemic sign of disease, detected as substantial changes in APP serum concentrations and most disease states involving inflammatory reactions give rise to APP responses. To obtain a detailed picture of the general utility of porcine APPs to detect...... gondii) and one viral (porcine respiratory and reproductive syndrome virus) infection and one aseptic inflammation. Immunochemical analyses of seven APPs, four positive (C-reactive protein (CRP), haptoglobin (Hp), pig major acute phase protein (pigMAP) and serum amyloid A (SAA)) and three negative...

  17. Correlations for the partition behavior of proteins in aqueous two-phase systems

    DEFF Research Database (Denmark)

    Schmidt, A.S.; Andrews, B.A.; Asenjo, J.A.

    1996-01-01

    to its high hydrophilicity. In the case of subtilisin and trypsin inhibitor, their high concentrations in the top phase were due to their hydrophobic nature (hydrophobic interaction with PEG) and small size (negligible steric exclusion). The maximum concentration in the bottom phase for trypsin inhibitor...... of the overall protein concentration, by the ratio between the ''saturation'' equations of the two individual phases. Better correlations were obtained when an empirical sigmoidal Boltzmann equation was fitted to the data, since in virtually all cases the partition coefficient is constant at low protein...

  18. [Determination of ciprofloxacin in human serum and urine by reversed-phase HPLC].

    Science.gov (United States)

    Qin, Y; Liang, D

    1993-03-01

    A sensitive and rapid method for the determination of ciprofloxacin using enoxacin as the internal standard was reported. High-performance liquid chromatograph model 344 (Beckman, USA) with a variable wavelength UV detector and reversed-phase Ultrasphere-ODS column (5 microns, 250 x 4.6 mm) was used. Serum or urine sample preparation involved addition phosphate buffer (pH 7.5) and aqueous solution of sodium lauryl sulfate, followed by chloroform extraction. The organic layer was removed and evaporated to dryness under an air stream in a 37 degrees C water bath. The residue was dissolved in 50 microliters mobile phase and 20 microliters injected. The mobile phase of 0.02 mol/L acetate buffer (pH 3.0) -acetonitril-dimethylformamide-10% aqueous solution of tetrabutyl ammonium hydroxide (88:6.5:5:0.5) was pumped at 0.9 ml/min through the column. The detector operated at 0.01 aufs and the wavelength was set at 276 nm. The retention times for ciprofloxacin and enoxacin were 7.31 min and 5.59 min, respectively. In serum, standard curve was linear in the concentration range of 0.75 to 24 mumol/L, the detective limit was 0.2 mumol/L, extraction recovery was 69-74%, within-day CV was less than 5%, and inter-day CV was less than 6%.

  19. A ''reverse'' solid-phase radio-immuno-assay for IgM-antibodies to hepatitis A virus

    International Nuclear Information System (INIS)

    Meurman, O.H.; Matter, L.; Krishna, R.V.; Krech, U.H.

    1981-01-01

    A ''reverse'' solid-phase radio-immuno-assay for IgM antibodies to hepatitis A virus (HAV) was developed. Anti-human IgM immunoglobulins were bound on the wells of polyvinylchloride microtiter plates. Serum specimens were incubated in the anti-human IgM coated wells and bound IgM antibodies were then assayed for antigen specificity by subsequent incubations with HAV antigen and 125 I-labelled human anti-HAV IgG. The test showed a high sensitivity and specificity for anti-HAV IgM antibodies. No false-positive reactions were observed either in the sera from patients with hepatobiliary disorders other than HAV infection or in the sera containing both rheumatoid factor and anti-HAV IgG antibodies. In acute HAV infections specific IgM antibodies were present already in the first specimens taken within a few days after the onset of jaundice. The persistence of the IgM antibodies was from 4 to 6 months. IgM antibody titers up to 1,000,000 were observed in the acute phase of HAV infection. In routine diagnostic work the titration of the sera was not necessary, since a reliable qualitative result was obtained by testing the sera in a single dilution of 1:100. A similar ''reverse'' immuno-assay principle may be adaptable for the diagnostic determination of IgM antibodies to different viral and microbial antigens. (author)

  20. Microscopic Investigation of Reversible Nanoscale Surface Size Dependent Protein Conjugation

    Directory of Open Access Journals (Sweden)

    Michael A. Carpenter

    2009-05-01

    Full Text Available Aβ1-40 coated 20 nm gold colloidal nanoparticles exhibit a reversible color change as pH is externally altered between pH 4 and 10. This reversible process may contain important information on the initial reversible step reported for the fibrillogenesis of Aβ (a hallmark of Alzheimer’s disease. We examined this reversible color change by microscopic investigations. AFM images on graphite surfaces revealed the morphology of Aβ aggregates with gold colloids. TEM images clearly demonstrate the correspondence between spectroscopic features and conformational changes of the gold colloid.

  1. Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electrospray mass spectrometry

    NARCIS (Netherlands)

    Carol, J.; Gorseling, M.C.J.K.; Jong, C.F. de; Lingeman, H.; Kientz, C.E.; Baar, B.L.M. van; Irth, H.

    2005-01-01

    A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid

  2. Retention of nucleic acids in ion-pair reversed-phase high-performance liquid chromatography depends not only on base composition but also on base sequence.

    Science.gov (United States)

    Qiao, Jun-Qin; Liang, Chao; Wei, Lan-Chun; Cao, Zhao-Ming; Lian, Hong-Zhen

    2016-12-01

    The study on nucleic acid retention in ion-pair reversed-phase high-performance liquid chromatography mainly focuses on size-dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double-stranded DNAs were investigated on silica-based C 18 stationary phase by ion-pair reversed-phase high-performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self-dimerization have weaker retention than those not prone to self-dimerization but with the same base composition. However, homo-oligonucleotides are suitable for the size-dependent separation as a special case of oligonucleotides. For double-stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double-stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion-pair reversed-phase high-performance liquid chromatography. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. [Separation and determination of eight plant hormones by reversed-phase high performance liquid chromatography].

    Science.gov (United States)

    Fang, N; Hou, S; Shao, X; He, Y; Zhao, G

    1998-09-01

    In this paper, reversed-phase high performance liquid chromatographic technique was used for the separation and determination of eight plant hormones. Methanol-water-acetic acid system was chosen as the mobile phase. The effects of different separation conditions, such as the methanol and acetic acid concentrations in mobile phase, on the retention behaviours of eight plant hormones in this system were studied. The general trends in retention behaviours could be correlated to the methanol concentration in mobile phase. The experimental results showed that the optimum separation was achieved with following gradient elution condition: 0-3 minutes, 70% (water percentage in mobile phase), 3-13 minutes, 70%-20%, 13-48 minutes, 20%. Benzene was added to be as the internal standard. Under this experimental condition, the eight plant hormones could be separated completely and detected quantitatively at 260 nm within 16 minutes. The calibration curves for the eight compounds gave linearity over a wide range. The correlation coefficients of each components were r(ZT) = 0.9971, r(GAs) = 0.9999, r(K) = 0.9997, r(BA) = 0.9995, r(IAA) = 0.9998, r(IPA) = 0.9982, r(IBA) = 0.9995 and r(NAA) = 0.9995. The method is rapid, simple and efficient. It is a suitable method for the accurate determination of gibberellic acid (GA) and alpha-naphthaleneacetic acid (alpha-NAA) in products for agricultural use.

  4. Parameter dependence of resonant spin torque magnetization reversal

    International Nuclear Information System (INIS)

    Fricke, L.; Serrano-Guisan, S.; Schumacher, H.W.

    2012-01-01

    We numerically study ultra fast resonant spin torque (ST) magnetization reversal in magnetic tunneling junctions (MTJ) driven by current pulses having a direct current (DC) and a resonant alternating current (AC) component. The precessional ST dynamics of the single domain MTJ free layer cell are modeled in the macro spin approximation. The energy efficiency, reversal time, and reversal reliability are investigated under variation of pulse parameters like direct and AC current amplitude, AC frequency and AC phase. We find a range of AC and direct current amplitudes where robust resonant ST reversal is obtained with faster switching time and reduced energy consumption per pulse compared to purely direct current ST reversal. However, for a certain range of AC and direct current amplitudes a strong dependence of the reversal properties on AC frequency and phase is found. Such regions of unreliable reversal must be avoided for ST memory applications.

  5. Parameter dependence of resonant spin torque magnetization reversal

    Science.gov (United States)

    Fricke, L.; Serrano-Guisan, S.; Schumacher, H. W.

    2012-04-01

    We numerically study ultra fast resonant spin torque (ST) magnetization reversal in magnetic tunneling junctions (MTJ) driven by current pulses having a direct current (DC) and a resonant alternating current (AC) component. The precessional ST dynamics of the single domain MTJ free layer cell are modeled in the macro spin approximation. The energy efficiency, reversal time, and reversal reliability are investigated under variation of pulse parameters like direct and AC current amplitude, AC frequency and AC phase. We find a range of AC and direct current amplitudes where robust resonant ST reversal is obtained with faster switching time and reduced energy consumption per pulse compared to purely direct current ST reversal. However, for a certain range of AC and direct current amplitudes a strong dependence of the reversal properties on AC frequency and phase is found. Such regions of unreliable reversal must be avoided for ST memory applications.

  6. Two-step preparation of nano-scaled magnetic chitosan particles using Triton X-100 reversed-phase water-in-oil microemulsion system

    International Nuclear Information System (INIS)

    Zhou, Zhengkun; Jiang, Feihong; Lee, Tung-Ching; Yue, Tianli

    2013-01-01

    Highlights: •A new two-step route for nano-scaled magnetic chitosan particles preparation. •Triton X-100 reversed-phase microemulsion system was used for chitosan coating. •Narrow size distribution of magnetic chitosan nanoparticles was achieved. •Quantitative evaluation of recoverability for the magnetic chitosan nanoparticles. -- Abstract: A new two-step route for the preparation of nano-scaled magnetic chitosan particles has been developed, different from reported one-step in situ preparation and two-step preparation method of reversed-phase suspension, Triton X-100 reversed-phase water-in-oil microemulsion encapsulation method was employed in coating the pre-prepared Fe 3 O 4 nanoparticles with chitosan. The resultant magnetic chitosan particles owned a narrow size distribution ranging from 50 to 92 nm. X-ray diffraction patterns (XRD) indicated that the chitosan coating procedure did not change the spinal structure of Fe 3 O 4 magnetic nanoparticles. The results of Fourier transform infrared (FTIR) analysis and thermogravimetric analysis (TGA) demonstrated that the chitosan was coated on Fe 3 O 4 nanoparticles and its average mass content was ∼50%. The saturated magnetization of the magnetic Fe 3 O 4 /chitosan nanoparticles reached 18.62 emu/g, meanwhile, the nanoparticles showed the characteristics of superparamagnetism. The magnetic chitosan nanoparticles showed a high recoverability of 99.99% in 10 min when pH exceeded 4. The results suggested that the as-prepared magnetic chitosan particles were nano-scaled with a narrow size distribution and a high recoverability

  7. Liquid crystalline phase behavior of protein fibers in water: experiments versus theory.

    Science.gov (United States)

    Jung, Jin-Mi; Mezzenga, Raffaele

    2010-01-05

    We have developed a new method allowing the study of the thermodynamic phase behavior of mesoscopic colloidal systems consisting of amyloid protein fibers in water, obtained by heat denaturation and aggregation of beta-lactoglobulin, a dairy protein. The fibers have a cross section of about 5.2 nm and two groups of polydisperse contour lengths: (i) long fibers of 1-20 microm, showing semiflexible behavior, and (ii) short rods of 100-200 nm long, obtained by cutting the long fibers via high-pressure homogenization. At pH 2 without salt, these fibers are highly charged and stable in water. We have studied the isotropic-nematic phase transition for both systems and compared our results with the theoretical values predicted by Onsager's theory. The experimentally measured isotropic-nematic phase transition was found to occur at 0.4% and at 3% for the long and short fibers, respectively. For both systems, this phase transition occurs at concentrations more than 1 order of magnitude lower than what is expected based on Onsager's theory. Moreover, at low enough pH, no intermediate biphasic region was observed between the isotropic phase and the nematic phase. The phase diagrams of both systems (pH vs concentration) showed similar, yet complex and rich, phase behavior. We discuss the possible physical fundamentals ruling the phase diagram as well as the discrepancy we observe for the isotropic-nematic phase transition between our experimental results and the predicted theoretical results. Our work highlights that systems formed by water-amyloid protein fibers are way too complex to be understood based solely on Onsager's theories. Experimental results are revisited in terms of the Flory's theory (1956) for suspensions of rods, which allows accounting for rod-solvent hydrophobic interactions. This theoretical approach allows explaining, on a semiquantitative basis, most of the discrepancies observed between the experimental results and Onsager's predictions. The sources of

  8. Targeted proteomics as a tool for porcine acute phase proteins measurements

    DEFF Research Database (Denmark)

    Marco-Ramell, Anna; Bassols, Anna; Bislev, Stine Lønnerup

    2013-01-01

    . Selected reaction monitoring (SRM), a targeted quantitative proteomic technique, may be used as an alternative to commercial kits for the measurement and validation of target proteins. Acute phase proteins (APPs) are widely recognized inflammation and infection biomarkers (Eckersall, 2010...

  9. Two-dimensional high-performance thin-layer chromatography of tryptic bovine albumin digest using normal- and reverse-phase systems with silanized silica stationary phase.

    Science.gov (United States)

    Gwarda, Radosław Łukasz; Dzido, Tadeusz Henryk

    2013-10-18

    Among many advantages of planar techniques, two-dimensional (2D) separation seems to be the most important for analysis of complex samples. Here we present quick, simple and efficient two-dimensional high-performance thin-layer chromatography (2D HPTLC) of bovine albumin digest using commercial HPTLC RP-18W plates (silica based stationary phase with chemically bonded octadecyl ligands of coverage density 0.5μmol/m(2) from Merck, Darmstadt). We show, that at low or high concentration of water in the mobile phase comprised methanol and some additives the chromatographic systems with the plates mentioned demonstrate normal- or reversed-phase liquid chromatography properties, respectively, for separation of peptides obtained. These two systems show quite different separation selectivity and their combination into 2D HPTLC process provides excellent separation of peptides of the bovine albumin digest. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Phased reversibility under the current French disposal concept

    International Nuclear Information System (INIS)

    Hoorelbeke, J.-M.

    2000-01-01

    The French law of 30 December 1991 and the implementing decrees provide for taking into account the reversibility in the study of geological disposal. This takes place within the framework of a 15 year research program. The research in this field implies both the assessment of technological possibilities for retrieving waste packages safely from the repository and the assessment of the consequence of delaying the closure of the repositories on the long term safety. This research program aims at proposing to the decision makers, by the year 2006, an open range of relevant options with regards to reversibility. (author)

  11. Pig-MAP, porcine acute phase proteins and standardisation of assays in Europe

    DEFF Research Database (Denmark)

    Alava, M.A.; Gonzalez-Ramon, N.; Heegaard, Peter M. H.

    1997-01-01

    The pattern of plasma proteins changes greatly following infection, inflammation or tissue injury. The concentration of some proteins referred to as acute phase proteins (APPs) significantly increases within hours or days after the onset of these processes. In contrast, the concentration of other...... during the inflammation. In addition to CRP and Hp, a serum alpha(2)-globulin was observed to be the major acute phase (MAP) protein in pigs. Pig-MAP is a new mammalian plasma protein, which is the pig counterpart of a recently cloned human serum protein denominated PK-120 or MRP. Pig-MAP shows promise...... for the presence of infectious, inflammatory and pathological lesions in animals. The ability to monitor the APP concentration in serum of pigs will improve the quality and safety of the meat produced as well as provide important diagnostic information for animal health and welfare. The serum concentration of APP...

  12. Multiple scales and phases in discrete chains with application to folded proteins

    Science.gov (United States)

    Sinelnikova, A.; Niemi, A. J.; Nilsson, Johan; Ulybyshev, M.

    2018-05-01

    Chiral heteropolymers such as large globular proteins can simultaneously support multiple length scales. The interplay between the different scales brings about conformational diversity, determines the phase properties of the polymer chain, and governs the structure of the energy landscape. Most importantly, multiple scales produce complex dynamics that enable proteins to sustain live matter. However, at the moment there is incomplete understanding of how to identify and distinguish the various scales that determine the structure and dynamics of a complex protein. Here we address this impending problem. We develop a methodology with the potential to systematically identify different length scales, in the general case of a linear polymer chain. For this we introduce and analyze the properties of an order parameter that can both reveal the presence of different length scales and can also probe the phase structure. We first develop our concepts in the case of chiral homopolymers. We introduce a variant of Kadanoff's block-spin transformation to coarse grain piecewise linear chains, such as the C α backbone of a protein. We derive analytically, and then verify numerically, a number of properties that the order parameter can display, in the case of a chiral polymer chain. In particular, we propose that in the case of a chiral heteropolymer the order parameter can reveal traits of several different phases, contingent on the length scale at which it is scrutinized. We confirm that this is the case with crystallographic protein structures in the Protein Data Bank. Thus our results suggest relations between the scales, the phases, and the complexity of folding pathways.

  13. Characterisation of UV-cured acrylate networks by means of hydrolysis followed by aqueous size-exclusion combined with reversed-phase chromatography

    NARCIS (Netherlands)

    Peters, R.; Litvinov, V. M.; Steeman, P.; Dias, A. A.; Mengerink, Y.; van Benthem, R.; de Koster, C. G.; van der Wal, S. J.; Schoenmakers, P.

    2007-01-01

    UV-cured networks prepared from mixtures of di-functional (polyethylene-glycol di-acrylate) and mono-functional (2-ethylhexyl acrylate) acrylates were analysed after hydrolysis, by aqueous size-exclusion chromatography coupled to on-line reversed-phase liquid-chromatography. The mean network density

  14. Reverse Zymography: Overview and Pitfalls.

    Science.gov (United States)

    Sharma, Kanika; Bhattacharyya, Debasish

    2017-01-01

    Reverse zymography is a technique by which protease inhibitor(s) in a sample could be electrophoretically separated in a substrate-impregnated acrylamide gel and their relative abundance could be semi-quantified. The gel after electrophoresis is incubated with a protease when the impregnated substrate and all other proteins of the sample are degraded into small peptides except the inhibitor(s) that show clear bands against a white background. Since reverse zymography cannot distinguish between a protease inhibitor and a protein that is resistant against proteolysis, the results should be confirmed from inhibition of protease activity by solution state assay.

  15. Facile preparation of an alternating copolymer-based high molecular shape-selective organic phase for reversed-phase liquid chromatography.

    Science.gov (United States)

    Mallik, Abul K; Noguchi, Hiroki; Rahman, Mohammed Mizanur; Takafuji, Makoto; Ihara, Hirotaka

    2018-06-22

    The synthesis of a new alternating copolymer-grafted silica phase is described for the separation of shape-constrained isomers of polycyclic aromatic hydrocarbons (PAHs) and tocopherols in reversed-phase high-performance liquid chromatography (RP-HPLC). Telomerization of the monomers (octadecyl acrylate and N-methylmaleimide) was carried out with a silane coupling agent; 3-mercaptopropyltrimethoxysilane (MPS), and the telomer (T) was grafted onto porous silica surface to prepare the alternating copolymer-grafted silica phase (Sil-alt-T). The new hybrid material was characterized by elemental analyses, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy, and solid-state 13 C and 29 Si cross-polarization magic-angle spinning (CP/MAS) NMR spectroscopy. The results of 13 C CP/MAS NMR demonstrated that the alkyl chains of the grafted polymers in Sil-alt-T remained disordered, amorphous, and mobile represented by gauche conformational form. Separation abilities and molecular-shape selectivities of the prepared organic phase were evaluated by the separation of PAHs isomers and Standard Reference Material 869b, Column Selectivity Test Mixture for Liquid Chromatography, respectively and compared with commercially available octadecylsilylated silica (ODS) and C 30 columns as well as previously reported alternating copolymer-based column. The effectiveness of this phase is also demonstrated by the separation of tocopherol isomers. Oriented functional groups along the polymer main chains and cavity formations are investigated to be the driving force for better separation with multiple-interactions with the solutes. One of the advantages of the Sil-alt-T phase to that of the previously reported phase is the synthesis of the telomer first and then immobilized onto silica surface. In this case, the telomer was characterized easily with simple spectroscopic techniques and the molecular mass and polydispersity index of the telomer were determined by size exclusion

  16. Purification of lignans from Fructus Arctii using off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography.

    Science.gov (United States)

    Yang, Bichao; Xin, Huaxia; Wang, Feier; Cai, Jianfeng; Liu, Yanfang; Fu, Qing; Jin, Yu; Liang, Xinmiao

    2017-08-01

    As a common traditional Chinese medicine, Fructus Arctii has important clinical medical values. Its main components are lignans, which are difficult to separate and analyze because of the complex composition, similar chemical structures, and close properties. In this study, an off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography method, as well as an effective sample pretreatment method based on hydrophilic interaction chromatography material, was developed to enrich the minor lignan fractions and obtain high-purity compounds. In total, 12 high-purity compounds were isolated from Fructus Arctii. Their structures were identified by using high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, which showed that all were lignans and that most of them were isomers. The results demonstrated the effective off-line two-dimensional supercritical fluid chromatography/reversed-phase liquid chromatography method for the purification of lignans from Fructus Arctii. The separation protocol established here will be beneficial for the separation of complex samples from other kinds of natural products. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves

    OpenAIRE

    Zeb, Alam; Ullah, Fareed

    2017-01-01

    Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight ca...

  18. Performance comparison of three-phase flux reversal permanent magnet motors in BLDC and BLAC operation mode

    International Nuclear Information System (INIS)

    Stumberger, B.; Stumberger, G.; Hadziselimovic, M.; Hamler, A.; Gorican, V.; Jesenik, M.; Trlep, M.

    2008-01-01

    The paper presents a comparison of torque capability and flux-weakening performance of three-phase flux reversal permanent magnet motors with surface and inset permanent magnets. Finite element analysis is employed to determine the performance of each motor in BLDC and BLAC operation mode. It is shown that the torque capability and flux-weakening performance of surface or inset permanent magnet configuration is strongly dependent on the stator teeth number/rotor pole number combination

  19. Multiple reversal olfactory learning in honeybees

    Directory of Open Access Journals (Sweden)

    Theo Mota

    2010-07-01

    Full Text Available In multiple reversal learning, animals trained to discriminate a reinforced from a non-reinforced stimulus are subjected to various, successive reversals of stimulus contingencies (e.g. A+ vs. B-, A- vs. B+, A+ vs. B-. This protocol is useful to determine whether or not animals learn to learn and solve successive discriminations faster (or with fewer errors with increasing reversal experience. Here we used the olfactory conditioning of proboscis extension reflex to study how honeybees Apis mellifera perform in a multiple reversal task. Our experiment contemplated four consecutive differential conditioning phases involving the same odors (A+ vs. B- to A- vs. B+ to A+ vs. B- to A- vs. B+. We show that bees in which the weight of reinforced or non-reinforced stimuli was similar mastered the multiple olfactory reversals. Bees which failed the task exhibited asymmetric responses to reinforced and non-reinforced stimuli, thus being unable to rapidly reverse stimulus contingencies. Efficient reversers did not improve their successive discriminations but rather tended to generalize their choice to both odors at the end of conditioning. As a consequence, both discrimination and reversal efficiency decreasedalong experimental phases. This result invalidates a learning-to-learn effect and indicates that bees do not only respond to the actual stimulus contingencies but rather combine these with an average of past experiences with the same stimuli.  

  20. Quantitative structure-retention relationships of pesticides in reversed-phase high-performance liquid chromatography

    International Nuclear Information System (INIS)

    Aschi, Massimiliano; D'Archivio, Angelo Antonio; Maggi, Maria Anna; Mazzeo, Pietro; Ruggieri, Fabrizio

    2007-01-01

    In this paper, a quantitative structure-retention relationships (QSRR) method is employed to predict the retention behaviour of pesticides in reversed-phase high-performance liquid chromatography (HPLC). A six-parameter nonlinear model is developed by means of a feed-forward artificial neural network (ANN) with back-propagation learning rule. Accurate description of the retention factors of 26 compounds including commonly used insecticides, herbicides and fungicides and some metabolites is successfully achieved. In addition to the acetonitrile content, included to describe composition of the water-acetonitrile mobile phase, the octanol-water partition coefficient (from literature) and four quantum chemical descriptors are considered to account for the effect of solute structure on the retention. These are: the total dipole moment, the mean polarizability, the anisotropy of polarizability and a descriptor of hydrogen bonding ability based on the atomic charges on hydrogen bond donor and acceptor chemical functionalities. The proposed nonlinear QSRR model exhibits a high degree of correlation between observed and computed retention factors and a good predictive performance in wide range of mobile phase composition (40-65%, v/v acetonitrile) that supports its application for the prediction of the chromatographic behaviour of unknown pesticides. A multilinear regression model based on the same six descriptors shows a significantly worse predictive capability

  1. Quantitative structure-retention relationships of pesticides in reversed-phase high-performance liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Aschi, Massimiliano [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy); D' Archivio, Angelo Antonio [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy)]. E-mail: darchivi@univaq.it; Maggi, Maria Anna [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy); Mazzeo, Pietro [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy); Ruggieri, Fabrizio [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy)

    2007-01-23

    In this paper, a quantitative structure-retention relationships (QSRR) method is employed to predict the retention behaviour of pesticides in reversed-phase high-performance liquid chromatography (HPLC). A six-parameter nonlinear model is developed by means of a feed-forward artificial neural network (ANN) with back-propagation learning rule. Accurate description of the retention factors of 26 compounds including commonly used insecticides, herbicides and fungicides and some metabolites is successfully achieved. In addition to the acetonitrile content, included to describe composition of the water-acetonitrile mobile phase, the octanol-water partition coefficient (from literature) and four quantum chemical descriptors are considered to account for the effect of solute structure on the retention. These are: the total dipole moment, the mean polarizability, the anisotropy of polarizability and a descriptor of hydrogen bonding ability based on the atomic charges on hydrogen bond donor and acceptor chemical functionalities. The proposed nonlinear QSRR model exhibits a high degree of correlation between observed and computed retention factors and a good predictive performance in wide range of mobile phase composition (40-65%, v/v acetonitrile) that supports its application for the prediction of the chromatographic behaviour of unknown pesticides. A multilinear regression model based on the same six descriptors shows a significantly worse predictive capability.

  2. Strategic study on energy-protein requirements for local sheep: 5. Ewes during lactation phase

    Directory of Open Access Journals (Sweden)

    I-W Mathius

    2004-03-01

    Full Text Available Thirty-six Javanese thin-tail ewes in the end of late pregnancy phase were set out to study the energy and crude protein requirements during the first eight-week of lactation phase. The ewes were penned individually in doors and randomly assigned to a 3 x 3 factorial arrangement, consisting of three levels of energy (low, medium and high and three levels of crude protein (low, medium and high diets with four ewes per treatment. The diets were pelleted and offered four times daily in approximately equal amount. Feed intake, nutrient digestibility, body weight and milk production were recorded. Results showed that, total lamb birth weights was not affected, but protein content on the ration treatments significantly altered (P0.05, while crude protein content on the ration highly significantly affected (P<0.01. Based on data recorded, the energy and protein requirements for ewes during lactation phase are highly significantly depended on ewes’ live weight, milk production and the ratio of energy metabolism and crude protein of the ration. It was concluded that in order to fulfil the crude protein and energy needs of the ewes during lactation phase, the ration given should contain crude protein and energy as much as 16% (based on dry matter and 13.4 MJ/kg dry matter respectively.

  3. Mobile phase effects on the retention on polar columns with special attention to the dual hydrophilic interaction-reversed-phase liquid chromatography mechanism, a review.

    Science.gov (United States)

    Jandera, Pavel; Hájek, Tomáš

    2018-01-01

    Hydrophilic interaction liquid chromatography on polar columns in aqueous-organic mobile phases has become increasingly popular for the separation of many biologically important compounds in chemical, environmental, food, toxicological, and other samples. In spite of many new applications appearing in literature, the retention mechanism is still controversial. This review addresses recent progress in understanding of the retention models in hydrophilic interaction liquid chromatography. The main attention is focused on the role of water, both adsorbed by the column and contained in the bulk mobile phase. Further, the theoretical retention models in the isocratic and gradient elution modes are discussed. The dual hydrophilic interaction liquid chromatography reversed-phase retention mechanism on polar columns is treated in detail, especially with respect to the practical use in one- and two-dimensional liquid chromatography separations. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Elution behavior of N-heterocyclic derivatives of mixed ruthenium(II)-sulfoxide complexes in reversed-phase high-performance liquid chromatography

    International Nuclear Information System (INIS)

    Toma, H.E.; Silva, D.O.

    1991-01-01

    The elution behaviour of a series of dichlorobis-(dimethylsulfoxide)bis(N-heterocyclic) ruthenium(II) complexes has been investigated using reversed-phase HPLC. Similar trends as those displayed by the free N-heterocyclic bases have been observed for the complexes, essentially reflecting the hydrophobic properties of the aromatic ligands. (orig.)

  5. Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

    Science.gov (United States)

    Periat, Aurélie; Kohler, Isabelle; Thomas, Aurélien; Nicoli, Raul; Boccard, Julien; Veuthey, Jean-Luc; Schappler, Julie; Guillarme, Davy

    2016-03-25

    Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples

  6. Separation of deuteriated isotopomers of dopamine by ion-pair reversed-phase high-performance liquid chromatography

    International Nuclear Information System (INIS)

    Masters, C.F.; Markey, S.P.; Mefford, I.N.; Duncan, M.W.

    1988-01-01

    The ion-pair reversed-phase separation of dopamine and deuterium-substituted dopamine isotopomers is described. Chromatographic parameters and deuterium isotope effects governing the resolution are examined and compared to the factors regulating the resolution are examined and compared to the factors regulating the resolution of the chemically distinct entities dopamine, norepinephrine, and epinephrine. The potential utility of the [ 2 H 7 ]dopamine, isotopomer as an internal standard for the high-performance liquid chromatography analysis of dopamine is demonstrated by using aluminum oxide extraction prior to chromatographic separation

  7. Plasma Membrane ATPase Activity following Reversible and Irreversible Freezing Injury 1

    Science.gov (United States)

    Iswari, S.; Palta, Jiwan P.

    1989-01-01

    Plasma membrane ATPase has been proposed as a site of functional alteration during early stages of freezing injury. To test this, plasma membrane was purified from Solanum leaflets by a single step partitioning of microsomes in a dextran-polyethylene glycol two phase system. Addition of lysolecithin in the ATPase assay produced up to 10-fold increase in ATPase activity. ATPase activity was specific for ATP with a Km around 0.4 millimolar. Presence of the ATPase enzyme was identified by immunoblotting with oat ATPase antibodies. Using the phase partitioning method, plasma membrane was isolated from Solanum commersonii leaflets which had four different degrees of freezing damage, namely, slight (reversible), partial (partially reversible), substantial and total (irreversible). With slight (reversible) damage the plasma membrane ATPase specific activity increased 1.5- to 2-fold and its Km was decreased by about 3-fold, whereas the specific activity of cytochrome c reductase and cytochrome c oxidase in the microsomes were not different from the control. However, with substantial (lethal, irreversible) damage, there was a loss of membrane protein, decrease in plasma membrane ATPase specific activity and decrease in Km, while cytochrome c oxidase and cytochrome c reductase were unaffected. These results support the hypothesis that plasma membrane ATPase is altered by slight freeze-thaw stress. Images Figure 1 Figure 2 PMID:16666856

  8. Evaluation of chromatographic conditions in reversed phase liquid chromatography-mass spectrometry systems for fingerprinting of polar and amphiphilic plant metabolites

    DEFF Research Database (Denmark)

    Nielsen, Nikoline Juul; Tomasi, Giorgio; Christensen, Jan H.

    2016-01-01

    Metabolic fingerprinting is a relatively young scientific discipline requiring robust, yet flexible and fit-for-purpose analytical methods. Here, we introduce a simple approach to select reversed phase LC systems with electrospray MS detection for fingerprinting of polar and amphiphilic plant met...

  9. An antiviral protein from Bougainvillea spectabilis roots; purification and characterisation.

    Science.gov (United States)

    Balasaraswathi, R; Sadasivam, S; Ward, M; Walker, J M

    1998-04-01

    An antiviral protein active against mechanical transmission of tomato spotted wilt virus was identified in the root tissues of Bougainvillea spectabilis Willd. Bougainvillea Antiviral Protein I (BAP I) was purified to apparent homogeneity from the roots of Bougainvillea by ammonium sulphate precipitation, CM- and DEAE-Sepharose chromatography and reverse phase HPLC. BAP I is a highly basic protein (pI value > 8.6) with an Mr of 28,000. The N-terminal sequence of BAP I showed homology with other plant antiviral proteins. Preliminary tests suggest that purified BAP I is capable of interfering with in vitro protein synthesis.

  10. High transmittance contrast in amorphous to hexagonal phase of Ge2Sb2Te5: Reversible NIR-window

    Science.gov (United States)

    Singh, Palwinder; Singh, A. P.; Kanda, Neetu; Mishra, Monu; Gupta, Govind; Thakur, Anup

    2017-12-01

    Ge2Sb2Te5 (GST) is one of the best phase change materials because of its splendid set of properties, viz., high thermal stability, fast crystallization speed, good endurance, scalability, and reliability. Phase transition [amorphous → face centered cubic (fcc) → hexagonal close packed (hcp)] of GST thin films with annealing was studied using X-ray diffraction. Thin films in amorphous, fcc, and hcp phases are highly, medium, and negligible transparent in the near infra-red region, respectively. The optical transmission in amorphous, fcc, and hcp phases is ˜92%, ˜46%, and ˜2%, respectively, at the wavelength of 2740 nm. At 2740 nm, a high transmission contrast (˜90%) is observed with phase transition from the amorphous to hcp phase. By utilizing large transmission contrast, it is demonstrated that GST can be availed as a potential candidate for reversible near infra-red-window. The sharp change in optical transmission with phase transition can be understood from the change in density of states in the valence band.

  11. Probing Protein Structure and Folding in the Gas Phase by Electron Capture Dissociation

    Science.gov (United States)

    Schennach, Moritz; Breuker, Kathrin

    2015-07-01

    The established methods for the study of atom-detailed protein structure in the condensed phases, X-ray crystallography and nuclear magnetic resonance spectroscopy, have recently been complemented by new techniques by which nearly or fully desolvated protein structures are probed in gas-phase experiments. Electron capture dissociation (ECD) is unique among these as it provides residue-specific, although indirect, structural information. In this Critical Insight article, we discuss the development of ECD for the structural probing of gaseous protein ions, its potential, and limitations.

  12. BRIEF COMMUNICATIONS: Compensation of phase distortions in a single-transit wavefront-reversal system with a degenerate four-photon interaction

    Science.gov (United States)

    Barashkov, M. S.; Matveev, I. N.; Petnikova, V. M.; Umnov, A. F.; Ustinov, N. D.; Shuvalov, Vladimir V.

    1982-11-01

    A proposal is made for a system designed to compensate phase distortions in a wavefront-reversal system in which some part of the signal radiation is deflected to pass through a spatial frequency filter and then acts as the pump; the remainder of such radiation acts as the signal. It is shown that a suitable selection of the pass band of the filter can ensure compensation of large-scale phase inhomogeneities in the system and can enable reconstruction of a small-scale structure of an object.

  13. Two-step preparation of nano-scaled magnetic chitosan particles using Triton X-100 reversed-phase water-in-oil microemulsion system

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Zhengkun; Jiang, Feihong [College of Food Science and Engineering, Northwest A and F University, Yangling, Shaanxi 712100 (China); Lee, Tung-Ching, E-mail: lee@aesop.rutgers.edu [Department of Food Science, Rutgers, the State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901 (United States); Yue, Tianli, E-mail: yuetl305@nwsuaf.edu.cn [College of Food Science and Engineering, Northwest A and F University, Yangling, Shaanxi 712100 (China)

    2013-12-25

    Highlights: •A new two-step route for nano-scaled magnetic chitosan particles preparation. •Triton X-100 reversed-phase microemulsion system was used for chitosan coating. •Narrow size distribution of magnetic chitosan nanoparticles was achieved. •Quantitative evaluation of recoverability for the magnetic chitosan nanoparticles. -- Abstract: A new two-step route for the preparation of nano-scaled magnetic chitosan particles has been developed, different from reported one-step in situ preparation and two-step preparation method of reversed-phase suspension, Triton X-100 reversed-phase water-in-oil microemulsion encapsulation method was employed in coating the pre-prepared Fe{sub 3}O{sub 4} nanoparticles with chitosan. The resultant magnetic chitosan particles owned a narrow size distribution ranging from 50 to 92 nm. X-ray diffraction patterns (XRD) indicated that the chitosan coating procedure did not change the spinal structure of Fe{sub 3}O{sub 4} magnetic nanoparticles. The results of Fourier transform infrared (FTIR) analysis and thermogravimetric analysis (TGA) demonstrated that the chitosan was coated on Fe{sub 3}O{sub 4} nanoparticles and its average mass content was ∼50%. The saturated magnetization of the magnetic Fe{sub 3}O{sub 4}/chitosan nanoparticles reached 18.62 emu/g, meanwhile, the nanoparticles showed the characteristics of superparamagnetism. The magnetic chitosan nanoparticles showed a high recoverability of 99.99% in 10 min when pH exceeded 4. The results suggested that the as-prepared magnetic chitosan particles were nano-scaled with a narrow size distribution and a high recoverability.

  14. Interface Promoted Reversible Mg Insertion in Nanostructured Tin-Antimony Alloys

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Yingwen; Shao, Yuyan; Parent, Lucas R.; Sushko, Maria L.; Li, Guosheng; Sushko, Petr; Browning, Nigel D.; Wang, Chong M.; Liu, Jun

    2015-11-11

    This paper demonstrates intermetallic compounds SnSb are highly active materials for reversibly hosting Mg ions. Compared with monometallic Sn and Sb, SnSb alloy exhibited exceptionally high reversible capacity (420 mAh/g), excellent rate capability and good cyclic stability. Mg insertion into pristine SnSb involves an activation process to complete, which induces particle breakdown and results in phase segregation to Sn-rich and Sb-rich phases. Both experimental analysis and DFT simulation suggest that the Sn-rich phase is particularly active and provides most of the capacity whereas the Sb-rich phase is not as active, and the interface between these two phases play a key role in promoting the formation and stabilization of the cubic Sn phase that is more favorable for fast and reversible Mg insertion. We further show that activated SnSb alloy has good compatibility with simple Mg electrolytes. Overall, this work could provide new approaches for designing materials capable of reversible Mg ion insertion and new opportunities for understanding Mg electrochemistry.

  15. Phase contrast imaging measurements of reversed shear Alfvén eigenmodes during sawteeth in Alcator C-Moda)

    Science.gov (United States)

    Edlund, E. M.; Porkolab, M.; Kramer, G. J.; Lin, L.; Lin, Y.; Wukitch, S. J.

    2009-05-01

    Reversed shear Alfvén eigenmodes (RSAEs) have been observed with the phase contrast imaging diagnostic and Mirnov coils during the sawtooth cycle in Alcator C-mod [M. Greenwald et al., Nucl. Fusion 45, S109 (2005)] plasmas with minority ion-cyclotron resonance heating. Both down-chirping RSAEs and up-chirping RSAEs have been observed during the sawtooth cycle. Experimental measurements of the spatial structure of the RSAEs are compared to theoretical models based on the code NOVA [C. Z. Cheng and M. S. Chance, J. Comput. Phys. 71, 124 (1987)] and used to derive constraints on the q profile. It is shown that the observed RSAEs can be understood by assuming a reversed shear q profile (up chirping) or a q profile with a local maximum (down chirping) with q ≈1.

  16. Comprehensive two-dimensional liquid chromatography: Ion chromatography × reversed-phase liquid chromatography for separation of low-molar-mass organic acids

    NARCIS (Netherlands)

    Brudin, S.S.; Shellie, R.A.; Haddad, P.R.; Schoenmakers, P.J.

    2010-01-01

    In the work presented here a novel approach to comprehensive two-dimensional liquid chromatography is evaluated. Ion chromatography is chosen for the first-dimension separation and reversed-phase liquid chromatography is chosen for the second-dimension separation mode. The coupling of these modes is

  17. Characterization of wide-pore reversed-phase columns for biopolymer separations. I. Single-parametric evaluation

    NARCIS (Netherlands)

    Bede, A; Rippel, G; Szepesy, L; Claessens, H.A.

    1996-01-01

    Nine prepacked wide-pore columns recommended for protein separation were studied. The stationary phases investigated were widely different with respect to the type of ligands. In order to characterize the columns, measurements were made with six different test mixtures containing two different

  18. Late Protein Synthesis-Dependent Phases in CTA Long-Term Memory: BDNF Requirement

    OpenAIRE

    Martínez-Moreno, Araceli; Rodríguez-Durán, Luis F.; Escobar, Martha L.

    2011-01-01

    It has been proposed that long-term memory persistence requires a late protein synthesis-dependent phase, even many hours after memory acquisition. Brain-derived neurotrophic factor (BDNF) is an essential protein synthesis product that has emerged as one of the most potent molecular mediators for long-term synaptic plasticity. Studies in the rat hippocampus have been shown that BDNF is capable to rescue the late-phase of long-term potentiation as well as the hippocampus-related long-term memo...

  19. Relationship between Acute Phase Proteins and Serum Fatty Acid Composition in Morbidly Obese Patients

    Science.gov (United States)

    Fernandes, Ricardo; Beserra, Bruna Teles Soares; Cunha, Raphael Salles Granato; Hillesheim, Elaine; Camargo, Carolina de Quadros; Pequito, Danielle Cristina Tonello; de Castro, Isabela Coelho; Fernandes, Luiz Cláudio; Nunes, Everson Araújo; Trindade, Erasmo Benício Santos de Moraes

    2013-01-01

    Background. Obesity is considered a low-grade inflammatory state and has been associated with increased acute phase proteins as well as changes in serum fatty acids. Few studies have assessed associations between acute phase proteins and serum fatty acids in morbidly obese patients. Objective. To investigate the relationship between acute phase proteins (C-Reactive Protein, Orosomucoid, and Albumin) and serum fatty acids in morbidly obese patients. Methods. Twenty-two morbidly obese patients were enrolled in this study. Biochemical and clinical data were obtained before bariatric surgery, and fatty acids measured in preoperative serum. Results. Orosomucoid was negatively correlated with lauric acid (P = 0.027) and eicosapentaenoic acid (EPA) (P = 0.037) and positively with arachidonic acid (AA) (P = 0.035), AA/EPA ratio (P = 0.005), and n-6/n-3 polyunsaturated fatty acids ratio (P = 0.035). C-Reactive Protein (CRP) was negatively correlated with lauric acid (P = 0.048), and both CRP and CRP/Albumin ratio were negatively correlated with margaric acid (P = 0.010, P = 0.008, resp.). Albumin was positively correlated with EPA (P = 0.027) and margaric acid (P = 0.008). Other correlations were not statistically significant. Conclusion. Our findings suggest that serum fatty acids are linked to acute phase proteins in morbidly obese patients. PMID:24167354

  20. A Novel Drug Delivery Vesicle Development to Reverse Neurodegeneration: Analysis of the Interactions among Protein, Graphene Oxide and Liposome

    Science.gov (United States)

    Miraz, Md Alamin

    In this study, Liposome was decorated with graphene oxide (GO) to synthesize fully-biocompatible theranostic vesicle that can carry bovine serum albumin (BSA) as a model protein. Graphene oxide has been studied as one of the most promising platforms for promoting the growth and repair of neurons. Our graphene oxide based structure could account for the high efficiency of protein loading and deliver to the damaged neuron cell which can reverse the neurodegeneration associated with Alzheimer's disease. The resultant vesicle exhibited high stability in aqueous solution. We investigated the protein adsorption capacity and protein interaction to carbon-based nanomaterials. The Liposome, graphene oxide and bovine serum albumin (BSA) are all biocompatible and hence will not trigger an immune response in vivo.

  1. Simulation of Far-Field Superresolution Fluorescence Imaging with Two-Color One-Photon Excitation of Reversible Photoactivatable Protein

    International Nuclear Information System (INIS)

    Wang Chen; Qiao Ling-Ling; Mao Zheng-Le

    2011-01-01

    We propose to achieve far-field super-resolution imaging by using offset two-color one-photon (2C1P) excitation of reversible photoactivatable fluorescence proteins. Due to the distinctive photoswitching performance of the proteins, such as dronpa, the fluorescence emission will only come from the overlapped region of activation beam and excitation beam. The analysis solution of rate equation shows that the resolution of offset 2C1P microscope is 'engineered' by laser power of excitation and activation beams and the power ratio between them. Superior lateral and transverse resolution is theoretically demonstrated compared with conventional fluorescence scanning microscopy. (fundamental areas of phenomenology(including applications))

  2. Chemical reactions in reverse micelle systems

    Science.gov (United States)

    Matson, Dean W.; Fulton, John L.; Smith, Richard D.; Consani, Keith A.

    1993-08-24

    This invention is directed to conducting chemical reactions in reverse micelle or microemulsion systems comprising a substantially discontinuous phase including a polar fluid, typically an aqueous fluid, and a microemulsion promoter, typically a surfactant, for facilitating the formation of reverse micelles in the system. The system further includes a substantially continuous phase including a non-polar or low-polarity fluid material which is a gas under standard temperature and pressure and has a critical density, and which is generally a water-insoluble fluid in a near critical or supercritical state. Thus, the microemulsion system is maintained at a pressure and temperature such that the density of the non-polar or low-polarity fluid exceeds the critical density thereof. The method of carrying out chemical reactions generally comprises forming a first reverse micelle system including an aqueous fluid including reverse micelles in a water-insoluble fluid in the supercritical state. Then, a first reactant is introduced into the first reverse micelle system, and a chemical reaction is carried out with the first reactant to form a reaction product. In general, the first reactant can be incorporated into, and the product formed in, the reverse micelles. A second reactant can also be incorporated in the first reverse micelle system which is capable of reacting with the first reactant to form a product.

  3. Reversed-Phase UHPLC Enantiomeric Separation of Rasagiline Salts Using a Chiralpak® AGP Column

    Directory of Open Access Journals (Sweden)

    Nagarajan Balaji

    2017-07-01

    Full Text Available We report the first rapid ultra-high performance liquid chromatographic (UHPLC enantiomeric reversed-phase separation of rasagiline mesylate and its tartrate salts using a Chiralpak® AGP column (50 mm × 2.1 mm, 5 μm as a stationary phase. This method was developed as an alternative to the usage of previously reported normal-phase chiral LC columns for isomer separation. Our method is based on an isocratic approach using a mixture of ammonium acetate and isopropyl alcohol (90:10, v/v as the mobile phase (0.6 mL/min flow rate. The detection limit (at a detection wavelength of 210 nm and quantification limit for the rasagiline enantiomers were 0.06 and 0.2 μg/mL, respectively. This method is compatible with the UHPLC-MS technique. The successful separation of rasagiline and its enantiomer was confirmed by determining the corresponding specific optical rotation values. Our method will be applicable for detecting rasagiline enantiomers during the control of manufacturing processes, and for use in rapid analysis for quality control in pharmaceutical industry to obtain optically pure pharmaceutical substances. This method was validated in terms of its precision, limit of detection, limit of quantification, linearity, accuracy, robustness, ruggedness, specificity, forced degradation, and solution stability, according to International Council on Harmonization Validation Guidelines Q2 (R1.

  4. Experience-dependent phase-reversal of hippocampal neuron firing during REM sleep.

    Science.gov (United States)

    Poe, G R; Nitz, D A; McNaughton, B L; Barnes, C A

    2000-02-07

    The idea that sleep could serve a cognitive function has remained popular since Freud stated that dreams were "not nonsense" but a time to sort out experiences [S. Freud, Letter to Wilhelm Fliess, May 1897, in The Origins of Psychoanalysis - Personal Letters of Sigmund Freud, M. Bonaparte, A. Freud, E. Kris (Eds.), Translated by E. Mosbacher, J. Strachey, Basic Books and Imago Publishing, 1954]. Rapid eye movement (REM) sleep, which is associated with dream reports, is now known to be is important for acquisition of some tasks [A. Karni, D. Tanne, B.S. Rubenstein, J.J.M. Askenasy, D. Sagi, Dependence on REM sleep of overnight improvement of a perceptual skill, Science 265 (1994) 679-682; C. Smith, Sleep states and learning: a review of the animal literature, Biobehav. Rev. 9 (1985) 157-168]; although why this is so remains obscure. It has been proposed that memories may be consolidated during REM sleep or that forgetting of unnecessary material occurs in this state [F. Crick, G. Mitchison, The function of dream sleep, Nature 304 (1983) 111-114; D. Marr, Simple memory: a theory for archicortex, Philos. Trans. R. Soc. B. 262 (1971) 23-81]. We studied the firing of multiple single neurons in the hippocampus, a structure that is important for episodic memory, during familiar and novel experiences and in subsequent REM sleep. Cells active in familiar places during waking exhibited a reversal of firing phase relative to local theta oscillations in REM sleep. Because firing-phase can influence whether synapses are strengthened or weakened [C. Holscher, R. Anwyl, M.J. Rowan, Stimulation on the positive phase of hippocampal theta rhythm induces long-term potentiation that can be depotentiated by stimulation on the negative phase in area CA1 in vivo, J. Neurosci. 15 (1977) 6470-6477; P.T. Huerta, J.E. Lisman, Bidirectional synaptic plasticity induced by a single burst during cholinergic theta oscillation in CA1 in vitro, Neuron 15 (1995) 1053-1063; C. Pavlides, Y

  5. Spectroscopic Analysis of 10MAG/LDAO Reverse Micelles to Determine Characteristic Properties and Behavioral Extrema

    Science.gov (United States)

    Berg, Joshua; Mawson, Cara; Norris, Zach; Nucci, Nathaniel

    Reverse micelles are spontaneously organizing complexes of surfactant that encapsulate a nanoscale pool of water in a bulk non-polar solvent. Reverse micelle (RM) mixtures have a wide range of applications, including biophysical investigation of protein systems. A new RM mixture composed of decyl-1-monoglycerol (10MAG) and lauryldimethylammonium-N-oxide (LDAO) was recently described. This mixture has the potential to prove more widely applicable for use of RMs in applications that involve encapsulation of macromolecules, yet little is known about the phase behavior or size of reverse micelles created by this mixture. Data describing such behaviors for this mixture are presented here. We have used dynamic light scattering (DLS) and fluorescence spectroscopy to investigate the size and partitioning behavior of RMs in varying mixtures of 10MAG, LDAO, water, pentane, and hexanol. These data demonstrate that the 10MAG/LDAO RM mixture exhibits markedly different phase and RM size behavior than that of commonly used RM surfactant mixtures. The implications of these findings for use of the 10MAG/LDAO mix for RM applications will also be addressed. Funding provided by Rowan University.

  6. Unique Features of Halophilic Proteins.

    Science.gov (United States)

    Arakawa, Tsutomu; Yamaguchi, Rui; Tokunaga, Hiroko; Tokunaga, Masao

    2017-01-01

    Proteins from moderate and extreme halophiles have unique characteristics. They are highly acidic and hydrophilic, similar to intrinsically disordered proteins. These characteristics make the halophilic proteins soluble in water and fold reversibly. In addition to reversible folding, the rate of refolding of halophilic proteins from denatured structure is generally slow, often taking several days, for example, for extremely halophilic proteins. This slow folding rate makes the halophilic proteins a novel model system for folding mechanism analysis. High solubility and reversible folding also make the halophilic proteins excellent fusion partners for soluble expression of recombinant proteins.

  7. Simultaneous determination of 18α-glycyrrhetinic acid and 18β-glycyrrhetinic acid in Glycyrrhiza glabra root by reversed phase high-performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Ambika Chamoli

    2016-01-01

    Full Text Available Background: The aim of the present research work is to develop a high-performance liquid chromatography (HPLC method for simultaneous analysis of 18α-glycyrrhetinic acid (18α-GA and 18β-GA (18β-GA of Glycyrrhiza glabra. Materials and Methods: About 20 μL aliquots of each 18α-GA and 18β-GA were analyzed using reversed-phase C-18 column. The mobile phase was acetonitrile:tetrahydrofuran:water (10:80:10, v/v/v. The run time was 10 min at flow rate of 1 ml/min. Ultraviolet detection was carried out at 254 nm. Results: 18α-GA and 18β-GA were well resolved in reversed phase C-18 column using mobile phase acetonitrile: tetrahydrofuran: water (10:80:10, v/v/v, pH 7.9. The Rtof 18α-GA and 18β-GA was detected at 2.091 and 2.377 min, respectively. Conclusion: The developed chromatography method could be extended for potential quantification or simultaneous determination of these markers in plant as well as in herbal formulation.

  8. Extensively Reversible Thermal Transformations of a Bistable, Fluorescence-Switchable Molecular Solid: Entry into Functional Molecular Phase-Change Materials.

    Science.gov (United States)

    Srujana, P; Radhakrishnan, T P

    2015-06-15

    Functional phase-change materials (PCMs) are conspicuously absent among molecular materials in which the various attributes of inorganic solids have been realized. While organic PCMs are primarily limited to thermal storage systems, the amorphous-crystalline transformation of materials like Ge-Sb-Te find use in advanced applications such as information storage. Reversible amorphous-crystalline transformations in molecular solids require a subtle balance between robust supramolecular assembly and flexible structural elements. We report novel diaminodicyanoquinodimethanes that achieve this transformation by interlinked helical assemblies coupled with conformationally flexible alkoxyalkyl chains. They exhibit highly reversible thermal transformations between bistable (crystalline/amorphous) forms, along with a prominent switching of the fluorescence emission energy and intensity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Determination of phenyl-N-methylcarbamates and their hydrolysis products in water, using solid-phase extraction and reversed-phase liquid chromatography with UV and electrospray mass spectrometric detection

    International Nuclear Information System (INIS)

    El Atrache, L.L.; Sabbah, S.

    2003-01-01

    In this study, eight phenyl-N-methylcarbamates (PNMCs) were considered. Reversed-phase LC was set up for UV and mass spectrometry (MS) detection mode. Gradient elution was used, and the mobile phase was composed of acetonitrile and water. UV-vis was performed at 220 nm. The method was tested with different reversed-phase columns. Comparison between chromatographic parameters: retention time (t R ), resolution (R S ), and selectivity (α) was established. Hydrolysis kinetics of three of the PNMCs were reported. The major hydrolysis products were determined by LC-UV, and the effect of pH on hydrolysis was also studied. Also, chromatographic separation of a mixture of PNMCs and four of their hydrolysis products was carried out. The preconcentration of 12 studied solutes was realized by solid-phase extraction. C18 extraction cartridges of 1 g were used to extract solutes from a 100 mL volume of tap and surface water spiked at 10 μg/L. The recoveries were, respectively, between 68-86% and 62-83% with relative a standard deviation of less than 11%. Limits of detection (LODs) and limits of quantitation (LOQs) ranged, respectively, from 1-4 μg/L and from 4-10 μg/L. Since standard UV detection does not provide adequate selectivity for water samples, an electrospray (ES)-MS instrument equipped with a triple quadrupole mass filter was used. MS data acquisition was performed by a time-scheduled, selected-ion monitoring (SIM) program. Limits of quantitation gave values between 0.1-0.5 μg/L. (author)

  10. Floral reversion mechanism in longan (Dimocarpus longan Lour.) revealed by proteomic and anatomic analyses.

    Science.gov (United States)

    You, Xiangrong; Wang, Lingxia; Liang, Wenyu; Gai, Yonghong; Wang, Xiaoyan; Chen, Wei

    2012-02-02

    Two-dimensional gel electrophoresis (2-DE) was used to analyze the proteins related to floral reversion in Dimocarpus longan Lour. Proteins were extracted from buds undergoing the normal process of flowering and from those undergoing floral reversion in three developing stages in D. longan. Differentially expressed proteins were identified from the gels after 2-DE analysis, which were confirmed using matrix-assisted laser desorption/ionization-time of flying-mass spectroscopy and protein database search. A total of 39 proteins, including 18 up-regulated and 21 down-regulated proteins, were classified into different categories, such as energy and substance metabolism, protein translation, secondary metabolism, phytohormone, cytoskeleton structure, regulation, and stress tolerance. Among these, the largest functional class was associated with primary metabolism. Down-regulated proteins were involved in photosynthesis, transcription, and translation, whereas up-regulated proteins were involved in respiration. Decreased flavonoid synthesis and up-regulated GA20ox might be involved in the floral reversion process. Up-regulated 14-3-3 proteins played a role in the regulation of floral reversion in D. longan by responding to abiotic stress. Observations via transmission electron microscopy revealed the ultrastructure changes in shedding buds undergoing floral reversion. Overall, the results provided insights into the molecular basis for the floral reversion mechanism in D. longan. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Alterations in brain Protein Kinase A activity and reversal of morphine tolerance by two fragments of native Protein Kinase A inhibitor peptide (PKI).

    Science.gov (United States)

    Dalton, George D; Smith, Forrest L; Smith, Paul A; Dewey, William L

    2005-04-01

    Two peptide fragments of native Protein Kinase A inhibitor (PKI), PKI-(6-22)-amide and PKI-(Myr-14-22)-amide, significantly reversed low-level morphine antinociceptive tolerance in mice. The inhibition of Protein Kinase A (PKA) activity by both peptide fragments was then measured in specific brain regions (thalamus, periaqueductal gray (PAG), and medulla) and in lumbar spinal cord (LSC), which in previous studies have been shown to play a role in morphine-induced analgesia. In drug naive animals, cytosolic PKA activity was greater than particulate PKA activity in each region, while cytosolic and particulate PKA activities were greater in thalamus and PAG compared to medulla and LSC. The addition of both peptides to homogenates from each region completely abolished cytosolic and particulate PKA activities in vitro. Following injection into the lateral ventricle of the brain of drug naive mice and morphine-tolerant mice, both peptides inhibited PKA activity in the cytosolic, but not the particulate fraction of LSC. In addition, cytosolic and particulate PKA activities were inhibited by both peptides in thalamus. These results demonstrate that the inhibition of PKA reverses morphine tolerance. Moreover, the inhibition of PKA activity in specific brain regions and LSC from morphine-tolerant mice by PKI analogs administered i.c.v. is evidence that PKA plays a role in morphine tolerance.

  12. Development and validation of a rapid reversed-phase HPLC method for the determination of the non-nucleoside reverse transcriptase inhibitor dapivirine from polymeric nanoparticles.

    Science.gov (United States)

    das Neves, José; Sarmento, Bruno; Amiji, Mansoor M; Bahia, Maria Fernanda

    2010-06-05

    The objective of this work was to develop and validate a rapid reversed-phase (RP) high-performance liquid chromatography (HPLC) method for the in vitro pharmaceutical characterization of dapivirine-loaded polymeric nanoparticles. Chromatographic runs were performed on a RP C18 column with a mobile phase comprising acetonitrile-0.5% (w/v) triethanolamine solution in isocratic mode (80:20, v/v) at a flow rate of 1 ml/min. Dapivirine was detected at a wavelength of 290 nm. The method was shown to be specific, linear in the range of 1-50 microg/ml (R(2)=0.9998), precise at the intra-day and inter-day levels as reflected by the relative standard deviation values (less than 0.85%), accurate (recovery rate of 100.17+/-0.35%), and robust to changes in the mobile phase and column brand. The detection and quantitation limits were 0.08 and 0.24 microg/ml, respectively. The method was successfully used to determine the loading capacity and association efficiency of dapivirine in poly(lactic-co-glycolic acid)-based nanoparticles and its in vitro release. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  13. Greening Reversed-Phase Liquid Chromatography Methods Using Alternative Solvents for Pharmaceutical Analysis

    Directory of Open Access Journals (Sweden)

    Moussa Yabré

    2018-05-01

    Full Text Available The greening of analytical methods has gained increasing interest in the field of pharmaceutical analysis to reduce environmental impacts and improve the health safety of analysts. Reversed-phase high-performance liquid chromatography (RP-HPLC is the most widely used analytical technique involved in pharmaceutical drug development and manufacturing, such as the quality control of bulk drugs and pharmaceutical formulations, as well as the analysis of drugs in biological samples. However, RP-HPLC methods commonly use large amounts of organic solvents and generate high quantities of waste to be disposed, leading to some issues in terms of ecological impact and operator safety. In this context, greening HPLC methods is becoming highly desirable. One strategy to reduce the impact of hazardous solvents is to replace classically used organic solvents (i.e., acetonitrile and methanol with greener ones. So far, ethanol has been the most often used alternative organic solvent. Others strategies have followed, such as the use of totally aqueous mobile phases, micellar liquid chromatography, and ionic liquids. These approaches have been well developed, as they do not require equipment investments and are rather economical. This review describes and critically discusses the recent advances in greening RP-HPLC methods dedicated to pharmaceutical analysis based on the use of alternative solvents.

  14. Extensive Mutagenesis of the Conserved Box E Motif in Duck Hepatitis B Virus P Protein Reveals Multiple Functions in Replication and a Common Structure with the Primer Grip in HIV-1 Reverse Transcriptase

    OpenAIRE

    Wang, Yong-Xiang; Luo, Cheng; Zhao, Dan; Beck, Jürgen; Nassal, Michael

    2012-01-01

    Hepadnaviruses, including the pathogenic hepatitis B virus (HBV), replicate their small DNA genomes through protein-primed reverse transcription, mediated by the terminal protein (TP) domain in their P proteins and an RNA stem-loop, ϵ, on the pregenomic RNA (pgRNA). No direct structural data are available for P proteins, but their reverse transcriptase (RT) domains contain motifs that are conserved in all RTs (box A to box G), implying a similar architecture; however, experimental support for...

  15. Reverse zymography alone does not confirm presence of a protease inhibitor.

    Science.gov (United States)

    Dutta, Sangita; Bhattacharyya, Debasish

    2013-03-01

    Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in reverse zymography against a protease used for digestion of the gel need not be an inhibitor; it might be resistant to degradation by the protease. We demonstrate that in reverse zymography, avidin, streptavidin and the leaf extract of Catharanthus roseus behave like inhibitors of proteases like papain, ficin, bromelain extracts from pineapple leaf, stem and fruit and trypsin. Still, they do not act as inhibitors of those proteases when enzyme assays were done in solution. In reverse zymography, the extract of pineapple crown leaf shows two major inhibitor bands against its own proteases. Identification of these proteins from sequences derived from MALDI TOF MS analysis indicated that they are fruit and stem bromelains. Avidin, streptavidin and bromelains are 'kinetically stable proteins' that are usually resistant to proteolysis. Thus, it is recommended that identification of an inhibitor of a protease by reverse zymography should be supported by independent assay methods for confirmation.

  16. Analyte induced water adsorbability in gas phase biosensors: the influence of ethinylestradiol on the water binding protein capacity.

    Science.gov (United States)

    Snopok, Borys; Kruglenko, Ivanna

    2015-05-07

    An ultra-sensitive gas phase biosensor/tracer/bio-sniffer is an emerging technology platform designed to provide real-time information on air-borne analytes, or those in liquids, through classical headspace analysis. The desired bio-sniffer measures gaseous 17α- ethinylestradiol (ETED) as frequency changes on a quartz crystal microbalance (QCM), which is a result of the interactions of liquid sample components in the headspace (ETED and water) with a biorecognition layer. The latter was constructed by immobilization of polyclonal antiserum against a phenolic A-ring of estrogenic receptors through protein A. The QCM response exhibited stretched exponential kinetics of negative frequency shifts with reversible and "irreversible" components of mass uptake onto the sensor surface in static headspace conditions when exposed to water solutions of ETED over the sensor working range, from 10(-10) to 10(-17) g L(-1). It was shown that the variations in the QCM response characteristics are due to the change of the water-binding capacity of the sensing layer induced by protein transformations initiated by the binding of ETED molecules. This result is well correlated with the natural physiological function of estrogens in controlling the homeostasis of body fluids in living beings.

  17. Protein content of leaf-cutting ant queens before the nuptial flight and during the post-claustral phase

    Directory of Open Access Journals (Sweden)

    Edypo Jacob Silva

    2014-12-01

    Full Text Available Protein content of leaf-cutting ant queens before the nuptial flight and during the post-claustral phase. This study evaluated the crude protein content of queens of Atta sexdens before the nuptial flight and after the claustral phase in laboratory and field colonies. The hypothesis was that protein is used for survival of the queen and for early colony growth during the claustral phase. Additionally, the nest morphology, live biomass and adult population of field colonies were evaluated. Crude protein was determined by digestion of the organic material with sulfuric acid at high temperatures. The mean crude protein content was 123.23 ± 11.20 mg for females before the nuptial flight and 70.44 ± 12.21 mg for laboratory-reared queens after the claustral phase. The post-claustral crude protein content of field-collected queen was 55.90 ± 9.18 mg. With respect to the loss of crude protein as a function of duration of the claustral phase, laboratory-reared queens lost 52.79 mg and field-collected queens lost 67.33 mg compared to females before the nuptial flight. A positive linear correlation was observed between the weight of field-collected queens (256.4 ± 36.3 mg and colony biomass (13.02 ± 9.12 g, but there was no correlation between biomass and nest depth (13.11 ± 3.82 cm. As expected, the present results support the hypothesis that protein is used for survival of the queen and for early colony growth, as demonstrated by the reduction in crude protein content as a function of duration of the claustral phase. To our knowledge, this is the first study to provide data of the dynamics of protein reserves in leaf-cutting ant queens during the claustral phase.

  18. Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion

    DEFF Research Database (Denmark)

    Thams, P; Capito, K; Hedeskov, C J

    1990-01-01

    and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1...

  19. Development of an on-line mixed-mode gel liquid chromatography×reversed phase liquid chromatography method for separation of water extract from Flos Carthami.

    Science.gov (United States)

    Wang, Yu-Qing; Tang, Xu; Li, Jia-Fu; Wu, Yun-Long; Sun, Yu-Ying; Fang, Mei-Juan; Wu, Zhen; Wang, Xiu-Min; Qiu, Ying-Kun

    2017-10-13

    A novel on-line comprehensive two-dimensional liquid chromatography (2D-LC) method by coupling mixed-mode gel liquid chromatography (MMG-LC) with reversed phase liquid chromatography (RPLC) was developed. A mixture of 17 reference compounds was used to study the separation mechanism. A crude water extract of Flos Carthami was applied to evaluate the performance of the novel 2D-LC system. In the first dimension, the extract was eluted with a gradient of water/methanol over a cross-linked dextran gel Sephadex LH-20 column. Meanwhile, the advantages of size exclusion, reversed phase partition and adsorption separation mechanism were exploited before further on-line reversed phase purification on the second dimension. This novel on-line mixed-mode Sephadex LH-20×RPLC method provided higher peak resolution, sample processing ability (2.5mg) and better orthogonality (72.9%) versus RPLC×RPLC and hydrophilic interaction liquid chromatography (HILIC)×RPLC. To the best of our knowledge, this is the first report of a mixed-mode Sephadex LH-20×RPLC separation method with successful applications in on-line mode, which might be beneficial for harvesting targets from complicated medicinal plants. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. QUANTITATIVE ION-PAIR EXTRACTION OF 4(5)-METHYLIMIDAZOLE FROM CARAMEL COLOR AND ITS DETERMINATION BY REVERSED-PHASE ION-PAIR LIQUID-CHROMATOGRAPHY

    DEFF Research Database (Denmark)

    Thomsen, Mohens; Willumsen, Dorthe

    1981-01-01

    A procedure for quantitative ion-pair extraction of 4(5)-methylimidazole from caramel colour using bis(2-ethylhexyl)phosphoric acid as ion-pairing agent has been developed. Furthermore, a reversed-phase ion-pair liquid chromatographic separation method has been established to analyse the content...

  1. Research on Single-Phase PWM Converter with Reverse Conducting IGBT Based on Loss Threshold Desaturation Control

    Directory of Open Access Journals (Sweden)

    Xianjin Huang

    2017-11-01

    Full Text Available In the application of vehicle power supply and distributed power generation, there are strict requirements for the pulse width modulation (PWM converter regarding power density and reliability. When compared with the conventional insulated gate bipolar transistor (IGBT module, the Reverse Conducting-Insulated Gate Bipolar Transistor (RC-IGBT with the same package has a lower thermal resistance and higher current tolerance. By applying the gate desaturation control, the reverse recovery loss of the RC-IGBT diode may be reduced. In this paper, a loss threshold desaturation control method is studied to improve the output characteristics of the single-phase PWM converter with a low switching frequency. The gate desaturation control characteristics of the RC-IGBT’s diode are studied. A proper current limit is set to avoid the ineffective infliction of the desaturation pulse, while the bridge arm current crosses zero. The expectation of optimized loss decrease is obtained, and the better performance for the RC-IGBTs of the single-phase PWM converter is achieved through the optimized desaturation pulse distribution. Finally, the improved predictive current control algorithm that is applied to the PWM converter with RC-IGBTs is simulated, and is operated and tested on the scaled reduced power platform. The results prove that the gate desaturation control with the improved predictive current algorithm may effectively improve the RC-IGBT’s characteristics, and realize the stable output of the PWM converter.

  2. Development and validation of reversed-phase HPLC gradient method for the estimation of efavirenz in plasma.

    Directory of Open Access Journals (Sweden)

    Shweta Gupta

    Full Text Available Efavirenz is an anti-viral agent of non-nucleoside reverse transcriptase inhibitor category used as a part of highly active retroviral therapy for the treatment of infections of human immune deficiency virus type-1. A simple, sensitive and rapid reversed-phase high performance liquid chromatographic gradient method was developed and validated for the determination of efavirenz in plasma. The method was developed with high performance liquid chromatography using Waters X-Terra Shield, RP18 50 x 4.6 mm, 3.5 μm column and a mobile phase consisting of phosphate buffer pH 3.5 and Acetonitrile. The elute was monitored with the UV-Visible detector at 260 nm with a flow rate of 1.5 mL/min. Tenofovir disoproxil fumarate was used as internal standard. The method was validated for linearity, precision, accuracy, specificity, robustness and data obtained were statistically analyzed. Calibration curve was found to be linear over the concentration range of 1-300 μg/mL. The retention times of efavirenz and tenofovir disoproxil fumarate (internal standard were 5.941 min and 4.356 min respectively. The regression coefficient value was found to be 0.999. The limit of detection and the limit of quantification obtained were 0.03 and 0.1 μg/mL respectively. The developed HPLC method can be useful for quantitative pharmacokinetic parameters determination of efavirenz in plasma.

  3. Determination of phenolic derivatives of antipyrine in plasma with solid-phase extraction and high-performance liquid chromatography-atmospheric-pressure chemical ionization mass spectrometry

    NARCIS (Netherlands)

    Coolen, S.A.J.; Ligor, T.; Lieshout, van H.P.M.; Huf, F.A.

    1999-01-01

    This manuscript describes a method to determine antipyrine and its phenolic derivatives in plasma by reversed-phase high-performance liquid chromatography–mass spectrometry (RP-HPLC–MS). The sample pretreatment consisted of a C18 solid-phase extraction (SPE), to remove the salts and proteins. The

  4. Fontes protéicas suplementadas com aminoácidos e minerais para a tilápia do Nilo durante a reversão sexual Protein sources supplemented with amino acids and minerals to Nile tilapia during sex reversal phase

    Directory of Open Access Journals (Sweden)

    Fábio Meurer

    2005-02-01

    Full Text Available Objetivou-se, no presente experimento, comparar o efeito da origem da fonte protéica e da suplementação com lisina e metionina ou cálcio e fósforo para a tilápia do Nilo (Oreochromis niloticus, durante a reversão sexual. Foram utilizadas 200 larvas, distribuídas em 20 aquários de 12 litros, em um delineamento inteiramente casualizado em quatro tratamentos e cinco repetições. A temperatura pela manhã e à tarde, o oxigênio dissolvido, pH e condutividade elétrica da água foram, respectivamente, de 22,6 ± 0,1ºC, 24,1±0,4ºC; 6,77 ± 0,38 mg/L, 7,75 ± 0,16; e 173,4 ± 2 mS/m. Os tratamentos foram compostos por quatro rações isoprotéicas e isoenergéticas: uma somente com ingredientes de origem animal (FA; outra com ingredientes de origem vegetal (FV e duas com ingredientes de origem vegetal, uma com adição dos aminoácidos lisina e metionina (FAA e outra com cálcio e fósforo (FP. Os valores médios finais de peso e comprimento dos animais submetidos à ração FA foi superior aos demais tratamentos, seguido pelo FP e FV, que não diferiram da FAA. O tratamento FA apresentou os maior valores de biomassa final, seguido do FP e FAA, que foram semelhantes ao FV. O fator de condição dos peixes do tratamento FA foi superior aos FAA e FV, porém o FP foi semelhante aos demais. A sobrevivência dos tratamentos FA, FV e FP foram semelhantes entre si e superiores ao FAA. Para a fase de reversão sexual da tilápia do Nilo, rações formuladas com ingredientes de origem animal foram mais eficientes que as formuladas com ingredientes de origem vegetal, mesmo quando estas foram suplementadas com lisina e metionina ou cálcio e fósforo.This work aimed to evaluate the effect of protein source origin and lysine and methionine or phosphorus and calcium supplementation to Nile tilapia during sex reversal phase. Two hundred fries were assigned to a completely randomized design with four treatments and five replications, in twenty 12 L

  5. Purification and Identification of Membrane Proteins from Urinary Extracellular Vesicles using Triton X-114 Phase Partitioning.

    Science.gov (United States)

    Hu, Shuiwang; Musante, Luca; Tataruch, Dorota; Xu, Xiaomeng; Kretz, Oliver; Henry, Michael; Meleady, Paula; Luo, Haihua; Zou, Hequn; Jiang, Yong; Holthofer, Harry

    2018-01-05

    Urinary extracellular vesicles (uEVs) have become a promising source for biomarkers accurately reflecting biochemical changes in kidney and urogenital diseases. Characteristically, uEVs are rich in membrane proteins associated with several cellular functions like adhesion, transport, and signaling. Hence, membrane proteins of uEVs should represent an exciting protein class with unique biological properties. In this study, we utilized uEVs to optimize the Triton X-114 detergent partitioning protocol targeted for membrane proteins and proceeded to their subsequent characterization while eliminating effects of Tamm-Horsfall protein, the most abundant interfering protein in urine. This is the first report aiming to enrich and characterize the integral transmembrane proteins present in human urinary vesicles. First, uEVs were enriched using a "hydrostatic filtration dialysis'' appliance, and then the enriched uEVs and lysates were verified by transmission electron microscopy. After using Triton X-114 phase partitioning, we generated an insoluble pellet fraction and aqueous phase (AP) and detergent phase (DP) fractions and analyzed them with LC-MS/MS. Both in- and off-gel protein digestion methods were used to reveal an increased number of membrane proteins of uEVs. After comparing with the identified proteins without phase separation as in our earlier publication, 199 different proteins were detected in DP. Prediction of transmembrane domains (TMDs) from these protein fractions showed that DP had more TMDs than other groups. The analyses of hydrophobicity revealed that the GRAVY score of DP was much higher than those of the other fractions. Furthermore, the analysis of proteins with lipid anchor revealed that DP proteins had more lipid anchors than other fractions. Additionally, KEGG pathway analysis showed that the DP proteins detected participate in endocytosis and signaling, which is consistent with the expected biological functions of membrane proteins. Finally

  6. Wavefront reversal in a copper vapor active medium

    Energy Technology Data Exchange (ETDEWEB)

    Bunkin, F.V.; Savranskii, V.V.; Shafeev, G.A.

    1981-09-01

    Wavefront reversal in the resonator of a copper vapor laser was observed. The frequencies of the signal and reversed waves were the same. The dependence of the reversed signal power on the input signal power had a threshold. Photographs were obtained of the reconstructed image of an object when a distorting phase plate was inserted in the resonator.

  7. Arsenic-containing fatty acids and hydrocarbons in marine oils - Determination using reversed-phase HPLC-ICP-MS and HPLC-qTOF-MS

    DEFF Research Database (Denmark)

    Sele, Veronika; Sloth, Jens Jørgen; Holmelid, Bjarte

    2014-01-01

    ; dimethylarsinate (DMA), triphenylarsinoxide (Ph3AsO) and a synthesized arsenic-containing hydrocarbon (AsHC) (dimethylarsinoyl nonadecane; C21H43AsO). The observed variation in signal intensity for arsenic during the gradient elution profile in reversed-phase HPLC was compensated for by determining the time...

  8. Solid phase radioimmunoassays for human C-reactive protein

    International Nuclear Information System (INIS)

    Shine, B.; Beer, F.C. de; Pepys, M.B.

    1981-01-01

    Two new, rapid and sensitive radioimmunoassays for human C-reactive protein (CRP) have been established using antiserum coupled to magnetizable cellulose particles, which facilitate phase separation. A single antibody method, using solid phase anti-CRP, provides a sensitivity of 50 μg/l with a 1-h incubation time and intra- and inter-assay coefficients of variation of 10%. A double antibody method, using fluid phase rabbit anti-CRP serum and solid phase sheep anti-rabbit IgG serum, provides a sensitivity of 3 μg/l with an overnight incubation and intra- and inter-assay coefficients of variation of 10%. Among 468 sera from normal adult volunteer blood donors the median CRP concentration was 800 μg/l, interquartile range 340-1700 μg/l and range 70-29,000 μg/l. Ninety percent of samples contained less than 3 mg/l and 99% less than 10 mg/l. Low levels (14-650 μg/l) of CRP were detected both in amniotic fluids and in cerebrospinal fluids. (Auth.)

  9. Hydrolysis of Whey Protein Isolate with Bacillus licheniformis Protease: Fractionation and Identification of Aggregating Peptides

    NARCIS (Netherlands)

    Creusot, N.P.; Gruppen, H.

    2007-01-01

    The objective of this work was to identify the dominant aggregating peptides from a whey protein hydrolysate (degree of hydrolysis of 6.8%) obtained with Bacillus licheniformis protease. The aggregating peptides were fractionated with preparative reversed-phase chromatography and identified with

  10. Phase formation of physically associating polymer blends

    International Nuclear Information System (INIS)

    Tanaka, Fumihiko

    1993-01-01

    Polymers exhibit a variety of condensed phases when some of their segments are capable of forming weak bonds which can be created and destroyed by thermal motion. Transition from one phase to another caused by such 'segment association' is reversible by the change of the temperature and the concentration, so that it is called 'reversible phase transition'. What types of reversible phase formation are possible for a given associative interaction? What is the most fundamental laws which govern the competition between molecular association and phase separation? This paper surveys, as typical examples of reversible phases, macroscopic phase separation, microphase formation, solvation, gelation, etc. from the unified point of view, and explores the possibility of new condensed phases caused by their mutual interference. (author)

  11. Coating Nanoparticles with Plant-Produced Transferrin-Hydrophobin Fusion Protein Enhances Their Uptake in Cancer Cells

    DEFF Research Database (Denmark)

    Reuter, Lauri J.; Shahbazi, Mohammad-Ali; Makila, Ermei M.

    2017-01-01

    can be expressed in Nicotiana benthamiana plants as a fusion with Trichoderma reesei hydrophobins HFBI, HFBII, or HFBIV. Transferrin-HFBIV was further expressed in tobacco BY-2 suspension cells. Both partners of the fusion protein retained their functionality; the hydrophobin moiety enabled migration...... to a surfactant phase in an aqueous two-phase system, and the transferrin moiety was able to reversibly bind iron. Coating porous silicon nanoparticles with the fusion protein resulted in uptake of the nanoparticles in human cancer cells. This study provides a proof-of concept for the functionalization...

  12. Optimizing gradient conditions in online comprehensive two-dimensional reversed-phase liquid chromatography by use of the linear solvent strength model

    DEFF Research Database (Denmark)

    Græsbøll, Rune; Janssen, Hans-Gerd; Christensen, Jan H.

    2017-01-01

    The linear solvent strength model was used to predict coverage in online comprehensive two-dimensional reversed-phase liquid chromatography. The prediction model uses a parallelogram to describe the separation space covered with peaks in a system with limited orthogonality. The corners of the par......The linear solvent strength model was used to predict coverage in online comprehensive two-dimensional reversed-phase liquid chromatography. The prediction model uses a parallelogram to describe the separation space covered with peaks in a system with limited orthogonality. The corners...... of the parallelogram are assumed to behave like chromatographic peaks and the position of these pseudo-compounds was predicted. A mix of 25 polycyclic aromatic compounds were used as a test. The precision of the prediction, span 0-25, was tested by varying input parameters, and was found to be acceptable with root...... factors were low, or when gradient conditions affected parameters not included in the model, e.g. second dimension gradient time affects the second dimension equilibration time. The concept shows promise as a tool for gradient optimization in online comprehensive two-dimensional liquid chromatography...

  13. Usefulness of acute phase proteins for monitoring development of ...

    African Journals Online (AJOL)

    Background: Serum levels of acute phase proteins (APP) have been used to diagnose and follow up treatment of liver diseases. This study was carried out to determine the usefulness of APP to predict development of hepatocellular carcinoma (HCC) among Hepatitis B virus (HBV) carriers. Study design: In a prospective ...

  14. Utility of reversed phase high performance liquid chromatography for on-line yield determination of radiochemical separations: Studies with cobalt

    International Nuclear Information System (INIS)

    Subramanian, S.; Woittiez, J.R.W.

    1993-01-01

    This article indicates the potentials of high performance liquid chromatography (HPLC) as a radiochemical technique for multielement separation of neutron irradiated samples. The focus lies on the convenience to use the detector signal of the eluted components to indicate the chemical yield of the analyte, which has often proved to be a crucial step in radiochemical separations. Two signals have been utilized. The UV signal of the metal-ligand complexes separated by reversed phase HPLC and the radioactive response as a result of sample irradiation of carrier-tracer addition. Change in ratio is discussed between the two signals, if any, for a specific sample. Losses of metal as much as 65% were simulated and corrected using the individual UV response. The method promises improved accuracy for elemental analysis despite losses suffered during the various chemical steps. The procedure omits the necessity of additional analytical steps for yield determination. The present article aims at the chromatographic part of the study. Cobalt as cobalt diethyldithiocarbamate has been used to demonstrate the viability of the concept. The separation was developed on c C18 reverse phase analytical column and optimized on a semi preparative one

  15. A new method to describe two-phase solvent extraction based on net transport potential derived as linear combinations of forward and reverse constituents

    International Nuclear Information System (INIS)

    Nabeshima, Masahiro

    1998-01-01

    With the view to avoiding the difficulties encountered in estimating thermodynamic activities of the multiple chemical species in two-phase liquid system, a set of forward, reverse, net and total transport potentials are defined to represent the chemical state of a transferring solute during transient using bulk concentrations. The net transport potential corresponds to that in the conventional two-film model of diffusion-controlled processes. The overall driving forces of mass transport are redefined as the derivatives of the relevant transport potentials differentiated with respect to a state variable newly defined in terms of the bulk concentrations of the solute contained in both phases. Net and total quantities, i.e. transport potentials, overall driving forces and the molar fluxes are obtained as linear combinations of those for forward and reverse directions. The topical features presented by these quantities and their mutual relations are discussed in detail. The experimental new overall transport coefficient for U(VI) varied in accord with the changes in the theoretical net transport potential and overall driving force. The present method permits describing the extractive mass transport consistently both to forward and reverse directions of transport. (author)

  16. Memory formation in reversal learning of the honeybee

    Directory of Open Access Journals (Sweden)

    Ravit Hadar

    2010-12-01

    Full Text Available In reversal learning animals are first trained with a differential learning protocol, where they learn to respond to a reinforced odor (CS+ and not to respond to a nonreinforced odor (CS-. Once they respond correctly to this rule, the contingencies of the conditioned stimuli are reversed, and animals learn to adjust their response to the new rule. This study investigated the effect of a protein synthesis inhibitor (emetine on the memory formed after reversal learning in the honeybee Apis mellifera. Two groups of bees were studied: summer bees and winter bees, each yielded different results. Blocking protein synthesis in summer bees inhibits consolidation of the excitatory learning following reversal learning whereas it blocked the consolidation of the inhibitory learning in winter bees. These findings suggest that excitatory and inhibitory learning may involve different molecular processes in bees, which are seasonally dependent.

  17. Steganographic optical image encryption system based on reversible data hiding and double random phase encoding

    Science.gov (United States)

    Chuang, Cheng-Hung; Chen, Yen-Lin

    2013-02-01

    This study presents a steganographic optical image encryption system based on reversible data hiding and double random phase encoding (DRPE) techniques. Conventional optical image encryption systems can securely transmit valuable images using an encryption method for possible application in optical transmission systems. The steganographic optical image encryption system based on the DRPE technique has been investigated to hide secret data in encrypted images. However, the DRPE techniques vulnerable to attacks and many of the data hiding methods in the DRPE system can distort the decrypted images. The proposed system, based on reversible data hiding, uses a JBIG2 compression scheme to achieve lossless decrypted image quality and perform a prior encryption process. Thus, the DRPE technique enables a more secured optical encryption process. The proposed method extracts and compresses the bit planes of the original image using the lossless JBIG2 technique. The secret data are embedded in the remaining storage space. The RSA algorithm can cipher the compressed binary bits and secret data for advanced security. Experimental results show that the proposed system achieves a high data embedding capacity and lossless reconstruction of the original images.

  18. Influence of preliminary deformation and phase strengthening on γ reversible α transformation kinetics in cerium under pressure

    International Nuclear Information System (INIS)

    Larionov, L.V.; Livshits, L.D.; Peresada, G.I.; AN SSSR, Moscow. Inst. Fiziki Zemli)

    1985-01-01

    Using the methods of piezo- and resistometry the influence of preliminary plastic deformation, phase transformation induced strengthening and heat treatment on kinetics of γ reversible α transformation in cerium is studied. It is shown, that the used methods of preliminary treatment of sample material do not change pressure value of γ → α and α →γ transformation initiation and do not affect hysteresis value, but affect considerably its kinetics. Preliminary plastic deformation and structural strengthening increase the average formation rate of a new phase. According to the data of tensile tests, structural strengthening, apprearing as a result of one cycle of γ → α → γ transformation, increases cerium strength characteristics 6y 30-50% with simultaneous decrease in plasticity. Metallographic studies confirm martensitic character of γ → α → γ transformations in cerium

  19. Heightened systemic levels of neutrophil and eosinophil granular proteins in pulmonary tuberculosis and reversal following treatment.

    Science.gov (United States)

    Moideen, Kadar; Kumar, Nathella Pavan; Nair, Dina; Banurekha, Vaithilingam V; Bethunaickan, Ramalingam; Babu, Subash

    2018-04-09

    Granulocytes are activated during tuberculosis (TB) infection and act as immune effector cells and granulocyte responses are implicated in TB pathogenesis. Plasma levels of neutrophil and eosinophil granular proteins provide an indirect measure of degranulation. In this study, we wanted to examine the levels of neutrophil and eosinophil granular proteins in individuals with pulmonary tuberculosis (PTB) and to compare them with the levels in latent TB (LTB) individuals. Hence, we measured the plasma levels of myeloperoxidase (MPO), neutrophil elastase, and proteinase-3; major basic protein (MBP), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP) and eosinophil peroxidase (EPX) in these individuals. Finally, we also measured the levels of all of these parameters in PTB individuals following anti-tuberculosis (ATT) treatment. Our data reveal that PTB individuals are characterized by significantly higher plasma levels of MPO, elastase, human proteinase 3 as well as MBP and EDN in comparison to LTB individuals. Our data also reveal that ATT resulted in reversal of all of these changes, indicating an association with TB disease. Finally, our data show that the systemic levels of MPO and proteinase-3 can significantly discriminate PTB from LTB individuals. Thus, our data suggest that neutrophil and eosinophil granular proteins could play a potential role in the innate immune response and therefore, the pathogenesis of pulmonary TB. Copyright © 2018 American Society for Microbiology.

  20. Analysis of Theobromine and Related Compounds by Reversed Phase High-Performance Liquid Chromatography with Ultraviolet Detection: An Update (1992–2011

    Directory of Open Access Journals (Sweden)

    Sandra Aparecida de Assis

    2011-01-01

    Full Text Available Theobromine and its related compounds, such as caffeine and theophylline, are secondary metabolites that belong to the alkaloids and have economic and cultural importance. These alkaloids have demonstrated stimulatory effects on the central nervous, gastrointestinal, cardiovascular, renal and respiratory systems, resulting in 'energy arousal', increased motivation to work, increased alertness and increased cognitive function. Several analytical methods have been used to analyse these compounds, but reversed phase high-performance liquid chromatography (RP-HPLC is the most commonly applied because of its efficiency, sensitivity, specificity and speed. This review describes the analyses of theobromine-related compounds by RP-HPLC with ultraviolet detection (UV in four sources: food, beverages, biological fluids and plants. Many RP-HPLC methods have been developed and optimized for the detection and quantification of these natural compounds. Elution under isocratic conditions is the most frequent method, with a water, methanol and acetonitrile mixture modified with acetic, phosphoric or formic acid as the mobile phase. For xanthine analysis, the use of reversed phase high-performance liquid chromatography with an ultraviolet/diode array detector (UV/DAD is particularly suitable as derivation is not required; it allows the analysis of absorbance at all wavelengths, it is simple and rapid.

  1. Reclaimable Thermally Reversible Polymers for AM Feedstock, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Cornerstone Research Group Inc. (CRG) proposes to design and develop thermally-reversible polymeric materials that will function as reprocessable thermosetting...

  2. Offline solid-phase extraction for preconcentration of pharmaceuticals and personal care products in environmental water and their simultaneous determination using the reversed phase high-performance liquid chromatography method.

    Science.gov (United States)

    G Archana; Dhodapkar, Rita; Kumar, Anupama

    2016-09-01

    The present study reports a precise and simple offline solid-phase extraction (SPE) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous determination of five representative and commonly present pharmaceuticals and personal care products (PPCPs), a new class of emerging pollutants in the aquatic environment. The target list of analytes including ciprofloxacin, acetaminophen, caffeine benzophenone and irgasan were separated by a simple HPLC method. The column used was a reversed-phase C18 column, and the mobile phase was 1 % acetic acid and methanol (20:80 v/v) under isocratic conditions, at a flow rate of 1 mL min(-1). The analytes were separated and detected within 15 min using the photodiode array detector (PDA). The linearity of the calibration curves were obtained with correlation coefficients 0.98-0.99.The limit of detection (LOD), limit of quantification (LOQ), precision, accuracy and ruggedness demonstrated the reproducibility, specificity and sensitivity of the developed method. Prior to the analysis, the SPE was performed using a C18 cartridge to preconcentrate the targeted analytes from the environmental water samples. The developed method was applied to evaluate and fingerprint PPCPs in sewage collected from a residential engineering college campus, polluted water bodies such as Nag river and Pili river and the influent and effluent samples from a sewage treatment plant (STP) situated at Nagpur city, in the peak summer season. This method is useful for estimation of pollutants present in microquantities in the surface water bodies and treated sewage as compared to nanolevel pollutants detected by mass spectrometry (MS) detectors.

  3. Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves

    Science.gov (United States)

    Zeb, Alam; Ullah, Fareed

    2017-04-01

    Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight carotenoids, four pheophytins and two chlorophylls. Lutein (806.0 µg/g), chlorophyll b' (410.0 µg/g), chlorophyll a (162.4 µg/g), 9'-Z-neoxanthin (142.8 µg/g) and all-E-violaxanthin (82.2 µg/g)) were present in higher amounts. The relatively high amounts of lutein may be one of the key indicator of beneficial antioxidant properties. The phenolic profile revealed a total of thirteen compounds, namely p-hydroxybenzoic acid, chlorogenic acid, caftaric acid, kaempferol glycosides, p-coumaric acid, rosmarinic acid, 5-caffeoylquinic acid, and quercetin glycosides. Kaempferol-3-sophorotrioside (58.7 mg/g), chlorogenic acid (28.5 mg/g), 5-O-caffeoylquinic acid (18.7 mg/g), coumaric acid (11.2 mg/g) and its derivative (33.1 mg/g) were present in high amounts. These results suggest that the reversed phase HPLC profiling of adiantum leaves provides a better understanding in to the actual composition of bioactive compounds, which may be responsible for possible medicinal properties. Adiantum leaves rich in important bioactive phytochemicals can be used as a potential source of nutraceuticals or as a functional food ingredient.

  4. Comparison of colorimetric m ethods for the quantification of model proteins in aqueous two-phase systems

    OpenAIRE

    Glyk, Anna; Heinisch, Sandra L.; Scheper, Thomas; Beutel, Sascha

    2015-01-01

    In the current study, the quantification of different model proteins in the presence of typical aqueous two-phase system components was investigated by using the Bradford and bicinchoninic acid (BCA) assays. Each phase-forming component above 1 and 5 wt% had considerable effects on the protein quantification in both assays, respectively, resulting in diminished protein recoveries/absorption values by increasing poly(ethylene glycol) (PEG)/salt concentration and PEG molecular weight. Therefore...

  5. Reversible Rigidity Control Using Low Melting Temperature Alloys

    Science.gov (United States)

    Shan, Wanliang; Lu, Tong; Majidi, Carmel

    2013-03-01

    Inspired by nature, materials able to achieve rapid rigidity changes have important applications for human body protection in military and many other areas. This talk presents the fabrication and design of soft-matter technologies that exhibit rapid reversible rigidity control. Fabricated with a masked deposition technique, the soft-matter composite contains liquid-phase and phase-changing metal alloys embedded in a soft and highly stretchable elastomer. The composite material can reversibly change its rigidity by three orders of magnitude and sustain large deformation.

  6. Cluster analysis of historical and modern hard red spring wheat cultivars based on parentage and HPLC analysis of gluten forming proteins

    Science.gov (United States)

    In this study, 30 hard red spring (HRS) wheat cultivars released between 1910 and 2013 were analyzed to determine how they cluster in terms of parentage and protein data, analyzed by reverse-phase HPLC (RP-HPLC) of gliadins, and size-exclusion HPLC (SE-HPLC) of unreduced proteins. Dwarfing genes in...

  7. Simultaneous determination of F-beta-alanine and beta-alanine in plasma and urine with dual-column reversed-phase high-performance liquid chromatography

    NARCIS (Netherlands)

    van Kuilenburg, A. B.; Stroomer, A. E.; Peters, G. J.; van Gennip, A. H.

    2001-01-01

    F-beta-Alanine and beta-alanine were detected in plasma and urine samples with fluorescence detection of orthophthaldialdehyde derivatives of F-beta-alanine and beta-alanine after separation with dual-column reversed-phase HPLC. The detection limits of F-beta-alanine and beta-alanine in the HPLC

  8. Diazonium modification of porous graphitic carbon with catechol and amide groups for hydrophilic interaction and attenuated reversed phase liquid chromatography.

    Science.gov (United States)

    Iverson, Chad D; Zhang, Ya; Lucy, Charles A

    2015-11-27

    Porous graphitic carbon (PGC) is an increasingly popular and attractive phase for HPLC on account of its chemical and thermal stability, and its unique separation mechanism. However, native PGC is strongly hydrophobic and in some instances excessively retentive. As part of our effort to build a library of hydrophilic covalently modified PGC phases, we functionalized PGC with catechol and amide groups by means of aryl diazonium chemistry to produce two new phases. Successful grafting was confirmed by X-ray photoelectron spectroscopy (XPS). Under HILIC conditions, the Catechol-PGC showed up to 5-fold increased retention relative to unmodified PGC and selectivity that differed from four other HILIC phases. Under reversed phase conditions, the Amide-PGC reduced the retentivity of PGC by almost 90%. The chromatographic performance of Catechol-PGC and Amide-PGC is demonstrated by separations of nucleobases, nucleosides, phenols, alkaline pharmaceuticals, and performance enhancing stimulants. These compounds had retention factors (k) ranging from 0.5 to 13. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Phase behaviour of disordered proteins underlying low density and high permeability of liquid organelles

    Science.gov (United States)

    Wei, Ming-Tzo; Elbaum-Garfinkle, Shana; Holehouse, Alex S.; Chen, Carlos Chih-Hsiung; Feric, Marina; Arnold, Craig B.; Priestley, Rodney D.; Pappu, Rohit V.; Brangwynne, Clifford P.

    2017-11-01

    Many intracellular membraneless organelles form via phase separation of intrinsically disordered proteins (IDPs) or regions (IDRs). These include the Caenorhabditis elegans protein LAF-1, which forms P granule-like droplets in vitro. However, the role of protein disorder in phase separation and the macromolecular organization within droplets remain elusive. Here, we utilize a novel technique, ultrafast-scanning fluorescence correlation spectroscopy, to measure the molecular interactions and full coexistence curves (binodals), which quantify the protein concentration within LAF-1 droplets. The binodals of LAF-1 and its IDR display a number of unusual features, including 'high concentration' binodal arms that correspond to remarkably dilute droplets. We find that LAF-1 and other in vitro and intracellular droplets are characterized by an effective mesh size of ∼3-8 nm, which determines the size scale at which droplet properties impact molecular diffusion and permeability. These findings reveal how specific IDPs can phase separate to form permeable, low-density (semi-dilute) liquids, whose structural features are likely to strongly impact biological function.

  10. Reversible switching in phase-change materials

    OpenAIRE

    Wojciech Wełnic; Matthias Wuttig

    2008-01-01

    Phase-change materials are successfully employed in optical data storage and are becoming a promising candidate for future electronic storage applications. Despite the increasing technological interest, many fundamental properties of these materials remain poorly understood. However, in the last few years the understanding of the material properties of phase-change materials has increased significantly. At the same time, great advances have been achieved in technological applications in elect...

  11. OCT-4 expression in follicular and luteal phase endometrium: a pilot study

    Directory of Open Access Journals (Sweden)

    Huber Johannes C

    2010-04-01

    Full Text Available Abstract Background The stem cell marker Octamer-4 (OCT-4 is expressed in human endometrium. Menstrual cycle-dependency of OCT-4 expression has not been investigated to date. Methods In a prospective, single center cohort study of 98 women undergoing hysteroscopy during the follicular (n = 49 and the luteal (n = 40 phases of the menstrual cycle, we obtained endometrial samples. Specimens were investigated for OCT-4 expression on the mRNA and protein levels using reverse transcriptase polymerase chain reaction (RT-PCR and immunohistochemistry. Expression of OCT-4 was correlated to menstrual cycle phase. Results Of 89 women sampled, 49 were in the follicular phase and 40 were in the luteal phase. OCT-4 mRNA was detected in all samples. Increased OCT-4 mRNA levels in the follicular and luteal phases was found in 35/49 (71% and 27/40 (68% of women, respectively (p = 0.9. Increased expression of OCT-4 protein was identified in 56/89 (63% samples. Increased expression of OCT-4 protein in the follicular and luteal phases was found in 33/49 (67% and 23/40 (58% of women, respectively (p = 0.5. Conclusions On the mRNA and protein levels, OCT-4 is not differentially expressed during the menstrual cycle. Endometrial OCT-4 is not involved in or modulated by hormone-induced cyclical changes of the endometrium.

  12. Mixed reverse micelles facilitated downstream processing of lipase involving water-oil-water liquid emulsion membrane.

    Science.gov (United States)

    Bhowal, Saibal; Priyanka, B S; Rastogi, Navin K

    2014-01-01

    Our earlier work for the first time demonstrated that liquid emulsion membrane (LEM) containing reverse micelles could be successfully used for the downstream processing of lipase from Aspergillus niger. In the present work, we have attempted to increase the extraction and purification fold of lipase by using mixed reverse micelles (MRM) consisting of cationic and nonionic surfactants in LEM. It was basically prepared by addition of the internal aqueous phase solution to the organic phase followed by the redispersion of the emulsion in the feed phase containing enzyme, which resulted in globules of water-oil-water (WOW) emulsion for the extraction of lipase. The optimum conditions for maximum lipase recovery (100%) and purification fold (17.0-fold) were CTAB concentration 0.075 M, Tween 80 concentration 0.012 M, at stirring speed of 500 rpm, contact time 15 min, internal aqueous phase pH 7, feed pH 9, KCl concentration 1 M, NaCl concentration 0.1 M, and ratio of membrane emulsion to feed volume 1:1. Incorporation of the nonionic surfactant (e.g., Tween 80) resulted in remarkable improvement in the purification fold (3.1-17.0) of the lipase. LEM containing a mixture of nonionic and cationic surfactants can be successfully used for the enhancement in the activity recovery and purification fold during downstream processing of enzymes/proteins. © 2014 American Institute of Chemical Engineers.

  13. Two-dimensional chromatographic analysis using three second-dimension columns for continuous comprehensive analysis of intact proteins.

    Science.gov (United States)

    Zhu, Zaifang; Chen, Huang; Ren, Jiangtao; Lu, Juan J; Gu, Congying; Lynch, Kyle B; Wu, Si; Wang, Zhe; Cao, Chengxi; Liu, Shaorong

    2018-03-01

    We develop a new two-dimensional (2D) high performance liquid chromatography (HPLC) approach for intact protein analysis. Development of 2D HPLC has a bottleneck problem - limited second-dimension (second-D) separation speed. We solve this problem by incorporating multiple second-D columns to allow several second-D separations to be proceeded in parallel. To demonstrate the feasibility of using this approach for comprehensive protein analysis, we select ion-exchange chromatography as the first-dimension and reverse-phase chromatography as the second-D. We incorporate three second-D columns in an innovative way so that three reverse-phase separations can be performed simultaneously. We test this system for separating both standard proteins and E. coli lysates and achieve baseline resolutions for eleven standard proteins and obtain more than 500 peaks for E. coli lysates. This is an indication that the sample complexities are greatly reduced. We see less than 10 bands when each fraction of the second-D effluents are analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), compared to hundreds of SDS-PAGE bands as the original sample is analyzed. This approach could potentially be an excellent and general tool for protein analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Optimization of the reversed-phase high-performance liquid chromatographic separation of synthetic estrogenic and progestogenic steroids using the multi-criteria decision making method

    NARCIS (Netherlands)

    Smilde, A. K.; Bruins, C. H.; Doornbos, D. A.; Vink, J.

    1987-01-01

    The optimization of the reversed-phase high-performance liquid chromatographic separation of a mixture of ethynylestradiol, desogestrel and three related compounds is described. A procedure is used that allows the prediction of the capacity factors of each individual synthetic steroid, depending on

  15. Sequence-dependent separation of trinucleotides by ion-interaction reversed-phase liquid chromatography A structure-retention study assisted by soft-modelling and molecular dynamics

    Czech Academy of Sciences Publication Activity Database

    Mikulášek, K.; Jaroň, Kamil S.; Kulhánek, P.; Bittová, M.; Havliš, J.

    2016-01-01

    Roč. 1469, October (2016), s. 88-95 ISSN 0021-9673 Institutional support: RVO:68081766 Keywords : Sequence-dependent separation * Ion-interaction reversed-phase liquid chromatography * Trinucleotides * Oligonucleotide sequence isomers * QSRR * Molecular dynamics Subject RIV: CE - Biochemistry Impact factor: 3.981, year: 2016

  16. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    Science.gov (United States)

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen JT; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-01-01

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates. DOI: http://dx.doi.org/10.7554/eLife.05338.001 PMID:25824290

  17. Determination of gas phase protein ion densities via ion mobility analysis with charge reduction.

    Science.gov (United States)

    Maisser, Anne; Premnath, Vinay; Ghosh, Abhimanyu; Nguyen, Tuan Anh; Attoui, Michel; Hogan, Christopher J

    2011-12-28

    We use a charge reduction electrospray (ESI) source and subsequent ion mobility analysis with a differential mobility analyzer (DMA, with detection via both a Faraday cage electrometer and a condensation particle counter) to infer the densities of single and multiprotein ions of cytochrome C, lysozyme, myoglobin, ovalbumin, and bovine serum albumin produced from non-denaturing (20 mM aqueous ammonium acetate) and denaturing (1 : 49.5 : 49.5, formic acid : methanol : water) ESI. Charge reduction is achieved through use of a Po-210 radioactive source, which generates roughly equal concentrations of positive and negative ions. Ions produced by the source collide with and reduce the charge on ESI generated drops, preventing Coulombic fissions, and unlike typical protein ESI, leading to gas-phase protein ions with +1 to +3 excess charges. Therefore, charge reduction serves to effectively mitigate any role that Coulombic stretching may play on the structure of the gas phase ions. Density inference is made via determination of the mobility diameter, and correspondingly the spherical equivalent protein volume. Through this approach it is found that for both non-denaturing and denaturing ESI-generated ions, gas-phase protein ions are relatively compact, with average densities of 0.97 g cm(-3) and 0.86 g cm(-3), respectively. Ions from non-denaturing ESI are found to be slightly more compact than predicted from the protein crystal structures, suggesting that low charge state protein ions in the gas phase are slightly denser than their solution conformations. While a slight difference is detected between the ions produced with non-denaturing and denaturing ESI, the denatured ions are found to be much more dense than those examined previously by drift tube mobility analysis, in which charge reduction was not employed. This indicates that Coulombic stretching is typically what leads to non-compact ions in the gas-phase, and suggests that for gas phase

  18. Extraction and determination of biogenic amines in fermented sausages and other meat products using reversed-phase-HPLC.

    Science.gov (United States)

    Straub, B; Schollenberger, M; Kicherer, M; Luckas, B; Hammes, W P

    1993-09-01

    A convenient method is described for the analysis of biogenic amines (BA) by means of reversed-phase-HPLC. The method is characterized by multi-channel UV detection (diodearray), subsequent post-column derivatization with o-phthaldialdehyde and 3-mercaptopropionic acid, and fluorescence detection. For the analysis of meat products and especially fermented sausages an optimized perchloric acid extraction process was introduced to determine putrescine, cadaverine, histamine, tyramine and 2-phenylethylamine. BA recoveries from meat ranged between 96 and 113% with a detection limit for amines of 0.5 mg/kg.

  19. Time course of protein synthesis-dependent phase of olfactory memory in the cricket Gryllus bimaculatus.

    Science.gov (United States)

    Matsumoto, Yukihisa; Noji, Sumihare; Mizunami, Makoto

    2003-04-01

    The cricket Gryllus bimaculatus forms a stable olfactory memory that lasts for practically a lifetime. As a first step to elucidate the cellular mechanisms of olfactory learning and memory retention in crickets, we studied the dependency of memory retention on the de novo brain protein synthesis by injecting the protein synthesis inhibitor cycloheximide (CHX) into the head capsule. Injection of CHX inhibited (3)H-leucine incorporation into brain proteins by > 90% for 3 hr. Crickets were trained to associate peppermint odor with water (reward) and vanilla odor with saline solution (non-reward) and were injected with CHX before or at different times after training. Their odor preferences were tested at 2 hr, 1 day and 4 days after training. Memory retention at 2 hr after training was unaffected by CHX injection. However, the level of retention at 1 day and 4 days after training was lowered when CHX was injected 1 hour before training or at 1 hr or 6 hr after training. To study the time course of the development of CHX-sensitive memory phase, crickets that had been injected with CHX at 1 hr after training were tested at different times from 2 to 12 hr after training. The level of retention was unaffected up to 4 hr after training but significantly lowered at 5 hr after training, and the CHX-sensitive memory phase developed gradually during the next several hours. CHX dissociates two phases of olfactory memory in crickets: earlier protein synthesis-independent phase ( 5 hr) protein synthesis-dependent phase.

  20. Acute phase protein response during acute ruminal acidosis in cattle

    DEFF Research Database (Denmark)

    Danscher, A. M.; Thoefner, M. B.; Heegaard, Peter M. H.

    2011-01-01

    The aim of the study was to describe the acute phase protein and leukocyte responses in dairy heifers during acute, oligofructose-induced ruminal acidosis. The study included 2 trials involving oral oligofructose overload (17g/kg BW) to nonpregnant Danish Holstein heifers. Trial 1 included 12...... performed.Heifers receiving oligofructose developed a profound ruminal and systemic acidosis (in Trial 1 and 2 lowest ruminal pH was 4.3±0.2 and 3.8±0.02, respectively, and minimum SBE was −9.3±4.1 and −8.9±2.8, respectively). In Trial 1, SAA concentrations were higher than baseline concentrations on all...... than control heifers at 18 and 24h after overload (max. 13.7±4.3 billions/L). Feeding had no effect on plasma fibrinogen concentrations or WBC in Trial 1.Acute ruminal and systemic acidosis caused by oligofructose overload resulted in distinct acute phase protein and leukocyte responses in dairy...

  1. Determination of free urinary cortisol in cushing's syndrome using reversed-phase high performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Eduardo Kinio Sugawara

    2010-01-01

    Full Text Available Determination of free urinary cortisol is a test of choice in the diagnosis of Cushing's syndrome. In this study, cortisol was quantified using reversed-phase high-performance liquid chromatography (RP-HPLC in urine samples previously extracted with ether and using triamcinolone acetonide as internal standard (IS. A BDS-Hypersil-C18® column, water-acetonitrile (72:28; v/v, with a flow rate of 1.0 mL/min and detection at 243 nm were used. This method showed to be both effective and efficient, with sensitivity and linearity ranging from 2.50 to 150 μg/L, and can be used in substitution to the radioimmunoassay technique within this concentration range.

  2. An integrated top-down and bottom-up strategy for characterization protein isoforms and modifications

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Si; Tolic, Nikola; Tian, Zhixin; Robinson, Errol W.; Pasa-Tolic, Ljiljana

    2011-04-15

    Bottom-up and top-down strategies are two commonly used methods for mass spectrometry (MS) based protein identification; each method has its own advantages and disadvantages. In this chapter, we describe an integrated top-down and bottom-up approach facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs a high resolution reversed phase (RP) LC separation coupled with LC eluent fraction collection and concurrent on-line MS with a high field (12 Tesla) Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. Protein elusion profiles and tentative modified protein identification are made using detected intact protein mass in conjunction with bottom-up protein identifications from the enzymatic digestion and analysis of corresponding LC fractions. Specific proteins of biological interest are incorporated into a target ion list for subsequent off-line gas-phase fragmentation that uses an aliquot of the original collected LC fraction, an aliquot of which was also used for bottom-up analysis.

  3. Rapid determination of human globin chains using reversed-phase high-performance liquid chromatography.

    Science.gov (United States)

    Wan, Jun-Hui; Tian, Pei-Ling; Luo, Wei-Hao; Wu, Bing-Yi; Xiong, Fu; Zhou, Wan-Jun; Wei, Xiang-Cai; Xu, Xiang-Min

    2012-07-15

    Reversed-phase high-performance liquid chromatography (RP-HPLC) of human globin chains is an important tool for detecting thalassemias and hemoglobin variants. The challenges of this method that limit its clinical application are a long analytical time and complex sample preparation. The aim of this study was to establish a simple, rapid and high-resolution RP-HPLC method for the separation of globin chains in human blood. Red blood cells from newborns and adults were diluted in deionized water and injected directly onto a micro-jupiter C18 reversed-phase column (250 mm × 4.6 mm) with UV detection at 280 nm. Under the conditions of varying pH or the HPLC gradient, the globin chains (pre-β, β, δ, α, (G)γ and (A)γ) were denatured and separated from the heme groups in 12 min with a retention time coefficient of variation (CV) ranging from 0.11 to 1.29% and a peak area CV between 0.32% and 4.86%. Significant differences (P<0.05) among three groups (normal, Hb H and β thalassemia) were found in the area ratio of α/pre-β+β applying the rapid elution procedure, while P≥0.05 was obtained between the normal and α thalassemia silent/trait group. Based on the ANOVA results, receiver operating characteristic (ROC) curve analysis of the δ/β and α/pre-β+β area ratios showed a sensitivity of 100.0%, and a specificity of 100.0% for indicating β thalassemia carriers, and a sensitivity of 96.6% and a specificity of 89.6% for the prediction of hemoglobin H (Hb H) disease. The proposed cut-off was 0.026 of δ/β for β thalassemia carriers and 0.626 of α/pre-β+β for Hb H disease. In addition, abnormal hemoglobin hemoglobin E (Hb E) and Hb Westmead (Hb WS) were successfully identified using this RP-HPLC method. Our experience in developing this RP-HPLC method for the rapid separation of human globin chains could be of use for similar work. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Unique Reversible Crystal-to-Crystal Phase Transition – Structural and Functional Properties of Fused Ladder Thienoarenes

    KAUST Repository

    Abe, Yuichiro

    2017-08-15

    Donor-acceptor type molecules based on fused ladder thienoarenes, indacenodithiophene (IDT) and dithienocyclopenta-thienothiophene (DTCTT), coupled with benzothiadiazole, are prepared and their solid-state structures are investigated. They display a rich variety of solid phases ranging from amorphous glass states to crystalline states, upon changes in the central aromatic core and side group structures. Most notably, the DTCTT-based derivatives showed reversible crystal-to-crystal phase transitions in heating and cooling cycles. Unlike what has been seen in π−conjugated molecules variable temperature XRD revealed that structural change occurs continuously during the transition. A columnar self-assembled structure with slip-stacked π−π interaction is proposed to be involved in the solid-state. This research provides the evidence of unique structural behavior of the DTCTT-based molecules through the detailed structural analysis. This unique structural transition paves the way for these materials to have self-healing of crystal defects, leading to improved optoelectronic properties.

  5. Unique Reversible Crystal-to-Crystal Phase Transition – Structural and Functional Properties of Fused Ladder Thienoarenes

    KAUST Repository

    Abe, Yuichiro; Savikhin, Victoria; Yin, Jun; Grimsdale, Andrew C.; Soci, Cesare; Toney, Michael F.; Lam, Yeng Ming

    2017-01-01

    Donor-acceptor type molecules based on fused ladder thienoarenes, indacenodithiophene (IDT) and dithienocyclopenta-thienothiophene (DTCTT), coupled with benzothiadiazole, are prepared and their solid-state structures are investigated. They display a rich variety of solid phases ranging from amorphous glass states to crystalline states, upon changes in the central aromatic core and side group structures. Most notably, the DTCTT-based derivatives showed reversible crystal-to-crystal phase transitions in heating and cooling cycles. Unlike what has been seen in π−conjugated molecules variable temperature XRD revealed that structural change occurs continuously during the transition. A columnar self-assembled structure with slip-stacked π−π interaction is proposed to be involved in the solid-state. This research provides the evidence of unique structural behavior of the DTCTT-based molecules through the detailed structural analysis. This unique structural transition paves the way for these materials to have self-healing of crystal defects, leading to improved optoelectronic properties.

  6. Application of acute phase protein measurements in veterinary clinical chemistry

    DEFF Research Database (Denmark)

    Petersen, Henning; Nielsen, J. P.; Heegaard, Peter M. H.

    2004-01-01

    The body's early defence in response to trauma, inflammation or infection, the acute phase response, is a complex set of systemic reactions seen shortly after exposure to a triggering event. One of the many components is an acute phase protein response in which increased hepatic synthesis leads t...... A and their possible use as non-specific indicators of health in large animal veterinary medicine such as in the health status surveillance of pigs at the herd level, for the detection of mastitis in dairy cattle and for the prognosis of respiratory diseases in horses....

  7. Experimental determination and thermodynamic modeling of phase equilibrium and protein partitioning in aqueous two-phase systems containing biodegradable salts

    International Nuclear Information System (INIS)

    Perez, Brenda; Malpiedi, Luciana Pellegrini; Tubío, Gisela; Nerli, Bibiana; Alcântara Pessôa Filho, Pedro de

    2013-01-01

    Highlights: ► Binodal data of systems (water + polyethyleneglycol + sodium) succinate are reported. ► Pitzer model describes the phase equilibrium of systems formed by polyethyleneglycol and biodegradable salts satisfactorily. ► This simple thermodynamic framework was able to predict the partitioning behaviour of model proteins acceptably well. - Abstract: Phase diagrams of sustainable aqueous two-phase systems (ATPSs) formed by polyethyleneglycols (PEGs) of different average molar masses (4000, 6000, and 8000) and sodium succinate are reported in this work. Partition coefficients (Kps) of seven model proteins: bovine serum albumin, catalase, beta-lactoglobulin, alpha-amylase, lysozyme, pepsin, urease and trypsin were experimentally determined in these systems and in ATPSs formed by the former PEGs and other biodegradable sodium salts: citrate and tartrate. An extension of Pitzer model comprising long and short-range term contributions to the excess Gibbs free energy was used to describe the (liquid + liquid) equilibrium. Comparison between experimental and calculated tie line data showed mean deviations always lower than 3%, thus indicating a good correlation. The partition coefficients were modeled by using the same thermodynamic approach. Predicted and experimental partition coefficients correlated quite successfully. Mean deviations were found to be lower than the experimental uncertainty for most of the assayed proteins.

  8. Optical reversible programmable Boolean logic unit.

    Science.gov (United States)

    Chattopadhyay, Tanay

    2012-07-20

    Computing with reversibility is the only way to avoid dissipation of energy associated with bit erase. So, a reversible microprocessor is required for future computing. In this paper, a design of a simple all-optical reversible programmable processor is proposed using a polarizing beam splitter, liquid crystal-phase spatial light modulators, a half-wave plate, and plane mirrors. This circuit can perform 16 logical operations according to three programming inputs. Also, inputs can be easily recovered from the outputs. It is named the "reversible programmable Boolean logic unit (RPBLU)." The logic unit is the basic building block of many complex computational operations. Hence the design is important in sense. Two orthogonally polarized lights are defined here as two logical states, respectively.

  9. Artificial neural network modelling of retention of pesticides in various octadecylsiloxane-bonded reversed-phase columns and water-acetonitrile mobile phase

    Energy Technology Data Exchange (ETDEWEB)

    D' Archivio, Angelo Antonio, E-mail: angeloantonio.darchivio@univaq.it [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy); Maggi, Maria Anna; Mazzeo, Pietro; Ruggieri, Fabrizio [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy)

    2009-07-30

    Previously, retention of 26 pesticides in the reversed-phase column Gemini (Phenomenex) and water-acetonitrile mobile phase was modelled using a feed-forward artificial neural network (ANN) learned by error back-propagation, accounting for both the effect of solute structure and mobile phase composition. To this end, log K{sub ow} of solutes and four quantum chemical molecular descriptors (the dipole moment, the mean polarizability, the anisotropy of the polarizability and an hydrogen-bonding descriptor based on the atomic charges located on the acid and basic functional groups) and acetonitrile % (v/v) in the eluent (%ACN) were used as ANN inputs. The above ANN-based approach is here tested on further five similar octadecylsiloxane-bonded columns in water-acetonitrile mobile phase within the %ACN range 30-70%. A quite good predictive performance evaluated on three external solutes in the whole %ACN range is observed, prediction errors being lower than {+-}0.1 log k units or slightly higher although still within {+-}0.15 log k units. On the other hand, multilinear regression used in place of ANN provides a more diffuse and non-uniform residual distribution for all the investigated columns. ANN multiple-column retention prediction is attempted by adding to the above variables a column descriptor defined as the average retention of calibration solutes extrapolated to 100% water. This more general model is built using 16 solutes and five 5-{mu}m columns in calibration, while its predictive performance is tested on the remaining 10 compounds. Under these conditions, prediction errors are generally within {+-}0.2 log k units regardless of the kind of column. The possibility of cross-column prediction is evaluated by column leave-one-out cross-validation within the five 5-{mu}m stationary phases and on a 4-{mu}m external column. This analysis reveals that accuracy of retention prediction for unknown solutes in unknown columns is acceptable provided that the external

  10. Artificial neural network modelling of retention of pesticides in various octadecylsiloxane-bonded reversed-phase columns and water-acetonitrile mobile phase

    International Nuclear Information System (INIS)

    D'Archivio, Angelo Antonio; Maggi, Maria Anna; Mazzeo, Pietro; Ruggieri, Fabrizio

    2009-01-01

    Previously, retention of 26 pesticides in the reversed-phase column Gemini (Phenomenex) and water-acetonitrile mobile phase was modelled using a feed-forward artificial neural network (ANN) learned by error back-propagation, accounting for both the effect of solute structure and mobile phase composition. To this end, log K ow of solutes and four quantum chemical molecular descriptors (the dipole moment, the mean polarizability, the anisotropy of the polarizability and an hydrogen-bonding descriptor based on the atomic charges located on the acid and basic functional groups) and acetonitrile % (v/v) in the eluent (%ACN) were used as ANN inputs. The above ANN-based approach is here tested on further five similar octadecylsiloxane-bonded columns in water-acetonitrile mobile phase within the %ACN range 30-70%. A quite good predictive performance evaluated on three external solutes in the whole %ACN range is observed, prediction errors being lower than ±0.1 log k units or slightly higher although still within ±0.15 log k units. On the other hand, multilinear regression used in place of ANN provides a more diffuse and non-uniform residual distribution for all the investigated columns. ANN multiple-column retention prediction is attempted by adding to the above variables a column descriptor defined as the average retention of calibration solutes extrapolated to 100% water. This more general model is built using 16 solutes and five 5-μm columns in calibration, while its predictive performance is tested on the remaining 10 compounds. Under these conditions, prediction errors are generally within ±0.2 log k units regardless of the kind of column. The possibility of cross-column prediction is evaluated by column leave-one-out cross-validation within the five 5-μm stationary phases and on a 4-μm external column. This analysis reveals that accuracy of retention prediction for unknown solutes in unknown columns is acceptable provided that the external column is not very

  11. Wave-front reversal in a copper-vapor active medium

    Energy Technology Data Exchange (ETDEWEB)

    Bunkin, F.V.; Savranskii, V.V.; Shafeev, G.A.

    1981-09-01

    The implementation of wave-front reversal in a copper-vapor laser resonator is reported. The frequencies of the signal wave and the reversed wave are the same, and the dependence of reversed-signal power on input-signal power has a threshold character. Photographs of the reconstructed object image upon insertion of a distorting phase plate into the resonator are presented.

  12. Cell viability and protein turnover in nongrowing Bacillus megaterium at sporulation suppressing temperature.

    Science.gov (United States)

    Kucerová, H; Strnadová, M; Ludvík, J; Chaloupka, J

    1999-01-01

    In Bacillus megaterium, a temperature that suppresses sporulation (43 degrees C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40-41 degrees C). Here we show that, when cells grown at 35 degrees C and transferred to a sporulation medium, were subjected to shifts between 35 degrees C and the sporulation suppressing temperature (SST, 43 degrees C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35 degrees C for 2-3 h (T2-T3), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T4-T5. Protein turnover rate increased again after T2 and up to T8 60-70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T3 no asymmetric septa were observed and the amount of heat-resistant spores at T8 was by 4-5 orders lower than at 35 degrees C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T3 but then its velocity sharply dropped and the amount of degraded protein at T8 corresponded to slightly more than one-half of that found at 35 degrees C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the

  13. Isolation and characterization of a reserve protein from the seeds of Opuntia ficus-indica (Cactaceae

    Directory of Open Access Journals (Sweden)

    Uchoa A.F.

    1998-01-01

    Full Text Available We describe here the isolation and characterization of a major albumin from the seeds of Opuntia ficus-indica (Cactaceae. This protein has a molecular mass of 6.5 kDa and was isolated by a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of this protein was determined and it was shown to have similarities with the amino acid composition of several proteins from the 2S albumin storage protein family. The N-terminal amino acid sequence of this protein is Asp-Pro-Tyr-Trp-Glu-Gln-Arg.

  14. 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...

  15. A Reverse-phase Protein Microarray-based Screen Identifies Host Signaling Dynamics upon Burkholderia spp. Infection

    Science.gov (United States)

    2015-07-27

    total protein in each sample was quantified by Bradford assay (Bio-Rad). RAW264.7 cell lysate preparations were boiled for 10 min with NuPAGE LDS Sample...RPMA assays , cells were harvested, washed with PBS, and then lysed in a mixture of T-PER Reagent (Thermo Scientific) and 2X Tris-Glycine SDS sample... assay (RIPA) buffer (Thermo Scientific) containing complete protease inhibitor cocktail (Roche), and phosphatase inhibitors (Roche). The amount of

  16. Reversed Phase HPLC-DAD Profiling of Carotenoids, Chlorophylls and Phenolic Compounds in Adiantum capillus-veneris Leaves

    Directory of Open Access Journals (Sweden)

    Alam Zeb

    2017-04-01

    Full Text Available Adiantum capillus-veneris is important endangered fern species with several medicinal properties. In this study, the leaves samples were extracted and separated using reversed phase HPLC with DAD for carotenoids, chlorophylls and phenolic compounds. Separation of carotenoids and chlorophylls were carried out using a tertiary gradient system of water, MTBE and methanol-water, while a binary gradient system of methanol-water-acetic acid was used for phenolic profiling. Results revealed eight carotenoids, four pheophytins, and two chlorophylls. Lutein (806.0 μg/g, chlorophyll b′ (410.0 μg/g, chlorophyll a (162.4 μg/g, 9′-Z-neoxanthin (142.8 μg/g and all-E-violaxanthin (82.2 μg/g were present in higher amounts. The relatively high amounts of lutein may be one of the key indicator of beneficial antioxidant properties. The phenolic profile revealed a total of 13 compounds, namely 4-hydroxybenzoic acid, chlorogenic acid, caftaric acid, kaempferol glycosides, p-coumaric acid, rosmarinic acid, 5-caffeoylquinic acid, and quercetin glycosides. Kaempferol-3-sophorotrioside (58.7 mg/g, chlorogenic acid (28.5 mg/g, 5-O-caffeoylquinic acid (18.7 mg/g, coumaric acid (11.2 mg/g, and its derivative (33.1 mg/g were present in high amounts. These results suggest that the reversed phase HPLC profiling of Adiantum leaves provides a better understanding in to the actual composition of bioactive compounds, which may be responsible for the potential medicinal properties. Adiantum leaves rich in important bioactive phytochemicals can be used as a possible source of nutraceuticals or as a functional food ingredient.

  17. Simultaneous separation and analysis of water- and fat-soluble vitamins on multi-modal reversed-phase weak anion exchange material by HPLC-UV.

    Science.gov (United States)

    Dabre, Romain; Azad, Nazanin; Schwämmle, Achim; Lämmerhofer, Michael; Lindner, Wolfgang

    2011-04-01

    Several methods for the separation of vitamins on HPLC columns were already validated in the last 20 years. However, most of the techniques focus on separating either fat- or water-soluble vitamins and only few methods are intended to separate lipophilic and hydrophilic vitamins simultaneously. A mixed-mode reversed-phase weak anion exchange (RP-WAX) stationary phase was developed in our laboratory in order to address such mixture of analytes with different chemical characteristics, which are difficult to separate on standard columns. The high versatility in usage of the RP-WAX chromatographic material allowed a baseline separation of ten vitamins within a single run, seven water-soluble and three fat-soluble, using three different chromatographic modes: some positively charged vitamins are eluted in ion exclusion and ion repulsion modes whereas the negatively charged molecules are eluted in the ion exchange mechanism. The non-charged molecules are eluted in a classical reversed-phase mode, regarding their polarities. The method was validated for the vitamin analysis in tablets, evaluating selectivity, robustness, linearity, accuracy, and precision. The validated method was finally employed for the analysis of the vitamin content of some commercially available supplement tablets. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. High-resolution ultrahigh-pressure long column reversed-phase liquid chromatography for top-down proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Yufeng; Tolic, Nikola; Piehowski, Paul D.; Shukla, Anil K.; Kim, Sangtae; Zhao, Rui; Qu, Yi; Robinson, E. W.; Smith, Richard D.; Pasa-Tolic, Ljiljana

    2017-05-01

    We report development of an approach providing high-resolution RPLC of proteins and its utility for mass spectrometry-based top-down proteomics. A chromatographic peak capacity of ~450 was achieved for proteins and large polypeptides having MWs up to 43 kDa in the context of proteomics applications. RPLC column lengths from 20 to 200 cm, particle sizes from 1.5 to 5 m, bonding alkyl chains from C1 to C2, C4, C8, and C18, and particle surface structures that spanned porous, superficially porous (porous shell, core-shell), and nonporous were investigated at pressures up to14K psi. Column length was found as the most important factor for >20 kDa proteins in gradient RPLC, and shortening column length degraded RPLC resolution and sensitivity regardless of the size and surface structure of the packing particles used. The alkyl chains bonded to the silica particle surface significantly affected the RPLC recovery and efficiency, and short alkyl C1-C4 phases provided higher sensitivity and resolution than C8 and C18 phases. Long gradient separations (e.g., >10 hours) with long columns (e.g., 100 cm) were particularly effective in conjunction with use of high accuracy mass spectrometers (e.g., the Orbitrap Elite) for top-down proteomics with improved proteoform coverage by allowing multiple HCD, CID, and ETD dissociation modes. It was also found that HCD produced small fragments useful for proteoform identification, while low energy CID and ETD often complemented HCD by providing large fragments.

  19. Controlled in meso phase crystallization--a method for the structural investigation of membrane proteins.

    Directory of Open Access Journals (Sweden)

    Jan Kubicek

    Full Text Available We investigated in meso crystallization of membrane proteins to develop a fast screening technology which combines features of the well established classical vapor diffusion experiment with the batch meso phase crystallization, but without premixing of protein and monoolein. It inherits the advantages of both methods, namely (i the stabilization of membrane proteins in the meso phase, (ii the control of hydration level and additive concentration by vapor diffusion. The new technology (iii significantly simplifies in meso crystallization experiments and allows the use of standard liquid handling robots suitable for 96 well formats. CIMP crystallization furthermore allows (iv direct monitoring of phase transformation and crystallization events. Bacteriorhodopsin (BR crystals of high quality and diffraction up to 1.3 Å resolution have been obtained in this approach. CIMP and the developed consumables and protocols have been successfully applied to obtain crystals of sensory rhodopsin II (SRII from Halobacterium salinarum for the first time.

  20. Determination of mycophenolic acid in mest products using mixed mode reversed phase-anion exchange clean-up and liquid chromatography-high-resolution mass spectrometry

    DEFF Research Database (Denmark)

    Sørensen, Louise Marie; Nielsen, Kristian Fog; Jacobsen, Thomas

    2008-01-01

    A method for determination of mycophenolic acid (MPA) in dry-cured ham, fermented sausage and liver pate is described. MPA was extracted from meat with bicarbonate-acetonitrile, further cleaned-up by mixed mode reversed phase-anion exchange and detected using a LC-MS system with electrospray...

  1. Purification of an Immunoadjuvant Saponin Fraction from Quillaja brasiliensis Leaves by Reversed-Phase Silica Gel Chromatography.

    Science.gov (United States)

    Yendo, Anna C A; de Costa, Fernanda; Kauffmann, Carla; Fleck, Juliane D; Gosmann, Grace; Fett-Neto, Arthur G

    2017-01-01

    Saponins include a large variety of molecules that find several applications in pharmacology. The use of Quillaja saponaria saponins as immunological adjuvants in vaccines is of interest due to their capacity to stimulate both humoral and cellular responses. The congener species Q. brasiliensis has saponins with chemical similarities and adjuvant activity comparable to that of Q. saponaria fraction Quil-A ® , with additional advantages of showing lower toxicity and reduced hemolytic activity. Here we describe in detail the methods for preparing the aqueous extract from Q. brasiliensis leaves, as well as the purification of the bioactive saponin fraction QB-90 using silica reversed-phase chromatography.

  2. Characterization of a new antifungal non-specific lipid transfer protein (nsLTP) from sugar beet leaves

    DEFF Research Database (Denmark)

    Kristensen, A K; Brunstedt, J; Madsen, M T

    2000-01-01

    A novel protein (IWF5) comprising 92 amino acids has been purified from the intercellular washing fluid of sugar beet leaves using cation exchange chromatography and reversed phase high performance liquid chromatography. Based on amino acid sequence homology, including the presence of eight...... cysteines at conserved positions, the protein can be classified as a member of the plant family of non-specific lipid transfer proteins (nsLTPs). The protein is 47% identical to IWF1, an antifungal nsLTP previously isolated from leaves of sugar beet. A potential site for N-linked glycosylation present...

  3. GRASP/Ada (Graphical Representations of Algorithms, Structures, and Processes for Ada): The development of a program analysis environment for Ada. Reverse engineering tools for Ada, task 1, phase 2

    Science.gov (United States)

    Cross, James H., II

    1990-01-01

    The study, formulation, and generation of structures for Ada (GRASP/Ada) are discussed in this second phase report of a three phase effort. Various graphical representations that can be extracted or generated from source code are described and categorized with focus on reverse engineering. The overall goal is to provide the foundation for a CASE (computer-aided software design) environment in which reverse engineering and forward engineering (development) are tightly coupled. Emphasis is on a subset of architectural diagrams that can be generated automatically from source code with the control structure diagram (CSD) included for completeness.

  4. Reversed phase propagation for hyperbolic surface waves

    DEFF Research Database (Denmark)

    Repän, Taavi; Novitsky, Andrey; Willatzen, Morten

    2018-01-01

    Magnetic properties can be used to control phase propagation in hyperbolic metamaterials. However, in the visible spectrum magnetic properties are difficult to obtain. We discuss hyperbolic surface waves allowing for a similar control over phase, achieved without magnetic properties....

  5. [Separation and purification of the components in Trachelospermum jasminoides by two dimensional hydrophilic interaction liquid chromatography- reversed-phase liquid chromatography].

    Science.gov (United States)

    Jia, Youmei; Cai, Jianfeng; Xin, Huaxia; Feng, Jiatao; Fu, Yanhui; Fu, Qing; Jin, Yu

    2017-06-08

    A preparative two dimensional hydrophilic interaction liquid chromatography/reversed-phase liquid chromatography (Pre-2D-HILIC/RPLC) method was established to separate and purify the components in Trachelospermum jasminoides . The pigments and strongly polar components were removed from the crude extract after the active carbon decolorization and solid phase extraction processes. A Click XIon column (250 mm×20 mm, 10 μm) was selected as stationary phase and water-acetonitrile as mobile phases in the first dimensional HILIC. Finally, 15 fractions were collected under UV-triggered mode. In the second dimensional RPLC, a C18 column (250 mm×20 mm, 5 μm) was selected and water-acetonitrile was used as mobile phases. As a result, 14 compounds with high purity were obtained, which were further identified by mass spectrometry (MS) and nuclear magnetic resonance (NMR). Finally, 11 lignan compounds and three flavonoid compounds were obtained. The method has a good orthogonality, and can improve the resolution and the peak capacity. It is significant for the separation of complex components from Trachelospermum jasminoides .

  6. Probing the Binding Interfaces of Protein Complexes Using Gas-Phase H/D Exchange Mass Spectrometry

    DEFF Research Database (Denmark)

    Mistarz, Ulrik H; Brown, Jeffery M; Haselmann, Kim F

    2016-01-01

    Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub-milliseco......Fast gas-phase hydrogen/deuterium exchange mediated by ND3 gas and measured by mass spectrometry (gas-phase HDX-MS) is a largely unharnessed, fast, and sensitive method for probing primary- and higher-order polypeptide structure. Labeling of heteroatom-bound non-amide hydrogens in a sub......-millisecond time span after electrospray ionization by ND3 gas can provide structural insights into protein conformers present in solution. Here, we have explored the use of gas-phase HDX-MS for probing the higher-order structure and binding interfaces of protein complexes originating from native solution...

  7. HIV infection and drugs of abuse: role of acute phase proteins.

    Science.gov (United States)

    Samikkannu, Thangavel; Rao, Kurapati V K; Arias, Adriana Y; Kalaichezian, Aarthi; Sagar, Vidya; Yoo, Changwon; Nair, Madhavan P N

    2013-09-17

    HIV infection and drugs of abuse such as methamphetamine (METH), cocaine, and alcohol use have been identified as risk factors for triggering inflammation. Acute phase proteins such as C-reactive protein (CRP) and serum amyloid A (SAA) are the biomarkers of inflammation. Hence, the interactive effect of drugs of abuse with acute phase proteins in HIV-positive subjects was investigated. Plasma samples were utilized from 75 subjects with METH use, cocaine use, alcohol use, and HIV-positive alone and HIV-positive METH, cocaine, and alcohol users, and age-matched control subjects. The plasma CRP and SAA levels were measured by ELISA and western blot respectively and the CD4 counts were also measured. Observed results indicated that the CRP and SAA levels in HIV-positive subjects who are METH, cocaine and alcohol users were significantly higher when compared with either drugs of abuse or HIV-positive alone. The CD4 counts were also dramatically reduced in HIV-positive with drugs of abuse subjects compared with only HIV-positive subjects. These results suggest that, in HIV-positive subjects, drugs of abuse increase the levels of CRP and SAA, which may impact on the HIV infection and disease progression.

  8. Nonclassical nucleation pathways in protein crystallization.

    Science.gov (United States)

    Zhang, Fajun

    2017-11-08

    Classical nucleation theory (CNT), which was established about 90 years ago, has been very successful in many research fields, and continues to be the most commonly used theory in describing the nucleation process. For a fluid-to-solid phase transition, CNT states that the solute molecules in a supersaturated solution reversibly form small clusters. Once the cluster size reaches a critical value, it becomes thermodynamically stable and favored for further growth. One of the most important assumptions of CNT is that the nucleation process is described by one reaction coordinate and all order parameters proceed simultaneously. Recent studies in experiments, computer simulations and theory have revealed nonclassical features in the early stage of nucleation. In particular, the decoupling of order parameters involved during a fluid-to-solid transition leads to the so-called two-step nucleation mechanism, in which a metastable intermediate phase (MIP) exists between the initial supersaturated solution and the final crystals. Depending on the exact free energy landscapes, the MIPs can be a high density liquid phase, mesoscopic clusters, or a pre-ordered state. In this review, we focus on the studies of nonclassical pathways in protein crystallization and discuss the applications of the various scenarios of two-step nucleation theory. In particular, we focus on protein solutions in the presence of multivalent salts, which serve as a model protein system to study the nucleation pathways. We wish to point out the unique features of proteins as model systems for further studies.

  9. Nonclassical nucleation pathways in protein crystallization

    Science.gov (United States)

    Zhang, Fajun

    2017-11-01

    Classical nucleation theory (CNT), which was established about 90 years ago, has been very successful in many research fields, and continues to be the most commonly used theory in describing the nucleation process. For a fluid-to-solid phase transition, CNT states that the solute molecules in a supersaturated solution reversibly form small clusters. Once the cluster size reaches a critical value, it becomes thermodynamically stable and favored for further growth. One of the most important assumptions of CNT is that the nucleation process is described by one reaction coordinate and all order parameters proceed simultaneously. Recent studies in experiments, computer simulations and theory have revealed nonclassical features in the early stage of nucleation. In particular, the decoupling of order parameters involved during a fluid-to-solid transition leads to the so-called two-step nucleation mechanism, in which a metastable intermediate phase (MIP) exists between the initial supersaturated solution and the final crystals. Depending on the exact free energy landscapes, the MIPs can be a high density liquid phase, mesoscopic clusters, or a pre-ordered state. In this review, we focus on the studies of nonclassical pathways in protein crystallization and discuss the applications of the various scenarios of two-step nucleation theory. In particular, we focus on protein solutions in the presence of multivalent salts, which serve as a model protein system to study the nucleation pathways. We wish to point out the unique features of proteins as model systems for further studies.

  10. Integration of carboxyl modified magnetic particles and aqueous two-phase extraction for selective separation of proteins.

    Science.gov (United States)

    Gai, Qingqing; Qu, Feng; Zhang, Tao; Zhang, Yukui

    2011-07-15

    Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Isolation of 62 kda protein with antioxidant properties from natural honey

    International Nuclear Information System (INIS)

    Mohammed, S.E.A.R.

    2014-01-01

    Fourteen natural honey samples from Libya, Sudan and Pakistan were evaluated for their antioxidant activity by employing 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical assay. The scavenging activity of honey samples were in the range of 18-32% when compared to control. A 62 kDa protein was isolated from honey by gel filtration chromatography followed by reverse phase HPLC showed significant radical scavenging activity. The research pointed out the antioxidative role of honey proteins and possibility of their contribution to the therapeutic value of the natural honey. (author)

  12. Preparation of a novel dual-function strong cation exchange/hydrophobic interaction chromatography stationary phase for protein separation.

    Science.gov (United States)

    Zhao, Kailou; Yang, Li; Wang, Xuejiao; Bai, Quan; Yang, Fan; Wang, Fei

    2012-08-30

    We have explored a novel dual-function stationary phase which combines both strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC) characteristics. The novel dual-function stationary phase is based on porous and spherical silica gel functionalized with ligand containing sulfonic and benzyl groups capable of electrostatic and hydrophobic interaction functionalities, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed. As a result, it can be employed to separate proteins with SCX and HIC modes, respectively. The resolution and selectivity of the dual-function stationary phase were evaluated under both HIC and SCX modes with standard proteins and can be comparable to that of conventional IEC and HIC columns. More than 96% of mass and bioactivity recoveries of proteins can be achieved in both HIC and SCX modes, respectively. The results indicated that the novel dual-function column could replace two individual SCX and HIC columns for protein separation. Mixed retention mechanism of proteins on this dual-function column based on stoichiometric displacement theory (SDT) in LC was investigated to find the optimal balance of the magnitude of electrostatic and hydrophobic interactions between protein and the ligand on the silica surface in order to obtain high resolution and selectivity for protein separation. In addition, the effects of the hydrophobicity of the ligand of the dual-function packings and pH of the mobile phase used on protein separation were also investigated in detail. The results show that the ligand with suitable hydrophobicity to match the electrostatic interaction is very important to prepare the dual-function stationary phase, and a better resolution and selectivity can be obtained at pH 6.5 in SCX mode. Therefore, the dual-function column can replace two individual SCX and HIC columns for protein separation and be used to set up two-dimensional liquid

  13. Two-phase aqueous micellar systems: an alternative method for protein purification

    Directory of Open Access Journals (Sweden)

    Rangel-Yagui C. O.

    2004-01-01

    Full Text Available Two-phase aqueous micellar systems can be exploited in separation science for the extraction/purification of desired biomolecules. This article reviews recent experimental and theoretical work by Blankschtein and co-workers on the use of two-phase aqueous micellar systems for the separation of hydrophilic proteins. The experimental partitioning behavior of the enzyme glucose-6-phosphate dehydrogenase (G6PD in two-phase aqueous micellar systems is also reviewed and new results are presented. Specifically, we discuss very recent work on the purification of G6PD using: i a two-phase aqueous micellar system composed of the nonionic surfactant n-decyl tetra(ethylene oxide (C10E4, and (ii a two-phase aqueous mixed micellar system composed of C10E4 and the cationic surfactant decyltrimethylammonium bromide (C10TAB. Our results indicate that the two-phase aqueous mixed (C10E4/C10TAB micellar system can improve significantly the partitioning behavior of G6PD relative to that observed in the two-phase aqueous C10E4 micellar system.

  14. A rapid hydrolysis method and DABS-Cl derivatization for complete amino acid analysis of octreotide acetate by reversed phase HPLC.

    Science.gov (United States)

    Akhlaghi, Yousef; Ghaffari, Solmaz; Attar, Hossein; Alamir Hoor, Amir

    2015-11-01

    Octreotide as a synthetic cyclic octapeptide is a somatostatin analog with longer half-life and more selectivity for inhibition of the growth hormone. The acetate salt of octreotide is currently used for medical treatment of somatostatin-related disorders such as endocrine and carcinoid tumors, acromegaly, and gigantism. Octreotide contains both cysteine and tryptophan residues which make the hydrolysis part of its amino acid analysis procedure very challenging. The current paper introduces a fast and additive-free method which preserves tryptophan and cysteine residues during the hydrolysis. Using only 6 M HCl, this hydrolysis process is completed in 30 min at 150 °C. This fast hydrolysis method followed by pre-column derivatization of the released amino acids with 4-N,N-dimethylaminoazobenzene-4'-sulfonyl chloride (DABS-Cl) which takes only 20 min, makes it possible to do the complete amino acid analysis of an octreotide sample in a few hours. The highly stable-colored DABS-Cl derivatives can be detected in 436 nm in a reversed phase chromatographic system, which eliminates spectral interferences to a great extent. The amino acid analysis of octreotide acetate including hydrolysis, derivatization, and reversed phase HPLC determination was validated according to International Conference of Harmonization (ICH) guidelines.

  15. Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Xueqin; Wang, Yuzhi, E-mail: wyzss@hnu.edu.cn; Zeng, Qun; Chen, Jing; Huang, Yanhua; Xu, Kaijia

    2014-03-01

    Graphical abstract: - Highlights: • A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been synthesized. • Functional guanidinium ionic liquid aqueous two-phase systems have been first designed for the purification of protein. • Mechanisms and performances of the process were researched. • Simple, green, safety and presents better purified ability than ordinary process. • A potential efficient platform for protein purification and related studies. - Abstract: A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by {sup 1}H nuclear magnetic resonance ({sup 1}H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV–vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV–vis spectrophotometer, Fourier-transform infrared

  16. Poly(dodecyl methacrylate) as solvent of paraffins for phase change materials and thermally reversible light scattering films.

    Science.gov (United States)

    Puig, Julieta; Williams, Roberto J J; Hoppe, Cristina E

    2013-09-25

    Paraffins are typical organic phase change materials (PCM) used for latent heat storage. For practical applications they must be encapsulated to prevent leakage or agglomeration during fusion. In this study it is shown that eicosane (C20H42 = C20) in the melted state could be dissolved in the hydrophobic domains of poly(dodecyl methacrylate) (PDMA) up to concentrations of 30 wt %, avoiding the need of encapsulation. For a 30 wt % solution, the heat of phase change was close to 69 J/g, a reasonable value for its use as a PCM. The fully converted solution remained transparent at 80 °C with no evidence of phase separation but became opaque by cooling as a consequence of paraffin crystallization. Heating above the melting temperature regenerated a transparent material. A high contrast ratio and abrupt transition between opaque and transparent states was observed for the 30 wt % blends, with a transparent state at 35 °C and an opaque state at 23 °C. This behavior was completely reproducible during consecutive heating/cooling cycles, indicating the possible use of this material as a thermally reversible light scattering (TRLS) film.

  17. Circulating microbial products and acute phase proteins as markers of pathogenesis in lymphatic filarial disease.

    Directory of Open Access Journals (Sweden)

    R Anuradha

    Full Text Available Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+ or without (CP Ag- active infection; with clinically asymptomatic infections (INF; and in those without infection (endemic normal [EN]. Comparisons between the two actively infected groups (CP Ag+ compared to INF and those without active infection (CP Ag- compared to EN were used preliminarily to identify markers of pathogenesis. Thereafter, we tested for group effects among all the four groups using linear models on the log transformed responses of the markers. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein, acute phase proteins (haptoglobin and serum amyloid protein-A, and inflammatory cytokines (IL-1β, IL-12, and TNF-α are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins.

  18. Phase behaviour and in vitro hydrolysis of wheat starch in mixture with whey protein.

    Science.gov (United States)

    Yang, Natasha; Liu, Yingting; Ashton, John; Gorczyca, Elisabeth; Kasapis, Stefan

    2013-04-15

    Network formation of whey protein isolate (WPI) with increasing concentrations of native wheat starch (WS) has been examined. Small deformation dynamic oscillation in shear and modulated temperature differential scanning calorimetry enabled analysis of binary mixtures at the macro- and micromolecular level. Following heat induced gelation, textural hardness was measured by undertaking compression tests. Environmental scanning electron microscopy provided tangible information on network morphology of polymeric constituents. Experiments involving in vitro starch digestion also allowed for indirect assessment of phase topology in the binary mixture. The biochemical component of this work constitutes an attempt to utilise whey protein as a retardant to the enzymatic hydrolysis of starch in a model system with α-amylase enzyme. During heating, rheological profiles of binary mixtures exhibited dramatic increases in G' at temperatures more closely related to those observed for single whey protein rather than pure starch. Results from this multidisciplinary approach of analysis, utilising rheology, calorimetry and microscopy, argue for the occurrence of phase separation phenomena in the gelled systems. There is also evidence of whey protein forming the continuous phase with wheat starch being the discontinuous filler, an outcome that is explored in the in vitro study of the enzymatic hydrolysis of starch. Copyright © 2012. Published by Elsevier Ltd.

  19. Inhibiting Heat-Shock Protein 90 Reverses Sensory Hypoalgesia in Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Michael J Urban

    2010-07-01

    Full Text Available Increasing the expression of Hsp70 (heat-shock protein 70 can inhibit sensory neuron degeneration after axotomy. Since the onset of DPN (diabetic peripheral neuropathy is associated with the gradual decline of sensory neuron function, we evaluated whether increasing Hsp70 was sufficient to improve several indices of neuronal function. Hsp90 is the master regulator of the heat-shock response and its inhibition can up-regulate Hsp70. KU-32 (N-{7-[(2R, 3R, 4S, 5R-3, 4-dihydroxy-5-methoxy-6, 6-dimethyl-tetrahydro-2H-pyran-2-yloxy]-8-methyl-2-oxo-2H-chromen-3-yl}acetamide was developed as a novel, novobiocin-based, C-terminal inhibitor of Hsp90 whose ability to increase Hsp70 expression is linked to the presence of an acetamide substitution of the prenylated benzamide moiety of novobiocin. KU-32 protected against glucose-induced death of embryonic DRG (dorsal root ganglia neurons cultured for 3 days in vitro. Similarly, KU-32 significantly decreased neuregulin 1-induced degeneration of myelinated Schwann cell DRG neuron co-cultures prepared from WT (wild-type mice. This protection was lost if the co-cultures were prepared from Hsp70.1 and Hsp70.3 KO (knockout mice. KU-32 is readily bioavailable and was administered once a week for 6 weeks at a dose of 20 mg/kg to WT and Hsp70 KO mice that had been rendered diabetic with streptozotocin for 12 weeks. After 12 weeks of diabetes, both WT and Hsp70 KO mice developed deficits in NCV (nerve conduction velocity and a sensory hypoalgesia. Although KU-32 did not improve glucose levels, HbA1c (glycated haemoglobin or insulin levels, it reversed the NCV and sensory deficits in WT but not Hsp70 KO mice. These studies provide the first evidence that targeting molecular chaperones reverses the sensory hypoalgesia associated with DPN.

  20. Dynamical similarity of geomagnetic field reversals.

    Science.gov (United States)

    Valet, Jean-Pierre; Fournier, Alexandre; Courtillot, Vincent; Herrero-Bervera, Emilio

    2012-10-04

    No consensus has been reached so far on the properties of the geomagnetic field during reversals or on the main features that might reveal its dynamics. A main characteristic of the reversing field is a large decrease in the axial dipole and the dominant role of non-dipole components. Other features strongly depend on whether they are derived from sedimentary or volcanic records. Only thermal remanent magnetization of lava flows can capture faithful records of a rapidly varying non-dipole field, but, because of episodic volcanic activity, sequences of overlying flows yield incomplete records. Here we show that the ten most detailed volcanic records of reversals can be matched in a very satisfactory way, under the assumption of a common duration, revealing common dynamical characteristics. We infer that the reversal process has remained unchanged, with the same time constants and durations, at least since 180 million years ago. We propose that the reversing field is characterized by three successive phases: a precursory event, a 180° polarity switch and a rebound. The first and third phases reflect the emergence of the non-dipole field with large-amplitude secular variation. They are rarely both recorded at the same site owing to the rapidly changing field geometry and last for less than 2,500 years. The actual transit between the two polarities does not last longer than 1,000 years and might therefore result from mechanisms other than those governing normal secular variation. Such changes are too brief to be accurately recorded by most sediments.

  1. Reversal learning and resurgence of operant behavior in zebrafish (Danio rerio).

    Science.gov (United States)

    Kuroda, Toshikazu; Mizutani, Yuto; Cançado, Carlos R X; Podlesnik, Christopher A

    2017-09-01

    Zebrafish are used extensively as vertebrate animal models in biomedical research for having such features as a fully sequenced genome and transparent embryo. Yet, operant-conditioning studies with this species are scarce. The present study investigated reversal learning and resurgence of operant behavior in zebrafish. A target response (approaching a sensor) was reinforced in Phase 1. In Phase 2, the target response was extinguished while reinforcing an alternative response (approaching a different sensor). In Phase 3, extinction was in effect for the target and alternative responses. Reversal learning was demonstrated when responding tracked contingency changes between Phases 1 and 2. Moreover, resurgence occurred in 10 of 13 fish in Phase 3: Target response rates increased transiently and exceeded rates of an unreinforced control response. The present study provides the first evidence with zebrafish supporting reversal learning between discrete operant responses and a laboratory model of relapse. These findings open the possibility to assessing genetic influences of operant behavior generally and in models of relapse (e.g., resurgence, renewal, reinstatement). Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Naltrexone Reverses Ethanol Preference and Protein Kinase C Activation in Drosophila melanogaster

    Directory of Open Access Journals (Sweden)

    Rajeswari Koyyada

    2018-03-01

    Full Text Available Alcohol use disorder (AUD is a major health, social and economic problem for which there are few effective treatments. The opiate antagonist naltrexone is currently prescribed clinically with mixed success. We have used naltrexone in an established behavioral assay (CAFE in Drosophila melanogaster that measures the flies' preference for ethanol-containing food. We have confirmed that Drosophila exposed to ethanol develop a preference toward this drug and we demonstrate that naltrexone, in a dose dependant manner, reverses the ethanol-induced ethanol preference. This effect is not permanent, as preference for alcohol returns after discontinuing naltrexone. Additionally, naltrexone reduced the alcohol-induced increase in protein kinase C activity. These findings are of interest because they confirm that Drosophila is a useful model for studying human responses to addictive drugs. Additionally because of the lack of a closely conserved opiate system in insects, our results could either indicate that a functionally related system does exist in insects or that in insects, and potentially also in mammals, naltrexone binds to alternative sites. Identifying such sites could lead to improved treatment strategies for AUD.

  3. Naltrexone Reverses Ethanol Preference and Protein Kinase C Activation in Drosophila melanogaster

    Science.gov (United States)

    Koyyada, Rajeswari; Latchooman, Nilesh; Jonaitis, Julius; Ayoub, Samir S.; Corcoran, Olivia; Casalotti, Stefano O.

    2018-01-01

    Alcohol use disorder (AUD) is a major health, social and economic problem for which there are few effective treatments. The opiate antagonist naltrexone is currently prescribed clinically with mixed success. We have used naltrexone in an established behavioral assay (CAFE) in Drosophila melanogaster that measures the flies' preference for ethanol-containing food. We have confirmed that Drosophila exposed to ethanol develop a preference toward this drug and we demonstrate that naltrexone, in a dose dependant manner, reverses the ethanol-induced ethanol preference. This effect is not permanent, as preference for alcohol returns after discontinuing naltrexone. Additionally, naltrexone reduced the alcohol-induced increase in protein kinase C activity. These findings are of interest because they confirm that Drosophila is a useful model for studying human responses to addictive drugs. Additionally because of the lack of a closely conserved opiate system in insects, our results could either indicate that a functionally related system does exist in insects or that in insects, and potentially also in mammals, naltrexone binds to alternative sites. Identifying such sites could lead to improved treatment strategies for AUD. PMID:29593550

  4. Purification of reversibly oxidized proteins (PROP reveals a redox switch controlling p38 MAP kinase activity.

    Directory of Open Access Journals (Sweden)

    Dennis J Templeton

    2010-11-01

    Full Text Available Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.

  5. Retention prediction of highly polar ionizable solutes under gradient conditions on a mixed-mode reversed-phase and weak anion-exchange stationary phase.

    Science.gov (United States)

    Balkatzopoulou, P; Fasoula, S; Gika, H; Nikitas, P; Pappa-Louisi, A

    2015-05-29

    In the present work the retention of three highly polar and ionizable solutes - uric acid, nicotinic acid and ascorbic acid - was investigated on a mixed-mode reversed-phase and weak anion-exchange (RP/WAX) stationary phase in buffered aqueous acetonitrile (ACN) mobile phases. A U-shaped retention behavior was observed for all solutes with respect to the eluent organic modifier content studied in a range of 5-95% (v/v). This retention behavior clearly demonstrates the presence of a HILIC-type retention mechanism at ACN-rich hydro-organic eluents and an RP-like retention at aqueous-rich hydro-organic eluents. Hence, this column should be promising for application under both RP and HILIC gradient elution modes. For this reason, a series of programmed elution runs were carried out with increasing (RP) and decreasing (HILIC) organic solvent concentration in the mobile phase. This dual gradient process was successfully modeled by two retention models exhibiting a quadratic or a cubic dependence of the logarithm of the solute retention factor (lnk) upon the organic modifier volume fraction (φ). It was found that both models produced by gradient retention data allow the prediction of solute retention times for both types of programmed elution on the mixed-mode column. Four, in the case of the quadratic model, or five, in the case of the cubic model, initial HILIC- and RP-type gradient runs gave satisfactory retention predictions of any similar kind elution program, even with different flow rate, with an overall error of only 2.5 or 1.7%, respectively. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Acute phase proteins as diagnostic markers in horses with colic

    DEFF Research Database (Denmark)

    Pihl, Tina; Scheepers, Elrien; Sanz, Macarena

    2016-01-01

    Objective – To investigate the diagnostic potential of the concentrations of acute-phase proteins serum amyloid A (SAA), haptoglobin (Hp), and fibrinogen in blood and peritoneal fluid (PF) for differentiating horses within flammatory colic (entero-colitis and peritonitis) from those with surgical...... of the model to correctly differentiate inflammatory from surgical colic was 86% determined as area under the receiver operating characteristic curve. Adding blood parameters (WBC, PCV, total plasma protein, lactate, SAA, Hp, and fibrinogen concentrations) to the logistic model based on clinical parameters...... revealed that only WBC and SAA and fibrinogen concentrations improved the model. With SAA included in the model no additional blood parameters improved the model, and the final model had an area under the curve of 90%. Addition of PF parameters (hemolysis, total protein concentration, WBC, SAA, or Hp...

  7. Singular anti-RNA virus-directed proteins.

    Directory of Open Access Journals (Sweden)

    Rayanade R

    2000-07-01

    Full Text Available AIMS: To additionally purify and characterise the anti-RNA virus-directed protein termed p14. MATERIALS AND METHODS: Antiviral assays of p14 against RNA and DNA viruses were carried out and its antigenic similarities with chicken interferon (CIFN were studied. HPLC-Reverse Phase of p14 was performed to further purify p14. RESULTS: p14 showed antiviral activity against RNA viruses only and not against DNA viruses. It was antigenically distinct from CIFN. Purification of p14 yielded three proteins with antiviral activity, which had different physico-chemical properties than those described for interferons. CONCLUSIONS: The data presented on the antiviral, immunological and physico-chemical properties, establish the unique nature of p14 vis-á-vis those of interferons.

  8. Roles of beta-turns in protein folding: from peptide models to protein engineering.

    Science.gov (United States)

    Marcelino, Anna Marie C; Gierasch, Lila M

    2008-05-01

    Reverse turns are a major class of protein secondary structure; they represent sites of chain reversal and thus sites where the globular character of a protein is created. It has been speculated for many years that turns may nucleate the formation of structure in protein folding, as their propensity to occur will favor the approximation of their flanking regions and their general tendency to be hydrophilic will favor their disposition at the solvent-accessible surface. Reverse turns are local features, and it is therefore not surprising that their structural properties have been extensively studied using peptide models. In this article, we review research on peptide models of turns to test the hypothesis that the propensities of turns to form in short peptides will relate to the roles of corresponding sequences in protein folding. Turns with significant stability as isolated entities should actively promote the folding of a protein, and by contrast, turn sequences that merely allow the chain to adopt conformations required for chain reversal are predicted to be passive in the folding mechanism. We discuss results of protein engineering studies of the roles of turn residues in folding mechanisms. Factors that correlate with the importance of turns in folding indeed include their intrinsic stability, as well as their topological context and their participation in hydrophobic networks within the protein's structure.

  9. Magnetic reversal processes and critical thickness in FePt/α-Fe/FePt trilayers

    International Nuclear Information System (INIS)

    Guo, N.L.; Zhao, G.P.; Zhang, H.W.; Zhou, X.L.; Deng, Y.

    2011-01-01

    Magnetic reversal processes of a FePt/α-Fe/FePt trilayer system with in-plane easy axes have been investigated within a micromagnetic approach. It is found that the magnetic reversal process consists of three steps: nucleation of a prototype of domain wall in the soft phase, the evolution as well as the motion of the domain wall from the soft to the hard phase and finally, the magnetic reversal of the hard phase. For small soft layer thickness L s , the three steps are reduced to one single step, where the magnetizations in the two phases reverses simultaneously and the hysteresis loops are square with nucleation as the coercivity mechanism. As L s increases, both nucleation and pinning fields decrease. In the meantime, the single-step reversal expands to a standard three-step one and the coercivity mechanism changes from nucleation to pinning. The critical thickness where the coercivity mechanism alters, could be derived analytically, which is found to be inversely proportional to the square root of the crystalline anisotropy of the hard phase. Such a scaling law might provide an easy way to test the present theory. Further increase of L s leads to the change of the coercivity mechanism from pinning to nucleation. - Highlights: → Analytical critical thickness scales with square root of anisotropy of hard phase. → Reversal process determined nucleation, motion and depinning of domain walls. → Coercivity mechanism is nucleation and pinning for thin and thick soft layers. → Microscopic and macroscopic hysteresis loops calculated.

  10. Enhancing the lateral-flow immunoassay for detection of proteins using an aqueous two-phase micellar system.

    Science.gov (United States)

    Mashayekhi, Foad; Le, Alexander M; Nafisi, Parsa M; Wu, Benjamin M; Kamei, Daniel T

    2012-10-01

    The lateral-flow (immuno)assay (LFA) has been widely investigated for the detection of molecular, macromolecular, and particle targets at the point-of-need due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements. However, for some analytes, such as certain proteins, the detection limit of LFA is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay, and needs to be improved. One solution for improving the detection limit of LFA is to concentrate the target protein in a solution prior to the detection step. In this study, a novel approach was used in the context of an aqueous two-phase micellar system comprised of the nonionic surfactant Triton X-114 to concentrate a model protein, namely transferrin, prior to LFA. Proteins have been shown to partition, or distribute, fairly evenly between the two phases of an aqueous two-phase system, which in turn results in their limited concentration in one of the two phases. Therefore, larger colloidal gold particles decorated with antibodies for transferrin were used in the concentration step to bind to transferrin and aid its partitioning into the top, micelle-poor phase. By manipulating the volume ratio of the two coexisting micellar phases and combining the concentration step with LFA, the transferrin detection limit of LFA was improved by tenfold from 0.5 to 0.05 μg/mL in a predictive manner. In addition to enhancing the sensitivity of LFA, this universal concentration method could also be used to improve other detection assays.

  11. Ageing, fragility and the reversibility window in bulk alloy glasses

    International Nuclear Information System (INIS)

    Chakravarty, S; Georgiev, D G; Boolchand, P; Micoulaut, M

    2005-01-01

    Non-reversing relaxation enthalpies (ΔH nr ) at glass transitions T g (x) in the P x Ge x Se 1-2x ternary display wide, sharp and deep global minima (∼0) in the 0.09 g s become thermally reversing. In this reversibility window, glasses are found not to age, in contrast to ageing observed for fragile glass compositions outside the window. Thermal reversibility and lack of ageing seem to be paradigms of self-organization which molecular glasses share with protein structures which repetitively and reversibly change conformation near T g and the folding temperature respectively. (letter to the editor)

  12. Rapid micro-scale proteolysis of proteins for MALDI-MS peptide mapping using immobilized trypsin

    Science.gov (United States)

    Gobom, Johan; Nordhoff, Eckhard; Ekman, Rolf; Roepstorff, Peter

    1997-12-01

    In this study we present a rapid method for tryptic digestion of proteins using micro-columns with enzyme immobilized on perfusion chromatography media. The performance of the method is exemplified with acyl-CoA-binding protein and reduced carbamidomethylated bovine serum albumin. The method proved to be significantly faster and yielded a better sequence coverage and an improved signal-to-noise ratio for the MALDI-MS peptide maps, compared to in-solution- and on-target digestion. Only a single sample transfer step is required, and therefore sample loss due to adsorption to surfaces is reduced, which is a critical issue when handling low picomole to femtomole amounts of proteins. An example is shown with on-column proteolytic digestion and subsequent elution of the digest into a reversed-phase micro-column. This is useful if the sample contains large amounts of salt or is too diluted for MALDI-MS analysis. Furthermore, by step-wise elution from the reversedphase column, a complex digest can be fractionated, which reduces signal suppression and facilitates data interpretation in the subsequent MS-analysis. The method also proved useful for consecutive digestions with enzymes of different cleavage specificity. This is exemplified with on-column tryptic digestion, followed by reversed-phase step-wise elution, and subsequent on-target V8 protease digestion.

  13. [[Chiral separation of five arylpropionic acid drugs and determination of their enantiomers in pharmaceutical preparations by reversed-phase high performance liquid chromatography with cellulose-tris-(4-methylbenzoate) stationary phase

    Science.gov (United States)

    Luo, An; Wan, Qiang; Fan, Huajun; Chen, Zhi; Wu, Xuehao; Huang, Xiaowen; Zang, Linquan

    2014-09-01

    Chromatographic behaviors for enantiomeric separation of arylpropionic acid drugs were systematically developed by reversed phase-high performance liquid chromatography (RP-HPLC) using cellulose-tris-(4-methylbenzoate) (CTMB) as chiral stationary phase (CSP). The effects of the composition of the mobile phase, additives and temperature on chiral separation of flurbiprofen, pranoprofen, naproxen, ibuprofen and loxoprofen were further investigated. The enantiomers had been successfully separated on CSP of CTMB by the mobile phase of methanol-0.1% (v/v) formic acid except naproxen by acetonitrile-0.1% (v/v) formic acid at 25 °C. The mechanisms of the racemic resolution for the above mentioned five drugs are discussed thermodynamically and structurally. The resolutions between respective enantiomers for arylpropionic acid drugs on CTMB had significant differences due to their chromatographic behaviors. The order of resolutions ranked pranoprofen, loxoprofen, flurbiprofen, ibuprofen and naproxen. The method established has been successfully applied to the determination of the enantiomers of the five drugs in commercial preparations under the optimized conditions. It proved that the method is simple, reliable and accurate.

  14. ACUTE PHASE PROTEIN INCREASE IN HIGH ALTITUDE MOUNTAINEERS

    Directory of Open Access Journals (Sweden)

    Tolga Saka

    Full Text Available ABSTRACT Introduction: Many middle-aged Turks go hiking in mountains to breathe some fresh air or to maintain fitness. Objective: This study investigated the effects of regular high altitude mountain climbing on the metabolic and hematological responses of mountaineers. Methods: Hematological and biochemical parameters were studied, as well as some hormonal values of 21 mountaineers and 16 healthy age-matched sedentary volunteers. Results: The neutrophil to lymphocyte ratio (NLR was significantly lower (p<0.04 in mountaineers compared with the sedentary group. Total protein (p<0.001 and albumin (p<0.001 were lower, while the levels of ferritin (p<0.04, creatine (p<0.03 and creatine phosphokinase (p<0.01 were higher in mountaineers. Other hematological and biochemical parameters, i.e., erythrocytes, leukocytes, hemoglobin and hematocrit, did not change significantly. Conclusion: Our results show that regular exposure to high altitude increased the serum levels of some acute phase proteins with anti-inflammatory properties.

  15. Naphthalocyanine-based time reversal mirror at 800 nm

    International Nuclear Information System (INIS)

    Galaup, Jean-Pierre; Fraigne, Sebastien; Le Goueet, Jean-Louis; Likforman, Jean-Pierre; Joffre, Manuel

    2004-01-01

    We performed pulse shaping and time reversal experiments using spectral holography based on persistent spectral hole burning in free-base naphthalocyanine-doped films. The application of a new pulse re-compression scheme based on a programmable hole burning material acting as a time reversal mirror is considered. In this work, we adapted the Fourier transform spectral interferometry technique for measuring the amplitude and phase of photon echo signals produced by diffraction of femtosecond pulses on a spectral hologram. We therefore demonstrated that we could control the pulses diffracted from the hologram by shaping and then characterizing these pulses in both amplitude and phase by spectral interferometry

  16. Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein.

    Science.gov (United States)

    Ding, Xueqin; Wang, Yuzhi; Zeng, Qun; Chen, Jing; Huang, Yanhua; Xu, Kaijia

    2014-03-07

    A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by (1)H nuclear magnetic resonance ((1)H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV-vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV-vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins. Copyright © 2014 Elsevier B.V. All rights

  17. Development of Direct Reversed-Phase High Performance liquid chromatographic method for quantitative determination of gabapentin in pharmaceutical dosage

    International Nuclear Information System (INIS)

    Hassan, W.; Zaman, B.; Rahman, S.; Rahman, A.U.; Ali, N.; Mohammadzai, I.U.

    2012-01-01

    The objective of the present work was to develop and validate a rapid analytical method for quantitative determination of Gabapentin in pharmaceutical dosage tablets and capsules. An accurate, simple, and sensitive reversed-phase high performance liquid chromatographic (HPLC) method, UV detection at 215 nm and flow rate at 1.0 ml/min has been developed. Isocratic elution was used instead of gradient elution to reduce the time and cost of serial analysis. The mobile phase was a mixture of water and methanol (HPLC grade). The retention time (Rt) of Gabapentin was 4.681 +- 0.013 minutes. Recovery, Precision, accuracy, and linearity were determined for the stated method. The calibration curve was linear and the correlation coefficient was 0.9996. There was no chromatographic interference from other excipients present in dosage form. The method was validated appropriately and successfully used for determination of Gabapentin in Pharmaceutical formulations. (author)

  18. A green deep eutectic solvent-based aqueous two-phase system for protein extracting

    International Nuclear Information System (INIS)

    Xu, Kaijia; Wang, Yuzhi; Huang, Yanhua; Li, Na; Wen, Qian

    2015-01-01

    Highlights: • A strategy for the protein purification with a deep eutectic solvent(DES)-based aqueous two-phase system. • Choline chloride-glycerin DES was selected as the extraction solvent. • Bovine serum albumin and trypsin were used as the analytes. • Aggregation phenomenon was detected in the mechanism research. - Abstract: As a new type of green solvent, deep eutectic solvent (DES) has been applied for the extraction of proteins with an aqueous two-phase system (ATPS) in this work. Four kinds of choline chloride (ChCl)-based DESs were synthesized to extract bovine serum albumin (BSA), and ChCl-glycerol was selected as the suitable extraction solvent. Single factor experiments have been done to investigate the effects of the extraction process, including the amount of DES, the concentration of salt, the mass of protein, the shaking time, the temperature and PH value. Experimental results show 98.16% of the BSA could be extracted into the DES-rich phase in a single-step extraction under the optimized conditions. A high extraction efficiency of 94.36% was achieved, while the conditions were applied to the extraction of trypsin (Try). Precision, repeatability and stability experiments were studied and the relative standard deviations (RSD) of the extraction efficiency were 0.4246% (n = 3), 1.6057% (n = 3) and 1.6132% (n = 3), respectively. Conformation of BSA was not changed during the extraction process according to the investigation of UV–vis spectra, FT-IR spectra and CD spectra of BSA. The conductivity, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to explore the mechanism of the extraction. It turned out that the formation of DES–protein aggregates play a significant role in the separation process. All the results suggest that ChCl-based DES-ATPS are supposed to have the potential to provide new possibilities in the separation of proteins

  19. Reverse gyrase functions in genome integrity maintenance by protecting DNA breaks in vivo

    DEFF Research Database (Denmark)

    Han, Wenyuan; Feng, Xu; She, Qunxin

    2017-01-01

    Reverse gyrase introduces positive supercoils to circular DNA and is implicated in genome stability maintenance in thermophiles. The extremely thermophilic crenarchaeon Sulfolobus encodes two reverse gyrase proteins, TopR1 (topoisomerase reverse gyrase 1) and TopR2, whose functions in thermophilic...... and subsequent DNA degradation. The former occurred immediately after drug treatment, leading to chromosomal DNA degradation that concurred with TopR1 degradation, followed by chromatin protein degradation and DNA-less cell formation. To gain a further insight into TopR1 function, the expression of the enzyme...

  20. Reversible Age-Related Phenotypes Induced during Larval Quiescence in C. elegans

    Science.gov (United States)

    Roux, Antoine E.; Langhans, Kelley; Huynh, Walter; Kenyon, Cynthia

    2017-01-01

    Summary Cells can enter quiescent states in which cell cycling and growth are suspended. We find that during a long developmental arrest (quiescence) induced by starvation, newly-hatched C. elegans acquire features associated with impaired proteostasis and aging: mitochondrial fission, ROS production, protein aggregation, decreased proteotoxic-stress resistance, and at the organismal level, decline of mobility and high mortality. All signs of aging but one, the presence of protein aggregates, were reversed upon return to development induced by feeding. The endoplasmic reticulum receptor IRE-1 is completely required for recovery, and the downstream transcription factor XBP-1, as well as a protein kinase, KGB-1, are partially required. Interestingly, kgb-1(−) mutants that do recover fail to reverse aging-like mitochondrial phenotypes and have a short adult lifespan. Our study describes the first pathway that reverses phenotypes of aging at the exit of prolonged quiescence. PMID:27304510

  1. The Role of Reversed Equatorial Zonal Transport in Terminating an ENSO Event

    Science.gov (United States)

    Chen, H. C.; Hu, Z. Z.; Huang, B.; Sui, C. H.

    2016-02-01

    In this study, we demonstrate that a sudden reversal of anomalous equatorial zonal current at the peaking ENSO phase triggers the rapid termination of an ENSO event. Throughout an ENSO cycle, the anomalous equatorial zonal current is strongly controlled by the concavity of the anomalous thermocline meridional structure near the equator. During the ENSO developing phase, the anomalous zonal current in the central and eastern Pacific generally enhances the ENSO growth through its zonal SST advection. In the mature phase of ENSO, however, the equatorial thermocline depth anomalies are reflected in the eastern Pacific and slowly propagate westward off the equator in both hemispheres. As a result, the concavity of the thermocline anomalies near the equator is reversed, i.e., the off-equatorial thermocline depth anomalies become higher than that on the equator for El Niño events and lower for La Niño events. This meridional change of thermocline structure reverses zonal transport rapidly in the central-to-eastern equatorial Pacific, which weakens the ENSO SST anomalies by reversed advection. More importantly, the reversed zonal mass transport weakens the existing zonal tilting of equatorial thermocline and suppresses the thermocline feedback. Both processes are concentrated in the eastern equatorial Pacific and can be effective on subseasonal time scales. These current reversal effects are built-in to the ENSO peak phase and independent of the zonal wind effect on thermocline slope. It functions as an oceanic control on ENSO evolution during both El Niño and La Niña events.

  2. Dialysis water treated by reverse osmosis decreases the levels of C-reactive protein in uremic patients

    Directory of Open Access Journals (Sweden)

    F.S. Thomé

    2005-05-01

    Full Text Available Atherosclerosis is a major complication of chronic renal failure. Microinflammation is involved in atherogenesis and is associated with uremia and dialysis. The role of dialysate water contamination in inducing inflammation has been debated. Our aim was to study inflammatory markers in patients on chronic dialysis, before and 3 to 6 months after switching the water purification system from deionization to reverse osmosis. Patients had demographic, clinical and nutritional information collected and blood drawn for determination of albumin, ferritin, C-reactive protein (CRP, interleukin-6, and tumor necrosis factor-alpha in both situations. Acceptable levels of water purity were less than 200 colony-forming units of bacteria and less than 1 ng/ml of endotoxin. Sixteen patients died. They had higher median CRP (26.6 vs 11.2 mg/dl, P = 0.007 and lower median albumin levels (3.1 vs 3.9 g/l, P < 0.05 compared to the 31 survivors. Eight patients were excluded because of obvious inflammatory conditions. From the 23 remaining patients (mean age ± SD: 51.3 ± 13.9 years, 18 had a decrease in CRP after the water treatment system was changed. Overall, median CRP was lower with reverse osmosis than with deionization (13.2 vs 4.5 mg/l, P = 0.022, N = 23. There was no difference in albumin, cytokines, subjective global evaluation, or clinical and biochemical parameters. In conclusion, uremic patients presented a clinically significant reduction in CRP levels when dialysate water purification system switched from deionization to reverse osmosis. It is possible that better water treatments induce less inflammation and eventually less atherosclerosis in hemodialysis patients.

  3. LC-H-1 NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems

    DEFF Research Database (Denmark)

    Mortensen, R. W.; Corcoran, O.; Cornett, Claus

    2001-01-01

    . In both systems the elution order for the 2-, 3- and 4-O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC-H-1 NMR [U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I...

  4. Designing the Reverse Supply Chain

    DEFF Research Database (Denmark)

    Gobbi, Chiara

    2011-01-01

    Purpose – The purpose of this paper is to explore the impact of the product residual value (PRV) and the loss of value over time of returned products in the reverse supply chain configuration. It also examines whether or not the distinction of Fisher's functional and innovative products holds...... that allows for recapturing most of the PRV. These notions have then been tested by analyzing two reverse supply chains with a case study research methodology. Findings – The findings show that low PRV is associated with second-class recovery options (recycling and energy recovery) and that high PRV...... is associated with first-class recovery options (reconditioning and remarketing). When the recovery option is recycling, time is not relevant, the primary objective is cost reduction (efficiency), the chain is centralized, and actors and phases of the reverse chain are determined by the specificity...

  5. Incorporation of ionic liquid into porous polymer monoliths to enhance the separation of small molecules in reversed-phase high-performance liquid chromatography.

    Science.gov (United States)

    Wang, Jiafei; Bai, Ligai; Wei, Zhen; Qin, Junxiao; Ma, Yamin; Liu, Haiyan

    2015-06-01

    An ionic liquid was incorporated into the porous polymer monoliths to afford stationary phases with enhanced chromatographic performance for small molecules in reversed-phase high-performance liquid chromatography. The effect of the ionic liquid in the polymerization mixture on the performance of the monoliths was studied in detail. While monoliths without ionic liquid exhibited poor resolution and low efficiency, the addition of ionic liquid to the polymerization mixture provides highly increased resolution and high efficiency. The chromatographic performances of the monoliths were demonstrated by the separations of various small molecules including aromatic hydrocarbons, isomers, and homologues using a binary polar mobile phase. The present column efficiency reached 27 000 plates/m, which showed that the ionic liquid monoliths are alternative stationary phases in the separation of small molecules by high-performance liquid chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Analytical method development and validation of simultaneous estimation of rabeprazole, pantoprazole, and itopride by reverse-phase high-performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Senthamil Selvan Perumal

    2014-12-01

    Full Text Available A simple, selective, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC method for the simultaneous estimation of rabeprazole (RP, pantoprazole (PP, and itopride (IP has been developed. The compounds were well separated on a Phenomenex C18 (Luna column (250 mm × 4.6 mm, dp = 5 μm with C18 guard column (4 mm × 3 mm × 5 μm with a mobile phase consisting of buffer containing 10 mM potassium dihydrogen orthophosphate (adjusted to pH 6.8: acetonitrile (70:30 v/v at a flow rate of 1.0 mL/min and ultraviolet detection at 288 nm. The retention time of RP, PP, and IP were 5.35, 7.92, and 11.16 minutes, respectively. Validation of the proposed method was carried out according to International Conference on Harmonisation (ICH guidelines. Linearity range was obtained for RP, PP, and IP over the concentration range of 2.5–25, 1–30, and 3–35 μg/mL and the r2 values were 0.994, 0.978, and 0.991, respectively. The calculated limit of detection (LOD values were 1, 0.3, and 1 μg/mL and limit of quantitation (LOQ values were 2.5, 1, and 3 μg/mL for RP, PP, and IP correspondingly. Thus, the current study showed that the developed reverse-phase liquid chromatography method is sensitive and selective for the estimation of RP, PP, and IP in combined dosage form.

  7. Analytical method development and validation of simultaneous estimation of rabeprazole, pantoprazole, and itopride by reverse-phase high-performance liquid chromatography.

    Science.gov (United States)

    Perumal, Senthamil Selvan; Ekambaram, Sanmuga Priya; Raja, Samundeswari

    2014-12-01

    A simple, selective, rapid, and precise reverse-phase high-performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of rabeprazole (RP), pantoprazole (PP), and itopride (IP) has been developed. The compounds were well separated on a Phenomenex C 18 (Luna) column (250 mm × 4.6 mm, dp = 5 μm) with C 18 guard column (4 mm × 3 mm × 5 μm) with a mobile phase consisting of buffer containing 10 mM potassium dihydrogen orthophosphate (adjusted to pH 6.8): acetonitrile (70:30 v/v) at a flow rate of 1.0 mL/min and ultraviolet detection at 288 nm. The retention time of RP, PP, and IP were 5.35, 7.92, and 11.16 minutes, respectively. Validation of the proposed method was carried out according to International Conference on Harmonisation (ICH) guidelines. Linearity range was obtained for RP, PP, and IP over the concentration range of 2.5-25, 1-30, and 3-35 μg/mL and the r 2 values were 0.994, 0.978, and 0.991, respectively. The calculated limit of detection (LOD) values were 1, 0.3, and 1 μg/mL and limit of quantitation (LOQ) values were 2.5, 1, and 3 μg/mL for RP, PP, and IP correspondingly. Thus, the current study showed that the developed reverse-phase liquid chromatography method is sensitive and selective for the estimation of RP, PP, and IP in combined dosage form. Copyright © 2014. Published by Elsevier B.V.

  8. Strengthening of the DNA-protein complex during stationary phase aging of cell cultures

    International Nuclear Information System (INIS)

    Khokhlov, A.N.; Chirkova, E.Yu.; Gorin, A.I.

    1986-01-01

    The possibility of accumulation of cross-linkages in the DNA-protein complex was studied during stationary phase aging of cells in culture. Chinese hamster cells were used in the experiments, along with human fibroblasts. 3 H-thymidine, 14 C-valine, and 14 C-leucine were added to the medium. The quantity of protein firmly bound with DNA was judged from the value of the coefficient 14 C/ 3 H determined with allowance for penetration of counting from the 14 C-channel into the 3 H-channel. The authors maintain that the results presented in this paper provide further evidence of the value of stationary phase cell cultures for the study of the mechanisms of aging and also of some of the general principles underlying hereditary pathology

  9. Reversed phase liquid chromatography with UV absorbance and flame ionization detection using a water mobile phase and a cyano propyl stationary phase Analysis of alcohols and chlorinated hydrocarbons.

    Science.gov (United States)

    Quigley, W W; Ecker, S T; Vahey, P G; Synovec, R E

    1999-10-01

    The development of liquid chromatography with a commercially available cyano propyl stationary phase and a 100% water mobile phase is reported. Separations were performed at ambient temperature, simplifying instrumental requirements. Excellent separation efficiency using a water mobile phase was achieved, for example N=18 800, or 75 200 m(-1), was obtained for resorcinol, at a retention factor of k'=4.88 (retention time of 9.55 min at 1 ml min(-1) for a 25 cmx4.6 mm i.d. column, packed with 5 mum diameter particles with the cyano propyl stationary phase). A separation via reversed phase liquid chromatography (RP-LC) with a 100% water mobile phase of six phenols and related compounds was compared to a separation of the same compounds by traditional RP-LC, using octadecylsilane (ODS), i.e. C18, bound to silica and an aqueous mobile phase modified with acetonitrile. Nearly identical analysis time was achieved for the separation of six phenols and related compounds using the cyano propyl stationary phase with a 100% water mobile phase, as compared to traditional RP-LC requiring a relatively large fraction of organic solvent modifier in the mobile phase (25% acetonitrile:75% water). Additional understanding of the retention mechanism with the 100% water mobile phase was obtained by relating measured retention factors of aliphatic alcohols, phenols and related compounds, and chlorinated hydrocarbons to their octanol:water partition coefficients. The retention mechanism is found to be consistent with a RP-LC mechanism coupled with an additional retention effect due to residual hydroxyl groups on the cyano propyl stationary phase. Advantages due to a 100% water mobile phase for the chemical analysis of alcohol mixtures and chlorinated hydrocarbons are reported. By placing an absorbance detector in-series and preceding a novel drop interface to a flame ionization detector (FID), selective detection of a separated mixture of phenols and related compounds and aliphatic

  10. Proteomic analysis of growth phase-dependent expression of Legionella pneumophila proteins which involves regulation of bacterial virulence traits.

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Hayashi

    Full Text Available Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE combined with Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-TOF-MS. Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs. Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase.

  11. Flux loss during the equilibrium phase of field-reversed configurations

    International Nuclear Information System (INIS)

    Tuszewski, M.; Armstrong, W.T.; Bartsch, R.R.; Chrien, R.E.; Cochrane, J.C. Jr.; Kewish, R.W. Jr.; Klingner, P.; Linford, R.K.; McKenna, K.F.; Rej, D.J.; Sherwood, E.G.; Siemon, R.E.

    1982-01-01

    Field-reversed configurations are consistently formed at low filling pressures in the FRX-C device, with decay time of the trapped flux after formation much larger than the stable period. This contrasts with previous experimental observations

  12. Flux loss during the equilibrium phase of field-reversed configurations

    Energy Technology Data Exchange (ETDEWEB)

    Tuszewski, M.; Armstrong, W.T.; Bartsch, R.R.; Chrien, R.E.; Cochrane, J.C. Jr.; Kewish, R.W. Jr.; Klingner, P.; Linford, R.K.; McKenna, K.F.; Rej, D.J.; Sherwood, E.G.; Siemon, R.E.

    1982-10-01

    Field-reversed configurations are consistently formed at low filling pressures in the FRX-C device, with decay time of the trapped flux after formation much larger than the stable period. This contrasts with previous experimental observations.

  13. Reversible martensitic ω-α transformation in Ti and Zr

    International Nuclear Information System (INIS)

    Al'shevskij, Yu.L.; Kul'nitskij, B.A.; Konyaev, Yu.S.; Rojtburt, A.L.

    1985-01-01

    The mechanism of phase transformation in pure Ti and Zr in samples with initial ω-structure, produced as a result of heating to 1400 K with subsequent increase in pressure up to 8 GPa and a sharp decrease in temperature and pressure to normal values, has been studies. As a result of α → β → ω transformation, occurring in the process of the above-mentioned treatment, grains of ω-phase with perfect rystal structure are formed. With ω-phase heating to 650 K formation of α-phase takes place, and it is accompanied by the appearance of characteristic martensitic relief on the sample surface. Subsequent application of high pressure results in the reverse α → ω transformation with the disappearance of surface relief.The data suggest that direct ω → α transformation while heating and reverse α → ω transformation under pressure are realized according to martensitic mechanism

  14. Upregulating reverse cholesterol transport with cholesteryl ester transfer protein inhibition requires combination with the LDL-lowering drug berberine in dyslipidemic hamsters.

    Science.gov (United States)

    Briand, François; Thieblemont, Quentin; Muzotte, Elodie; Sulpice, Thierry

    2013-01-01

    This study aimed to investigate whether cholesteryl ester transfer protein inhibition promotes in vivo reverse cholesterol transport in dyslipidemic hamsters. In vivo reverse cholesterol transport was measured after an intravenous injection of (3)H-cholesteryl-oleate-labeled/oxidized low density lipoprotein particles ((3)H-oxLDL), which are rapidly cleared from plasma by liver-resident macrophages for further (3)H-tracer egress in plasma, high density lipoprotein (HDL), liver, and feces. A first set of hamsters made dyslipidemic with a high-fat and high-fructose diet was treated with vehicle or torcetrapib 30 mg/kg (TOR) over 2 weeks. Compared with vehicle, TOR increased apolipoprotein E-rich HDL levels and significantly increased (3)H-tracer appearance in HDL by 30% over 72 hours after (3)H-oxLDL injection. However, TOR did not change (3)H-tracer recovery in liver and feces, suggesting that uptake and excretion of cholesterol deriving from apolipoprotein E-rich HDL is not stimulated. As apoE is a potent ligand for the LDL receptor, we next evaluated the effects of TOR in combination with the LDL-lowering drug berberine, which upregulates LDL receptor expression in dyslipidemic hamsters. Compared with TOR alone, treatment with TOR+berberine 150 mg/kg resulted in lower apolipoprotein E-rich HDL levels. After (3)H-oxLDL injection, TOR+berberine significantly increased (3)H-tracer appearance in fecal cholesterol by 109%. Our data suggest that cholesteryl ester transfer protein inhibition alone does not stimulate reverse cholesterol transport in dyslipidemic hamsters and that additional effects mediated by the LDL-lowering drug berberine are required to upregulate this process.

  15. [Simultaneous Determination of Three Kinds of Effective Constituents in Cannabis Plants by Reversed-phase HPLC].

    Science.gov (United States)

    Fu, Q; Shu, Z; Deng, K; Luo, X; Zeng, C G

    2016-08-01

    To establish a high performance liquid chromatographic (HPLC) method for simultaneous determination of three effective constituents, including tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in Cannabis plants. A C₁₈ column was used in this study, and acetonitrile-phosphate buffer (0.015 mol/L KH₂PO₄) was used as mobile phase at a flow rate of 1.0 mL/min. At a detection wavelength of 220 mm, UV absorption spectra were collected at the wavelength range of 190-400 nm, and the spectra and retention time were counted as qualitative evidence. THC, CBD and CBN could be well separated by this method. Three components had good linear relationship in the range of 0.4-40 μg/mL ( R ²≥0.999 3). The recoveries were over 87%. The limits of detection were 1.8 ng, 2.0 ng and 1.3 ng, respectively. The relative standard deviation (RSD) were less than 5% for both inter-day and intra-day precisions. Reversed-phase HPLC method is simple, rapid and accurate, and it is suitable for the qualitative and quantitative detection of THC, CBD and CBN in Cannabis plants. Copyright© by the Editorial Department of Journal of Forensic Medicine

  16. Determination of trace elements in acid rain by reversed phase extraction chromatography and neutron activation

    International Nuclear Information System (INIS)

    Rao, R.R.; Goski, D.G.; Chatt, A.

    1991-01-01

    A preconcentration neutron activation analysis (PNAA) method has been developed for the simultaneous determination of selected trace elements in acid rain and other water samples. The method consists of preconcentration of the elements by reversed phase extraction chromatography using oxine-loaded Amberlite XAD-2 resin. Nearly 100% recoveries were obtained for Co, Cu, Hg, V and Zn at pH 6.0 and for Cd at pH 7.0. Manganese gave incomplete recoveries at the pH range of 4.0-8.0 studies. Various factors that can influence preconcentration of the elements have been investigated in detail. The precision and accuracy of measurements have been evaluated by analyzing certified reference materials. The detection limits have been found to be of the order of ppb. The PNAA method has been applied to a number of acid rain and other water samples

  17. True and measured outgassing rates of a vacuum chamber with a reversibly adsorbed phase

    Energy Technology Data Exchange (ETDEWEB)

    Akaishi, K. [National Inst. for Fusion Science, Toki, Gifu (Japan); Nakasuga, M. [Graduate School of Energy Science, Kyoto Univ., Uji, Kyoto (Japan); Funato, Y. [Suzuka National College of Technology, Suzuka, Mie (Japan)

    2000-03-01

    A pump down model for a vacuum chamber with a reversibly adsorbed phase is presented. The outgassing equation which predicts the variation of coverage at the wall surface of a vacuum chamber with time is derived. Then the measured and the true outgassing rates are defined. The theoretical measured outgassing rate shows only a very weak dependence for pumping speed. This prediction is opposite to the experimental result that the measured outgassing rate depends significantly on pumping speed. It is discussed that the experimental measured outgassing rate must be described as the product of the effective pumping speed and the measured pressure in the pumped chamber, in which the measured pressure is equivalent to the equilibrium pressure of the wall surface described by the equilibrium adsorption isotherm as a function of the shifted surface coverage {theta}-{delta}{theta} by a small coverage {delta}{theta} from the coverage of a pumping point {delta}. (author)

  18. True and measured outgassing rates of a vacuum chamber with a reversibly adsorbed phase

    International Nuclear Information System (INIS)

    Akaishi, K.; Nakasuga, M.; Funato, Y.

    2000-03-01

    A pump down model for a vacuum chamber with a reversibly adsorbed phase is presented. The outgassing equation which predicts the variation of coverage at the wall surface of a vacuum chamber with time is derived. Then the measured and the true outgassing rates are defined. The theoretical measured outgassing rate shows only a very weak dependence for pumping speed. This prediction is opposite to the experimental result that the measured outgassing rate depends significantly on pumping speed. It is discussed that the experimental measured outgassing rate must be described as the product of the effective pumping speed and the measured pressure in the pumped chamber, in which the measured pressure is equivalent to the equilibrium pressure of the wall surface described by the equilibrium adsorption isotherm as a function of the shifted surface coverage θ-Δθ by a small coverage Δθ from the coverage of a pumping point Δ. (author)

  19. Directional absorption by phased arrays of plasmonic nanoantennae probed with time-reversed Fourier microscopy

    International Nuclear Information System (INIS)

    Lozano, Gabriel; Barten, Tommy; Grzela, Grzegorz; Rivas, Jaime Gómez

    2014-01-01

    We demonstrate that an ordered array of aluminum nanopyramids, behaving as a phased array of optical antennae, strongly modifies light absorption in thin layers of dye molecules. Photoluminescence measurements as a function of the illumination angle are performed using a time-reversed Fourier microscope. This technique enables a variable-angle plane-wave illumination of nanostructures in a microscope-based setup. Our measurements reveal an enhancement of the light conversion in certain directions of illumination, which indicate the efficient diffractive coupling between the free space radiation and the surface plasmons. Numerical simulations confirm that surface modes supported by the periodic array enhance the intensity of the pump field in the space between particles, where the dye molecules are located, yielding a directional plasmonic-mediated enhancement of the optical absorption. This combined experimental and numerical characterization of the angular dependence of light absorption in nanostructures can be beneficial for the design and optimization of devices in which the harvesting of light plays a major role. (paper)

  20. Reversed Phase Column HPLC-ICP-MS Conditions for Arsenic Speciation Analysis of Rice Flour.

    Science.gov (United States)

    Narukawa, Tomohiro; Matsumoto, Eri; Nishimura, Tsutomu; Hioki, Akiharu

    2015-01-01

    New measurement conditions for arsenic speciation analysis of rice flour were developed using HPLC-ICP-MS equipped with a reversed phase ODS column. Eight arsenic species, namely, arsenite [As(III)], arsenate [As(V)], monomethylarsonic acid (MMAA), dimethylarsinic acid (DMAA), trimethylarsine oxide (TMAO), tetramethylarsonium (TeMA), arsenobetaine (AsB) and arsenocholine (AsC), were separated and determined under the proposed conditions. In particular, As(III) and MMAA and DMAA and AsB were completely separated using a newly proposed eluent containing ammonium dihydrogen phosphate. Importantly, the sensitivity changes, in particular those of As(V) and As(III) caused by coexisting elements and by complex matrix composition, which had been problematical in previously reported methods, were eliminated. The new eluent can be applied to C8, C18 and C30 ODS columns with the same effectiveness and with excellent repeatability. The proposed analytical method was successfully applied to extracts of rice flour certified reference materials.

  1. Development of anion-exchange/reversed-phase high performance liquid chromatography-inductively coupled plasma-mass spectrometry methods for the speciation of bio-available iodine and bromine from edible seaweed.

    Science.gov (United States)

    Romarís-Hortas, Vanessa; Bermejo-Barrera, Pilar; Moreda-Piñeiro, Antonio

    2012-05-04

    Anion exchange high performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry has been novelly applied to assess inorganic (iodide and iodate) and organic (3-iodotyrosine - MIT, and 3,5-diiodotyrosine - DIT) iodine species in a single chromatographic run. The optimized operating conditions (Dionex IonPac AS7, gradient elution with 175 mM ammonium nitrate plus 15% (v/v) methanol, pH 3.8, as a mobile phase and flow rates within the 0.5-1.5 mL min(-1) range) have also been used to perform inorganic bromine speciation analysis (bromide and bromate). The developed method has been applied for determining the bio-available contents of iodine and bromine species in dialyzates from edible seaweed. Reverse phase high performance liquid chromatography (Zorbax Eclipse XDB-C8, gradient elution with 0.2% (m/m) acetic acid, and 0.2% (m/m) acetic acid in methanol, as mobile phases, and a constant flow rate of 0.75 mL min(-1)) also hyphenated with inductively coupled plasma-mass spectrometry was used to confirm the presence of organic iodine species (MIT and DIT) in the dialyzates. The verification of the presence of iodinated amino acids (MIT and DIT) in the extracts was also performed by reverse phase high performance liquid chromatography-electrospray ionization-mass spectrometry (LTQ Orbitrap). The developed methods have provided good repeatability (RSD values lower than 10% for both anion exchange and reverse phase separations) and analytical recoveries within the 90-105% range for all cases. The in vitro bio-availability method consisted of a simulated gastric and an intestinal digestion/dialysis (10 kDa molecular weight cut-off - MWCO) two-stage procedure. Iodide and MIT were the main bio-available species quantified, whereas bromide was the major bromine species found in the extracts. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Quantification of Protein Hydration, Glass Transitions, and Structural Relaxations of Aqueous Protein and Carbohydrate-Protein Systems.

    Science.gov (United States)

    Roos, Yrjö H; Potes, Naritchaya

    2015-06-11

    Water distribution and miscibility of carbohydrate and protein components in biological materials and their structural contributions in concentrated solids are poorly understood. In the present study, structural relaxations and a glass transition of protein hydration water and antiplasticization of the hydration water at low temperatures were measured using dynamic mechanical analysis (DMA) and differential scanning calorimetry (DSC) for bovine whey protein (BWP), aqueous glucose-fructose (GF), and their mixture. Thermal transitions of α-lactalbumin and β-lactoglobulin components of BWP included water-content-dependent endothermic but reversible dehydration and denaturation, and exothermic and irreversible aggregation. An α-relaxation assigned to hydration water in BWP appeared at water-content-dependent temperatures and increased to over the range of 150-200 K at decreasing water content and in the presence of GF. Two separate glass transitions and individual fractions of unfrozen water of ternary GF-BWP-water systems contributed to uncoupled α-relaxations, suggesting different roles of protein hydration water and carbohydrate vitrification in concentrated solids during freezing and dehydration. Hydration water in the BWP fraction of GF-BWP systems was derived from equilibrium water sorption and glass transition data of the GF fraction, which gave a significant universal method to quantify (i) protein hydration water and (ii) the unfrozen water in protein-carbohydrate systems for such applications as cryopreservation, freezing, lyophilization, and dehydration of biological materials. A ternary supplemented phase diagram (state diagram) established for the GF-BWP-water system can be used for the analysis of the water distribution across carbohydrate and protein components in such applications.

  3. Determination of caffeine, theobromine, and theophylline in standard reference material 2384, baking chocolate, using reversed-phase liquid chromatography.

    Science.gov (United States)

    Thomas, Jeanice Brown; Yen, James H; Schantz, Michele M; Porter, Barbara J; Sharpless, Katherine E

    2004-06-02

    A rapid and selective isocratic reversed-phase liquid chromatographic method has been developed at the National Institute of Standards and Technology to simultaneously measure caffeine, theobromine, and theophylline in a food-matrix standard reference material (SRM) 2384, Baking Chocolate. The method uses isocratic elution with a mobile phase composition (volume fractions) of 10% acetronitrile/90% water (pH adjusted to 2.5 using acetic acid) at a flow rate of 1.5 mL/min with ultraviolet absorbance detection (274 nm). Total elution time for these analytes is less than 15 min. Concentration levels of caffeine, theobromine, and theophylline were measured in single 1-g samples taken from each of eight bars of chocolate over an eight-day period. Samples were defatted with hexane, and beta-hydroxyethyltheophylline was added as the internal standard. The repeatability for the caffeine, theobromine, and theophylline measurements was 5.1, 2.3, and 1.9%, respectively. The limit of quantitation for all analytes was theobromine, and theophylline in SRM 2384.

  4. Rescue of cAMP response element-binding protein signaling reversed spatial memory retention impairments induced by subanesthetic dose of propofol.

    Science.gov (United States)

    Zhang, Hao; Zhang, Shao-Bo; Zhang, Qing-Qing; Liu, Meng; He, Xing-Ying; Zou, Zui; Sun, Hai-Jing; You, Zhen-Dong; Shi, Xue-Yin

    2013-07-01

    The intravenous anesthetic propofol caused episodic memory impairments in human. We hypothesized propofol caused episodic-like spatial memory retention but not acquisition impairments in rats and rescuing cAMP response element-binding protein (CREB) signaling using selective type IV phosphodiesterase (PDEIV) inhibitor rolipram reversed these effects. Male Sprague-Dawley rats were randomized into four groups: control; propofol (25 mg/kg, intraperitoneal); rolipram; and rolipram + propofol (pretreatment of rolipram 25 min before propofol, 0.3 mg/kg, intraperitoneal). Sedation and motor coordination were evaluated 5, 15, and 25 min after propofol injection. Invisible Morris water maze (MWM) acquisition and probe test (memory retention) were performed 5 min and 24 h after propofol injection. Visible MWM training was simultaneously performed to resist nonspatial effects. Hippocampal CREB signaling was detected 5 min, 50 min, and 24 h after propofol administration. Rolipram did not change propofol-induced anesthetic/sedative states or impair motor skills. No difference was found on the latency to the platform during the visible MWM. Propofol impaired spatial memory retention but not acquisition. Rolipram reversed propofol-induced spatial memory impairments and suppression on cAMP levels, CaMKIIα and CREB phosphorylation, brain-derived neurotropic factor (BDNF) and Arc protein expression. Propofol caused spatial memory retention impairments but not acquisition inability possibly by inhibiting CREB signaling. © 2013 John Wiley & Sons Ltd.

  5. HALT & REVERSE: Hsf1 activators lower cardiomyocyt damage; towards a novel approach to REVERSE atrial fibrillation.

    Science.gov (United States)

    Lanters, Eva A H; van Marion, Denise M S; Kik, Charles; Steen, Herman; Bogers, Ad J J C; Allessie, Maurits A; Brundel, Bianca J J M; de Groot, Natasja M S

    2015-11-05

    Atrial fibrillation is a progressive arrhythmia, the exact mechanism underlying the progressive nature of recurrent AF episodes is still unknown. Recently, it was found that key players of the protein quality control system of the cardiomyocyte, i.e. Heat Shock Proteins, protect against atrial fibrillation progression by attenuating atrial electrical and structural remodeling (electropathology). HALT & REVERSE aims to investigate the correlation between electropathology, as defined by endo- or epicardial mapping, Heat Shock Protein levels and development or recurrence of atrial fibrillation following pulmonary vein isolation, or electrical cardioversion or cardiothoracic surgery. This study is a prospective observational study. Three separate study groups are defined: (1) cardiothoracic surgery, (2) pulmonary vein isolation and (3) electrical cardioversion. An intra-operative high-resolution epicardial (group 1) or endocardial (group 2) mapping procedure of the atria is performed to study atrial electropathology. Blood samples for Heat Shock Protein determination are obtained at baseline and during the follow-up period at 3 months (group 2), 6 months (groups 1 and 2) and 1 year (group 1 and 2). Tissue samples of the right and left atrial appendages in patients in group 1 are analysed for Heat Shock Protein levels and for tissue characteristics. Early post procedural atrial fibrillation is detected by continuous rhythm monitoring, whereas late post procedural atrial fibrillation is documented by either electrocardiogram or 24-h Holter registration. HALT & REVERSE aims to identify the correlation between Heat Shock Protein levels and degree of electropathology. The study outcome will contribute to novel diagnostic tools for the early recognition of clinical atrial fibrillation. Rotterdam Medical Ethical Committee MEC-2014-393, Dutch Trial Registration NTR4658.

  6. The practical management of bleedings during treatment with direct oral anticoagulants: the emergency reversal therapy

    Directory of Open Access Journals (Sweden)

    Luca Masotti

    2013-12-01

    Full Text Available Bleeding represents the most feared complication of the new oral anticoagulants, direct oral anticoagulants (DOACs, as well as all the antithrombotic therapies. During the acute phase of bleeding in patients taking anticoagulants, restoration of an effective hemostasis represents the cornerstone of practical management. While vitamin K antagonists are effectively and promptly reversed by specific antidotes such as prothrombin complex concentrates (PCCs, fresh frozen plasma or vitamin K, it is still not clear how to manage the urgent reversal of DOACs during life-threatening or major bleedings due to the lack of specific antidotes. However, in vitro and ex vivo studies have suggested some potential strategies to reverse DOACs in clinical practice, other than general support measures that are always recommended. Activated charcoal could be used in subjects with DOAC-related bleedings presenting to the emergency department within two hours of the last oral intake. Non-activated or activated PCCs (FEIBA and recombinant activated Factor VII (raFVII seem to be the optimal strategy for urgent reversal of dabigatran, while non-activated PCCs seem to have efficacy in reversing rivaroxaban. Due to its low plasma protein binding, dabigatran could be also dialyzed in urgent cases. Clinically relevant non-major bleedings and minor bleedings should be treated with general and local measures, respectively, and, when necessary, with dose delay or drug withdrawal. In this article, the Authors describe the practical approach to bleedings occurring during DOACs treatment.

  7. Determination of adrenaline, noradrenaline and corticosterone in rodent blood by ion pair reversed phase UHPLC-MS/MS.

    Science.gov (United States)

    Bergh, Marianne Skov-Skov; Bogen, Inger Lise; Andersen, Jannike Mørch; Øiestad, Åse Marit Leere; Berg, Thomas

    2018-01-01

    A novel ion pair reversed phase ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of the stress hormones adrenaline, noradrenaline and corticosterone in rodent blood was developed and fully validated. Separations were performed on an Acquity HSS T3 column (2.1mm i.d.×100mm, 1.8μm) with gradient elution and a runtime of 5.5min. The retention of adrenaline and noradrenaline was substantially increased by employing the ion pair reagent heptafluorobutyric acid (HFBA). Ion pair reagents are usually added to the mobile phase only, but we demonstrate for the first time that including HFBA to the sample reconstitution solvent as well, has a major impact on the chromatography of these compounds. The stability of adrenaline and corticosterone in rodent blood was investigated using the surrogate analytes adrenaline-d 3 and corticosterone-d 8 . The applicability of the described method was demonstrated by measuring the concentration of stress hormones in rodent blood samples. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The porcine acute phase protein response to acute clinical and subclinical experimental infection with Streptococcus suis

    DEFF Research Database (Denmark)

    Sørensen, Nanna Skall; Tegtmeier, C.; Andresen, Lars Ole

    2006-01-01

    The pig acute phase protein (APP) response to experimental Streptococcus suis (S. suis) infection was mapped by the measurement of the positive APPs C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin (Hp) and major acute phase protein (pig-MAP) and the negative APPs albumin...... and apolipoprotein (Apo) A-I. The aim was to elucidate the differences in the acute phase behaviour of the individual APPs during a typical bacterial septicaemic, infection. Pigs were inoculated subcutaneously with live S. suis serotype 2 and blood was sampled before and on various days post inoculation (p...... the experiment with maximum levels around 10 times the day 0-levels, and pig-MAP was elevated on days 1-12 p.i. with peak levels of around seven times the day 0-levels. Apo A-I was decreased from days 1 to 8 and showed minimum levels of about 40% of day 0-levels around 1-2 days p.i. No clear pattern of changes...

  9. Repletion of branched chain amino acids reverses mTORC1 signaling but not improved metabolism during dietary protein dilution

    DEFF Research Database (Denmark)

    Maida, Adriano; Chan, Jessica S K; Sjøberg, Kim Anker

    2017-01-01

    OBJECTIVE: Dietary protein dilution (PD) has been associated with metabolic advantages such as improved glucose homeostasis and increased energy expenditure. This phenotype involves liver-induced release of FGF21 in response to amino acid insufficiency; however, it has remained unclear whether...... dietary dilution of specific amino acids (AAs) is also required. Circulating branched chain amino acids (BCAAs) are sensitive to protein intake, elevated in the serum of obese humans and mice and thought to promote insulin resistance. We tested whether replenishment of dietary BCAAs to an AA-diluted (AAD......) diet is sufficient to reverse the glucoregulatory benefits of dietary PD. METHODS: We conducted AA profiling of serum from healthy humans and lean and high fat-fed or New Zealand obese (NZO) mice following dietary PD. We fed wildtype and NZO mice one of three amino acid defined diets: control, total...

  10. Reversible and irreversible magnetization of the Chevrel-phase superconductor PbMo6S8

    International Nuclear Information System (INIS)

    Zheng, D.N.; Ramsbottom, H.D.; Hampshire, D.P.

    1995-01-01

    Magnetic measurements have been carried out on the hot-isostatically-pressed Chevrel-phase superconductor PbMo 6 S 8 at temperatures from 4.2 K to T c and for magnetic fields up to 12 T. The results show that for the PbMo 6 S 8 compound there is a wide magnetically reversible region, between the irreversibility field B irr and the upper critical field B c2 , on the isothermal magnetic hysteresis curves. The B irr (T) line, i.e., the irreversibility line, was found to obey a power-law expression: B irr =B * (1-T/T c ) α with α∼1.5. Magnetic relaxation measurements revealed that the flux-creep effect in the material studied is substantial and is greater than those observed in conventional metallic alloys, but smaller than in high-temperature superconductors. The existence of the irreversibility line and pronounced flux-creep effect in PbMo 6 S 8 is attributed to the short coherence length of the material. From the reversible magnetization data, the values of the penetration depth, the coherence length, and the critical fields are obtained together with the Ginzburg-Landau parameter κ. At 4.2 K, the critical current density J c is 10 9 A m -2 at zero field, and decreases to 2x10 8 A m -2 at 10 T. Pinning force curves measured at different temperatures obey a Kramer-scaling law of the form: F p (=J c xB)∝b 1/2 (1-b) 2 , which indicates that the J c is limited by one predominant flux-pinning mechanism

  11. Reversed-phase liquid chromatography coupled on-line with capillary gas chromatography use of an anion-exchange membrane to remove an ion-pair reagent from the eluent.

    NARCIS (Netherlands)

    Brinkman, U.A.T.; Goosens, E.C.; de Jong, D.; de Jong, G.J.; Beerthuizen, I.M.

    1995-01-01

    In order to enable the coupling of reversed-phase liquid chromatography (RPLC) with capillary gas chromatography (GC), the performance of an anion-exchange micromembrane device has been studied to remove the ion-pair reagent methanesulphonic acid from an acetonitrile/water LC eluent. The regenerant

  12. Acute phase and transport protein synthesis in simulated infection in undernourished men using uniformly labelled Spirulina Platensis

    International Nuclear Information System (INIS)

    Reeds, P.J.; Opekun, A.; Jahoor, F.; Wong, W.W.; Klein, P.D.

    1994-01-01

    Although it has been known for many years that injury and infection lead to body nitrogen loss, the reason has remained obscure. In this paper, we develop the argument that the processes that are activated during infection demand the provision of specific amino acids which have to be supplied from body protein. In particular, we show that the positive acute phase proteins are very rich in the aromatic amino acids and the exaggerated use of these amino acids (phenylalanine, tryptophan and tyrosine) in acute phase protein synthesis lead to an endogenous ''amino acid imbalance'' which restricts the use of other amino acids for tissue protein synthesis. Minimally invasive protocols, involving the administration of 15 N and 13 C-labelled amino acids for studying whole body nitrogen turnover, amino acid oxidation and plasma protein synthesis are described. (author). 22 refs, 3 tabs

  13. A direct comparison of protein structure in the gas and solution phase: the Trp-cage

    DEFF Research Database (Denmark)

    Patriksson, Alexandra; Adams, Christopher M; Kjeldsen, Frank

    2007-01-01

    Molecular dynamics simulations of zwitterions of the Trp-cage protein in the gas phase show that the most stable ion in vacuo has preserved the charge locations acquired in solution. A direct comparison of the gas and solution-phase structures reveals that, despite the similarity in charge location...

  14. Protein-mediated antagonism between HIV reverse transcriptase ligands nevirapine and MgATP.

    Science.gov (United States)

    Zheng, Xunhai; Mueller, Geoffrey A; DeRose, Eugene F; London, Robert E

    2013-06-18

    Nonnucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open conformation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. To investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains. Such probes would be expected to be sensitive to changes in the local environment depending on the fractions of open and closed RT. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of NVP. The exchange kinetics of the M63 resonance are fast on the chemical shift timescale, but become slow in the presence of NVP due to the slow binding of RT with the inhibitor. The simplest model consistent with this behavior involves a rapid open/closed equilibrium coupled with a slow interaction of the inhibitor with the open conformation. Studies of RT in the presence of both NVP and MgATP indicate a strong negative cooperativity. Binding of MgATP reduces the fraction of RT bound to NVP, as indicated by the intensity of the NVP-perturbed M230 resonance, and enhances the dissociation rate constant of the NVP, resulting in an increase of the open/closed interconversion rate, so that the M63 resonance moves into the fast/intermediate-exchange regime. Protein-mediated interactions appear to explain most of the affinity variation of NVP for RT. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  15. Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

    Science.gov (United States)

    Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

    1993-08-01

    Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

  16. LIQUID CHROMATOGRAPHIC / MASS SPECTROMETRIC DETERMINATION OF MORPHINE, CODEINE AND COCAINE IN HUMAN SERA USING A NEW INTERNAL SURFACE REVERSED PHASE COLUMN

    OpenAIRE

    Hattori, Hideki; Arinobu, Tetsuya; Iwai, Masae; Suzuki, Osamu; Seno, Hiroshi

    2003-01-01

    A new internal surface reversed phase column (Shim-pack MAYI-ODS) was used for analysis of morphine, codeine and cocaine in human sera by LC/MS/MS. The column enabled direct injection of crude biological samples without pretreatment realizing a rapid analytical procedure. Therecoveries of morphine, codeine and cocaine spiked into human sera were 50-60 %. The regression equations for morphine, codeine and cocaine in sera, using atropine as internal standard, showed good linearity in the ranges...

  17. Advanced 3D tools used in reverse engineering and ray tracing simulation of phased array inspection of turbine components with complex geometry

    International Nuclear Information System (INIS)

    Daks, W.; Kovacshazy, C.; Mair, D.; Ciorau, P.

    2002-01-01

    This paper outlines the practical aspects of reverse engineering and the integration of multiple pieces of software (Drafting, CNC Machining, Ray Tracing, Inspection Simulation Scenario and Phased Array UT Analysis), in order to inspect turbine components comprised of complex geometry. The CNC software, Mastercam, and design software, CADKEY/FastSURF, were used to validate the phased-array automated and manual inspection of blade root, rotor steeples and disk-blade rim attachment. The integration of a 3D part in the software engine, Imagine 3D and SimScan, as well as Tomoview analysis (specimen feature) is based on CADKEY Developer Kit - IGES/SAT file format. A generic Ray Tracing simulation for multi-probe beam was integrated into Imagine 3D. Representative examples of reference blocks and mock-ups, UT simulation and phased-array data comparison are presented. (author)

  18. Solid-phase synthesis of an apoptosis-inducing tetrapeptide mimicking the Smac protein

    DEFF Research Database (Denmark)

    Le Quement, Sebastian Thordal; Ishøy, Mette; Petersen, Mette Terp

    2011-01-01

    An approach for the solid-phase synthesis of apoptosis-inducing Smac peptidomimetics is presented. Using a Rink linker strategy, tetrapeptides mimicking the N-4-terminal residue of the Smac protein [(N-Me)AVPF sequence] were synthesized on PEGA resin in excellent purities and yields. Following two...

  19. Complete separation of racemic p-boronophenylalanine by high performance liquid chromatography with crown ether-coated reversed-phase packings

    International Nuclear Information System (INIS)

    Yoshino, K.; Mieda, S.; Maruyama, T.; Mori, Y.; Mishima, Y.; Ichihashi, M.

    1992-01-01

    Since the L-form of p-boronophenylalanine (p-bpa) has been shown to be more efficiently incorporated into melanoma cells than racemic p-bpa separation of racemic p-bpa into its stereoisomers is an important subject. One of the preparative methods used to resolve racemic p-bpa involves the use of α-chymotrypsin. However, there has been a problem in that optical purity of resolved L- or D-p-bpa products was not easily determined. In this paper, the authors describe a method which can be used to confirm the optical purity of b-pba using high performance liquid chromatography (HPLC) with crown ether-coated reversed-phase packings

  20. Reverse Transfection Using Gold Nanoparticles

    Science.gov (United States)

    Yamada, Shigeru; Fujita, Satoshi; Uchimura, Eiichiro; Miyake, Masato; Miyake, Jun

    Reverse transfection from a solid surface has the potential to deliver genes into various types of cell and tissue more effectively than conventional methods of transfection. We present a method for reverse transfection using a gold colloid (GC) as a nanoscaffold by generating nanoclusters of the DNA/reagentcomplex on a glass surface, which could then be used for the regulation of the particle size of the complex and delivery of DNA into nuclei. With this method, we have found that the conjugation of gold nanoparticles (20 nm in particle size) to the pEGFP-N1/Jet-PEI complex resulted in an increase in the intensity of fluorescence of enhanced green fluorescent protein (EGFP) (based on the efficiency of transfection) from human mesenchymal stem cells (hMSCs), as compared with the control without GC. In this manner, we constructed a method for reverse transfection using GC to deliver genes into the cells effectively.

  1. Determination of modafinil in plasma and urine by reversed phase high-performance liquid-chromatography.

    Science.gov (United States)

    Schwertner, Harvey A; Kong, Suk Bin

    2005-03-09

    Modafinil (Provigil) is a new wake-promoting drug that is being used for the management of excessive sleepiness in patients with narcolepsy. It has pharmacological properties similar to that of amphetamine, but without some of the side effects associated with amphetamine-like stimulants. Since modafinil has the potential to be abused, accurate drug-screening methods are needed for its analysis. In this study, we developed a high-performance liquid-chromatographic procedure (HPLC) for the quantitative analysis of modafinil in plasma and urine. (Phenylthio)acetic acid was used as an internal standard for the analysis of both plasma and urine. Modafinil was extracted from urine and plasma with ethyl acetate and ethyl acetate-acetic acid (100:1, v/v), respectively, and analyzed on a C18 reverse phase column with methanol-water-acetic acid (500:500:1, v/v) as the mobile phase. Recoveries from urine and plasma were 80.0 and 98.9%, respectively and the limit of quantitation was 0.1 microg/mL at 233 nm. Forty-eight 2-h post-dose urine samples from sham controls and from individuals taking 200 or 400 mg of modafinil were analyzed without knowledge of drug administration. All 16-placebo urine samples and all 32 2-h post-dose urine samples were correctly classified. The analytical procedure is accurate and reproducible and can be used for therapeutic drug monitoring, pharmacokinetic studies, and drug abuse screening.

  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. Reversible Unfolding of Rhomboid Intramembrane Proteases.

    Science.gov (United States)

    Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

    2016-03-29

    Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Protein synthesis during the initial phase of the temperature-induced bleaching response in Euglena gracilis

    International Nuclear Information System (INIS)

    Ortiz, W.

    1990-01-01

    Growing cultures of photoheterotrophic Euglena gracilis experience an increase in chlorophyll accumulation during the initial phase of the temperature-induced bleaching response suggesting an increase in the synthesis of plastid components at the bleaching temperature of 33 degree C. A primary goal of this work was to establish whether an increase in the synthesis of plastid proteins accompanies the observed increase in chlorophyll accumulation. In vivo pulse-labeling experiments with [ 35 S]sodium sulfate were carried out with cells grown at room temperature or at 33 degree C. The synthesis of a number of plastid polypeptides of nucleocytoplasmic origin, including some presumably novel polypeptides, increased in cultures treated for 15 hours at 33 degree C. In contrast, while synthesis of thylakoid proteins by the plastid protein synthesis machinery decreased modestly, synthesis of the large subunit of the enzyme ribulosebisphosphate carboxylase was strongly affected at the elevated temperature. Synthesis of novel plastid-encoded polypeptides was not induced at the bleaching temperature. It is concluded that protein synthesis in plastids declines during the initial phase of the temperature response in Euglena despite an overall increase in cellular protein synthesis and an increase in chlorophyll accumulation per cell

  5. Characterization of Isomeric Glycans by Reversed Phase Liquid Chromatography-Electronic Excitation Dissociation Tandem Mass Spectrometry

    Science.gov (United States)

    Tang, Yang; Wei, Juan; Costello, Catherine E.; Lin, Cheng

    2018-04-01

    The occurrence of numerous structural isomers in glycans from biological sources presents a severe challenge for structural glycomics. The subtle differences among isomeric structures demand analytical methods that can provide structural details while working efficiently with on-line glycan separation methods. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool for mixture analysis, the commonly utilized collision-induced dissociation (CID) method often does not generate a sufficient number of fragments at the MS2 level for comprehensive structural characterization. Here, we studied the electronic excitation dissociation (EED) behaviors of metal-adducted, permethylated glycans, and identified key spectral features that could facilitate both topology and linkage determinations. We developed an EED-based, nanoscale, reversed phase (RP)LC-MS/MS platform, and demonstrated its ability to achieve complete structural elucidation of up to five structural isomers in a single LC-MS/MS analysis. [Figure not available: see fulltext.

  6. First interactions between hydrogen and stress-induced reverse transformation of Ni-Ti superelastic alloy

    Science.gov (United States)

    Yokoyama, Ken'ichi; Hashimoto, Tatsuki; Sakai, Jun'ichi

    2017-11-01

    The first dynamic interactions between hydrogen and the stress-induced reverse transformation have been investigated by performing an unloading test on a Ni-Ti superelastic alloy subjected to hydrogen charging under a constant applied strain in the elastic deformation region of the martensite phase. Upon unloading the specimen, charged with a small amount of hydrogen, no change in the behaviour of the stress-induced reverse transformation is observed in the stress-strain curve, although the behaviour of the stress-induced martensite transformation changes. With increasing amount of hydrogen charging, the critical stress for the reverse transformation markedly decreases. Eventually, for a larger amount of hydrogen charging, the reverse transformation does not occur, i.e. there is no recovery of the superelastic strain. The residual martensite phase on the side surface of the unloaded specimen is confirmed by X-ray diffraction. Upon training before the unloading test, the properties of the reverse transformation slightly recover after ageing in air at room temperature. The present study indicates that to change the behaviour of the reverse transformation a larger amount of hydrogen than that for the martensite transformation is necessary. In addition, it is likely that a substantial amount of hydrogen in solid solution more strongly suppresses the reverse transformation than hydrogen trapped at defects, thereby stabilising the martensite phase.

  7. Phase diagram of SnTe-CdSe cross-section of SnTe+CdSe reversible SnSe+CdTe ternary reciprocal system

    International Nuclear Information System (INIS)

    Dubrovin, I.V.; Budennaya, L.D.; Mizetskaya, I.B.; Sharkina, Eh.V.

    1986-01-01

    Phase equilibrium diagram of SnTe-CdSe cross-section of Sn, Cd long Te, Se ternary reciprocal system is investigated using the methods of differential thermal, X-ray phase, and microstructural analyses. Maximum length of solid solutions on the base of SnTe corresponds to approximately 14 mol.% at 1050 K and approximately 3 mol.% of CdSe at 670 K. Region of solid solutions on the base of CdSe corresponds to less than 1 mol.% of SnTe at room temperature. SnTe-CdSe cross-section is not a quasibinar one. Equilibrium is shifted to the left in the SnTe+CdSe reversible SnSe+CdTe reciprocal system

  8. Partitioning the proteome: phase separation for targeted analysis of membrane proteins in human post-mortem brain.

    Directory of Open Access Journals (Sweden)

    Jane A English

    Full Text Available Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.

  9. Similarities between long interspersed element-1 (LINE-1) reverse transcriptase and telomerase.

    Science.gov (United States)

    Kopera, Huira C; Moldovan, John B; Morrish, Tammy A; Garcia-Perez, Jose Luis; Moran, John V

    2011-12-20

    Long interspersed element-1 (LINE-1 or L1) retrotransposons encode two proteins (ORF1p and ORF2p) that contain activities required for conventional retrotransposition by a mechanism termed target-site primed reverse transcription. Previous experiments in XRCC4 or DNA protein kinase catalytic subunit-deficient CHO cell lines, which are defective for the nonhomologous end-joining DNA repair pathway, revealed an alternative endonuclease-independent (ENi) pathway for L1 retrotransposition. Interestingly, some ENi retrotransposition events in DNA protein kinase catalytic subunit-deficient cells are targeted to dysfunctional telomeres. Here we used an in vitro assay to detect L1 reverse transcriptase activity to demonstrate that wild-type or endonuclease-defective L1 ribonucleoprotein particles can use oligonucleotide adapters that mimic telomeric ends as primers to initiate the reverse transcription of L1 mRNA. Importantly, these ribonucleoprotein particles also contain a nuclease activity that can process the oligonucleotide adapters before the initiation of reverse transcription. Finally, we demonstrate that ORF1p is not strictly required for ENi retrotransposition at dysfunctional telomeres. Thus, these data further highlight similarities between the mechanism of ENi L1 retrotransposition and telomerase.

  10. Comparison of Separation of Seed Oil Triglycerides Containing Isomeric Conjugated Octadecatrienoic Acid Moieties by Reversed-Phase HPLC

    Directory of Open Access Journals (Sweden)

    Anh Van Nguyen

    2017-12-01

    Full Text Available Relative retention analysis and increment approach were applied for the comparison of triglycerides (TGs retention of a broad set of plant seed oils with isomeric conjugated octadecatrienoic acids (CLnA by reversed-phase HPLC for “propanol-2-acetonitrile” mobile phases and Kromasil 100-5C18 stationary phase with diode array detection (DAD and mass spectrometric (MS detection. The subjects of investigation were TGs of seed oils: Calendula officinalis, Catalpa ovata, Jacaranda mimosifolia, Centranthus ruber, Momordica charantia, Trichosanthes anguina, Punica granatum, Thladiantha dubia, Valeriana officinalis, and Vernicia montana. It was found that a sequence of elution of TGs of the same types is the same without any inversions for full range of mobile phase compositions: punicic (C18:39Z11E13Z < jacaric (C18:38Z10E12Z < catalpic (C18:39E11E13Z < α-eleostearic (C18:39Z11E13E < calendic (C18:38E10E12Z < β-eleostearic (C18:39E11E13E < all-E calendic (C18:38E10E12E acids. TGs and fatty acid compositions were calculated for all oil samples. Regularities of solute retentions as a function of isomeric conjugated octadecatrienoic acid moiety structure are discussed. Thus, it was proven that it is possible to differentiate TGs of complex composition with moieties of all natural CLnA by retention control accomplished by electronic spectra comparison, even though there are only three types of electronic-vibration spectra for seven isomeric CLnA.

  11. Supplemental and highly-elevated tocopherol doses differentially regulate allergic inflammation: reversibility of α-tocopherol and γ-tocopherol's effects

    Science.gov (United States)

    McCary, Christine A.; Abdala-Valencia, Hiam; Berdnikovs, Sergejs; Cook-Mills, Joan M.

    2011-01-01

    We have reported that supplemental doses of the α- and γ-tocopherol isoforms of vitamin E decrease and increase, respectively, allergic lung inflammation. We have now assessed whether these effects of tocopherols are reversible. For these studies, mice were treated with antigen and supplemental tocopherols in a first phase of treatment followed by a 4 week clearance phase and then the mice received a second phase of antigen and tocopherol treatments. The pro-inflammatory effects of supplemental levels of γ-tocopherol in phase 1 were only partially reversed by supplemental α-tocopherol in phase 2 but were completely reversed by raising α-tocopherol levels 10-fold in phase 2. When γ-tocopherol levels were increased 10-fold (highly-elevated tocopherol) so that the lung tissue γ-tocopherol levels were equal to the lung tissue levels of supplemental α-tocopherol, γ-tocopherol reduced leukocyte numbers in the lung lavage fluid. In contrast to the lung lavage fluid, highly-elevated levels of γ-tocopherol increased inflammation in the lung tissue. These regulatory effects of highly-elevated tocopherols on tissue inflammation and lung lavage fluid were reversible in a second phase of antigen challenge without tocopherols. In summary, the pro-inflammatory effects of supplemental γ-tocopherol on lung inflammation were partially reversed by supplemental levels of α-tocopherol but were completely reversed by highly-elevated-levels of α-tocopherol. Also, highly-elevated levels of γ-tocopherol were inhibitory and reversible in lung lavage but, importantly, were pro-inflammatory in lung tissue sections. These results have implications for future studies with tocopherols and provide a new context in which to review vitamin E studies in the literature. PMID:21317387

  12. The Reversal Effect and Its Mechanisms of Tetramethylpyrazine on Multidrug Resistance in Human Bladder Cancer.

    Directory of Open Access Journals (Sweden)

    Shanshan Wang

    Full Text Available Chemotherapy is an important strategy for the treatment of bladder cancer. However, the main problem limiting the success of chemotherapy is the development of multidrug resistance (MDR. To improve the management of bladder cancer, it is an urgent matter to search for strategies to reverse MDR. We chose three kinds of herbal medicines including ginsenoside Rh2, (--Epigallocatechin gallate (EGCG and Tetramethylpyrazine (TMP to detect their effects on bladder cancer. Reversal effects of these three herbal medicines for drug resistance in adriamycin (ADM-resistant Pumc-91 cells (Pumc-91/ADM were assessed by Cell Counting Kit-8 (CCK-8 cell proliferation assay system. The mechanisms of reversal effect for TMP were explored in Pumc-91/ADM and T24/DDP cells. After Pumc-91/ADM and T24/DDP cells were treated with TMP, cell cycle distribution analysis was performed by flow cytometry. The expression of MRP1, GST, BCL-2, LRP and TOPO-II was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR, immunefluorescence assay and western blot. It was observed that TMP was capable of enhancing the cytotoxicity of anticancer agents on Pumc-91/ADM cells in response to ADM, however Rh2 and EGCG were unable to. The reversal effect of TMP was also demonstrated in T24/DDP cells. Moreover, the treatment with TMP in Pumc-91/ADM and T24/DDP cells led to an increased of G1 phase accompanied with a concomitant decrease of cell numbers in S phase. Compared to the control group, an obvious decrease of MRP1, GST, BCL-2 and an increase of TOPO-II were shown in TMP groups with a dose-dependency in mRNA and protein levels. However, there was no difference on LRP expression between TMP groups and the control group. TMP could effectively reverse MDR of Pumc-91/ADM and T24/DDP cells and its mechanisms might be correlated with the alteration of MRP1, GST, BCL-2 and TOPO-II. TMP might be a potential candidate for reversing drug resistance in bladder cancer

  13. Reversible thyristor converters of brushless synchronous compensators

    Directory of Open Access Journals (Sweden)

    А.М.Galynovskiy

    2013-12-01

    Full Text Available Behavior of models of three-phase-to-single-phase rotary reversible thyristor converters of brushless synchronous compensators in a circuit simulation system is analyzed. It is shown that combined control mode of opposite-connected thyristors may result in the exciter armature winding short circuits both at the thyristor feed-forward and lagging current delay angles. It must be taken into consideration when developing brushless compensator excitation systems.

  14. Evaluations of in vitro metabolism, drug-drug interactions mediated by reversible and time-dependent inhibition of CYPs, and plasma protein binding of MMB4 DMS.

    Science.gov (United States)

    Hong, S Peter; Lusiak, Bozena D; Burback, Brian L; Johnson, Jerry D

    2013-01-01

    1,1'-Methylenebis[4-[(hydroxyimino)methyl]-pyridinium] (MMB4) dimethanesulfonate (DMS) is a bisquaternary pyridinium aldoxime that reactivates acetylcholinesterase inhibited by organophosphorus nerve agent. Drug metabolism and plasma protein binding for MMB4 DMS were examined using various techniques and a wide range of species. When (14)C-MMB4 DMS was incubated in liver microsomes, 4-pyridine aldoxime (4-PA) and an additional metabolite were detected in all species tested. Identity of the additional metabolite was postulated to be isonicotinic acid (INA) based on liquid chromatography with a tandem mass spectrometry analysis, which was confirmed by comparison with authentic INA. Formation of INA was dependent on species, with the highest level found in monkey liver microsomes. The MMB4 DMS exhibited reversible inhibition in a concentration-dependent manner toward cytochrome P450 1A2 (CYP1A2), CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in human liver microsomes showing the highest inhibition for CYP2D6. Human recombinant CYPs were used to evaluate inhibitory curves more adequately and determine detailed kinetic constants for reversible inhibition and potential time-dependent inhibition (TDI). The MMB4 DMS exhibited reversible inhibition toward human-recombinant CYP2D6 with an inhibition constant (K i) value of 66.6 µmol/L. Based on the k inact/K I values, MMB4 DMS was found to exhibit the most potent TDI toward CYP2D6. The MMB4 DMS at 5 different concentrations was incubated in plasma for 5 hours using an equilibrium dialysis device. For all species tested, there were no concentration-dependent changes in plasma protein binding, ranging from 10% to 17%. These results suggest that MMB4 was not extensively bound to plasma protein, and there were no overt species-related differences in the extent of MMB4 bound to plasma protein.

  15. Local particle flux reversal under strongly sheared flow

    International Nuclear Information System (INIS)

    Terry, P.W.; Newman, D.E.; Ware, A.S.

    2003-01-01

    The advection of electron density by turbulent ExB flow with linearly varying mean yields a particle flux that can reverse sign at certain locations along the direction of magnetic shear. The effect, calculated for strong flow shear, resides in the density-potential cross phase. It is produced by the interplay between the inhomogeneities of magnetic shear and flow shear, but subject to a variety of conditions and constraints. The regions of reversed flux tend to wash out if the turbulence consists of closely spaced modes of different helicities, but survive if modes of a single helicity are relatively isolated. The reversed flux becomes negligible if the electron density response is governed by electron scales while the eigenmode is governed by ion scales. The relationship of these results to experimentally observe flux reversals is discussed

  16. Development of an analytical method for the determination of histamine in fish by liquid chromatography of high performance in reverse phase

    International Nuclear Information System (INIS)

    Valverde Chavarria, Juan Carlos; Barquero Quiros, Mirian

    2004-01-01

    The histamine is a biogenic amine able to promote allergic intoxications in sensitive persons. For this reason is necessary to have a method of analysis with sensitivity, precision and accuracy able to quantify lower amounts than actual FDA Normative sets. A reverse phase chromatographic method was developed. To identify and quantify histamine through automatic generation of histamine derivative of OPA, using an ultraviolet detector, performance parameters of developed method were adequate to quantification of histamine amount lower than FDA Normative. (Author) [es

  17. Serum protein profiling by miniaturized solid-phase extraction and matrix-assisted laser desorption/ionization mass spectrometry

    DEFF Research Database (Denmark)

    Callesen, Anne K; Mohammed, Shabaz; Bunkenborg, Jakob

    2005-01-01

    for translation of MALDI-MS based diagnostic methods to clinical applications. We have investigated a number of MALDI matrices and several miniaturized solid-phase extraction (SPE) methods for serum protein concentration and desalting with the aim of generating reproducible, high-quality protein profiles by MALDI...

  18. Proteomic analysis of cerebrospinal fluid from children with central nervous system tumors identifies candidate proteins relating to tumor metastatic spread.

    Science.gov (United States)

    Spreafico, Filippo; Bongarzone, Italia; Pizzamiglio, Sara; Magni, Ruben; Taverna, Elena; De Bortoli, Maida; Ciniselli, Chiara M; Barzanò, Elena; Biassoni, Veronica; Luchini, Alessandra; Liotta, Lance A; Zhou, Weidong; Signore, Michele; Verderio, Paolo; Massimino, Maura

    2017-07-11

    Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

  19. Ginseng Total Saponins Reverse Corticosterone-Induced Changes in Depression-Like Behavior and Hippocampal Plasticity-Related Proteins by Interfering with GSK-3β-CREB Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Lin Chen

    2014-01-01

    Full Text Available This study aimed to explore the antidepressant mechanisms of ginseng total saponins (GTS in the corticosterone-induced mouse depression model. In Experiment 1, GTS (50, 25, and 12.5 mg kg−1 d−1, intragastrically were given for 3 weeks. In Experiment 2, the same doses of GTS were administrated after each corticosterone (20 mg kg−1 d−1, subcutaneously injection for 22 days. In both experiments, mice underwent a forced swimming test and a tail suspension test on day 20 and day 21, respectively, and were sacrificed on day 22. Results of Experiment 1 revealed that GTS (50 and 25 mg kg−1 d−1 exhibited antidepressant activity and not statistically altered hippocampal protein levels of brain-derived neurotrophic factor (BDNF and neurofilament light chain (NF-L. Results of Experiment 2 showed that GTS (50 and 25 mg kg−1 d−1 ameliorated depression-like behavior without normalizing hypercortisolism. The GTS treatments reversed the corticosterone-induced changes in mRNA levels of BDNF and NF-L, and protein levels of BDNF NF-L, phosphor-cAMP response element-binding protein (Ser133, and phosphor-glycogen synthase kinase-3β (Ser9 in the hippocampus. These findings imply that the effect of GTS on corticosterone-induced depression-like behavior may be mediated partly through interfering with hippocampal GSK-3β-CREB signaling pathway and reversing decrease of some plasticity-related proteins.

  20. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    International Nuclear Information System (INIS)

    Warren, Anna J.; Armour, Wes; Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R.; Horrell, Sam; McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf

    2013-01-01

    A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required

  1. Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging

    Energy Technology Data Exchange (ETDEWEB)

    Warren, Anna J. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Armour, Wes [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Oxford e-Research Centre, 7 Keble Road, Oxford OX1 3QG (United Kingdom); Axford, Danny; Basham, Mark; Connolley, Thomas; Hall, David R. [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); Horrell, Sam [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom); University of Liverpool, Liverpool L69 3BX (United Kingdom); McAuley, Katherine E.; Mykhaylyk, Vitaliy; Wagner, Armin; Evans, Gwyndaf, E-mail: gwyndaf.evans@diamond.ac.uk [Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE (United Kingdom)

    2013-07-01

    A comparison of X-ray diffraction and radiographic techniques for the location and characterization of protein crystals is demonstrated on membrane protein crystals mounted within lipid cubic phase material. The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

  2. Reversible bulk-phase change of anthroyl compounds for photopatterning based on photodimerization in the molten state and thermal back reaction.

    Science.gov (United States)

    Kihara, Hideyuki; Yoshida, Masaru

    2013-04-10

    As new organic materials for rewritable photopatterning, 2-anthroyl and 9-anthroyl ester compounds were synthesized. Their bulk-phase changes (we use "bulk-phase change" as complete phase change in a mass of a material neither in a surface nor in a small quantity in this study) triggered by photodimerization under melting conditions (melt-photodimerization) and subsequent thermal back reactions were investigated. All the anthroyl compounds exhibited melting points lower than ca. 160 °C, and they were nearly quantitatively converted to the corresponding photodimers by UV irradiation at temperatures of ∼5 °C higher than their respective melting points. We found that there were two kinds of bulk-phase change behaviors through the photoreaction. Two of the anthroyl compounds remained isotropic and lost fluidity during the melt-photodimerization. The obtained photodimers exhibited robust solid-state amorphous phases at room temperature. In contrast, the other three anthroyl compounds showed crystallization during the melt-photodimerization. The resulting photodimers changed from isotropic to crystalline phases, even at high temperature. Various experiments revealed that the bulk phase of the photodimers was affected not by the existence of regioisomers but by their fluidity at the photoirradiation temperature. The latter three photodimers retained enough fluidity, reflecting their high molecular mobilities at the photoirradiation temperature at which the isothermal crystallization occurred. The other two products were not able to crystallize due to low fluidity, resulting in amorphous phases. We also found that all the photodimers reverted to the corresponding monomers by thermal back reaction and recovered their initial photochemical and thermal properties. Using these reversible bulk-phase changes of the anthroyl compounds, we successfully demonstrated rewritable photopatterning in not only negative images but also positive ones, based on the optical contrast

  3. Cell cycle checkpoints: reversible when possible, irreversible when needed

    NARCIS (Netherlands)

    Krenning, L.

    2015-01-01

    Cell cycle checkpoints are reversible in nature, and can prevent progression into the next cell cycle phase if needed. In the case of DNA damage, cells can prevent progression from G1 into S phase, and from G2 into mitosis in the presence of DNA double strand breaks. Following DNA repair, these

  4. Non-reversible evolution of quantum chaotic system. Kinetic description

    International Nuclear Information System (INIS)

    Chotorlishvili, L.; Skrinnikov, V.

    2008-01-01

    It is well known that the appearance of non-reversibility in classical chaotic systems is connected with a local instability of phase trajectories relatively to a small change of initial conditions and parameters of the system. Classical chaotic systems reveal an exponential sensitivity to these changes. This leads to an exponential growth of initial error with time, and as the result after the statistical averaging over this error, the dynamics of the system becomes non-reversible. In spite of this, the question about the origin of non-reversibility in quantum case remains actual. The point is that the classical notion of instability of phase trajectories loses its sense during quantum consideration. The current work is dedicated to the clarification of the origin of non-reversibility in quantum chaotic systems. For this purpose we study a non-stationary dynamics of the chaotic quantum system. By analogy with classical chaos, we consider an influence of a small unavoidable error of the parameter of the system on the non-reversibility of the dynamics. It is shown in the Letter that due to the peculiarity of chaotic quantum systems, the statistical averaging over the small unavoidable error leads to the non-reversible transition from the pure state into the mixed one. The second part of the Letter is dedicated to the kinematic description of the chaotic quantum-mechanical system. Using the formalism of superoperators, a muster kinematic equation for chaotic quantum system was obtained from Liouville equation under a strict mathematical consideration

  5. Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: A quantitative proteomics approach.

    Science.gov (United States)

    Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J R; Santos, Romana

    2016-04-14

    Marine bioadhesives have unmatched performances in wet environments, being an inspiration for biomedical applications. In sea urchins specialized adhesive organs, tube feet, mediate reversible adhesion, being composed by a disc, producing adhesive and de-adhesive secretions, and a motile stem. After tube foot detachment, the secreted adhesive remains bound to the substratum as a footprint. Sea urchin adhesive is composed by proteins and sugars, but so far only one protein, Nectin, was shown to be over-expressed as a transcript in tube feet discs, suggesting its involvement in sea urchin adhesion. Here we use high-resolution quantitative mass-spectrometry to perform the first study combining the analysis of the differential proteome of an adhesive organ, with the proteome of its secreted adhesive. This strategy allowed us to identify 163 highly over-expressed disc proteins, specifically involved in sea urchin reversible adhesion; to find that 70% of the secreted adhesive components fall within five protein groups, involved in exocytosis and microbial protection; and to provide evidences that Nectin is not only highly expressed in tube feet discs but is an actual component of the adhesive. These results give an unprecedented insight into the molecular mechanisms underlying sea urchin adhesion, and opening new doors to develop wet-reliable, reversible, and ecological biomimetic adhesives. Sea urchins attach strongly but in a reversible manner to substratum, being a valuable source of inspiration for industrial and biomedical applications. Yet, the molecular mechanisms governing reversible adhesion are still poorly studied delaying the engineering of biomimetic adhesives. We used the latest mass spectrometry techniques to analyze the differential proteome of an adhesive organ and the proteome of its secreted adhesive, allowing us to uncover the key players in sea urchin reversible adhesion. We demonstrate, that Nectin, a protein previously pointed out as potentially

  6. Inulin in Medicinal Plants (IV) : Reversed-Phase High-Performance Liquid Chromatography of Inulin after Acetylation : Molecular-Weight Distribution of Inulin in Medicinal Plants

    OpenAIRE

    三野, 芳紀; 筒井, 聡美; 太田, 長世; YOSHIKI, MINO; SATOMI, TSUTSUI; NAGAYO, OTA; 大阪薬科大学; 大阪薬科大学; 大阪薬科大学; Osaka College of Pharmacy; Osaka College of Pharmacy; Osaka College of Pharmacy

    1985-01-01

    Reversed-phase high-performance liquid chromatography coupled with pre-acetylation enabled acculate molecular-weight assay of inulin in medicinal plants to be conducted. The results clearly showed that the molecular-weight distribution of inulin varied depending on the stage of growth: Small molecular weight inulin polymers were detected in large quantity in the earlier growth stage whereas large molecular weight inulin polymers at the flowering and post flowering period.

  7. Unidirectional transmission in 1D nonlinear photonic crystal based on topological phase reversal by optical nonlinearity

    Directory of Open Access Journals (Sweden)

    Chong Li

    2017-02-01

    Full Text Available We propose a scheme of unidirectional transmission in a 1D nonlinear topological photonic crystal based on the topological edge state and three order optical nonlinearity. The 1D photonic crystals consists of a nonlinear photonic crystal L and a linear photonic crystal R. In the backward direction, light is totally reflected for the photons transmission prohibited by the bandgap. While in the forward direction, light interacts with the nonlinear photonic crystal L by optical Kerr effect, bringing a topological phase reversal and results the topological edge mode arising at the interface which could transmit photons through the bandgaps both of the photonic crystal L and R. When the signal power intensity larger than a moderate low threshold value of 10.0 MW/cm2, the transmission contrast ratio could remain at 30 steadily.

  8. Characterization of a methionine-rich protein from the seeds of Cereus jamacaru Mill. (Cactaceae

    Directory of Open Access Journals (Sweden)

    T.C.F.R. Aragão

    2000-08-01

    Full Text Available We describe here the isolation and characterization of a major albumin from the seeds of Cereus jamacaru (Cactaceae, to which we gave the trivial name of cactin. This protein has a molecular mass of 11.3 kDa and is formed by a light chain (3.67 kDa and a heavy chain (7.63 kDa. This protein was isolated using a combination of gel filtration chromatography and reverse-phase HPLC. The amino acid composition of cactin was determined and found to resemble that of the 2S seed reserve protein from the Brazil nut, a protein remarkable for its high methionine content. The usefulness of cactin as a molecular marker in the taxonomy of the Cactaceae is discussed.

  9. Polycomb Group Proteins RING1A and RING1B Regulate the Vegetative Phase Transition in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jian Li

    2017-05-01

    Full Text Available Polycomb group (PcG protein-mediated gene silencing is a major regulatory mechanism in higher eukaryotes that affects gene expression at the transcriptional level. Here, we report that two conserved homologous PcG proteins, RING1A and RING1B (RING1A/B, are required for global H2A monoubiquitination (H2Aub in Arabidopsis. The mutation of RING1A/B increased the expression of members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL gene family and caused an early vegetative phase transition. The early vegetative phase transition observed in ring1a ring1b double mutant plants was dependent on an SPL family gene, and the H2Aub status of the chromatin at SPL locus was dependent on RING1A/B. Moreover, mutation in RING1A/B affected the miRNA156a-mediated vegetative phase transition, and RING1A/B and the AGO7-miR390-TAS3 pathway were found to additively regulate this transition in Arabidopsis. Together, our results demonstrate that RING1A/B regulates the vegetative phase transition in Arabidopsis through the repression of SPL family genes.

  10. Electrochemical deposition and characterization of zinc–nickel alloys deposited by direct and reverse current

    Directory of Open Access Journals (Sweden)

    JELENA B. BAJAT

    2005-12-01

    Full Text Available Zn–Ni alloys electrochemically deposited on steel under various deposition conditions were investigated. The alloys were deposited on a rotating disc electrode and on a steel panel from chloride solutions by direct and reverse current. The influence of reverse plating variables (cathodic and anodic current densities and their time duration on the composition, phase structure and corrosion properties were investigated. The chemical content and phase composition affect the anticorrosive properties of Zn–Ni alloys during exposure to a corrosive agent (3 % NaCl solution. It was shown that the Zn–Ni alloy electrodeposited by reverse current with a full period T = 1 s and r = 0.2 exhibits the best corrosion properties of all the investigated alloys deposited by reverse current.

  11. Routine phasing of coiled-coil protein crystal structures with AMPLE

    Directory of Open Access Journals (Sweden)

    Jens M. H. Thomas

    2015-03-01

    Full Text Available Coiled-coil protein folds are among the most abundant in nature. These folds consist of long wound α-helices and are architecturally simple, but paradoxically their crystallographic structures are notoriously difficult to solve with molecular-replacement techniques. The program AMPLE can solve crystal structures by molecular replacement using ab initio search models in the absence of an existent homologous protein structure. AMPLE has been benchmarked on a large and diverse test set of coiled-coil crystal structures and has been found to solve 80% of all cases. Successes included structures with chain lengths of up to 253 residues and resolutions down to 2.9 Å, considerably extending the limits on size and resolution that are typically tractable by ab initio methodologies. The structures of two macromolecular complexes, one including DNA, were also successfully solved using their coiled-coil components. It is demonstrated that both the ab initio modelling and the use of ensemble search models contribute to the success of AMPLE by comparison with phasing attempts using single structures or ideal polyalanine helices. These successes suggest that molecular replacement with AMPLE should be the method of choice for the crystallographic elucidation of a coiled-coil structure. Furthermore, AMPLE may be able to exploit the presence of a coiled coil in a complex to provide a convenient route for phasing.

  12. Deciphering the fluorescence resonance energy transfer from denatured transport protein to anthracene 1,5 disulphonate in reverse micellar environment

    Science.gov (United States)

    Singharoy, Dipti; Bhattacharya, Subhash Chandra

    2017-12-01

    Constrained environmental effect inside AOT reverse micellar media has been employed in this work to collect the information about energy transfer efficacy between sodium salt of anthracene 1,5 disulphonate (1,5-AS) with model transport proteins, bovine serum albumin (BSA), and human serum albumin (HSA). Steady state, time-resolved fluorescence and circular dichroism techniques have been used for this purpose and corresponding Fӧrster-type resonance energy transfer (FRET) from tryptophan residues to 1,5-AS indicates that 1,5-AS binds in the vicinity of the tryptophan residue (BSA and HSA) with equal strength. Indication of protein damage from fluorescence data and its confirmation has been measured from CD measurement. Molecular modeling study hereby plays a crucial role to predict the minimum energy docked conformation of the probe inside the protein environment. From the docked conformation the distance between 1,5-AS and tryptophan moiety of BSA/HSA has successfully explained the FRET possibility between them. A comparative modeling study between BSA and HSA with 1,5-AS assigning their binding site within specific amino acids plays a crucial role in support of the FRET study.

  13. Rapid Determination of Bile Acids in Bile from Various Mammals by Reversed-Phase Ultra-Fast Liquid Chromatography.

    Science.gov (United States)

    Si, Gu Leng Ri; Yao, Peng; Shi, Luwen

    2015-08-01

    A valid and efficient reversed-phase ultra-fast liquid chromatography method was developed for the simultaneous determination of 13 bile acids in the bile of three mammal species, including rat, pig and human gallstone patients. Chromatographic separation was performed with a Shim-pack XR-ODS column, and the mobile phase consisted of acetonitrile and potassium phosphate buffer (pH 2.6) at a flow rate of 0.5 mL min(-1). The linear detection range of most bile acids ranged from 2 to 600 ng µL(-1) with a good correlation coefficient (>0.9995). The precision of each bile acid was bile acids were separated in 15 min with satisfactory resolution, and the total analysis time was 18 min, including equilibration. The method was successfully applied in rapid screening of bile samples from the three mammals. Significant metabolic frameworks of bile acids among various species were observed, whereas considerable quantitative variations in both inter- and intraspecies were also observed, especially for gallstone patients. Our results suggest that detecting the change of bile acid profiles could be applied for the diagnosis of gallstone disease. © Crown copyright 2014.

  14. Protein and Peptide Gas-phase Structure Investigation Using Collision Cross Section Measurements and Hydrogen Deuterium Exchange

    Science.gov (United States)

    Khakinejad, Mahdiar

    Protein and peptide gas-phase structure analysis provides the opportunity to study these species outside of their explicit environment where the interaction network with surrounding molecules makes the analysis difficult [1]. Although gas-phase structure analysis offers a unique opportunity to study the intrinsic behavior of these biomolecules [2-4], proteins and peptides exhibit very low vapor pressures [2]. Peptide and protein ions can be rendered in the gas-phase using electrospray ionization (ESI) [5]. There is a growing body of literature that shows proteins and peptides can maintain solution structures during the process of ESI and these structures can persist for a few hundred milliseconds [6-9]. Techniques for monitoring gas-phase protein and peptide ion structures are categorized as physical probes and chemical probes. Collision cross section (CCS) measurement, being a physical probe, is a powerful method to investigate gas-phase structure size [3, 7, 10-15]; however, CCS values alone do not establish a one to one relation with structure(i.e., the CCS value is an orientationally averaged value [15-18]. Here we propose the utility of gas-phase hydrogen deuterium exchange (HDX) as a second criterion of structure elucidation. The proposed approach incudes extensive MD simulations to sample biomolecular ion conformation space with the production of numerous, random in-silico structures. Subsequently a CCS can be calculated for these structures and theoretical CCS values are compared with experimental values to produce a pool of candidate structures. Utilizing a chemical reaction model based on the gas-phase HDX mechanism, the HDX kinetics behavior of these candidate structures are predicted and compared to experimental results to nominate the best in-silico structures which match (chemically and physically) with experimental observations. For the predictive approach to succeed, an extensive technique and method development is essential. To combine CCS

  15. Acute phase proteins in bovine milk in an experimental model of Staphylococcus aureus subclinical mastitis

    DEFF Research Database (Denmark)

    Eckersall, P D; Young, F J; Nolan, A M

    2006-01-01

    and serum amyloid A increase in serum during mastitis. The concentrations of these proteins were determined in an experimental model using a field strain of Staphylococcus aureus to induce subclinical mastitis in dairy cows. The expression of mRNA coding for these proteins was assessed and the presence of M......The objectives were to establish the origin of 2 acute phase proteins in milk during subclinical bovine mastitis and to characterize the relationship between those proteins in milk and blood. Haptoglobin (Hp) and mammary-associated serum amyloid A (M-SAA3) appear in milk during mastitis, whereas Hp...

  16. Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist.

    Science.gov (United States)

    Rezelj, Veronica V; Överby, Anna K; Elliott, Richard M

    2015-05-01

    Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a

  17. Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

    DEFF Research Database (Denmark)

    Houen, G.; Olsen, D.T.; Hansen, P.R.

    2003-01-01

    A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated......, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates...... of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings...

  18. Elucidating the weak protein-protein interaction mechanisms behind the liquid-liquid phase separation of a mAb solution by different types of additives.

    Science.gov (United States)

    Wu, Guoliang; Wang, Shujing; Tian, Zhou; Zhang, Ning; Sheng, Han; Dai, Weiguo; Qian, Feng

    2017-11-01

    Liquid-liquid phase separation (LLPS) has long been observed during the physical stability investigation of therapeutic protein formulations. The buffer conditions and the presence of various excipients are thought to play important roles in the formulation development of monoclonal antibodies (mAbs). In this study, the effects of several small-molecule excipients (histidine, alanine, glycine, sodium phosphate, sodium chloride, sorbitol and sucrose) with diverse physical-chemical properties on LLPS of a model IgG1 (JM2) solutions were investigated by multiple techniques, including UV-vis spectroscopy, circular dichroism, differential scanning calorimetry/fluorimetry, size exclusion chromatography and dynamic light scattering. The LLPS of JM2 was confirmed to be a thermodynamic equilibrium process with no structural changes or irreversible aggregation of proteins. Phase diagrams of various JM2 formulations were constructed, suggesting that the phase behavior of JM2 was dependent on the solution pH, ionic strength and the presence of other excipients such as glycine, alanine, sorbitol and sucrose. Furthermore, we demonstrated that for this mAb, the interaction parameter (k D ) determined at low protein concentration appeared to be a good predictor for the occurrence of LLPS at high concentration. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Differential expression of proteins and genes in the lag phase of Lactococcus lactis subsp lactis grown in synthetic medium and reconstituted skim milk

    DEFF Research Database (Denmark)

    Larsen, N.; Boye, Mette; Jakobsen, Marianne

    2006-01-01

    We investigated protein and gene expression in the lag phase of Lactococcus lactis subsp. lactis CNRZ 157 and compared it to the exponential and stationary phases. By means of two-dimensional polyacrylamide gel electrophoresis, 28 highly expressed lag-phase proteins, implicated in nucleotide meta...

  20. Quantum vertex model for reversible classical computing.

    Science.gov (United States)

    Chamon, C; Mucciolo, E R; Ruckenstein, A E; Yang, Z-C

    2017-05-12

    Mappings of classical computation onto statistical mechanics models have led to remarkable successes in addressing some complex computational problems. However, such mappings display thermodynamic phase transitions that may prevent reaching solution even for easy problems known to be solvable in polynomial time. Here we map universal reversible classical computations onto a planar vertex model that exhibits no bulk classical thermodynamic phase transition, independent of the computational circuit. Within our approach the solution of the computation is encoded in the ground state of the vertex model and its complexity is reflected in the dynamics of the relaxation of the system to its ground state. We use thermal annealing with and without 'learning' to explore typical computational problems. We also construct a mapping of the vertex model into the Chimera architecture of the D-Wave machine, initiating an approach to reversible classical computation based on state-of-the-art implementations of quantum annealing.

  1. Emergence of the acute-phase protein hemopexin in jawed vertebrates

    OpenAIRE

    Dooley, Helen; Buckingham, E. Bryan; Criscitiello, Michael F.; Flajnik, Martin F.

    2010-01-01

    When released from damaged erythrocytes free heme not only provides a source of iron for invading bacteria but is also highly toxic due to its ability to catalyze free radical formation. Hemopexin (Hx) binds free heme with very high affinity and thus protects against heme toxicity, sequesters heme from pathogens, and helps conserve valuable iron. Hx is also an acute-phase serum protein (APP), whose expression is induced by inflammation. To date Hx has been identified as far back in phylogeny ...

  2. Nitrile group as infrared probe for the characterization of the conformation of bovine serum albumin solubilized in reverse micelles.

    Science.gov (United States)

    Xue, Luyan; Zou, Feixue; Zhao, Yin; Huang, Xirong; Qu, Yinbo

    2012-11-01

    Infrared spectroscopy is a powerful technique for structure characterization. For a protein hosted in a reversed micellar medium, the spectral features of the protein are always interfered by the IR absorption bands of the medium in addition to the congestion in their IR spectra. Fortunately, there is a transparent window in the 2500-2200 cm(-1) region. Incorporation of a vibrational probe with IR absorption frequencies in this region into proteins represents a promising strategy for the study of the conformation of a protein in a reverse micelle. In the present work, we incorporated 4-cyanobenzyl group (CN) into bovine serum albumin (BSA) via cysteine alkylation reactions under mild conditions. Circular dichroism spectroscopy showed that the CN modified BSA (CNBSA) could retain its conformation. When CNBSA was hosted in AOT reverse micelle, it was found that the nitrile group on BSA was sensitive to the conformational change of BSA induced by urea as an additive in the reverse micelle. The peak splitting of nitrile group was also observed when the size of AOT reverse micelle and the concentration of an electrolyte were varied. Obviously, the shift of the IR absorption peak and/or peak splitting of nitrile group on BSA are correlated with the change of BSA conformation in AOT reverse micelle. So we conclude that the nitrile infrared probe can be used to study protein conformation in a reverse micelle. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

    Science.gov (United States)

    Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

    1998-11-01

    Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

  4. Prediction of Protein-Protein Interactions by NanoLuc-Based Protein-Fragment Complementation Assay | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Emory has developed a new NanoLuc®-based protein-fragment complementation assay (NanoPCA) which allows the detection of novel protein-protein interactions (PPI). NanoPCA allows the study of PPI dynamics with reversible interactions.  Read the abstract. Experimental Approaches Read the detailed Experimetnal Approaches. 

  5. Hepatic acute-phase proteins control innate immune responses during infection by promoting myeloid-derived suppressor cell function

    NARCIS (Netherlands)

    Sander, L.E.; Sackett, S.D.; Dierssen, U.; Beraza, N.; Linke, R.; Müller, M.R.; Blander, J.M.; Tacke, F.; Trautwein, C.

    2010-01-01

    Acute-phase proteins (APPs) are an evolutionarily conserved family of proteins produced mainly in the liver in response to infection and inflammation. Despite vast pro- and antiinflammatory properties ascribed to individual APPs, their collective function during infections remains poorly defined.

  6. Hepatic acute phase proteins control innate immune responses during infection by promoting myeloid derived suppressor cell function

    NARCIS (Netherlands)

    Sander, Leif E.; Dutton Sackett, Sara; Dierssen, Uta; Beraza, Naiara; Linke, Reinhold P.; Muller, Michael; Magarian Blander, Julie; Tacke, Frank; Trautwein, Christian

    2010-01-01

    Acute phase proteins (APPs) are an evolutionarily conserved family of proteins produced mainly in the liver in response to infection and inflammation. Despite vast pro- and anti-inflammatory properties ascribed to individual APPs, their collective function during infections remains poorly defined.

  7. Modelling of the effect of solute structure and mobile phase pH and composition on the retention of phenoxy acid herbicides in reversed-phase high-performance liquid chromatography

    International Nuclear Information System (INIS)

    Aschi, Massimiliano; D'Archivio, Angelo Antonio; Mazzeo, Pietro; Pierabella, Mirko; Ruggieri, Fabrizio

    2008-01-01

    A feed-forward artificial neural network (ANN) learned by error back-propagation is used to generate a retention predictive model for phenoxy acid herbicides in isocratic reversed-phase high-performance liquid chromatography. The investigated solutes (18 compounds), apart from the most common herbicides of this class, include some derivatives of benzoic acid and phenylacetic acid structurally related to phenoxy acids, as a whole covering a pK a range between 2.3 and 4.3. A mixed model in terms of both solute descriptors and eluent attributes is built with the aim of predicting retention in water-acetonitrile mobile phases within a large range of composition (acetonitrile from 30% to 70%, v/v) and acidity (pH of water before mixing with acetonitrile ranging between 2 and 5). The set of input variables consists of solute pK a and quantum chemical molecular descriptors of both the neutral and dissociated form, %v/v of acetonitrile in the mobile phase and pH of aqueous phase before mixing with acetonitrile. After elimination of redundant variables, a nine-dimensional model is identified and its prediction ability is evaluated by external validation based on three solutes not involved in model generation and by cross-validation. A multilinear counterpart in terms of the same descriptors is seen to provide a noticeably poorer retention prediction

  8. Modelling of the effect of solute structure and mobile phase pH and composition on the retention of phenoxy acid herbicides in reversed-phase high-performance liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Aschi, Massimiliano [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy); D' Archivio, Angelo Antonio [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy)], E-mail: darchivi@univaq.it; Mazzeo, Pietro; Pierabella, Mirko; Ruggieri, Fabrizio [Dipartimento di Chimica, Ingegneria Chimica e Materiali, Universita degli Studi di L' Aquila, Via Vetoio, 67010 Coppito, L' Aquila (Italy)

    2008-06-02

    A feed-forward artificial neural network (ANN) learned by error back-propagation is used to generate a retention predictive model for phenoxy acid herbicides in isocratic reversed-phase high-performance liquid chromatography. The investigated solutes (18 compounds), apart from the most common herbicides of this class, include some derivatives of benzoic acid and phenylacetic acid structurally related to phenoxy acids, as a whole covering a pK{sub a} range between 2.3 and 4.3. A mixed model in terms of both solute descriptors and eluent attributes is built with the aim of predicting retention in water-acetonitrile mobile phases within a large range of composition (acetonitrile from 30% to 70%, v/v) and acidity (pH of water before mixing with acetonitrile ranging between 2 and 5). The set of input variables consists of solute pK{sub a} and quantum chemical molecular descriptors of both the neutral and dissociated form, %v/v of acetonitrile in the mobile phase and pH of aqueous phase before mixing with acetonitrile. After elimination of redundant variables, a nine-dimensional model is identified and its prediction ability is evaluated by external validation based on three solutes not involved in model generation and by cross-validation. A multilinear counterpart in terms of the same descriptors is seen to provide a noticeably poorer retention prediction.

  9. Flow reversal in combined laminar mixed convection heat and mass transfer with phase change in a vertical channel

    International Nuclear Information System (INIS)

    Oulaid, Othmane; Benhamou, Brahim; Galanis, Nicolas

    2010-01-01

    This paper, deals with a numerical study of the effects of buoyancy forces on an upward, steady state, laminar flow of humid air in a vertical parallel-plate channel. The plates are wetted by a thin liquid water film and maintained at a constant temperature which is lower than that of the air entering the channel. A 2D fully elliptical model, associated with the Boussinesq assumption, is used to take into account axial diffusion. The solution of this mathematical model is based on the finite volume method and the velocity-pressure coupling is handled by the SIMPLER algorithm. Numerical results show that buoyancy forces have a significant effect on the hydrodynamic, thermal and mass fraction fields. Additionally, these forces induce flow reversal for high air temperatures and mass fractions at the channel entrance. It is established that heat transfer associated with phase change is, sometimes, more significant than sensible heat transfer. Furthermore, this importance depends on the mass fraction gradient. The conditions for the existence of flow reversal are presented in charts and analytical expressions specifying the critical thermal Grashof number as a function of the Reynolds number for different values of the solutal Grashof number and different aspect ratios of the channel.

  10. Peripheral administration of antisense oligonucleotides targeting the amyloid-β protein precursor reverses AβPP and LRP-1 overexpression in the aged SAMP8 mouse brain.

    Science.gov (United States)

    Erickson, Michelle A; Niehoff, Michael L; Farr, Susan A; Morley, John E; Dillman, Lucy A; Lynch, Kristin M; Banks, William A

    2012-01-01

    The senescence accelerated mouse-prone 8 (SAMP8) mouse model of Alzheimer's disease has a natural mutation leading to age-related increases in the amyloid-β protein precursor (AβPP) and amyloid-β (Aβ) in the brain, memory impairment, and deficits in Aβ removal from the brain. Previous studies show that centrally administered antisense oligonucleotide directed against AβPP can decrease AβPP expression and Aβ production in the brains of aged SAMP8 mice, and improve memory. The same antisense crosses the blood-brain barrier and reverses memory deficits when injected intravenously. Here, we give 6 μg of AβPP or control antisense 3 times over 2 week intervals to 12 month old SAMP8 mice. Object recognition test was done 48 hours later, followed by removal of whole brains for immunoblot analysis of AβPP, low-density lipoprotein-related protein-1 (LRP-1), p-glycoprotein (Pgp), receptor for advanced glycation endproducts (RAGE), or ELISA of soluble Aβ(40). Our results show that AβPP antisense completely reverses a 30% age-associated increase in AβPP signal (p < 0.05 versus untreated 4 month old SAMP8). Soluble Aβ(40) increased with age, but was not reversed by antisense. LRP-1 large and small subunits increased significantly with age (147.7%, p < 0.01 and 123.7%, p < 0.05 respectively), and AβPP antisense completely reversed these increases (p < 0.05). Pgp and RAGE were not significantly altered with age or antisense. Antisense also caused improvements in memory (p < 0.001). Together, these data support the therapeutic potential of AβPP antisense and show a unique association between AβPP and LRP-1 expression in the SAMP8 mouse.

  11. Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis.

    Directory of Open Access Journals (Sweden)

    Takahiro Kawagishi

    2016-02-01

    Full Text Available Nelson Bay orthoreoviruses (NBVs are members of the fusogenic orthoreoviruses and possess 10-segmented double-stranded RNA genomes. NBV was first isolated from a fruit bat in Australia more than 40 years ago, but it was not associated with any disease. However, several NBV strains have been recently identified as causative agents for respiratory tract infections in humans. Isolation of these pathogenic bat reoviruses from patients suggests that NBVs have evolved to propagate in humans in the form of zoonosis. To date, no strategy has been developed to rescue infectious viruses from cloned cDNA for any member of the fusogenic orthoreoviruses. In this study, we report the development of a plasmid-based reverse genetics system free of helper viruses and independent of any selection for NBV isolated from humans with acute respiratory infection. cDNAs corresponding to each of the 10 full-length RNA gene segments of NBV were cotransfected into culture cells expressing T7 RNA polymerase, and viable NBV was isolated using a plaque assay. The growth kinetics and cell-to-cell fusion activity of recombinant strains, rescued using the reverse genetics system, were indistinguishable from those of native strains. We used the reverse genetics system to generate viruses deficient in the cell attachment protein σC to define the biological function of this protein in the viral life cycle. Our results with σC-deficient viruses demonstrated that σC is dispensable for cell attachment in several cell lines, including murine fibroblast L929 cells but not in human lung epithelial A549 cells, and plays a critical role in viral pathogenesis. We also used the system to rescue a virus that expresses a yellow fluorescent protein. The reverse genetics system developed in this study can be applied to study the propagation and pathogenesis of pathogenic NBVs and in the generation of recombinant NBVs for future vaccines and therapeutics.

  12. Acute phase protein concentrations in serum and milk from healthy cows, cows with clinical mastitis and cows with extramammary inflammatory conditions

    NARCIS (Netherlands)

    Nielsen, B.H.; Jacobsen, S.; Andersen, P.H.; Niewold, T.A.; Heegaard, P.M.H.

    2004-01-01

    The concentrations of the two acute phase proteins, serum amyloid A and haptoglobin, in serum and milk were compared in 10 cows with clinical mastitis, 11 cows with extramammary inflammatory conditions and 10 clinically healthy control cows. The concentrations of both acute phase proteins were

  13. Equilibrium-phase MR angiography: Comparison of unspecific extracellular and protein-binding gadolinium-based contrast media with respect to image quality.

    Science.gov (United States)

    Erb-Eigner, Katharina; Taupitz, Matthias; Asbach, Patrick

    2016-01-01

    The purpose of this study was to compare contrast and image quality of whole-body equilibrium-phase high-spatial-resolution MR angiography using a non-protein-binding unspecific extracellular gadolinium-based contrast medium with that of two contrast media with different protein-binding properties. 45 patients were examined using either 15 mL of gadobutrol (non-protein-binding, n = 15), 32 mL of gadobenate dimeglumine (weakly protein binding, n = 15) or 11 mL gadofosveset trisodium (protein binding, n = 15) followed by equilibrium-phase high-spatial-resolution MR-angiography of four consecutive anatomic regions. The time elapsed between the contrast injection and the beginning of the equilibrium-phase image acquisition in the respective region was measured and was up to 21 min. Signal intensity was measured in two vessels per region and in muscle tissue. Relative contrast (RC) values were calculated. Vessel contrast, artifacts and image quality were rated by two radiologists in consensus on a five-point scale. Compared with gadobutrol, gadofosveset trisodium revealed significantly higher RC values only when acquired later than 15 min after bolus injection. Otherwise, no significant differences between the three contrast media were found regarding vascular contrast and image quality. Equilibrium-phase high-spatial-resolution MR-angiography using a weakly protein-binding or even non-protein-binding contrast medium is equivalent to using a stronger protein-binding contrast medium when image acquisition is within the first 15 min after contrast injection, and allows depiction of the vasculature with high contrast and image quality. The protein-binding contrast medium was superior for imaging only later than 15 min after contrast medium injection. Copyright © 2015 John Wiley & Sons, Ltd.

  14. Theta, time reversal and temperature

    Energy Technology Data Exchange (ETDEWEB)

    Gaiotto, Davide [Perimeter Institute for Theoretical Physics,Waterloo, Ontario, N2L 2Y5 (Canada); Kapustin, Anton [Walter Burke Institute for Theoretical Physics, California Institute of Technology,Pasadena, CA 91125 (United States); Komargodski, Zohar [Department of Particle Physics and Astrophysics, Weizmann Institute of Science,Rehovot 76100 (Israel); Seiberg, Nathan [School of Natural Sciences, Institute for Advanced Study,Princeton, NJ 08540 (United States)

    2017-05-17

    SU(N) gauge theory is time reversal invariant at θ=0 and θ=π. We show that at θ=π there is a discrete ’t Hooft anomaly involving time reversal and the center symmetry. This anomaly leads to constraints on the vacua of the theory. It follows that at θ=π the vacuum cannot be a trivial non-degenerate gapped state. (By contrast, the vacuum at θ=0 is gapped, non-degenerate, and trivial.) Due to the anomaly, the theory admits nontrivial domain walls supporting lower-dimensional theories. Depending on the nature of the vacuum at θ=π, several phase diagrams are possible. Assuming area law for space-like loops, one arrives at an inequality involving the temperatures at which CP and the center symmetry are restored. We also analyze alternative scenarios for SU(2) gauge theory. The underlying symmetry at θ=π is the dihedral group of 8 elements. If deconfined loops are allowed, one can have two O(2)-symmetric fixed points. It may also be that the four-dimensional theory around θ=π is gapless, e.g. a Coulomb phase could match the underlying anomalies.

  15. Theta, time reversal and temperature

    International Nuclear Information System (INIS)

    Gaiotto, Davide; Kapustin, Anton; Komargodski, Zohar; Seiberg, Nathan

    2017-01-01

    SU(N) gauge theory is time reversal invariant at θ=0 and θ=π. We show that at θ=π there is a discrete ’t Hooft anomaly involving time reversal and the center symmetry. This anomaly leads to constraints on the vacua of the theory. It follows that at θ=π the vacuum cannot be a trivial non-degenerate gapped state. (By contrast, the vacuum at θ=0 is gapped, non-degenerate, and trivial.) Due to the anomaly, the theory admits nontrivial domain walls supporting lower-dimensional theories. Depending on the nature of the vacuum at θ=π, several phase diagrams are possible. Assuming area law for space-like loops, one arrives at an inequality involving the temperatures at which CP and the center symmetry are restored. We also analyze alternative scenarios for SU(2) gauge theory. The underlying symmetry at θ=π is the dihedral group of 8 elements. If deconfined loops are allowed, one can have two O(2)-symmetric fixed points. It may also be that the four-dimensional theory around θ=π is gapless, e.g. a Coulomb phase could match the underlying anomalies.

  16. Reversal of diabetic nephropathy by a ketogenic diet.

    Directory of Open Access Journals (Sweden)

    Michal M Poplawski

    Full Text Available Intensive insulin therapy and protein restriction delay the development of nephropathy in a variety of conditions, but few interventions are known to reverse nephropathy. Having recently observed that the ketone 3-beta-hydroxybutyric acid (3-OHB reduces molecular responses to glucose, we hypothesized that a ketogenic diet, which produces prolonged elevation of 3-OHB, may reverse pathological processes caused by diabetes. To address this hypothesis, we assessed if prolonged maintenance on a ketogenic diet would reverse nephropathy produced by diabetes. In mouse models for both Type 1 (Akita and Type 2 (db/db diabetes, diabetic nephropathy (as indicated by albuminuria was allowed to develop, then half the mice were switched to a ketogenic diet. After 8 weeks on the diet, mice were sacrificed to assess gene expression and histology. Diabetic nephropathy, as indicated by albumin/creatinine ratios as well as expression of stress-induced genes, was completely reversed by 2 months maintenance on a ketogenic diet. However, histological evidence of nephropathy was only partly reversed. These studies demonstrate that diabetic nephropathy can be reversed by a relatively simple dietary intervention. Whether reduced glucose metabolism mediates the protective effects of the ketogenic diet remains to be determined.

  17. Reversal of Diabetic Nephropathy by a Ketogenic Diet

    Science.gov (United States)

    Poplawski, Michal M.; Mastaitis, Jason W.; Isoda, Fumiko; Grosjean, Fabrizio; Zheng, Feng; Mobbs, Charles V.

    2011-01-01

    Intensive insulin therapy and protein restriction delay the development of nephropathy in a variety of conditions, but few interventions are known to reverse nephropathy. Having recently observed that the ketone 3-beta-hydroxybutyric acid (3-OHB) reduces molecular responses to glucose, we hypothesized that a ketogenic diet, which produces prolonged elevation of 3-OHB, may reverse pathological processes caused by diabetes. To address this hypothesis, we assessed if prolonged maintenance on a ketogenic diet would reverse nephropathy produced by diabetes. In mouse models for both Type 1 (Akita) and Type 2 (db/db) diabetes, diabetic nephropathy (as indicated by albuminuria) was allowed to develop, then half the mice were switched to a ketogenic diet. After 8 weeks on the diet, mice were sacrificed to assess gene expression and histology. Diabetic nephropathy, as indicated by albumin/creatinine ratios as well as expression of stress-induced genes, was completely reversed by 2 months maintenance on a ketogenic diet. However, histological evidence of nephropathy was only partly reversed. These studies demonstrate that diabetic nephropathy can be reversed by a relatively simple dietary intervention. Whether reduced glucose metabolism mediates the protective effects of the ketogenic diet remains to be determined. PMID:21533091

  18. Proteomic analysis of three gonad types of swamp eel reveals genes differentially expressed during sex reversal.

    Science.gov (United States)

    Sheng, Yue; Zhao, Wei; Song, Ying; Li, Zhigang; Luo, Majing; Lei, Quan; Cheng, Hanhua; Zhou, Rongjia

    2015-05-18

    A variety of mechanisms are engaged in sex determination in vertebrates. The teleost fish swamp eel undergoes sex reversal naturally and is an ideal model for vertebrate sexual development. However, the importance of proteome-wide scanning for gonad reversal was not previously determined. We report a 2-D electrophoresis analysis of three gonad types of proteomes during sex reversal. MS/MS analysis revealed a group of differentially expressed proteins during ovary to ovotestis to testis transformation. Cbx3 is up-regulated during gonad reversal and is likely to have a role in spermatogenesis. Rab37 is down-regulated during the reversal and is mainly associated with oogenesis. Both Cbx3 and Rab37 are linked up in a protein network. These datasets in gonadal proteomes provide a new resource for further studies in gonadal development.

  19. Reversible S-nitrosylation in an engineered azurin

    Energy Technology Data Exchange (ETDEWEB)

    Tian, Shiliang; Liu, Jing; Cowley, Ryan E.; Hosseinzadeh, Parisa; Marshall, Nicholas M.; Yu, Yang; Robinson, Howard; Nilges, Mark J.; Blackburn, Ninian J.; Solomon, Edward I.; Lu, Yi

    2016-04-25

    S-Nitrosothiols are known as reagents for NO storage and transportation and as regulators in many physiological processes. Although the S-nitrosylation catalysed by haem proteins is well known, no direct evidence of S-nitrosylation in copper proteins has been reported. Here, we report reversible insertion of NO into a copper–thiolate bond in an engineered copper centre in Pseudomonas aeruginosa azurin by rational design of the primary coordination sphere and tuning its reduction potential by deleting a hydrogen bond in the secondary coordination sphere. The results not only provide the first direct evidence of S-nitrosylation of Cu(II)-bound cysteine in metalloproteins, but also shed light on the reaction mechanism and structural features responsible for stabilizing the elusive Cu(I)–S(Cys)NO species. The fast, efficient and reversible S-nitrosylation reaction is used to demonstrate its ability to prevent NO inhibition of cytochrome bo3 oxidase activity by competing for NO binding with the native enzyme under physiologically relevant conditions.

  20. Optical Time Reversal from Time-Dependent Epsilon-Near-Zero Media

    Science.gov (United States)

    Vezzoli, Stefano; Bruno, Vincenzo; DeVault, Clayton; Roger, Thomas; Shalaev, Vladimir M.; Boltasseva, Alexandra; Ferrera, Marcello; Clerici, Matteo; Dubietis, Audrius; Faccio, Daniele

    2018-01-01

    Materials with a spatially uniform but temporally varying optical response have applications ranging from magnetic field-free optical isolators to fundamental studies of quantum field theories. However, these effects typically become relevant only for time variations oscillating at optical frequencies, thus presenting a significant hurdle that severely limits the realization of such conditions. Here we present a thin-film material with a permittivity that pulsates (uniformly in space) at optical frequencies and realizes a time-reversing medium of the form originally proposed by Pendry [Science 322, 71 (2008), 10.1126/science.1162087]. We use an optically pumped, 500 nm thick film of epsilon-near-zero (ENZ) material based on Al-doped zinc oxide. An incident probe beam is both negatively refracted and time reversed through a reflected phase-conjugated beam. As a result of the high nonlinearity and the refractive index that is close to zero, the ENZ film leads to time reversed beams (simultaneous negative refraction and phase conjugation) with near-unit efficiency and greater-than-unit internal conversion efficiency. The ENZ platform therefore presents the time-reversal features required, e.g., for efficient subwavelength imaging, all-optical isolators and fundamental quantum field theory studies.