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Sample records for core protein characterization

  1. Structural characterization of Mumps virus fusion protein core

    International Nuclear Information System (INIS)

    Liu Yueyong; Xu Yanhui; Lou Zhiyong; Zhu Jieqing; Hu Xuebo; Gao, George F.; Qiu Bingsheng; Rao Zihe; Tien, Po

    2006-01-01

    The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar charecterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins

  2. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Jian [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Zhang, Huaidong [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Gong, Rui, E-mail: gongr@wh.iov.cn [CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072 (China); State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071 (China)

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.

  3. Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W

    International Nuclear Information System (INIS)

    Gong Rui; Peng Xiaoxue; Kang Shuli; Feng Huixing; Huang Jianying; Zhang Wentao; Lin Donghai; Tien Po; Xiao Gengfu

    2005-01-01

    Syncytin is a captive retroviral envelope protein, possibly involved in the formation of the placental syncytiotrophoblast layer generated by trophoblast cell fusion at the maternal-fetal interface. We found that syncytin and type I viral envelope proteins shared similar structural profiling, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR). We expressed the predicted regions of NHR (41 aa) and CHR (34 aa) in syncytin as a native single chain (named 2-helix protein) to characterize it. 2-helix protein exists as a trimer and is highly α-helix, thermo-stable, and denatured by low pH. NHR and CHR could form a protease-resistant complex. The complex structure built by the molecular docking demonstrated that NHR and CHR associated in an antiparallel manner. Overall, the 2-helix protein could form a thermo-stable coiled coil trimer. The fusion core structure of syncytin was first demonstrated in endogenous retrovirus. These results support the explanation how syncytin mediates cytotrophoblast cell fusion involved in placental morphogenesis

  4. Heparan sulfate proteoglycan from the extracellular matrix of human lung fibroblasts. Isolation, purification, and core protein characterization

    International Nuclear Information System (INIS)

    Heremans, A.; Cassiman, J.J.; Van den Berghe, H.; David, G.

    1988-01-01

    Confluent cultured human lung fibroblasts were labeled with 35SO4(2-). After 48 h of labeling, the pericellular matrix was prepared by Triton X-100 and deoxycholate extraction of the monolayers. Heparan sulfate proteoglycan (HSPG) accounted for nearly 80% of the total matrix [35S]proteoglycans. After solubilization in 6 M guanidinium HCl and cesium chloride density gradient centrifugation, the majority (78%) of these [35S] HSPG equilibrated at an average buoyant density of 1.35 g/ml. This major HSPG fraction was purified by ion-exchange chromatography on Mono Q and by gel filtration on Sepharose CL-4B, and further characterized by gel electrophoresis and immunoblotting. Intact [35S]HSPG eluted with Kav 0.1 from Sepharose CL-4B, whereas the protein-free [35S]heparan sulfate chains, obtained by alkaline borohydride treatment of the proteoglycan fractions, eluted with Kav 0.45 (Mr approximately 72,000). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, core (protein) preparations, obtained by heparitinase digestion of 125I-labeled HSPG fractions, yielded one major labeled band with apparent molecular mass of approximately 300 kDa. Reduction with beta-mercaptoethanol slightly increased the apparent Mr of the labeled band, suggesting a single polypeptide structure and the presence of intrachain disulfide bonds. Immunoadsorption experiments and immunostaining of electrophoretically separated heparitinase-digested core proteins with monoclonal antibodies raised against matrix and cell surface-associated HSPG suggested that the major matrix-associated HSPG of cultured human lung fibroblasts is distinct from the HSPG that are anchored in the membranes of these cells. Binding studies suggested that this matrix HSPG interacts with several matrix components, both through its glycosaminoglycan chains and through its heparitinase-resistant core. (Abstract Truncated)

  5. Multifunctional Fe3O4/Au core/satellite nanocubes: an efficient chemical synthesis, characterization and functionalization of streptavidin protein.

    Science.gov (United States)

    Abbas, Mohamed; RamuluTorati, Sri; Kim, CheolGi

    2017-02-14

    A novel and efficient chemical approach for the synthesis of Fe 3 O 4 /Au core/satellite nanocubes is reported. In a one-pot reaction, metallic Au nanodots were successfully deposited on the polyvinylpyrrolidone (PVP) functionalized Fe 3 O 4 nanocube surface for the fabrication of a core/satellite structure (Fe 3 O 4 /Au) by the reduction of HAuCl 4 using ammonia. Transmission electron microscopy and energy dispersive spectroscopy mapping revealed that small Au nanodots of about 2 nm average size decorated the surface of Fe 3 O 4 nanocubes. X-ray diffraction data was used to confirm the formation of both the phases of a cubic inverse spinel structure for Fe 3 O 4 and a bcc structure for Au in the core/satellite structure of Fe 3 O 4 /Au nanocubes. The magnetic properties of the seed Fe 3 O 4 nanocubes and Fe 3 O 4 /Au core/satellite nanocubes were measured by using a superconducting quantum interference device at 300 K. For biological application purposes, the as-synthesized Fe 3 O 4 /Au core/satellite nanocubes were functionalized by cysteamine followed by successful immobilization of streptavidin protein as confirmed through the fluorescence confocal microscopy images.

  6. Random close packing in protein cores.

    Science.gov (United States)

    Gaines, Jennifer C; Smith, W Wendell; Regan, Lynne; O'Hern, Corey S

    2016-03-01

    Shortly after the determination of the first protein x-ray crystal structures, researchers analyzed their cores and reported packing fractions ϕ ≈ 0.75, a value that is similar to close packing of equal-sized spheres. A limitation of these analyses was the use of extended atom models, rather than the more physically accurate explicit hydrogen model. The validity of the explicit hydrogen model was proved in our previous studies by its ability to predict the side chain dihedral angle distributions observed in proteins. In contrast, the extended atom model is not able to recapitulate the side chain dihedral angle distributions, and gives rise to large atomic clashes at side chain dihedral angle combinations that are highly probable in protein crystal structures. Here, we employ the explicit hydrogen model to calculate the packing fraction of the cores of over 200 high-resolution protein structures. We find that these protein cores have ϕ ≈ 0.56, which is similar to results obtained from simulations of random packings of individual amino acids. This result provides a deeper understanding of the physical basis of protein structure that will enable predictions of the effects of amino acid mutations to protein cores and interfaces of known structure.

  7. Core-shell microparticles for protein sequestration and controlled release of a protein-laden core.

    Science.gov (United States)

    Rinker, Torri E; Philbrick, Brandon D; Temenoff, Johnna S

    2017-07-01

    Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85+19% vs. 9.0+4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic

  8. CHARACTERIZING AND MODELING FERRITE-CORE PROBES

    International Nuclear Information System (INIS)

    Sabbagh, Harold A.; Murphy, R. Kim; Sabbagh, Elias H.; Aldrin, John C.

    2010-01-01

    In this paper, we accurately and carefully characterize a ferrite-core probe that is widely used for aircraft inspections. The characterization starts with the development of a model that can be executed using the proprietary volume-integral code, VIC-3D(c), and then the model is fitted to measured multifrequency impedance data taken with the probe in freespace and over samples of a titanium alloy and aluminum. Excellent results are achieved, and will be discussed.

  9. Differential Stoichiometry among Core Ribosomal Proteins

    Directory of Open Access Journals (Sweden)

    Nikolai Slavov

    2015-11-01

    Full Text Available Understanding the regulation and structure of ribosomes is essential to understanding protein synthesis and its dysregulation in disease. While ribosomes are believed to have a fixed stoichiometry among their core ribosomal proteins (RPs, some experiments suggest a more variable composition. Testing such variability requires direct and precise quantification of RPs. We used mass spectrometry to directly quantify RPs across monosomes and polysomes of mouse embryonic stem cells (ESC and budding yeast. Our data show that the stoichiometry among core RPs in wild-type yeast cells and ESC depends both on the growth conditions and on the number of ribosomes bound per mRNA. Furthermore, we find that the fitness of cells with a deleted RP-gene is inversely proportional to the enrichment of the corresponding RP in polysomes. Together, our findings support the existence of ribosomes with distinct protein composition and physiological function.

  10. Random close packing in protein cores

    OpenAIRE

    Gaines, Jennifer C.; Smith, W. Wendell; Regan, Lynne; O'Hern, Corey S.

    2015-01-01

    Shortly after the determination of the first protein x-ray crystal structures, researchers analyzed their cores and reported packing fractions $\\phi \\approx 0.75$, a value that is similar to close packing equal-sized spheres. A limitation of these analyses was the use of `extended atom' models, rather than the more physically accurate `explicit hydrogen' model. The validity of using the explicit hydrogen model is proved by its ability to predict the side chain dihedral angle distributions obs...

  11. Expression and partial characterization of an ice binding protein from a bacterium isolated at a depth of 3,519 meters in the Vostok ice core, Antarctica

    Directory of Open Access Journals (Sweden)

    Amanda Marie Achberger

    2011-12-01

    Full Text Available Cryopreservation of microorganisms in ancient glacial ice is possible if lethal levels of macromolecular damage are not incurred and cellular integrity is not compromised via intracellular ice formation or recrystallization. Previously, a bacterium (isolate 3519-10 recovered from a depth of 3,519 meters below the surface in the Vostok ice core was shown to secrete an IBP that inhibits the recrystallization of ice. To explore the advantage that IBPs confer to ice-entrapped cells, experiments were designed to examine the expression of 3519-10’s IBP gene and protein at different temperatures, assess the effect of the IBP on bacterial viability in ice, and determine how the IBP influences the physical structure of the ice. Total RNA isolated from cultures grown between 4 to 25⁰C and analyzed by reverse transcription-PCR indicated constitutive expression of the IBP gene. SDS-PAGE analysis of 3519-10’s extracellular proteins also revealed a polypeptide of the predicted size of the 54 kDa IBP at all temperatures tested. In the presence of 100 µg mL-1 of extracellular protein from 3519-10, the survival of Escherichia coli was increased by greater than 34-fold after freeze-thaw cycling. Microscopic analysis of ice formed in the presence of the IBP indicated that per mm2 field of view, there were ~5 times as many crystals as in ice formed in the presence of washed 3519-10 cells and non-IBP producing bacteria, and ~10 times as many crystals as in filtered deionized water. Presumably, the effect that the IBP has on bacterial viability and ice crystal structure is due to its activity as an inhibitor of ice recrystallization. A myriad of molecular adaptations are likely to play a role in bacterial persistence under frozen conditions, but the ability of 3519-10’s IBP to control ice crystal structure, and thus the liquid vein network within the ice, may provide one explanation for its successful survival deep within the Antarctic ice sheet for

  12. Characterizing the Core via K-Core Covers

    NARCIS (Netherlands)

    Sanchez, S.M.; Borm, P.E.M.; Estevez, A.

    2013-01-01

    This paper extends the notion of individual minimal rights for a transferable utility game (TU-game) to coalitional minimal rights using minimal balanced families of a specific type, thus defining a corresponding minimal rights game. It is shown that the core of a TU-game coincides with the core of

  13. The Expanded FindCore Method for Identification of a Core Atom Set for Assessment of Protein Structure Prediction

    Science.gov (United States)

    Snyder, David A.; Grullon, Jennifer; Huang, Yuanpeng J.; Tejero, Roberto; Montelione, Gaetano T.

    2014-01-01

    Maximizing the scientific impact of NMR-based structure determination requires robust and statistically sound methods for assessing the precision of NMR-derived structures. In particular, a method to define a core atom set for calculating superimpositions and validating structure predictions is critical to the use of NMR-derived structures as targets in the CASP competition. FindCore (D.A. Snyder and G.T. Montelione PROTEINS 2005;59:673–686) is a superimposition independent method for identifying a core atom set, and partitioning that set into domains. However, as FindCore optimizes superimposition by sensitively excluding not-well-defined atoms, the FindCore core may not comprise all atoms suitable for use in certain applications of NMR structures, including the CASP assessment process. Adapting the FindCore approach to assess predicted models against experimental NMR structures in CASP10 required modification of the FindCore method. This paper describes conventions and a standard protocol to calculate an “Expanded FindCore” atom set suitable for validation and application in biological and biophysical contexts. A key application of the Expanded FindCore method is to identify a core set of atoms in the experimental NMR structure for which it makes sense to validate predicted protein structure models. We demonstrate the application of this Expanded FindCore method in characterizing well-defined regions of 18 NMR-derived CASP10 target structures. The Expanded FindCore protocol defines “expanded core atom sets” that match an expert’s intuition of which parts of the structure are sufficiently well-defined to use in assessing CASP model predictions. We also illustrate the impact of this analysis on the CASP GDT assessment scores. PMID:24327305

  14. Hepatitis C Virus Core Protein Decreases Lipid Droplet Turnover

    Science.gov (United States)

    Harris, Charles; Herker, Eva; Farese, Robert V.; Ott, Melanie

    2011-01-01

    Steatosis is a frequent complication of hepatitis C virus infection. In mice, this condition is recapitulated by the expression of a single viral protein, the nucleocapsid core. Core localizes to the surface of lipid droplets (LDs) in infected liver cells through a process dependent on host diacylglycerol acyltransferase 1 (DGAT1), an enzyme that synthesizes triglycerides in the endoplasmic reticulum. Whether DGAT1 also plays a role in core-induced steatosis is uncertain. Here, we show that mouse embryonic fibroblasts isolated from DGAT1−/− mice are protected from core-induced steatosis, as are livers of DGAT1−/− mice expressing core, demonstrating that the steatosis is DGAT1-dependent. Surprisingly, core expression did not increase DGAT1 activity or triglyceride synthesis, thus excluding the possibility that core activates DGAT1 to cause steatosis. Instead, we find that DGAT1-dependent localization of core to LDs is a prerequisite for the steatogenic properties of the core. Using biochemical and immunofluorescence microscopy techniques, we show that the turnover of lipids in core-coated droplets is decreased, providing a physiological mechanism for core-induced steatosis. Our results support a bipartite model in which core first requires DGAT1 to gain access to LDs, and then LD-localized core interferes with triglyceride turnover, thus stabilizing lipid droplets and leading to steatosis. PMID:21984835

  15. Advanced core-analyses for subsurface characterization

    Science.gov (United States)

    Pini, R.

    2017-12-01

    The heterogeneity of geological formations varies over a wide range of length scales and represents a major challenge for predicting the movement of fluids in the subsurface. Although they are inherently limited in the accessible length-scale, laboratory measurements on reservoir core samples still represent the only way to make direct observations on key transport properties. Yet, properties derived on these samples are of limited use and should be regarded as sample-specific (or `pseudos'), if the presence of sub-core scale heterogeneities is not accounted for in data processing and interpretation. The advent of imaging technology has significantly reshaped the landscape of so-called Special Core Analysis (SCAL) by providing unprecedented insight on rock structure and processes down to the scale of a single pore throat (i.e. the scale at which all reservoir processes operate). Accordingly, improved laboratory workflows are needed that make use of such wealth of information by e.g., referring to the internal structure of the sample and in-situ observations, to obtain accurate parameterisation of both rock- and flow-properties that can be used to populate numerical models. We report here on the development of such workflow for the study of solute mixing and dispersion during single- and multi-phase flows in heterogeneous porous systems through a unique combination of two complementary imaging techniques, namely X-ray Computed Tomography (CT) and Positron Emission Tomography (PET). The experimental protocol is applied to both synthetic and natural porous media, and it integrates (i) macroscopic observations (tracer effluent curves), (ii) sub-core scale parameterisation of rock heterogeneities (e.g., porosity, permeability and capillary pressure), and direct 3D observation of (iii) fluid saturation distribution and (iv) the dynamic spreading of the solute plumes. Suitable mathematical models are applied to reproduce experimental observations, including both 1D and 3D

  16. Modulation of mitogen-activated protein kinase-activated protein kinase 3 by hepatitis C virus core protein

    DEFF Research Database (Denmark)

    Ngo, HT; Pham, Long; Kim, JW

    2013-01-01

    Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray...... inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA...... increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation....

  17. Prediction of Hydrophobic Cores of Proteins Using Wavelet Analysis.

    Science.gov (United States)

    Hirakawa; Kuhara

    1997-01-01

    Information concerning the secondary structures, flexibility, epitope and hydrophobic regions of amino acid sequences can be extracted by assigning physicochemical indices to each amino acid residue, and information on structure can be derived using the sliding window averaging technique, which is in wide use for smoothing out raw functions. Wavelet analysis has shown great potential and applicability in many fields, such as astronomy, radar, earthquake prediction, and signal or image processing. This approach is efficient for removing noise from various functions. Here we employed wavelet analysis to smooth out a plot assigned to a hydrophobicity index for amino acid sequences. We then used the resulting function to predict hydrophobic cores in globular proteins. We calculated the prediction accuracy for the hydrophobic cores of 88 representative set of proteins. Use of wavelet analysis made feasible the prediction of hydrophobic cores at 6.13% greater accuracy than the sliding window averaging technique.

  18. Colorful packages : fluorescent proteins in complex coacervate core micelles

    NARCIS (Netherlands)

    Nolles, Antsje

    2018-01-01

    This thesis explores the encapsulation of fluorescent proteins (FPs) into complex coacervate core micelles (C3Ms) and features the impact of this encapsulation on the biophysical properties of the FPs. In total eight different FPs were investigated originating from two different classes

  19. Analysis of core-periphery organization in protein contact networks reveals groups of structurally and functionally critical residues.

    Science.gov (United States)

    Isaac, Arnold Emerson; Sinha, Sitabhra

    2015-10-01

    The representation of proteins as networks of interacting amino acids, referred to as protein contact networks (PCN), and their subsequent analyses using graph theoretic tools, can provide novel insights into the key functional roles of specific groups of residues. We have characterized the networks corresponding to the native states of 66 proteins (belonging to different families) in terms of their core-periphery organization. The resulting hierarchical classification of the amino acid constituents of a protein arranges the residues into successive layers - having higher core order - with increasing connection density, ranging from a sparsely linked periphery to a densely intra-connected core (distinct from the earlier concept of protein core defined in terms of the three-dimensional geometry of the native state, which has least solvent accessibility). Our results show that residues in the inner cores are more conserved than those at the periphery. Underlining the functional importance of the network core, we see that the receptor sites for known ligand molecules of most proteins occur in the innermost core. Furthermore, the association of residues with structural pockets and cavities in binding or active sites increases with the core order. From mutation sensitivity analysis, we show that the probability of deleterious or intolerant mutations also increases with the core order. We also show that stabilization centre residues are in the innermost cores, suggesting that the network core is critically important in maintaining the structural stability of the protein. A publicly available Web resource for performing core-periphery analysis of any protein whose native state is known has been made available by us at http://www.imsc.res.in/ ~sitabhra/proteinKcore/index.html.

  20. Virus-producing cells determine the host protein profiles of HIV-1 virion cores

    Science.gov (United States)

    2012-01-01

    Background Upon HIV entry into target cells, viral cores are released and rearranged into reverse transcription complexes (RTCs), which support reverse transcription and also protect and transport viral cDNA to the site of integration. RTCs are composed of viral and cellular proteins that originate from both target and producer cells, the latter entering the target cell within the viral core. However, the proteome of HIV-1 viral cores in the context of the type of producer cells has not yet been characterized. Results We examined the proteomic profiles of the cores purified from HIV-1 NL4-3 virions assembled in Sup-T1 cells (T lymphocytes), PMA and vitamin D3 activated THP1 (model of macrophages, mMΦ), and non-activated THP1 cells (model of monocytes, mMN) and assessed potential involvement of identified proteins in the early stages of infection using gene ontology information and data from genome-wide screens on proteins important for HIV-1 replication. We identified 202 cellular proteins incorporated in the viral cores (T cells: 125, mMΦ: 110, mMN: 90) with the overlap between these sets limited to 42 proteins. The groups of RNA binding (29), DNA binding (17), cytoskeleton (15), cytoskeleton regulation (21), chaperone (18), vesicular trafficking-associated (12) and ubiquitin-proteasome pathway-associated proteins (9) were most numerous. Cores of the virions from SupT1 cells contained twice as many RNA binding proteins as cores of THP1-derived virus, whereas cores of virions from mMΦ and mMN were enriched in components of cytoskeleton and vesicular transport machinery, most probably due to differences in virion assembly pathways between these cells. Spectra of chaperones, cytoskeletal proteins and ubiquitin-proteasome pathway components were similar between viral cores from different cell types, whereas DNA-binding and especially RNA-binding proteins were highly diverse. Western blot analysis showed that within the group of overlapping proteins, the level of

  1. Proteins: Chemistry, Characterization, and Quality

    NARCIS (Netherlands)

    Sforza, S.; Tedeschi, T.; Wierenga, P.A.

    2016-01-01

    Proteins are one of the major macronutrients in food, and several traditional food commodities are good sources of proteins (meat, egg, milk and dairy products, fish, and soya). Proteins are polymers made by 20 different amino acids. They might undergo desired or undesired chemical or enzymatic

  2. Structural characterization of core-bradavidin in complex with biotin

    Science.gov (United States)

    Agrawal, Nitin; Määttä, Juha A. E.; Kulomaa, Markku S.; Hytönen, Vesa P.; Johnson, Mark S.; Airenne, Tomi T.

    2017-01-01

    Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 (“Brad-tag”) act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin–Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin. PMID:28426764

  3. Structural characterization of core-bradavidin in complex with biotin.

    Directory of Open Access Journals (Sweden)

    Nitin Agrawal

    Full Text Available Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 ("Brad-tag" act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala of core-bradavidin (CC mutant. Our data help us to further engineer the core-bradavidin-Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.

  4. Structural Characterization of Core Region in Erwinia amylovora Lipopolysaccharide

    Directory of Open Access Journals (Sweden)

    Angela Casillo

    2017-03-01

    Full Text Available Erwinia amylovora (E. amylovora is the first bacterial plant pathogen described and demonstrated to cause fire blight, a devastating plant disease affecting a wide range of species including a wide variety of Rosaceae. In this study, we reported the lipopolysaccharide (LPS core structure from E. amylovora strain CFBP1430, the first one for an E. amylovora highly pathogenic strain. The chemical characterization was performed on the mutants waaL (lacking only the O-antigen LPS with a complete LPS-core, wabH and wabG (outer-LPS core mutants. The LPSs were isolated from dry cells and analyzed by means of chemical and spectroscopic methods. In particular, they were subjected to a mild acid hydrolysis and/or a hydrazinolysis and investigated in detail by one and two dimensional Nuclear Magnetic Resonance (NMR spectroscopy and ElectroSpray Ionization Fourier Transform-Ion Cyclotron Resonance (ESI FT-ICR mass spectrometry.

  5. Fracture Characterization of Sandwich Face/Core Interfaces

    DEFF Research Database (Denmark)

    Manca, Marcello

    of load transfer between the faces and the core layer is lost, the debonds are considered as primary damage initiators. Under fatigue loading the debonds may evolve into cracks that cause a reduction in structural performance and consequent failure. At present most structural design is based on “life-time...... of sandwich structures is defects that are introduced in the manufacturing process. It is inevitable that areas of the face sheets will not fully adhere to the core resulting in defects known as “debonds”. Debonds can also be induced in-service due to e.g. localised impact loading or overloading. As the means...... such result it is important to devise new experimental and analytical techniques to establish the multi-mode fracture characteristics of sandwich plate structures and accordingly develop methods to inhibit defect propagation. This thesis deals with characterization of fracture between face and core...

  6. Fatigue Characterization of Fire Resistant Syntactic Foam Core Material

    Science.gov (United States)

    Hossain, Mohammad Mynul

    Eco-Core is a fire resistant material for sandwich structural application; it was developed at NC A&T State University. The Eco-Core is made of very small amount of phenolic resin and large volume of flyash by a syntactic process. The process development, static mechanical and fracture, fire and toxicity safety and water absorption properties and the design of sandwich structural panels with Eco-Core material was established and published in the literature. One of the important properties that is needed for application in transportation vehicles is the fatigue performance under different stress states. Fatigue data are not available even for general syntactic foams. The objective of this research is to investigate the fatigue performance of Eco-Core under three types of stress states, namely, cyclic compression, shear and flexure, then document failure modes, and develop empherical equations for predicting fatigue life of Eco-Core under three stress states. Compression-Compression fatigue was performed directly on Eco-Core cylindrical specimen, whereas shear and flexure fatigue tests were performed using sandwich beam made of E glass-Vinyl Ester face sheet and Eco-Core material. Compression-compression fatigue test study was conducted at two values of stress ratios (R=10 and 5), for the maximum compression stress (sigmamin) range of 60% to 90% of compression strength (sigmac = 19.6 +/- 0.25 MPa) for R=10 and 95% to 80% of compression strength for R=5. The failure modes were characterized by the material compliance change: On-set (2% compliance change), propagation (5%) and ultimate failure (7%). The number of load cycles correspond to each of these three damages were characterized as on-set, propagation and total lives. A similar approach was used in shear and flexure fatigue tests with stress ratio of R=0.1. The fatigue stress-number of load cycles data followed the standard power law equation for all three stress states. The constant of the equation were

  7. Core-shell magnetic nanowires fabrication and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Kalska-Szostko, B., E-mail: kalska@uwb.edu.pl [Institute of Chemistry, University of Bialystok, Ciolkowskiego 1K, 15-245 Bialystok (Poland); Faculty of Physics, University of Bialystok, Ciolkowskiego 1L, 15-245 Bialystok, Poland (Poland); Klekotka, U.; Satuła, D. [Institute of Chemistry, University of Bialystok, Ciolkowskiego 1K, 15-245 Bialystok (Poland); Faculty of Physics, University of Bialystok, Ciolkowskiego 1L, 15-245 Bialystok, Poland (Poland)

    2017-02-28

    Highlights: • New approach for nanowires modification are presented. • Physical and chemical characterization of the nanowires are shown. • Properties modulations as an effect of the surface layer composition are discussed. - Abstract: In this paper, a new way of the preparation of core-shell magnetic nanowires has been proposed. For the modification Fe nanowires were prepared by electrodeposition in anodic aluminium oxide matrixes, in first step. In second, by wetting chemical deposition, shell layers of Ag, Au or Cu were obtained. Resultant core-shell nanowires structure was characterized by X-ray diffraction, infrared spectroscopy, transmission electron microscopy, and energy dispersive x-ray. Whereas magnetic properties by Mössbauer spectroscopy.

  8. Preparation and recognition of surface molecularly imprinted core-shell microbeads for protein in aqueous solutions

    International Nuclear Information System (INIS)

    Lu Yan; Yan Changling; Gao Shuyan

    2009-01-01

    In this paper, a surface molecular imprinting technique was reported for preparing core-shell microbeads of protein imprinting, and bovine hemoglobin or bovine serum albumin were used as model proteins for studying the imprinted core-shell microbeads. 3-Aminophenylboronic acid (APBA) was polymerized onto the surface of polystyrene microbead in the presence of the protein templates to create protein-imprinted core-shell microbeads. The various samples were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) methods. The effect of pH on rebinding of the template hemoglobin, the specific binding and selective recognition were studied for the imprinted microbeads. The results show that the bovine hemoglobin-imprinted core-shell microbeads were successfully created. The shell was a sort of imprinted thin films with porous structure and larger surface areas. The imprinted microbeads have good selectivity for templates and high stability. Due to the recognition sites locating at or closing to the surface, these imprinted microbeads have good property of mass-transport. Unfortunately, the imprint technology was not successfully applied to imprinting bovine serum albumin (BSA).

  9. Preparation and recognition of surface molecularly imprinted core-shell microbeads for protein in aqueous solutions

    Energy Technology Data Exchange (ETDEWEB)

    Lu Yan, E-mail: yanlu2001@sohu.com [College of Chemistry and Environmental Science, Henan Normal University, 46 Jlanshe Road, Xinxiang 453007 (China); Yan Changling; Gao Shuyan [College of Chemistry and Environmental Science, Henan Normal University, 46 Jlanshe Road, Xinxiang 453007 (China)

    2009-04-01

    In this paper, a surface molecular imprinting technique was reported for preparing core-shell microbeads of protein imprinting, and bovine hemoglobin or bovine serum albumin were used as model proteins for studying the imprinted core-shell microbeads. 3-Aminophenylboronic acid (APBA) was polymerized onto the surface of polystyrene microbead in the presence of the protein templates to create protein-imprinted core-shell microbeads. The various samples were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and Brunauer-Emmett-Teller (BET) methods. The effect of pH on rebinding of the template hemoglobin, the specific binding and selective recognition were studied for the imprinted microbeads. The results show that the bovine hemoglobin-imprinted core-shell microbeads were successfully created. The shell was a sort of imprinted thin films with porous structure and larger surface areas. The imprinted microbeads have good selectivity for templates and high stability. Due to the recognition sites locating at or closing to the surface, these imprinted microbeads have good property of mass-transport. Unfortunately, the imprint technology was not successfully applied to imprinting bovine serum albumin (BSA).

  10. First principles design of a core bioenergetic transmembrane electron-transfer protein

    Energy Technology Data Exchange (ETDEWEB)

    Goparaju, Geetha; Fry, Bryan A.; Chobot, Sarah E.; Wiedman, Gregory; Moser, Christopher C.; Leslie Dutton, P.; Discher, Bohdana M.

    2016-05-01

    Here we describe the design, Escherichia coli expression and characterization of a simplified, adaptable and functionally transparent single chain 4-α-helix transmembrane protein frame that binds multiple heme and light activatable porphyrins. Such man-made cofactor-binding oxidoreductases, designed from first principles with minimal reference to natural protein sequences, are known as maquettes. This design is an adaptable frame aiming to uncover core engineering principles governing bioenergetic transmembrane electron-transfer function and recapitulate protein archetypes proposed to represent the origins of photosynthesis. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

  11. Homogeneous protein analysis by magnetic core-shell nanorod probes

    KAUST Repository

    Schrittwieser, Stefan

    2016-03-29

    Studying protein interactions is of vital importance both to fundamental biology research and to medical applications. Here, we report on the experimental proof of a universally applicable label-free homogeneous platform for rapid protein analysis. It is based on optically detecting changes in the rotational dynamics of magnetically agitated core-shell nanorods upon their specific interaction with proteins. By adjusting the excitation frequency, we are able to optimize the measurement signal for each analyte protein size. In addition, due to the locking of the optical signal to the magnetic excitation frequency, background signals are suppressed, thus allowing exclusive studies of processes at the nanoprobe surface only. We study target proteins (soluble domain of the human epidermal growth factor receptor 2 - sHER2) specifically binding to antibodies (trastuzumab) immobilized on the surface of our nanoprobes and demonstrate direct deduction of their respective sizes. Additionally, we examine the dependence of our measurement signal on the concentration of the analyte protein, and deduce a minimally detectable sHER2 concentration of 440 pM. For our homogeneous measurement platform, good dispersion stability of the applied nanoprobes under physiological conditions is of vital importance. To that end, we support our measurement data by theoretical modeling of the total particle-particle interaction energies. The successful implementation of our platform offers scope for applications in biomarker-based diagnostics as well as for answering basic biology questions.

  12. Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core

    International Nuclear Information System (INIS)

    Ma Guangpeng; Feng Youjun; Gao Feng; Wang Jinzi; Liu Cheng; Li Yijing

    2005-01-01

    Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host

  13. Modified ferrite core-shell nanoparticles magneto-structural characterization

    Science.gov (United States)

    Klekotka, Urszula; Piotrowska, Beata; Satuła, Dariusz; Kalska-Szostko, Beata

    2018-06-01

    In this study, ferrite nanoparticles with core-shell structures and different chemical compositions of both the core and shell were prepared with success. Proposed nanoparticles have in the first and second series magnetite core, and the shell is composed of a mixture of ferrites with Fe3+, Fe2+ and M ions (where M = Co2+, Mn2+ or Ni2+) with a general composition of M0.5Fe2.5O4. In the third series, the composition is inverted, the core is composed of a mixture of ferrites and as a shell magnetite is placed. Morphology and structural characterization of nanoparticles were done using Transmission Electron Microscopy (TEM), X-ray diffraction (XRD), and Infrared spectroscopy (IR). While room temperature magnetic properties were measured using Mössbauer spectroscopy (MS). It is seen from Mössbauer measurements that Co always increases hyperfine magnetic field on Fe atoms at RT, while Ni and Mn have opposite influences in comparison to pure Fe ferrite, regardless of the nanoparticles structure.

  14. Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments.

    Science.gov (United States)

    Barski, Michal; Brennan, Benjamin; Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M; Schwarz-Linek, Ulrich

    2017-09-15

    Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83-248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.

  15. Hepatitis B core protein as a therapeutic target.

    Science.gov (United States)

    Mak, Lung-Yi; Wong, Danny Ka-Ho; Seto, Wai-Kay; Lai, Ching-Lung; Yuen, Man Fung

    2017-12-01

    Chronic hepatitis B virus (HBV) infection is difficult to cure, due to the presence of covalently-closed-circular DNA and virus-mediated blunting of host immune response. Existing therapies with nucleos(t)ide analogue or pegylated-interferon are not sufficient to achieve a high rate of HBV surface antigen seroclearance, a more desirable treatment outcome. Novel therapeutic agents targeting alternative viral replication steps are being developed. In this review, we will discuss the hepatitis B core antigen (HBcAg) as a therapeutic target. Areas covered: The basic structure and fundamental functions of HBcAg including nucleocapsid assembly, pre-genomic RNA encapsidation, reverse transcription, virion formation, cccDNA amplification, immune response regulation, and HBx protein interaction will be reviewed. Most of these are identified as therapeutic targets and tested in in vitro and in vivo studies, although clinical trials are scanty. Among the different components, the core protein allosteric modulators (CpAM) have been most widely investigated and appear promising in clinical trials. Expert opinion: The multiple and essential functions of HBcAg for HBV life cycle are important and attractive targets for HBV therapeutic interventions. Controlled trials involving CpAM are awaited. Apart from CpAM, drugs directed against different functions of HBcAg may be further explored to maximize the chance of cure.

  16. Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation.

    Science.gov (United States)

    Zhao, Qiong; Hu, Zhanying; Cheng, Junjun; Wu, Shuo; Luo, Yue; Chang, Jinhong; Hu, Jianming; Guo, Ju-Tao

    2018-07-01

    Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly. IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which

  17. Characterizing the statistical properties of protein surfaces

    Science.gov (United States)

    Bak, Ji Hyun; Bitbol, Anne-Florence; Bialek, William

    Proteins and their interactions form the body of the signaling transduction pathway in many living systems. In order to ensure the accuracy as well as the specificity of signaling, it is crucial that proteins recognize their correct interaction partners. How difficult, then, is it for a protein to discriminate its correct interaction partner(s) from the possibly large set of other proteins it may encounter in the cell? An important ingredient of recognition is shape complementarity. The ensemble of protein shapes should be constrained by the need for maintaining functional interactions while avoiding spurious ones. To address this aspect of protein recognition, we consider the ensemble of proteins in terms of the shapes of their surfaces. We take into account the high-resolution structures of E.coli non-DNA-binding cytoplasmic proteins, retrieved from the Protein Data Bank. We aim to characterize the statistical properties of the protein surfaces at two levels: First, we study the intrinsic dimensionality at the level of the ensemble of the surface objects. Second, at the level of the individual surfaces, we determine the scale of shape variation. We further discuss how the dimensionality of the shape space is linked to the statistical properties of individual protein surfaces. Jhb and WB acknowledge support from National Science Foundation Grants PHY-1305525 and PHY-1521553. AFB acknowledges support from the Human Frontier Science Program.

  18. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wen-Ta [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Li, Hui-Chun [Department of Biochemistry, Tzu Chi University, Hualien, Taiwan (China); Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Lo, Shih-Yen, E-mail: losylo@mail.tcu.edu.tw [Graduate Institute of Medical Biotechnology, Tzu Chi University, Hualien, Taiwan (China); Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (China)

    2013-05-24

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway.

  19. Both core and F proteins of hepatitis C virus could enhance cell proliferation in transgenic mice

    International Nuclear Information System (INIS)

    Hu, Wen-Ta; Li, Hui-Chun; Lee, Shen-Kao; Ma, Hsin-Chieh; Yang, Chee-Hing; Chen, Hung-Ling; Lo, Shih-Yen

    2013-01-01

    Highlights: •HCV core and F proteins could induce hepatocyte proliferation in the transgenic mice. •β-Catenin signaling pathway was activated by core protein in the transgenic mice. •β-Catenin signaling pathway was activated by myc-F protein in the transgenic mice. •Expression of SMA protein was enhanced by core but not myc-F protein. -- Abstract: The role of the protein encoded by the alternative open reading frame (ARF/F/core+1) of the Hepatitis C virus (HCV) genome in viral pathogenesis remains unknown. The different forms of ARF/F/core+1 protein were labile in cultured cells, a myc-tag fused at the N-terminus of the F protein made it more stable. To determine the role of core and F proteins in HCV pathogenesis, transgenic mice with either protein expression under the control of Albumin promoter were generated. Expression of core protein and F protein with myc tag (myc-F) could be detected by Western blotting analysis in the livers of these mice. The ratio of liver to body weight is increased for both core and myc-F transgenic mice compared to that of wild type mice. Indeed, the proliferating cell nuclear antigen protein, a proliferation marker, was up-regulated in the transgenic mice with core or myc-F protein. Further analyses by microarray and Western blotting suggested that β-catenin signaling pathway was activated by either core or myc-F protein in the transgenic mice. These transgenic mice were further treated with either Diethynitrosamine (a tumor initiator) or Phenobarbital (a tumor promoter). Phenobarbital but not Diethynitrosamine treatment could increase the liver/body weight ratio of these mice. However, no tumor formation was observed in these mice. In conclusion, HCV core and myc-F proteins could induce hepatocyte proliferation in the transgenic mice possibly through β-catenin signaling pathway

  20. Characterization of interfacial waves in horizontal core-annular flow

    Science.gov (United States)

    Tripathi, Sumit; Bhattacharya, Amitabh; Singh, Ramesh; Tabor, Rico F.

    2016-11-01

    In this work, we characterize interfacial waves in horizontal core annular flow (CAF) of fuel-oil and water. Experimental studies on CAF were performed in an acrylic pipe of 15.5mm internal diameter, and the time evolution of the oil-water interface shape was recorded with a high speed camera for a range of different flow-rates of oil (Qo) and water (Qw). The power spectrum of the interface shape shows a range of notable features. First, there is negligible energy in wavenumbers larger than 2 π / a , where a is the thickness of the annulus. Second, for high Qo /Qw , there is no single dominant wavelength, as the flow in the confined annulus does not allow formation of a preferred mode. Third, for lower Qo /Qw , a dominant mode arises at a wavenumber of 2 π / a . We also observe that the power spectrum of the interface shape depends weakly on Qw, and strongly on Qo, perhaps because the net shear rate in the annulus appears to depend weakly on Qw as well. We also attempt to build a general empirical model for CAF by relating the interfacial stress (calculated via the mean pressure gradient) to the flow rate in the annulus, the annular thickness and the core velocity. Authors are thankful to Orica Mining Services (Australia) for the financial support.

  1. HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPARγ

    International Nuclear Information System (INIS)

    Kim, Kook Hwan; Hong, Sung Pyo; Kim, KyeongJin; Park, Min Jung; Kim, Kwang Jin; Cheong, JaeHun

    2007-01-01

    Hepatic steatosis is a common feature in patients with chronic hepatitis C virus (HCV) infection. HCV core protein plays an important role in the development of hepatic steatosis in HCV infection. Because SREBP1 (sterol regulatory element binding protein 1) and PPARγ (peroxisome proliferators-activated receptor γ) are involved in the regulation of lipid metabolism of hepatocyte, we sought to determine whether HCV core protein may impair the expression and activity of SREBP1 and PPARγ. In this study, it was demonstrated that HCV core protein increases the gene expression of SREBP1 not only in Chang liver, Huh7, and HepG2 cells transiently transfected with HCV core protein expression plasmid, but also in Chang liver-core stable cells. Furthermore, HCV core protein enhanced the transcriptional activity of SREBP1. In addition, HCV core protein elevated PPARγ transcriptional activity. However, HCV core protein had no effect on PPARγ gene expression. Finally, we showed that HCV core protein stimulates the genes expression of lipogenic enzyme and fatty acid uptake associated protein. Therefore, our finding provides a new insight into the mechanism of hepatic steatosis by HCV infection

  2. Electrokinetic characterization of whey protein separation

    DEFF Research Database (Denmark)

    Keiding, Kristian; Stougård, Anders; Christensen, Morten Lykkegaard

    Cross flow filtration of whey protein has been performed on 3 different membranes. The rejections have been determined by HPLC analysis of the feed and permeate. The pure membranes as well as the fouled membranes have been characterized by measurements of the streaming potential along the membrane...

  3. On characterization of anisotropic plant protein structures

    NARCIS (Netherlands)

    Krintiras, G.A.; Göbel, J.; Bouwman, W.G.; Goot, van der A.J.; Stefanidis, G.D.

    2014-01-01

    In this paper, a set of complementary techniques was used to characterize surface and bulk structures of an anisotropic Soy Protein Isolate (SPI)–vital wheat gluten blend after it was subjected to heat and simple shear flow in a Couette Cell. The structured biopolymer blend can form a basis for a

  4. The retrovirus MA and PreTM proteins follow immature MVL cores

    DEFF Research Database (Denmark)

    Andersen, Klaus Bahl

    2013-01-01

    Detergent can dissolve retrovirus, exept the immature core. Here we show that the Matrix protein (MA) and the Transmembrane protein in its immature form (PreTM) bind to the retrovirus core. These attachments explain the attachment in the virus particle and the dynamics of the ability to fuse with...

  5. Characterization and identification of microRNA core promoters in four model species.

    Directory of Open Access Journals (Sweden)

    Xuefeng Zhou

    2007-03-01

    Full Text Available MicroRNAs are short, noncoding RNAs that play important roles in post-transcriptional gene regulation. Although many functions of microRNAs in plants and animals have been revealed in recent years, the transcriptional mechanism of microRNA genes is not well-understood. To elucidate the transcriptional regulation of microRNA genes, we study and characterize, in a genome scale, the promoters of intergenic microRNA genes in Caenorhabditis elegans, Homo sapiens, Arabidopsis thaliana, and Oryza sativa. We show that most known microRNA genes in these four species have the same type of promoters as protein-coding genes have. To further characterize the promoters of microRNA genes, we developed a novel promoter prediction method, called common query voting (CoVote, which is more effective than available promoter prediction methods. Using this new method, we identify putative core promoters of most known microRNA genes in the four model species. Moreover, we characterize the promoters of microRNA genes in these four species. We discover many significant, characteristic sequence motifs in these core promoters, several of which match or resemble the known cis-acting elements for transcription initiation. Among these motifs, some are conserved across different species while some are specific to microRNA genes of individual species.

  6. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Directory of Open Access Journals (Sweden)

    Andrea Cerutti

    Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS, but no nuclear export signal (NES has yet been identified.We show here that the aa(109-133 region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126 in the identified NES or in the sequence encoding the mature core aa(1-173 significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  7. Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

    Science.gov (United States)

    Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

    2011-01-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

  8. Characterization of paralogous protein families in rice

    Directory of Open Access Journals (Sweden)

    Zhu Wei

    2008-02-01

    Full Text Available Abstract Background High gene numbers in plant genomes reflect polyploidy and major gene duplication events. Oryza sativa, cultivated rice, is a diploid monocotyledonous species with a ~390 Mb genome that has undergone segmental duplication of a substantial portion of its genome. This, coupled with other genetic events such as tandem duplications, has resulted in a substantial number of its genes, and resulting proteins, occurring in paralogous families. Results Using a computational pipeline that utilizes Pfam and novel protein domains, we characterized paralogous families in rice and compared these with paralogous families in the model dicotyledonous diploid species, Arabidopsis thaliana. Arabidopsis, which has undergone genome duplication as well, has a substantially smaller genome (~120 Mb and gene complement compared to rice. Overall, 53% and 68% of the non-transposable element-related rice and Arabidopsis proteins could be classified into paralogous protein families, respectively. Singleton and paralogous family genes differed substantially in their likelihood of encoding a protein of known or putative function; 26% and 66% of singleton genes compared to 73% and 96% of the paralogous family genes encode a known or putative protein in rice and Arabidopsis, respectively. Furthermore, a major skew in the distribution of specific gene function was observed; a total of 17 Gene Ontology categories in both rice and Arabidopsis were statistically significant in their differential distribution between paralogous family and singleton proteins. In contrast to mammalian organisms, we found that duplicated genes in rice and Arabidopsis tend to have more alternative splice forms. Using data from Massively Parallel Signature Sequencing, we show that a significant portion of the duplicated genes in rice show divergent expression although a correlation between sequence divergence and correlation of expression could be seen in very young genes. Conclusion

  9. Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV core DII protein.

    Directory of Open Access Journals (Sweden)

    Rodney K Lyn

    Full Text Available Host cell lipid droplets (LD are essential in the hepatitis C virus (HCV life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein's lipid binding domain II (DII-core induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV.

  10. RNA chaperoning and intrinsic disorder in the core proteins of Flaviviridae.

    Science.gov (United States)

    Ivanyi-Nagy, Roland; Lavergne, Jean-Pierre; Gabus, Caroline; Ficheux, Damien; Darlix, Jean-Luc

    2008-02-01

    RNA chaperone proteins are essential partners of RNA in living organisms and viruses. They are thought to assist in the correct folding and structural rearrangements of RNA molecules by resolving misfolded RNA species in an ATP-independent manner. RNA chaperoning is probably an entropy-driven process, mediated by the coupled binding and folding of intrinsically disordered protein regions and the kinetically trapped RNA. Previously, we have shown that the core protein of hepatitis C virus (HCV) is a potent RNA chaperone that can drive profound structural modifications of HCV RNA in vitro. We now examined the RNA chaperone activity and the disordered nature of core proteins from different Flaviviridae genera, namely that of HCV, GBV-B (GB virus B), WNV (West Nile virus) and BVDV (bovine viral diarrhoea virus). Despite low-sequence similarities, all four proteins demonstrated general nucleic acid annealing and RNA chaperone activities. Furthermore, heat resistance of core proteins, as well as far-UV circular dichroism spectroscopy suggested that a well-defined 3D protein structure is not necessary for core-induced RNA structural rearrangements. These data provide evidence that RNA chaperoning-possibly mediated by intrinsically disordered protein segments-is conserved in Flaviviridae core proteins. Thus, besides nucleocapsid formation, core proteins may function in RNA structural rearrangements taking place during virus replication.

  11. Core/shell particles containing liquid cores : morphology prediction, synthesis and characterization

    NARCIS (Netherlands)

    Zyl, van A.J.P.; Sanderson, R.D.; Wet-Roos, de D.; Klumperman, B.

    2003-01-01

    The ability to synthesize core/shell particles with distinct geometries is becoming increasingly important due to their potential applications. In this study structured particles with liquid cores and polymeric shells were synthesized by an in situ miniemulsion polymerization reaction. The resulting

  12. Characterization Of Core Sample Collected From The Saltstone Disposal Facility

    International Nuclear Information System (INIS)

    Cozzi, A.; Duncan, A.

    2010-01-01

    During the month of September 2008, grout core samples were collected from the Saltstone Disposal Facility, Vault 4, cell E. This grout was placed during processing campaigns in December 2007 from Deliquification, Dissolution and Adjustment Batch 2 salt solution. The 4QCY07 Waste Acceptance Criteria sample collected on 11/16/07 represents the salt solution in the core samples. Core samples were retrieved to initiate the historical database of properties of emplaced Saltstone and to demonstrate the correlation between field collected and laboratory prepared samples. Three samples were collected from three different locations. Samples were collected using a two-inch diameter concrete coring bit. In April 2009, the core samples were removed from the evacuated sample container, inspected, transferred to PVC containers, and backfilled with nitrogen. Samples furthest from the wall were the most intact cylindrically shaped cored samples. The shade of the core samples darkened as the depth of coring increased. Based on the visual inspection, sample 3-3 was selected for all subsequent analysis. The density and porosity of the Vault 4 core sample, 1.90 g/cm 3 and 59.90% respectively, were comparable to values achieved for laboratory prepared samples. X-ray diffraction analysis identified phases consistent with the expectations for hydrated Saltstone. Microscopic analysis revealed morphology features characteristic of cementitious materials with fly ash and calcium silicate hydrate gel. When taken together, the results of the density, porosity, x-ray diffraction analysis and microscopic analysis support the conclusion that the Vault 4, Cell E core sample is representative of the expected waste form.

  13. HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Yongsheng, E-mail: yongshengtanwhu@126.com; Li, Yan, E-mail: liyansd2@163.com

    2015-10-23

    This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 {sup low} and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96{sup ®}Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 {sup low}, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases

  14. HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

    International Nuclear Information System (INIS)

    Tan, Yongsheng; Li, Yan

    2015-01-01

    This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 "l"o"w and control cells were treated with different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96"®Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 "l"o"w, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases expression of NR4A1

  15. Synthesis and characterization of mesoporous silica core-shell particles

    Directory of Open Access Journals (Sweden)

    Milan Nikolić

    2010-06-01

    Full Text Available Core-shell particles were formed by deposition of primary silica particles synthesized from sodium silicate solution on functionalized silica core particles (having size of ~0.5 µm prepared by hydrolysis and condensation of tetraethylortosilicate. The obtained mesoporous shell has thickness of about 60 nm and consists of primary silica particles with average size of ~21 nm. Scanning electron microscopy and zeta potential measurements showed that continuous silica shell exists around functionalized core particles which was additionally proved by FTIR and TEM results.

  16. Homogeneous protein analysis by magnetic core-shell nanorod probes

    KAUST Repository

    Schrittwieser, Stefan; Pelaz, Beatriz; Parak, Wolfgang J; Lentijo Mozo, Sergio; Soulantica, Katerina; Dieckhoff, Jan; Ludwig, Frank; Altantzis, Thomas; Bals, Sara; Schotter, Joerg

    2016-01-01

    analyte protein size. In addition, due to the locking of the optical signal to the magnetic excitation frequency, background signals are suppressed, thus allowing exclusive studies of processes at the nanoprobe surface only. We study target proteins

  17. Identification & Characterization of Fungal Ice Nucleation Proteins

    Science.gov (United States)

    Scheel, Jan Frederik; Kunert, Anna Theresa; Kampf, Christopher Johannes; Mauri, Sergio; Weidner, Tobias; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2016-04-01

    Freezing of water at relatively warm subfreezing temperatures is dependent on ice nucleation catalysis facilitated by ice nuclei (IN). These IN can be of various origins and although extensive research was done and progress was achieved, the nature and mechanisms leading to an effective IN are to date still poorly understood. Some of the most important processes of our geosphere like the water cycle are highly dependent on effective ice nucleation at temperatures between -2°C - -8°C, a temperature range which is almost exclusively covered by biological IN (BioIN). BioIN are usually macromolecular structures of biological polymers. Sugars as well as proteins have been reported to serve as IN and the best characterized BioIN are ice nucleation proteins (IN-P) from gram negative bacteria. Fungal strains from Fusarium spp. were described to be effective IN at subfreezing temperatures up to -2°C already 25 years ago and more and more fungal species are described to serve as efficient IN. Fungal IN are also thought to be proteins or at least contain a proteinaceous compound, but to date the fungal IN-P primary structure as well as their coding genetic elements of all IN active fungi are unknown. The aim of this study is a.) to identify the proteins and their coding genetic elements from IN active fungi (F. acuminatum, F. avenaceum, M. alpina) and b.) to characterize the mechanisms by which fungal IN serve as effective IN. We designed an interdisciplinary approach using biological, analytical and physical methods to identify fungal IN-P and describe their biological, chemical, and physical properties.

  18. The effect of HCV Core protein on the expression of miR-150

    Directory of Open Access Journals (Sweden)

    Sayad Khanizadeh

    2016-09-01

    Full Text Available Background : Hepatitis C virus (HCV is considered as one of the major pathogenic agents of chronic liver diseases. Previous studies have shown that HCV proteins can interaction with gene regulatory networks such as microRNAs. The aim of this study was to investigate the effect of HCV core protein on the expression of miR-150 in a cell culture model. Materials and Methods: Plasmids expressing full HCV core protein was transfected into Huh7 cell lines while a GFP expressing plasmid employed as negative control. Subsequently, total RNA extracted and Real-Time PCR performed to measure the expression level of miR-150 expression. Moreover, trypan blue exclusion assay was performed to investigate the effect of core protein on cell viability. Results: The gene expression analysis of miR-150 in Huh7 cells showed that endogenous HCV core protein could significantly down regulation of miR-150 when compared to GFP control plasmid and normal cells (P<0.01. Beside, core protein induced no significant proliferative or cytotoxic effects on hepatic cells as determined by trypan blue exclusion assay (P<0.05. Conclusion: Our study suggests that HCV core protein can led to down regulation of miR-150 expression. This data revealed that HCV protein interactions with cell regulatory machinery may contribute to pathogenesis of chronic liver diseases.

  19. Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts.

    Science.gov (United States)

    Rajalakshmy, A R; Malathi, J; Madhavan, H N; Bhaskar, S; Iyer, G K

    2016-01-01

    Recent studies indicate that hepatitis C virus (HCV) proteins can mediate innate immune response and inflammation in conjunctival fibroblasts which contributes to the pathology of dry eye condition associated with chronic HCV infection. The present study investigates the phagocytic potential of human conjunctival fibroblasts (HCFj) for HCV core protein. HCFj cells were incubated with HCV core antigen for different periods of time, and fluorescent micrographs were taken to observe protein internalisation. HCFj cells were capable of internalising HCV core antigen within 1 h; this gives an insight into another molecular mechanism which may contribute towards HCV-associated conjunctival inflammation.

  20. Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

    Science.gov (United States)

    Shao, Yu-Yun; Hsieh, Min-Shu; Wang, Han-Yu; Li, Yong-Shi; Lin, Hang; Hsu, Hung-Wei; Huang, Chung-Yi; Hsu, Chih-Hung; Cheng, Ann-Lii

    2017-10-17

    Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones-HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

  1. Quantitative analysis of the interaction between the envelope protein domains and the core protein of human hepatitis B virus

    International Nuclear Information System (INIS)

    Choi, Kyoung-Jae; Lim, Chun-Woo; Yoon, Moon-Young; Ahn, Byung-Yoon; Yu, Yeon Gyu

    2004-01-01

    Interaction between preformed nucleocapsids and viral envelope proteins is critical for the assembly of virus particles in infected cells. The pre-S1 and pre-S2 and cytosolic regions of the human hepatitis B virus envelope protein had been implicated in the interaction with the core protein of nucleocapsids. The binding affinities of specific subdomains of the envelope protein to the core protein were quantitatively measured by both ELISA and BIAcore assay. While a marginal binding was detected with the pre-S1 or pre-S2, the core protein showed high affinities to pre-S with apparent dissociation constants (K D app ) of 7.3 ± 0.9 and 8.2 ± 0.4 μM by ELISA and BIAcore assay, respectively. The circular dichroism analysis suggested that conformational change occurs in pre-S through interaction with core protein. These results substantiate the importance of specific envelope domains in virion assembly, and demonstrate that the interaction between viral proteins can be quantitatively measured in vitro

  2. Characterization of the core poloidal flow at ASDEX Upgrade

    Science.gov (United States)

    Lebschy, Alexander

    2017-10-01

    An essential result from neoclassical (NC) theory is that the fluid poloidal rotation (upol) of the main ions is strongly damped by magnetic pumping and, therefore, expected to be small (theory has been found at both DIII-D and TCV. This is qualitatively consistent with the edge results from both Alcator C-Mod and ASDEX Upgrade (AUG). At AUG thanks to an upgrade of the core charge exchange recombination spectroscopy (CXRS) diagnostics, the core upol can be evaluated through the inboard-outboard asymmetry of the toroidal rotation with an accuracy of 0.5 - 1 km / s . This measurement also provides the missing ingredient to evaluate the core (E-> × B->) velocity (uE-> × B->) via the radial force balance equation. At AUG the core upol (0.35 × B-> determined from CXRS and the perpendicular velocity measured from turbulence propagation. The difference between these two quantities is the turbulent phase velocity. The gathered dataset indicates that the transition in the turbulence regime occurs after the saturation of the energy confinement time. The author thankfully acknowledges the financial support from the Helmholtz Association of German Research Centers through the Helmholtz Young Investigators Group program.

  3. Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM.

    Science.gov (United States)

    Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi; Zhang, Lihua; Goldman, Radoslav

    2018-02-07

    Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and

  4. Characterization of Factors affecting IASCC of PWR Core Internals

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Woo; Hwang, Seong Sik; Kim, Won Sam [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2008-09-15

    A lot works have been performed on IASCC in BWR. Recent efforts have been devoted to investigate IASCC in PWR, but the mechanism in PWR is not fully understood yet as compared with that in BWR due to a lack of data from laboratories and fields. Therefore it is strongly needed to review and analyse recent researches of IASCC in both BWR and PWR for establishing a proactive management technology for IASCC of core internals in Korean PWRs. This work is aimed to review mainly recent technical reports on IASCC of stainless steels for core internals in PWR. For comparison, the works on IASCC in BWR were also reviewed and briefly introduced in this report.

  5. Hepatitis C virus core protein induces hepatic steatosis via Sirt1-dependent pathway.

    Science.gov (United States)

    Zhang, Chuanhai; Wang, Jingjing; Zhang, Hanlin; Liu, Shunai; Lee, Hyuek Jong; Jin, Wanzhu; Cheng, Jun

    2018-05-01

    Hepatic steatosis is a common feature of patients with chronic hepatitis C. Previous reports have shown that the overexpression of hepatitis C virus core-encoding sequences (hepatitis C virus genotypes 3a and 1b) significantly induces intracellular triglyceride accumulation. However, the underlying mechanism has not yet been revealed. To investigate whether Sirt1 is involved in hepatitis C virus-mediated hepatic steatosis, the overexpression of hepatitis C virus core 1b protein and Sirt1 and the knockdown of Sirt1 in HepG2 cells were performed. To confirm the results of the cellular experiment liver-specific Sirt1 KO mice with lentivirus-mediated hepatitis C virus core 1b overexpression were studied. Our results show that hepatitis C virus core 1b protein overexpression led to the accumulation of triglycerides in HepG2 cells. Notably the expression of PPARγ2 was dramatically increased at both the mRNA and protein levels by hepatitis C virus core 1b overexpression. The protein expression of Sirt1 is an upstream regulator of PPARγ2 and was also significantly increased after core 1b overexpression. In addition, the overexpression or knockdown of Sirt1 expression alone was sufficient to modulate p300-mediated PPARγ2 deacetylation. In vivo studies showed that hepatitis C virus core protein 1b-induced hepatic steatosis was attenuated in liver-specific Sirt1 KO mice by downregulation of PPARγ2 expression. Sirt1 mediates hepatitis C virus core protein 1b-induced hepatic steatosis by regulation of PPARγ2 expression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

    Directory of Open Access Journals (Sweden)

    Xianjun Shen

    Full Text Available How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment. It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.

  7. Identification of Protein Complexes Using Weighted PageRank-Nibble Algorithm and Core-Attachment Structure.

    Science.gov (United States)

    Peng, Wei; Wang, Jianxin; Zhao, Bihai; Wang, Lusheng

    2015-01-01

    Protein complexes play a significant role in understanding the underlying mechanism of most cellular functions. Recently, many researchers have explored computational methods to identify protein complexes from protein-protein interaction (PPI) networks. One group of researchers focus on detecting local dense subgraphs which correspond to protein complexes by considering local neighbors. The drawback of this kind of approach is that the global information of the networks is ignored. Some methods such as Markov Clustering algorithm (MCL), PageRank-Nibble are proposed to find protein complexes based on random walk technique which can exploit the global structure of networks. However, these methods ignore the inherent core-attachment structure of protein complexes and treat adjacent node equally. In this paper, we design a weighted PageRank-Nibble algorithm which assigns each adjacent node with different probability, and propose a novel method named WPNCA to detect protein complex from PPI networks by using weighted PageRank-Nibble algorithm and core-attachment structure. Firstly, WPNCA partitions the PPI networks into multiple dense clusters by using weighted PageRank-Nibble algorithm. Then the cores of these clusters are detected and the rest of proteins in the clusters will be selected as attachments to form the final predicted protein complexes. The experiments on yeast data show that WPNCA outperforms the existing methods in terms of both accuracy and p-value. The software for WPNCA is available at "http://netlab.csu.edu.cn/bioinfomatics/weipeng/WPNCA/download.html".

  8. Characterization of membrane association of Rinderpest virus matrix protein

    International Nuclear Information System (INIS)

    Subhashri, R.; Shaila, M.S.

    2007-01-01

    Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M protein gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein

  9. Characterization of the coating and tablet core roughness by means of 3D optical coherence tomography.

    Science.gov (United States)

    Markl, Daniel; Wahl, Patrick; Pichler, Heinz; Sacher, Stephan; Khinast, Johannes G

    2018-01-30

    This study demonstrates the use of optical coherence tomography (OCT) to simultaneously characterize the roughness of the tablet core and coating of pharmaceutical tablets. OCT is a high resolution non-destructive and contactless imaging methodology to characterize structural properties of solid dosage forms. Besides measuring the coating thickness, it also facilitates the analysis of the tablet core and coating roughness. An automated data evaluation algorithm extracts information about coating thickness, as well as tablet core and coating roughness. Samples removed periodically from a pan coating process were investigated, on the basis of thickness and profile maps of the tablet core and coating computed from about 480,000 depth measurements (i.e., 3D data) per sample. This data enables the calculation of the root mean square deviation, the skewness and the kurtosis of the assessed profiles. Analyzing these roughness parameters revealed that, for the given coating formulation, small valleys in the tablet core are filled with coating, whereas coarse features of the tablet core are still visible on the final film-coated tablet. Moreover, the impact of the tablet core roughness on the coating thickness is analyzed by correlating the tablet core profile and the coating thickness map. The presented measurement method and processing could be in the future transferred to in-line OCT measurements, to investigate core and coating roughness during the production of film-coated tablets. Copyright © 2017. Published by Elsevier B.V.

  10. HCV Core Protein Uses Multiple Mechanisms to Induce Oxidative Stress in Human Hepatoma Huh7 Cells

    Science.gov (United States)

    Ivanov, Alexander V.; Smirnova, Olga A.; Petrushanko, Irina Y.; Ivanova, Olga N.; Karpenko, Inna L.; Alekseeva, Ekaterina; Sominskaya, Irina; Makarov, Alexander A.; Bartosch, Birke; Kochetkov, Sergey N.; Isaguliants, Maria G.

    2015-01-01

    Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGFβ1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1α. The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein. PMID:26035647

  11. Identification and characterization of secreted proteins in Eimeria tenella

    Science.gov (United States)

    Ramlee, Intan Azlinda; Firdaus-Raih, Mohd; Wan, Kiew-Lian

    2015-09-01

    Eimeria tenella is a protozoan parasite that causes coccidiosis, an economically important disease in the poultry industry. The characterization of proteins that are secreted by parasites have been shown to play important roles in parasite invasion and are considered to be potential control agents. In this study, 775 proteins potentially secreted by E. tenella were identified. These proteins were further filtered to remove mitochondrial proteins. Out of 763 putative secreted proteins, 259 proteins possess transmembrane domains while another 150 proteins have GPI (Glycosylphosphatidylinositol) anchors. Homology search revealed that 315 and 448 proteins have matches with known and hypothetical proteins in the database, respectively. Within this data set, previously characterized secretory proteins such as micronemes, rhoptry kinases and dense granules were detected.

  12. Characterization of HCoV-229E fusion core: Implications for structure basis of coronavirus membrane fusion

    International Nuclear Information System (INIS)

    Liu Cheng; Feng Youjun; Gao Feng; Zhang Qiangmin; Wang Ming

    2006-01-01

    Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection

  13. SDSL-ESR-based protein structure characterization.

    Science.gov (United States)

    Strancar, Janez; Kavalenka, Aleh; Urbancic, Iztok; Ljubetic, Ajasja; Hemminga, Marcus A

    2010-03-01

    As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be seen in the number of protein structures published in the Protein Data Bank. This is especially the case for less-ordered, more hydrophobic and more flexible protein systems. The lack of efficient methods for structure determination calls for urgent development of a new class of biophysical techniques. This work attempts to address this problem with a novel combination of site-directed spin labelling electron spin resonance spectroscopy (SDSL-ESR) and protein structure modelling, which is coupled by restriction of the conformational spaces of the amino acid side chains. Comparison of the application to four different protein systems enables us to generalize the new method and to establish a general procedure for determination of protein structure.

  14. Characterization of redox proteins using electrochemical methods

    OpenAIRE

    Verhagen, M.

    1995-01-01

    The use of electrochemical techniques in combination with proteins started approximately a decade ago and has since then developed into a powerfull technique for the study of small redox proteins. In addition to the determination of redox potentials, electrochemistry can be used to obtain information about the kinetics of electron transfer between proteins and about the dynamic behaviour of redox cofactors in proteins. This thesis describes the results of a study, initiated to get a ...

  15. A critical role for protein degradation in the nucleus accumbens core in cocaine reward memory.

    Science.gov (United States)

    Ren, Zhen-Yu; Liu, Meng-Meng; Xue, Yan-Xue; Ding, Zeng-Bo; Xue, Li-Fen; Zhai, Suo-Di; Lu, Lin

    2013-04-01

    The intense associative memories that develop between cocaine-paired contexts and rewarding stimuli contribute to cocaine seeking and relapse. Previous studies have shown impairment in cocaine reward memories by manipulating a labile state induced by memory retrieval, but the mechanisms that underlie the destabilization of cocaine reward memory are unknown. In this study, using a Pavlovian cocaine-induced conditioned place preference (CPP) procedure in rats, we tested the contribution of ubiquitin-proteasome system-dependent protein degradation in destabilization of cocaine reward memory. First, we found that polyubiquitinated protein expression levels and polyubiquitinated N-ethylmaleimide-sensitive fusion (NSF) markedly increased 15 min after retrieval while NSF protein levels decreased 1 h after retrieval in the synaptosomal membrane fraction in the nucleus accumbens (NAc) core. We then found that infusion of the proteasome inhibitor lactacystin into the NAc core prevented the impairment of memory reconsolidation induced by the protein synthesis inhibitor anisomycin and reversed the effects of anisomycin on NSF and glutamate receptor 2 (GluR2) protein levels in the synaptosomal membrane fraction in the NAc core. We also found that lactacystin infusion into the NAc core but not into the shell immediately after extinction training sessions inhibited CPP extinction and reversed the extinction training-induced decrease in NSF and GluR2 in the synaptosomal membrane fraction in the NAc core. Finally, infusions of lactacystin by itself into the NAc core immediately after each training session or before the CPP retrieval test had no effect on the consolidation and retrieval of cocaine reward memory. These findings suggest that ubiquitin-proteasome system-dependent protein degradation is critical for retrieval-induced memory destabilization.

  16. CHEMICAL AND PHYSICAL CHARACTERIZATION OF COLLAPSING LOW-MASS PRESTELLAR DENSE CORES

    Energy Technology Data Exchange (ETDEWEB)

    Hincelin, U. [Department of Chemistry, University of Virginia, Charlottesville, VA 22904 (United States); Commerçon, B. [Ecole Normale Supérieure de Lyon, CRAL, UMR 5574 du CNRS, Université Lyon I, 46 Allée d’Italie, F-69364 Lyon cedex 07 (France); Wakelam, V.; Hersant, F.; Guilloteau, S. [Univ. Bordeaux, LAB, UMR 5804, F-33270, Floirac (France); Herbst, E., E-mail: ugo.hincelin@gmail.com [Departments of Chemistry and Astronomy, University of Virginia, Charlottesville, VA 22904 (United States)

    2016-05-01

    The first hydrostatic core, also called the first Larson core, is one of the first steps in low-mass star formation as predicted by theory. With recent and future high-performance telescopes, the details of these first phases are becoming accessible, and observations may confirm theory and even present new challenges for theoreticians. In this context, from a theoretical point of view, we study the chemical and physical evolution of the collapse of prestellar cores until the formation of the first Larson core, in order to better characterize this early phase in the star formation process. We couple a state-of-the-art hydrodynamical model with full gas-grain chemistry, using different assumptions for the magnetic field strength and orientation. We extract the different components of each collapsing core (i.e., the central core, the outflow, the disk, the pseudodisk, and the envelope) to highlight their specific physical and chemical characteristics. Each component often presents a specific physical history, as well as a specific chemical evolution. From some species, the components can clearly be differentiated. The different core models can also be chemically differentiated. Our simulation suggests that some chemical species act as tracers of the different components of a collapsing prestellar dense core, and as tracers of the magnetic field characteristics of the core. From this result, we pinpoint promising key chemical species to be observed.

  17. Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein

    Energy Technology Data Exchange (ETDEWEB)

    Sekine, Shuichi; Ito, Konomi; Watanabe, Haruna; Nakano, Takafumi [Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 (Japan); Moriya, Kyoji; Shintani, Yoshizumi; Fujie, Hajime; Tsutsumi, Takeya; Miyoshi, Hideyuki; Fujinaga, Hidetake; Shinzawa, Seiko; Koike, Kazuhiko [Department of Internal Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Horie, Toshiharu, E-mail: t.horie@thu.ac.jp [Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675 (Japan)

    2015-02-01

    Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ({sup 59}Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca{sup 2+} uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein. - Highlights: • Iron accumulation in the livers of patients with hepatitis C virus (HCV) infection is thought to exacerbate oxidative stress. • The impact of iron overload on mitochondrial damage and ROS production in HCV core protein-expressing cells were examined. • Mitochondrial iron uptake was increased in the livers of HCV core protein-expressing transgenic mice. • Ca{sup 2+} uniporter-mediated mitochondrial accumulation of iron exacerbates

  18. Sub-core permeability and relative permeability characterization with Positron Emission Tomography

    Science.gov (United States)

    Zahasky, C.; Benson, S. M.

    2017-12-01

    This study utilizes preclinical micro-Positron Emission Tomography (PET) to image and quantify the transport behavior of pulses of a conservative aqueous radiotracer injected during single and multiphase flow experiments in a Berea sandstone core with axial parallel bedding heterogeneity. The core is discretized into streamtubes, and using the micro-PET data, expressions are derived from spatial moment analysis for calculating sub-core scale tracer flux and pore water velocity. Using the flux and velocity data, it is then possible to calculate porosity and saturation from volumetric flux balance, and calculate permeability and water relative permeability from Darcy's law. Full 3D simulations are then constructed based on this core characterization. Simulation results are compared with experimental results in order to test the assumptions of the simple streamtube model. Errors and limitations of this analysis will be discussed. These new methods of imaging and sub-core permeability and relative permeability measurements enable experimental quantification of transport behavior across scales.

  19. Preparation and characterization of antibacterial Au/C core-shell composite

    Energy Technology Data Exchange (ETDEWEB)

    Gao Yanhong [Department of Chemistry and Institute of Nanochemistry, Jinan University, 601 Huangpudadaoxi Road, Guangzhou 510632, Guangdong (China); Centers for Disease Control and Prevention of Guangdong Province, Guangzhou 510300, Guangdong (China); Zhang Nianchun [Department of Chemistry and Institute of Nanochemistry, Jinan University, 601 Huangpudadaoxi Road, Guangzhou 510632, Guangdong (China); Zhong Yuwen [Centers for Disease Control and Prevention of Guangdong Province, Guangzhou 510300, Guangdong (China); Cai Huaihong [Department of Chemistry and Institute of Nanochemistry, Jinan University, 601 Huangpudadaoxi Road, Guangzhou 510632, Guangdong (China); Liu Yingliang, E-mail: tliuyl@jnu.edu.cn [Department of Chemistry and Institute of Nanochemistry, Jinan University, 601 Huangpudadaoxi Road, Guangzhou 510632, Guangdong (China)

    2010-09-01

    An environment-friendly oxidation-reduction method was used to prepare Au/C core-shell composite using carbon as core and gold as shell. The chemical structures and morphologies of Au/C core-shell composite and carbon sphere were characterized by X-ray diffraction, transmission electron microscope, energy dispersion X-ray spectrometry (EDS) and X-ray photoelectron spectroscopy (XPS). The antibacterial properties of the Au/C core-shell composite against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Candida albicans (C. albicans) were examined by the disk diffusion assay and minimal inhibition concentration (MIC) methods. In addition, antibacterial ability of Au/C core-shell composite was observed by atomic force microscope. Results demonstrated that gold homogeneously supported on the surface of carbon spheres without aggregation and showed efficient antibacterial abilities.

  20. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    Science.gov (United States)

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  1. Characterization of tumour virus proteins, 2

    International Nuclear Information System (INIS)

    Higuchi, T.

    1977-01-01

    The structural protein in murine tumour virus P30 has been measured by radioiummunoassay. The titer of each serum was determined by using as antigen the purified Rauscher viral protein labeled with 125 iodine. Standard competition curve was constructed in order to determine the equivalent of protein to inhibit the precipitation reaction under limited antibody concentration. Competition by purified Kirsten virus suspension normal rat kidney cells, transformed-productive and transformed non-productive cells were measured in homologous and heterologous systems [pt

  2. Characterization of redox proteins using electrochemical methods

    NARCIS (Netherlands)

    Verhagen, M.

    1995-01-01

    The use of electrochemical techniques in combination with proteins started approximately a decade ago and has since then developed into a powerfull technique for the study of small redox proteins. In addition to the determination of redox potentials, electrochemistry can be used to obtain

  3. SDSL-ESR-based protein structure characterization

    NARCIS (Netherlands)

    Strancar, J.; Kavalenka, A.A.; Urbancic, I.; Ljubetic, A.; Hemminga, M.A.

    2010-01-01

    As proteins are key molecules in living cells, knowledge about their structure can provide important insights and applications in science, biotechnology, and medicine. However, many protein structures are still a big challenge for existing high-resolution structure-determination methods, as can be

  4. Identification and characterization of the surface proteins of Clostridium difficile

    International Nuclear Information System (INIS)

    Dailey, D.C.

    1988-01-01

    Several clostridial proteins were detected on the clostridial cell surface by sensitive radioiodination techniques. Two major proteins and six minor proteins comprised the radioiodinated proteins on the clostridial cell surface. Cellular fractionation of surface radiolabeled C. difficile determined that the radioiodinated proteins were found in the cell wall fraction of C. difficile and surprisingly were also present in the clostridial membrane. Furthermore, an interesting phenomenon of disulfide-crosslinking of the cell surface proteins of C. difficile was observed. Disulfide-linked protein complexes were found in both the membrane and cell wall fractions. In addition, the cell surface proteins of C. difficile were found to be released into the culture medium. In attempts to further characterize the clostridial proteins recombinant DNA techniques were employed. In addition, the role of the clostridial cell surface proteins in the interactions of C. difficile with human PMNs was also investigated

  5. Characterization of chickpea germplasm conserved in the Indian National Genebank and development of a core set using qualitative and quantitative trait data

    Directory of Open Access Journals (Sweden)

    Sunil Archak

    2016-10-01

    Full Text Available Chickpea is the third most important pulse crop as a source of dietary protein. Ever-increasing demand in Asian countries calls for breeding superior desi-type varieties, in turn necessitating the availability of characterized germplasm to breeders. The Indian National Genebank, located at the National Bureau of Plant Genetic Resources, New Delhi, conserves 14,651 accessions of chickpea. The entire set was characterized in a single large-scale experiment. High variation was observed for eight quantitative and 12 qualitative agro-morphological traits. Allelic richness procedure was employed to assemble a core set comprising 1103 accessions, 70.0% of which were of Indian origin. Comparable values of total variation explained by the first three principal components in the entire collection (51.1% and the core (52.4% together with conservation of nine pairwise r values among quantitative traits in the core collection and a coincidence rate around 99.7% indicated that the chickpea core was indeed an excellent representation of the entire chickpea collection in the National Genebank. The chickpea core exhibited greater diversity than the entire collection in agro-morphological traits, as assessed by higher variance and Shannon–Weaver diversity indices, indicating that the chickpea core maximized the phenotypic diversity available in the Indian chickpea germplasm. The chickpea core, comprising mainly indigenous desi genotypes, is expected to be an excellent resource for chickpea breeders. Information on the chickpea core can be accessed at http://www.nbpgr.ernet.in/pgrportal.

  6. Multiple TPR motifs characterize the Fanconi anemia FANCG protein.

    Science.gov (United States)

    Blom, Eric; van de Vrugt, Henri J; de Vries, Yne; de Winter, Johan P; Arwert, Fré; Joenje, Hans

    2004-01-05

    The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC proteins (FANCA, -C, -E, -F, -G, and -L). Except for FANCL, which has WD40 repeats and a RING finger domain, no significant domain structure has so far been recognized in any of the core complex proteins. By using a homology search strategy comparing the human FANCG protein sequence with its ortholog sequences in Oryzias latipes (Japanese rice fish) and Danio rerio (zebrafish) we identified at least seven tetratricopeptide repeat motifs (TPRs) covering a major part of this protein. TPRs are degenerate 34-amino acid repeat motifs which function as scaffolds mediating protein-protein interactions, often found in multiprotein complexes. In four out of five TPR motifs tested (TPR1, -2, -5, and -6), targeted missense mutagenesis disrupting the motifs at the critical position 8 of each TPR caused complete or partial loss of FANCG function. Loss of function was evident from failure of the mutant proteins to complement the cellular FA phenotype in FA-G lymphoblasts, which was correlated with loss of binding to FANCA. Although the TPR4 mutant fully complemented the cells, it showed a reduced interaction with FANCA, suggesting that this TPR may also be of functional importance. The recognition of FANCG as a typical TPR protein predicts this protein to play a key role in the assembly and/or stabilization of the nuclear FA protein core complex.

  7. In-house characterization of protein powder

    DEFF Research Database (Denmark)

    Hartmann, Christian Grundahl; Nielsen, Ole Faurskov; Ståhl, Kenny

    2010-01-01

    X-ray powder diffraction patterns of lysozyme and insulin were recorded on a standard in-house powder diffractometer. The experimental powder diffraction patterns were compared with patterns calculated from Protein Data Bank coordinate data. Good agreement was obtained by including straightforward...... to include calculated H-atom positions did not improve the overall fit and was abandoned. The method devised was shown to be a quick and convenient tool for distinguishing precipitates and polymorphs of proteins....

  8. Characterization of the fast neutron irradiation facility of the Portuguese Research Reactor after core conversion

    International Nuclear Information System (INIS)

    Marques, J.G.; Sousa, M.; Santos, J.P.; Fernandes, A.C.

    2011-01-01

    The fast neutron irradiation facility of the Portuguese Research Reactor was characterized after the reduction in uranium enrichment and rearrangement of the core configuration. In this work we report on the determination of the hardness parameter and the 1 MeV equivalent neutron flux along the facility, in the new irradiation conditions, following ASTM E722 standard.

  9. Synthesis, Characterization and Gold Loading of Polystyrene-Poly(pyridyl methacrylate) Core-Shell Latex Systems

    NARCIS (Netherlands)

    Oláh, A.; Hempenius, Mark A.; Vancso, Gyula J.

    2004-01-01

    In this research, novel 3-(2-pyridyl)propyl methacrylate and 3-(3-pyridyloxy)propyl methacrylate monomers were synthesized and emulsion polymerized on colloidal polystyrene seeds, resulting in core–shell latex systems. The cores and the core–shell particles were characterized by static light

  10. Hepatitis C Virus Core Protein Modulates Endoglin (CD105) Signaling Pathway for Liver Pathogenesis.

    Science.gov (United States)

    Kwon, Young-Chan; Sasaki, Reina; Meyer, Keith; Ray, Ranjit

    2017-11-01

    Endoglin is part of the TGF-β receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In a previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition (EMT) state and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV-associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of an HCV core-expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV-infected cells or viral core gene-transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase 1 (ALK1) Fc blocking peptide and endoglin small interfering RNA (siRNA). Further, follow-up from in vitro analysis suggested that the antiapoptosis Bcl2 protein, proliferation-related cyclin D1 protein, and CSC-associated Hes1, Notch1, Nanog, and Sox2 proteins are enhanced during infection or ectopic expression of HCV core protein. IMPORTANCE Endoglin plays a crucial role in fibrogenesis and angiogenesis and is an important protein for tumor growth, survival, and cancer cell metastasis. Endoglin enhances ALK1-SMAD1/5 signaling in different cell types, leading to increased proliferation and migration responses. We have observed endoglin expression on the HCV core-expressing cell surface of human hepatocyte origin and activation of phospho-SMAD1/5 and ID1 downstream signaling molecules. ID1 protein plays a role in CSC properties, and we found that

  11. Getting to the core of protein pharmaceuticals – comprehensive structure analysis by mass spectrometry

    DEFF Research Database (Denmark)

    Leurs, Ulrike; Mistarz, Ulrik Hvid; Rand, Kasper Dyrberg

    2015-01-01

    . Mass spectrometry has evolved as a powerful tool for the characterization of both primary and higher order structures of protein pharmaceuticals. Furthermore, the chemical and physical stability of protein drugs, as well as their pharmacokinetics are nowadays routinely determined by mass spectrometry...

  12. Simultaneous inhibition of aberrant cancer kinome using rationally designed polymer-protein core-shell nanomedicine.

    Science.gov (United States)

    Chandran, Parwathy; Gupta, Neha; Retnakumari, Archana Payickattu; Malarvizhi, Giridharan Loghanathan; Keechilat, Pavithran; Nair, Shantikumar; Koyakutty, Manzoor

    2013-11-01

    Simultaneous inhibition of deregulated cancer kinome using rationally designed nanomedicine is an advanced therapeutic approach. Herein, we have developed a polymer-protein core-shell nanomedicine to inhibit critically aberrant pro-survival kinases (mTOR, MAPK and STAT5) in primitive (CD34(+)/CD38(-)) Acute Myeloid Leukemia (AML) cells. The nanomedicine consists of poly-lactide-co-glycolide core (~250 nm) loaded with mTOR inhibitor, everolimus, and albumin shell (~25 nm thick) loaded with MAPK/STAT5 inhibitor, sorafenib and the whole construct was surface conjugated with monoclonal antibody against CD33 receptor overexpressed in AML. Electron microscopy confirmed formation of core-shell nanostructure (~290 nm) and flow cytometry and confocal studies showed enhanced cellular uptake of targeted nanomedicine. Simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells, was demonstrated using immunoblotting, cytotoxicity and apoptosis assays. This cell receptor plus multi-kinase targeted core-shell nanomedicine was found better specific and tolerable compared to current clinical regime of cytarabine and daunorubicin. These authors demonstrate simultaneous inhibition of critical kinases causing synergistic lethality against leukemic cells, without affecting healthy blood cells by using rationally designed polymer-protein core-shell nanomedicine, provoding an advanced method to eliminate cancer cells, with the hope of future therapeutic use. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

    Science.gov (United States)

    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  14. In-house characterization of protein powder

    DEFF Research Database (Denmark)

    Hartmann, Christian Grundahl; Harris, Pernille; Ståhl, Kenny

    2011-01-01

    . For safe identification of the crystal form the experimental patterns have to be compared with patterns calculated from known crystal structures. Very good agreement with Protein Data Bank data was obtained after including corrections for background, unit cell parameters, disordered bulk......Collecting protein powder diffraction data on standard in-house powder diffractometers requires careful handling of the samples. Specially designed sample holders combined with optimized collimation were found to be the key factors in improving the data quality and reducing the data collection time......-solvent, and geometric factors. The data collection and correction procedures were demonstrated by the identification of three different crystal forms of insulin....

  15. Parts Characterization for Tunable Protein Expression

    DEFF Research Database (Denmark)

    Klausen, Michael Schantz; Sommer, Morten Otto Alexander

    2018-01-01

    Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression. Construc......Flow-seq combines flexible genome engineering methods with flow cytometry-based cell sorting and deep DNA sequencing to enable comprehensive interrogation of genotype to phenotype relationships. One application is to study the effect of specific regulatory elements on protein expression...

  16. Electrophoretic characterization of crude leaf proteins in ...

    African Journals Online (AJOL)

    Administrator

    ground in an eppendorf tube with 100 µl of lysis buffer. The mixtures were allowed to settle inside the eppendorf immersed in an ice bath for 1 h, and the supernatants were fractionated by. 7.5% SDS-PAGE (Laemmili, 1970). RESULTS AND DISCUSSION. Protein distribution patterns in three cultivars of. Lycopersicon and ...

  17. Molecular and functional characterization of MICAL proteins

    NARCIS (Netherlands)

    Zhou, Y.

    2011-01-01

    Since their original identification in 2002, MICAL proteins have been implicated in various physiological and pathological processes including axon guidance, tight junction formation, spinal cord injury and cancer. MICALs mediate cell signaling via their unusual N-terminal monooxygenase (MO) domain

  18. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  19. Characterization of radiation-induced proteins in Deinococcus radiodurans

    International Nuclear Information System (INIS)

    Tanaka, A.; Watanabe, H.; Nozawa, R.; Hu, Q.; Kitayama, S.

    1992-01-01

    Induction of proteins after gamma-irradiation in Deinococcus radiodurans were investigated. 10 proteins were induced and about 15 proteins were reduced after irradiation with 6kGy. These proteins were classified to four groups by responses to gamma-rays, UV light, mitomycin C(MMC) treatment and heating. Additional studies were carried out for the characterization of two induced proteins. One protein was induced by gamma-rays, UV light as well as heating. This protein appeared to be a glycoprotein from its reaction with lectin. From the amino acid sequences of N-terminal and internal region, it was found that this protein is homologous to EF-Tu protein of E. coli. Meanwhile the other protein was induced not only by gamma-rays but also by UV light and MMC treatment. This protein seems to be a new enzyme as it has no homology to the known proteins which have ever been analyzed. No accumulations of these two proteins were observed in radiation sensitive strain of D. radiodurans and in both of E. coli and Bacillus pumilus, suggesting that induction of these two proteins would be specific for high resistant strain. (author)

  20. Characterization of the first core sample of neutralized current acid waste from double-shell tank 101-AZ

    International Nuclear Information System (INIS)

    Peterson, M.E.; Scheele, R.D.; Tingey, J.M.

    1989-09-01

    In FY 1989, Westinghouse Hanford Company (WHC) successfully obtained four core samples (totaling seven segments) of neutralized current acid waste (NCAW) from double-shell tanks (DSTs) 101-AZ and 102-AZ. A segment was a 19-in.-long and 1-in.-diameter cylindrical sample of waste. A core sample consisted of enough 19-in.-long segments to obtain the waste of interest. Three core samples were obtained from DST 101-AZ and one core sample from DST 102-AZ. Two DST 101-AZ core samples consisted of two segments per core, and the third core sample consisted of only one segment. The third core consisted of the solids from the bottom of the tank and was used to determine the relative abrasiveness of this NCAW. The DST 102-AZ core sample consisted of two segments. The core samples were transported to the Pacific Northwest Laboratory (PNL), where the waste was extruded from its sampler and extensively characterized. A characterization plan was followed that simulated the processing of the NCAW samples through retrieval, pretreatment and vitrification process steps. Physical, rheological, chemical and radiochemical properties were measured throughout the process steps. The characterization of the first core sample from DST 101-AZ was completed, and the results are provided in this report. The results for the other core characterizations will be reported in future reports. 3 refs., 13 figs., 10 tabs

  1. Preparation and Characterization of Myosin Proteins.

    Science.gov (United States)

    Caldwell, Elizabeth; Eftink, Maurice R.

    1985-01-01

    Students complete five experimental projects at the end of a senior-level biochemistry course which involves the isolation and characterization of myosin and its water-soluble subfragments. Procedures used and results obtained are provided for such projects as viscosity and ATPase measurements and gel electrophoresis experiments. (JN)

  2. A novel approach to preparing magnetic protein microspheres with core-shell structure

    International Nuclear Information System (INIS)

    Jiang Wei; Sun Zhendong; Li Fengsheng; Chen Kai; Liu Tianyu; Liu Jialing; Zhou Tianle; Guo Rui

    2011-01-01

    Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3 O 4 cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe 3 O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail. - Research Highlights: → Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method.→ The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3 O 4 cores and coated with globular bovine serum albumin (BSA).→ 57.8 wt% of approximately 10 nm superparamagnetic Fe 3 O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides the abundant functional groups.

  3. A novel approach to preparing magnetic protein microspheres with core-shell structure

    Energy Technology Data Exchange (ETDEWEB)

    Jiang Wei, E-mail: climentjw@126.co [National Special Superfine Powder Engineering Research Center, Nanjing University of Science and Technology, Nanjing 210094 (China); Sun Zhendong; Li Fengsheng [National Special Superfine Powder Engineering Research Center, Nanjing University of Science and Technology, Nanjing 210094 (China); Chen Kai; Liu Tianyu; Liu Jialing [Department of Physics, Nanjing University of Science and Technology, Nanjing 210094 (China); Zhou Tianle [Department of Materials Science and Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China); Guo Rui [Department of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094 (China)

    2011-03-15

    Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe{sub 3}O{sub 4} cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe{sub 3}O{sub 4} nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail. - Research Highlights: Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe{sub 3}O{sub 4} cores and coated with globular bovine serum albumin (BSA). 57.8 wt% of approximately 10 nm superparamagnetic Fe{sub 3}O{sub 4} nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides the abundant functional groups.

  4. Vaccine delivery system for tuberculosis based on nano-sized hepatitis B virus core protein particles

    Directory of Open Access Journals (Sweden)

    Dhanasooraj D

    2013-02-01

    Full Text Available Dhananjayan Dhanasooraj, R Ajay Kumar, Sathish MundayoorMycobacterium Research Group, Rajiv Gandhi Centre for Biotechnology, Kerala, IndiaAbstract: Nano-sized hepatitis B virus core virus-like particles (HBc-VLP are suitable for uptake by antigen-presenting cells. Mycobacterium tuberculosis antigen culture filtrate protein 10 (CFP-10 is an important vaccine candidate against tuberculosis. The purified antigen shows low immune response without adjuvant and tends to have low protective efficacy. The present study is based on the assumption that expression of these proteins on HBc nanoparticles would provide higher protection when compared to the native antigen alone. The cfp-10 gene was expressed as a fusion on the major immunodominant region of HBc-VLP, and the immune response in Balb/c mice was studied and compared to pure proteins, a mixture of antigens, and fusion protein-VLP, all without using any adjuvant. The humoral, cytokine, and splenocyte cell proliferation responses suggested that the HBc-VLP bearing CFP-10 generated an antigen-specific immune response in a Th1-dependent manner. By virtue of its self-adjuvant nature and ability to form nano-sized particles, HBc-VLPs are an excellent vaccine delivery system for use with subunit protein antigens identified in the course of recent vaccine research.Keywords: Mycobacterium tuberculosis, VLP, hepatitis B virus core particle, CFP-10, self-adjuvant, vaccine delivery

  5. Core/shell structured ZnO/SiO2 nanoparticles: Preparation, characterization and photocatalytic property

    International Nuclear Information System (INIS)

    Zhai Jing; Tao Xia; Pu Yuan; Zeng Xiaofei; Chen Jianfeng

    2010-01-01

    ZnO nanoparticles were prepared by a simple chemical synthesis route. Subsequently, SiO 2 layers were successfully coated onto the surface of ZnO nanoparticles to modify the photocatalytic activity in acidic or alkaline solutions. The obtained particles were characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive spectrometry (EDS) and zeta potential. It was found that ultrafine core/shell structured ZnO/SiO 2 nanoparticles were successfully obtained. The photocatalytic performance of ZnO/SiO 2 core/shell structured nanoparticles in Rhodamine B aqueous solution at varied pH value were also investigated. Compared with uncoated ZnO nanoparticles, core/shell structured ZnO/SiO 2 nanoparticles with thinner SiO 2 shell possess improved stability and relatively better photocatalytic activity in acidic or alkaline solutions, which would broaden its potential application in pollutant treatment.

  6. Characterization and interactome study of white spot syndrome virus envelope protein VP11.

    Directory of Open Access Journals (Sweden)

    Wang-Jing Liu

    Full Text Available White spot syndrome virus (WSSV is a large enveloped virus. The WSSV viral particle consists of three structural layers that surround its core DNA: an outer envelope, a tegument and a nucleocapsid. Here we characterize the WSSV structural protein VP11 (WSSV394, GenBank accession number AF440570, and use an interactome approach to analyze the possible associations between this protein and an array of other WSSV and host proteins. Temporal transcription analysis showed that vp11 is an early gene. Western blot hybridization of the intact viral particles and fractionation of the viral components, and immunoelectron microscopy showed that VP11 is an envelope protein. Membrane topology software predicted VP11 to be a type of transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal. Based on an immunofluorescence assay performed on VP11-transfected Sf9 cells and a trypsin digestion analysis of the virion, we conclude that, contrary to topology software prediction, the C-terminal of this protein is in fact inside the virion. Yeast two-hybrid screening combined with co-immunoprecipitation assays found that VP11 directly interacted with at least 12 other WSSV structural proteins as well as itself. An oligomerization assay further showed that VP11 could form dimers. VP11 is also the first reported WSSV structural protein to interact with the major nucleocapsid protein VP664.

  7. Identification and characterization of a core fucosidase from the bacterium Elizabethkingia meningoseptica.

    Science.gov (United States)

    Li, Tiansheng; Li, Mengjie; Hou, Linlin; Guo, Yameng; Wang, Lei; Sun, Guiqin; Chen, Li

    2018-01-26

    All reported α-l-fucosidases catalyze the removal of nonreducing terminal l-fucoses from oligosaccharides or their conjugates, while having no capacity to hydrolyze core fucoses in glycoproteins directly. Here, we identified an α-fucosidase from the bacterium Elizabethkingia meningoseptica with catalytic activity against core α-1,3-fucosylated substrates, and we named it core fucosidase I (cFase I). Using site-specific mutational analysis, we found that three acidic residues (Asp-242, Glu-302, and Glu-315) in the predicted active pocket are critical for cFase I activity, with Asp-242 and Glu-315 acting as a pair of classic nucleophile and acid/base residues and Glu-302 acting in an as yet undefined role. These findings suggest a catalytic mechanism for cFase I that is different from known α-fucosidase catalytic models. In summary, cFase I exhibits glycosidase activity that removes core α-1,3-fucoses from substrates, suggesting cFase I as a new tool for glycobiology, especially for studies of proteins with core fucosylation. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Characterization of interactions between inclusion membrane proteins from Chlamydia trachomatis

    Directory of Open Access Journals (Sweden)

    Emilie eGauliard

    2015-02-01

    Full Text Available Chlamydiae are obligate intracellular pathogens of eukaryotes. The bacteria grow in an intracellular vesicle called an inclusion, the membrane of which is heavily modified by chlamydial proteins called Incs (Inclusion membrane proteins. Incs represent 7-10% of the genomes of Chlamydia and, given their localization at the interface between the host and the pathogen, likely play a key role in the development and pathogenesis of the bacterium. However, their functions remain largely unknown. Here, we characterized the interaction properties between various Inc proteins of C. trachomatis, using a bacterial two-hybrid (BACTH method suitable for detecting interactions between integral membrane proteins. To validate this approach, we first examined the oligomerization properties of the well-characterized IncA protein and showed that both the cytoplasmic domain and the transmembrane region independently contribute to IncA oligomerization. We then analyzed a set of Inc proteins and identified novel interactions between these components. Two small Incs, IncF and Ct222, were found here to interact with many other Inc proteins and may thus represent interaction nodes within the inclusion membrane. Our data suggest that the Inc proteins may assemble in the membrane of the inclusion to form specific multi-molecular complexes in an hierarchical and temporal manner. These studies will help to better define the putative functions of the Inc proteins in the infectious process of Chlamydia.

  9. Vibrational characterization of hexagonal duct core assemblies under various support conditions

    International Nuclear Information System (INIS)

    Bartholf, L.W.; Julyk, L.J.; Ryan, J.A.

    1989-03-01

    Analysis of the dynamic response of advanced Liquid Metal Reactor (LMR) core internals to seismic excitation requires a significant number of simplifying assumptions and idealizations to economically meet the constraints of present-day computer limitations. Fluid coupling and nonlinearities associated with inter-assembly lateral support stiffness and clearances of a large cluster of core internal assemblies are some of the factors that complicate the analytical procedure (Moran, 1976). Well defined test data were needed to quantify these and other uncertainties associated with the use of analytical or numerical computer codes used in the seismic design and analysis of reactor cores. The purpose of the present experimental program was to supplement existing data, such as reported in (Sasaki and Muto, 1983), by developing vibrational characteristics of core assemblies over a range of parameters relative to LMR conceptual designs. The parameters selected for this program were variations in number and location of restraints, restraint-pad to duct-load-pad clearances, and input forcing frequency and g-level. Feature tests were conducted to characterize load pad stiffness and coefficient of restitution, and to calibrate load pads to measure inter-assembly across-flat impact loads. Simulated full-size LMR hexagonal duct core assemblies were used in vibration tests. A single assembly and a row of five assemblies were tested in air to establish modal characteristics and forced response behavior. 2 refs., 7 figs., 1 tab

  10. Preparation and Characterization of SiO2/SiCN Core-shell Ceramic Microspheres

    Directory of Open Access Journals (Sweden)

    ZHANG Hai-yuan

    2017-05-01

    Full Text Available The SiO2/PSN core-shell microspheres were prepared via an emulsion reaction combined with the polymer-derived ceramics (PDCs method using polysilazane (PSN in situ polymerization on the surface of SiO2 modified by silane coupling agents MPS, followed by pyrolysis process to obtain SiO2/SiCN core-shell ceramic microspheres. The effects of raw mass ratio, curing time and pyrolysis temperature on the formation and the morphology of core-shell microspheres were studied. The morphology, chemical composition and phase transformation were characterized by SEM, EDS, TEM, FT-IR and XRD. The results show that after reaction for 4h at 200℃, SiO2 completely coated PSN forms a core-shell microsphere with rough surface when the mass ratio of SiO2 and PSN is 1:4; when pyrolysis temperature is at 800-1200℃, amorphous SiO2/SiCN core-shell ceramic microspheres are prepared; at 1400℃, the amorphous phase partially crystallizes to produce SiO2, SiC and Si3N4 phase.

  11. Modified solvothermal synthesis and characterization of CdS/ZnS core/shell nanorods

    International Nuclear Information System (INIS)

    Baby Suganthi, A.R.; Sagayaraj, P.

    2013-01-01

    Core/shell CdS/ZnS nanorods were synthesized using a two-step solvothermal approach. The first step is the formation of CdS nanoparticles initiated using nucleation followed by growth through coalescence-exchange and particle coagulation. The second step leads to the formation of ZnS and further coalescence-exchange leading to deposition and growth of a ZnS shell around CdS nanoparticles. The structural, morphological and chemical studies were performed using X-ray diffraction, Energy Dispersive X-ray spectroscopy (EDX) Scanning electron Microscopy (SEM), UV–vis absorption spectra and Transmission Electron Microscopy (TEM), provide direct evidence for shell growth. The present synthesis provides a rational approach to the design of novel core/shell nanomaterials with appealing applications in optoelectronic devices. - Graphical abstract: From the resulting TEM images, the formation of core/shell could be observed. The apparent microscopy contrast between the CdS core and the ZnS shell offers evidence for the formation of CdS/ZnS core/shell nanostructures. It is clearly evident that the surfaces of the nanorods became rough after coating and also the diameter of the nanorod is seen increased up to 40–50 nm. Highlights: ► CdS/ZnS core/shell nanorods were synthesized using two-step solvothermal approach. ► The nanoparticles were characterized by XRD, EDX, SEM, UV–vis and TEM. ► SEM images revealed the surface roughness after ZnS shell growth. ► TEM microscopy offers evidence for the formation of core/shell nanostructures

  12. Preparation and characterization of nanodiamond cores coated with a thin Ni-Zn-P alloy film

    International Nuclear Information System (INIS)

    Wang Rui; Ye Weichun; Ma Chuanli; Wang Chunming

    2008-01-01

    Nanodiamond cores coated with a thin Ni-Zn-P alloy film were prepared by an electroless deposition method under the conditions of tin chloride sensitization and palladium chloride activation. The prepared materials were analyzed by Fourier transform infrared (FTIR) spectrometry and X-ray diffraction (XRD). The nanostructure of the materials was then characterized by transmission electron microscopy (TEM). The alloy film composition was characterized by Energy Dispersive X-ray (EDX) analysis. The results indicated the approximate composition 49.84%Ni-37.29%Zn-12.88%P was obtained

  13. Hepatitis C Virus core+1/ARF Protein Modulates the Cyclin D1/pRb Pathway and Promotes Carcinogenesis.

    Science.gov (United States)

    Moustafa, Savvina; Karakasiliotis, Ioannis; Mavromara, Penelope

    2018-05-01

    Viruses often encompass overlapping reading frames and unconventional translation mechanisms in order to maximize the output from a minimum genome and to orchestrate their timely gene expression. Hepatitis C virus (HCV) possesses such an unconventional open reading frame (ORF) within the core-coding region, encoding an additional protein, initially designated ARFP, F, or core+1. Two predominant isoforms of core+1/ARFP have been reported, core+1/L, initiating from codon 26, and core+1/S, initiating from codons 85/87 of the polyprotein coding region. The biological significance of core+1/ARFP expression remains elusive. The aim of the present study was to gain insight into the functional and pathological properties of core+1/ARFP through its interaction with the host cell, combining in vitro and in vivo approaches. Our data provide strong evidence that the core+1/ARFP of HCV-1a stimulates cell proliferation in Huh7-based cell lines expressing either core+1/S or core+1/L isoforms and in transgenic liver disease mouse models expressing core+1/S protein in a liver-specific manner. Both isoforms of core+1/ARFP increase the levels of cyclin D1 and phosphorylated Rb, thus promoting the cell cycle. In addition, core+1/S was found to enhance liver regeneration and oncogenesis in transgenic mice. The induction of the cell cycle together with increased mRNA levels of cell proliferation-related oncogenes in cells expressing the core+1/ARFP proteins argue for an oncogenic potential of these proteins and an important role in HCV-associated pathogenesis. IMPORTANCE This study sheds light on the biological importance of a unique HCV protein. We show here that core+1/ARFP of HCV-1a interacts with the host machinery, leading to acceleration of the cell cycle and enhancement of liver carcinogenesis. This pathological mechanism(s) may complement the action of other viral proteins with oncogenic properties, leading to the development of hepatocellular carcinoma. In addition, given that

  14. Functional characterization of Arabidopsis thaliana transthyretin-like protein.

    Science.gov (United States)

    Pessoa, João; Sárkány, Zsuzsa; Ferreira-da-Silva, Frederico; Martins, Sónia; Almeida, Maria R; Li, Jianming; Damas, Ana M

    2010-02-18

    Arabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL. The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein. The Arabidopsis thaliana transthyretin-like (TTL) protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

  15. Functional characterization of Arabidopsis thaliana transthyretin-like protein

    Directory of Open Access Journals (Sweden)

    Almeida Maria R

    2010-02-01

    Full Text Available Abstract Background Arabidopsis thaliana transthyretin-like (TTL protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU decarboxylase (N-terminal domain and 5-hydroxyisourate (5-HIU hydrolase (C-terminal domain. TTL is a member of the transthyretin-related protein family (TRP, which comprises a number of proteins with sequence homology to transthyretin (TTR and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL. Results The TTL gene was cloned and the protein expressed and purified for biochemical and functional characterization. The results show that TTL is composed of four subunits, with a moderately elongated shape. We also found evidence for 5-HIU hydrolase and OHCU decarboxylase activities in vitro, in the full-length protein. Conclusions The Arabidopsis thaliana transthyretin-like (TTL protein is a tetrameric bifunctional enzyme, since it has 5-HIU hydrolase and OHCU decarboxylase activities, which were simultaneously observed in vitro.

  16. Third-generation site characterization: Cryogenic core collection, nuclear magnetic resonance, and electrical resistivity

    Science.gov (United States)

    Kiaalhosseini, Saeed

    In modern contaminant hydrology, management of contaminated sites requires a holistic characterization of subsurface conditions. Delineation of contaminant distribution in all phases (i.e., aqueous, non-aqueous liquid, sorbed, and gas), as well as associated biogeochemical processes in a complex heterogeneous subsurface, is central to selecting effective remedies. Arguably, a factor contributing to the lack of success of managing contaminated sites effectively has been the limitations of site characterization methods that rely on monitoring wells and grab sediment samples. The overarching objective of this research is to advance a set of third-generation (3G) site characterization methods to overcome shortcomings of current site characterization techniques. 3G methods include 1) cryogenic core collection (C3) from unconsolidated geological subsurface to improve recovery of sediments and preserving key attributes, 2) high-throughput analysis (HTA) of frozen core in the laboratory to provide high-resolution, depth discrete data of subsurface conditions and processes, 3) resolution of non-aqueous phase liquid (NAPL) distribution within the porous media using a nuclear magnetic resonance (NMR) method, and 4) application of a complex resistivity method to track NAPL depletion in shallow geological formation over time. A series of controlled experiments were conducted to develop the C 3 tools and methods. The critical aspects of C3 are downhole circulation of liquid nitrogen via a cooling system, the strategic use of thermal insulation to focus cooling into the core, and the use of back pressure to optimize cooling. The C3 methods were applied at two contaminated sites: 1) F.E. Warren (FEW) Air Force Base near Cheyenne, WY and 2) a former refinery in the western U.S. The results indicated that the rate of core collection using the C3 methods is on the order of 30 foot/day. The C3 methods also improve core recovery and limits potential biases associated with flowing sands

  17. An α-Helical Core Encodes the Dual Functions of the Chlamydial Protein IncA*

    Science.gov (United States)

    Ronzone, Erik; Wesolowski, Jordan; Bauler, Laura D.; Bhardwaj, Anshul; Hackstadt, Ted; Paumet, Fabienne

    2014-01-01

    Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2. PMID:25324548

  18. An α-helical core encodes the dual functions of the chlamydial protein IncA.

    Science.gov (United States)

    Ronzone, Erik; Wesolowski, Jordan; Bauler, Laura D; Bhardwaj, Anshul; Hackstadt, Ted; Paumet, Fabienne

    2014-11-28

    Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. PanCoreGen - Profiling, detecting, annotating protein-coding genes in microbial genomes.

    Science.gov (United States)

    Paul, Sandip; Bhardwaj, Archana; Bag, Sumit K; Sokurenko, Evgeni V; Chattopadhyay, Sujay

    2015-12-01

    A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing the pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen - a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for a species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars - Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. PanCoreGen – profiling, detecting, annotating protein-coding genes in microbial genomes

    Science.gov (United States)

    Bhardwaj, Archana; Bag, Sumit K; Sokurenko, Evgeni V.

    2015-01-01

    A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen – a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars – Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study. PMID:26456591

  1. Fabrication and Characterization of ZnS/Diamond-Like Carbon Core-Shell Nanowires

    Directory of Open Access Journals (Sweden)

    Jung Han Kim

    2016-01-01

    Full Text Available We fabricated ZnS/diamond-like carbon (DLC core-shell heterostructure nanowire using a simple two-step process: the vapor-liquid-solid method combined with radio frequency plasma enhanced chemical vapor deposition (rf PECVD. As a core nanowire, ZnS nanowires with face-centered cubic structure were synthesized with a sputtered Au thin film, which exhibit a length and a diameter of ~10 μm and ~30–120 nm . After rf PECVD for DLC coating, The length and width of the dense ZnS/DLC core-shell nanowires were a range of ~10 μm  and 50–150 nm , respectively. In addition, ZnS/DLC core-shell nanowires were characterized with scanning transmission electron microscopy. From the results, the products have flat and uniform DLC coating layer on ZnS nanowire in spite of high residual stress induced by the high sp3 fraction. To further understanding of the DLC coating layer, Raman spectroscopy was employed with ZnS/DLC core-shell nanowires, which reveals two Raman bands at 1550 cm−1 (G peak and 1330 cm−1 (D peak. Finally, we investigated the optical properties from ultraviolet to infrared wavelength region using ultraviolet-visible (UV-Vis and Fourier transform infrared (FT-IR spectrometry. Related to optical properties, ZnS/DLC core-shell nanowires exhibit relatively lower absorbance and higher IR transmittance than that of ZnS nanowires.

  2. cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

    DEFF Research Database (Denmark)

    Wu, R R; Couchman, J R

    1997-01-01

    Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now....... The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser...

  3. A novel approach to preparing magnetic protein microspheres with core-shell structure

    Science.gov (United States)

    Jiang, Wei; Sun, Zhendong; Li, Fengsheng; Chen, Kai; Liu, Tianyu; Liu, Jialing; Zhou, Tianle; Guo, Rui

    2011-03-01

    Magnetic protein microspheres with core-shell structure were prepared through a novel approach based on the sonochemical method and the emulsion solvent evaporation method. The microspheres are composed of the oleic acid and undecylenic acid modified Fe 3O 4 cores and coated with globular bovine serum albumin (BSA). Under an optimized condition, up to 57.8 wt% of approximately 10 nm superparamagnetic Fe 3O 4 nanoparticles could be uniformly encapsulated into the BSA microspheres with the diameter of approximately 160 nm and the high saturation magnetization of 38.5 emu/g, besides of the abundant functional groups. The possible formation mechanism of magnetic microspheres was discussed in detail.

  4. TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends*

    Science.gov (United States)

    Izumi, Takashi; Shimizu, Shigeomi

    2016-01-01

    Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. PMID:27514743

  5. Characterization and Preparation of Broken Rice Proteins Modified by Proteases

    Directory of Open Access Journals (Sweden)

    Lixia Hou

    2010-01-01

    Full Text Available Broken rice is an underutilized by-product of milling. Proteins prepared from broken rice by treatments with alkaline protease and papain have been characterized with regard to nutritional and functional properties. The protein content and the protein recovery were 56.45 and 75.45 % for alkaline protease treatment, and 65.45 and 46.32 % for papain treatment, respectively. Protease treatment increased the lysine and valine content, leading to a more balanced amino acid profile. Broken rice proteins had high emulsifying capacity, 58.3–71.6 % at neutral pH, and adequate water holding capacity, ranging from 1.96 to 2.93 g/g of proteins. At pH=7.0, the broken rice protein had the highest water holding capacity and the best interfacial activities (emulsifying capacity, emulsifying stability, foaming capacity and foaming stability, which may be the result of the higher solubility at pH=7.0. The interfacial activities increased with the increase in the mass fraction of broken rice proteins. The proteins prepared by the papain treatment had higher water holding capacity (p>0.05, emulsifying capacity (p0.05 than alkaline protease treatment at the same pH or mass fraction. To test the fortification of food products with broken rice proteins, pork sausages containing the proteins were prepared. Higher yield of the sausages was obtained with the increased content of broken rice proteins, in the range of 2.0–9.0 %. The results indicate that broken rice proteins have potential to be used as the protein fortification ingredient for food products.

  6. Controlled-release and preserved bioactivity of proteins from (self-assembled core-shell double-walled microspheres

    Directory of Open Access Journals (Sweden)

    Yuan W

    2012-01-01

    Full Text Available Weien Yuan1,2, Zhenguo Liu11Department of Neurology, Xinhua Hospital, affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 2School of Pharmacy, Shanghai Jiao Tong University, Shanghai, People’s Republic of ChinaAbstract: In order to address preserved protein bioactivities and protein sustained-release problems, a method for preparing double-walled microspheres with a core (protein-loaded nanoparticles with a polymer-suspended granule system-formed core and a second shell (a polymer-formed shell for controlled drug release and preserved protein bioactivities has been developed using (solid-in-oil phase-in-hydrophilic oil-in-water (S/O/Oh/W phases. The method, based on our previous microsphere preparation method (solid-in-oil phase-in-hydrophilic oil-in-water (S/O/Oh/W, employs different concentric poly(D,L-lactide-co-glycolide, poly(D,L-lactide, and protein-loaded nanoparticles to produce a suspended liquid which then self-assembles to form shell-core microspheres in the hydrophilic oil phase, which are then solidified in the water phase. Variations in the preparation parameters allowed complete encapsulation by the shell phase, including the efficient formation of a poly(D,L-lactide shell encapsulating a protein-loaded nanoparticle-based poly(D,L-lactide-co-glycolide core. This method produces core-shell double-walled microspheres that show controlled protein release and preserved protein bioactivities for 60 days. Based upon these results, we concluded that the core-shell double-walled microspheres might be applied for tissue engineering and therapy for chronic diseases, etc.Keywords: protein delivery, protein stability, core-shell microspheres, dextran nanoparticles

  7. Partial characterization of GTP-binding proteins in Neurospora

    International Nuclear Information System (INIS)

    Hasunuma, K.; Miyamoto-Shinohara, Y.; Furukawa, K.

    1987-01-01

    Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [ 35 S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [ 35 S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin

  8. Synthesis and characterization of ZnSe:Fe/ZnSe core/shell nanocrystals

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Lin; Zhu, Jianguo, E-mail: yanglin_1028@163.com; Xiao, Dingquan

    2014-04-15

    High-quality ZnSe:Fe/ZnSe core/shell nanocrystals were prepared via a hydrothermal microemulsion technique. Effective surface passivation of monodisperse ZnSe:Fe nanocrystals is achieved by overcoating them with a ZnSe shell. The samples were characterized by means of XRD, EDX, TEM, PSD, XPS, photoluminescence, and Raman spectrum. The results show that the as-synthesized nanocrystals are cubic zinc blende ZnSe structure with high purity and the average particle size of ZnSe:Fe/ZnSe core/shell nanocrystal is larger than that of ZnSe:Fe core. The growth of ZnSe shell causes a small red shift in PL spectra, and then the PL quantum yield (QY) increases from 16% before shell growth to the maximum of 37% after increasing shell thickness up to 1.2 monolayers (ML). Moreover, both transverse optic (TO) and longitudinal optic (LO) phonon modes of ZnSe are shifted toward lower frequency as compared with the reported ones. -- Highlights: • ZnSe:Fe/ZnSe core/shell QDs were prepared by a hydrothermal microemulsion method. • ZnSe shell efficiently passivates surface defects by serving as a physical barrier. • The particle size and PL properties can be turned with the growth of ZnSe shell. • The luminescence efficiency and stability of QDs could be improved in this manner.

  9. High-temperature electrochemical characterization of Ru core Pt shell fuel cell catalyst

    Energy Technology Data Exchange (ETDEWEB)

    Bokach, D.; Fuente, J.L.G. de la; Tsypkin, M.; Ochal, P.; Tunold, R.; Sunde, S.; Seland, F. [Department of Materials Science and Engineering, Norwegian University of Science and Technology (NTNU), Sem Saelands veg 12, N-7491 Trondheim (Norway); Endsjoe, I.C. [Washington Mills AS, NO-7300 Orkanger (Norway)

    2011-12-15

    The electrooxidation of methanol was studied at elevated temperature and pressure by cyclic voltammetry and constant potential experiments at real fuel cell electrocatalysts. Ruthenium core and platinum shell nanoparticles were synthesized by a sequential polyol route, and characterized electrochemically by CO stripping at room temperature to quickly confirm the structure of the synthesized core-shell structure as compared to pure commercial Pt/C and Pt-Ru/C alloy catalysts. A significant promotional effect of Pt decorated Ru cores in the methanol oxidation was found at elevated temperatures and rather high-electrode potentials. A negative potential shift of the methanol oxidation peak is observed for the Ru rate at Pt/C core-shell catalyst at moderate temperatures, while a significant shift to positive potentials of the methanol oxidation peak occurs for Pt/C catalysts. The onset potential for methanol oxidation is lowered some 200 mV from room temperature and up to 120 C for all electrocatalysts, indicating that it is the thermal activity of water adsorption that dictates the onset potential. Direct methanol fuel cell experiments showed only small performance differences between Ru rate at Pt/C and Pt/C anode electrocatalysts, suggesting the necessity of render possible the formation of surface oxygen species at lower electrode potentials. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  10. Murine colon proteome and characterization of the protein pathways

    Directory of Open Access Journals (Sweden)

    Magdeldin Sameh

    2012-08-01

    Full Text Available Abstract Background Most of the current proteomic researches focus on proteome alteration due to pathological disorders (i.e.: colorectal cancer rather than normal healthy state when mentioning colon. As a result, there are lacks of information regarding normal whole tissue- colon proteome. Results We report here a detailed murine (mouse whole tissue- colon protein reference dataset composed of 1237 confident protein (FDR I and Mw ranged from 3–12 and 4–600 KDa, respectively. Gravy index scoring predicted 19.5% membranous and 80.5% globularly located proteins. GO hierarchies and functional network analysis illustrated proteins function together with their relevance and implication of several candidates in malignancy such as Mitogen- activated protein kinase (Mapk8, 9 in colorectal cancer, Fibroblast growth factor receptor (Fgfr 2, Glutathione S-transferase (Gstp1 in prostate cancer, and Cell division control protein (Cdc42, Ras-related protein (Rac1,2 in pancreatic cancer. Protein abundances calculated with 3 different algorithms (NSAF, PAF and emPAI provide a relative quantification under normal condition as guidance. Conclusions This highly confidence colon proteome catalogue will not only serve as a useful reference for further experiments characterizing differentially expressed proteins induced from diseased conditions, but also will aid in better understanding the ontology and functional absorptive mechanism of the colon as well.

  11. A quantitative characterization of the yeast heterotrimeric G protein cycle

    Science.gov (United States)

    Yi, Tau-Mu; Kitano, Hiroaki; Simon, Melvin I.

    2003-01-01

    The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Gα and Gβγ-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose–response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of α-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose–response curve and the downstream dose–response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast. PMID:12960402

  12. Characterization of the Eimeria maxima sporozoite surface protein IMP1

    Science.gov (United States)

    The purpose of this study was to characterize Eimeria maxima immunoprotective protein IMP1 that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transc...

  13. Induction and characterization of pathogenesis-related proteins in ...

    African Journals Online (AJOL)

    Furthermore, induced proteins were extracted from roots of inoculated and control tolerant (RO1054 and RO3015) and susceptible (RO2063) accessions at 8 dpi, and characterized by isoelectric focusing (IEF), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Chitinase ...

  14. Molecular characterization of capsid protein gene of potato virus X ...

    African Journals Online (AJOL)

    Molecular characterization of capsid protein gene of potato virus X from Pakistan. Arshad Jamal, Idrees Ahmad Nasir, Bushra Tabassum, Muhammad Tariq, Abdul Munim Farooq, Zahida Qamar, Mohsin Ahmad Khan, Nadeem Ahmad, Muhammad Shafiq, Muhammad Saleem Haider, M. Arshad Javed, Tayyab Husnain ...

  15. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

    Science.gov (United States)

    Guo, Fang; Zhao, Qiong; Cheng, Junjun; Qi, Yonghe; Su, Qing; Wei, Lai; Li, Wenhui; Chang, Jinhong

    2017-01-01

    Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B. PMID:28945802

  16. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.

    Science.gov (United States)

    Guo, Fang; Zhao, Qiong; Sheraz, Muhammad; Cheng, Junjun; Qi, Yonghe; Su, Qing; Cuconati, Andrea; Wei, Lai; Du, Yanming; Li, Wenhui; Chang, Jinhong; Guo, Ju-Tao

    2017-09-01

    Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.

  17. HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways.

    Directory of Open Access Journals (Sweden)

    Fang Guo

    2017-09-01

    Full Text Available Hepatitis B virus (HBV core protein assembles viral pre-genomic (pg RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs and sulfamoylbenzamides (SBAs, have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or "empty" capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.

  18. Radioimmunoassay and characterization of woodchuck hepatitis virus core antigen and antibody

    Energy Technology Data Exchange (ETDEWEB)

    Ponzetto, A; Cote, P J; Ford, E C; Engle, R; Gerin, J L; Cicmanec, J; Shapiro, M; Purcell, R H

    1985-06-01

    Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy and light cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection.

  19. DNA Three Way Junction Core Decorated with Amino Acids-Like Residues-Synthesis and Characterization

    Directory of Open Access Journals (Sweden)

    Claudia Addamiano

    2016-08-01

    Full Text Available Construction and physico-chemical behavior of DNA three way junction (3WJ functionalized by protein-like residues (imidazole, alcohol and carboxylic acid at unpaired positions at the core is described. One 5′-C(S-propargyl-thymidine nucleotide was specifically incorporated on each strand to react through a post synthetic CuACC reaction with either protected imidazolyl-, hydroxyl- or carboxyl-azide. Structural impacts of 5′-C(S-functionalization were investigated to evaluate how 3WJ flexibility/stability is affected.

  20. Identification and characterization of the pseudorabies virus UL43 protein

    International Nuclear Information System (INIS)

    Klupp, Barbara G.; Altenschmidt, Jan; Granzow, Harald; Fuchs, Walter; Mettenleiter, Thomas C.

    2005-01-01

    Among the least characterized herpesvirus membrane proteins are the homologs of UL43 of herpes simplex virus 1 (HSV-1). To identify and characterize the UL43 protein of pseudorabies virus (PrV), part of the open reading frame was expressed in Escherichia coli and used for immunization of a rabbit. The antiserum recognized in Western blots a 34-kDa protein in lysates of PrV infected cells and purified virions, demonstrating that the UL43 protein is a virion component. In indirect immunofluorescence analysis, the antiserum labeled vesicular structures in PrV infected cells which also contained glycoprotein B. To functionally analyze UL43, a deletion mutant was constructed lacking amino acids 23-332 of the 373aa protein. This mutant was only slightly impaired in replication as assayed by one-step growth kinetics, measurement of plaque sizes, and electron microscopy. Interestingly, the PrV UL43 protein was able to inhibit fusion induced by PrV glycoproteins in a transient expression-fusion assay to a similar extent as gM. Double mutant viruses lacking, in addition to UL43, the multiply membrane spanning glycoproteins K or M did not show a phenotype beyond that observed in the gK and gM single deletion mutants

  1. Green synthesis and characterization of Au@Pt core-shell bimetallic nanoparticles using gallic acid

    Science.gov (United States)

    Zhang, Guojun; Zheng, Hongmei; Shen, Ming; Wang, Lei; Wang, Xiaosan

    2015-06-01

    In this study, we developed a facile and benign green synthesis approach for the successful fabrication of well-dispersed urchin-like Au@Pt core-shell nanoparticles (NPs) using gallic acid (GA) as both a reducing and protecting agent. The proposed one-step synthesis exploits the differences in the reduction potentials of AuCl4- and PtCl62-, where the AuCl4- ions are preferentially reduced to Au cores and the PtCl62- ions are then deposited continuously onto the Au core surface as a Pt shell. The as-prepared Au@Pt NPs were characterized by transmission electron microscope (TEM); high-resolution transmission electron microscope (HR-TEM); scanning electron microscope (SEM); UV-vis absorption spectra (UV-vis); X-ray diffraction (XRD); Fourier transmission infrared spectra (FT-IR). We systematically investigated the effects of some experimental parameters on the formation of the Au@Pt NPs, i.e., the reaction temperature, the molar ratios of HAuCl4/H2PtCl6, and the amount of GA. When polyvinylpyrrolidone K-30 (PVP) was used as a protecting agent, the Au@Pt core-shell NPs obtained using this green synthesis method were better dispersed and smaller in size. The as-prepared Au@Pt NPs exhibited better catalytic activity in the reaction where NaBH4 reduced p-nitrophenol to p-aminophenol. However, the results showed that the Au@Pt bimetallic NPs had a lower catalytic activity than the pure Au NPs obtained by the same method, which confirmed the formation of Au@Pt core-shell nanostructures because the active sites on the surfaces of the Au NPs were covered with a Pt shell.

  2. Synthesis and characterization of diethylenetriaminepentaacetic acid-chitosan-coated cobalt ferrite core/shell nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Runhua, Qin [Department of Physics, North University of China, Taiyuan 030051 (China); National Special Superfine Powder Engineering Research Center, Nanjing University Science and Technology, Xiaolingwei 200, Nanjing 210094 (China); Li Fengsheng, E-mail: qinrunh@126.com [National Special Superfine Powder Engineering Research Center, Nanjing University Science and Technology, Xiaolingwei 200, Nanjing 210094 (China); Wei, Jiang; Mingyue, Chen [National Special Superfine Powder Engineering Research Center, Nanjing University Science and Technology, Xiaolingwei 200, Nanjing 210094 (China)

    2010-08-01

    Special diethylenetriaminepentaacetic acid (DTPA)-chitosan-coated cobalt ferrite core/shell nanoparticles have been synthesized via a novel zero-length emulsion crosslinking process and characterized via crosslinking degree, simultaneous thermogravimetric analysis and differential scanning calorimetry, X-ray diffractometry, Fourier transform infrared spectrometer, transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and vibration sample magnetometry. The experimental results showed that the CoFe{sub 2}O{sub 4} nanoparticles were really encapsulated with a DTPA-chitosan hybrid layer and the nanocomposites were proved to be nearly superparamagnetic with saturation magnetization of 26.6 emu g{sup -1}.

  3. SISGR - Design and Characterization of Novel Photocatalysts With Core-Shell Nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Zaera, Francisco [Univ. of California, Riverside, CA (United States). Dept. of Chemistry; Bardeen, Christopher J. [Univ. of California, Riverside, CA (United States). Dept. of Chemistry; Yin, Yadong [Univ. of California, Riverside, CA (United States). Dept. of Chemistry

    2017-03-15

    The overall goal of this project has been to develop new a new and novel class of well-characterized nanostructured Metal@TiO2 core-shell and yolk-shell photocatalysts to address two fundamental issues presently limiting this field: (1) the fast recombination of electron-hole pairs once generated by light absorption, and (2) the recombination of H2 and O2 on the metal surface once produced. These model samples are also used to study the fundamentals of the photocatalytic processes.

  4. Characterization of pathogenic germline mutations in human Protein Kinases

    Directory of Open Access Journals (Sweden)

    Orengo Christine A

    2011-07-01

    Full Text Available Abstract Background Protein Kinases are a superfamily of proteins involved in crucial cellular processes such as cell cycle regulation and signal transduction. Accordingly, they play an important role in cancer biology. To contribute to the study of the relation between kinases and disease we compared pathogenic mutations to neutral mutations as an extension to our previous analysis of cancer somatic mutations. First, we analyzed native and mutant proteins in terms of amino acid composition. Secondly, mutations were characterized according to their potential structural effects and finally, we assessed the location of the different classes of polymorphisms with respect to kinase-relevant positions in terms of subfamily specificity, conservation, accessibility and functional sites. Results Pathogenic Protein Kinase mutations perturb essential aspects of protein function, including disruption of substrate binding and/or effector recognition at family-specific positions. Interestingly these mutations in Protein Kinases display a tendency to avoid structurally relevant positions, what represents a significant difference with respect to the average distribution of pathogenic mutations in other protein families. Conclusions Disease-associated mutations display sound differences with respect to neutral mutations: several amino acids are specific of each mutation type, different structural properties characterize each class and the distribution of pathogenic mutations within the consensus structure of the Protein Kinase domain is substantially different to that for non-pathogenic mutations. This preferential distribution confirms previous observations about the functional and structural distribution of the controversial cancer driver and passenger somatic mutations and their use as a proxy for the study of the involvement of somatic mutations in cancer development.

  5. Structure of the protein core of the glypican Dally-like and localization of a region important for hedgehog signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Min-Sung; Saunders, Adam M.; Hamaoka, Brent Y.; Beachy, Philip A.; Leahy, Daniel J. (Stanford-MED); (JHU)

    2011-09-20

    Glypicans are heparan sulfate proteoglycans that modulate the signaling of multiple growth factors active during animal development, and loss of glypican function is associated with widespread developmental abnormalities. Glypicans consist of a conserved, approximately 45-kDa N-terminal protein core region followed by a stalk region that is tethered to the cell membrane by a glycosyl-phosphatidylinositol anchor. The stalk regions are predicted to be random coil but contain a variable number of attachment sites for heparan sulfate chains. Both the N-terminal protein core and the heparan sulfate attachments are important for glypican function. We report here the 2.4-{angstrom} crystal structure of the N-terminal protein core region of the Drosophila glypican Dally-like (Dlp). This structure reveals an elongated, {alpha}-helical fold for glypican core regions that does not appear homologous to any known structure. The Dlp core protein is required for normal responsiveness to Hedgehog (Hh) signals, and we identify a localized region on the Dlp surface important for mediating its function in Hh signaling. Purified Dlp protein core does not, however, interact appreciably with either Hh or an Hh:Ihog complex.

  6. An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

    Directory of Open Access Journals (Sweden)

    Marcin Michalik

    Full Text Available An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.

  7. Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

    Science.gov (United States)

    Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

    2018-02-15

    Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this

  8. Food protein-based phytosterol nanoparticles: fabrication and characterization.

    Science.gov (United States)

    Cao, Wen-Jun; Ou, Shi-Yi; Lin, Wei-Feng; Tang, Chuan-He

    2016-09-14

    The development of food-grade (nano)particles as a delivery system for poorly water soluble bioactives has recently attracted increasing attention. This work is an attempt to fabricate food protein-based nanoparticles as delivery systems for improving the water dispersion and bioaccessibility of phytosterols (PS) by an emulsification-evaporation method. The fabricated PS nanoparticles were characterized in terms of particle size, encapsulation efficiency (EE%) and loading amount (LA), and ξ-potential. Among all the test proteins, including soy protein isolate (SPI), whey protein concentrate (WPC) and sodium caseinate (SC), SC was confirmed to be the most suitable protein for the PS nano-formulation. Besides the type of protein, the particle size, EE% and LA of PS in the nanoparticles varied with the applied protein concentration in the aqueous phase and organic volume fraction. The freeze-dried PS nanoparticles with SC exhibited good water re-dispersion behavior and low crystallinity of PS. The LA of PS in the nanoparticles decreased upon storage, especially at high temperatures (e.g., >25 °C). The PS in the fabricated nanoparticles exhibited much better bioaccessibility than free PS. The findings would be of relevance for the fabrication of food-grade colloidal phytosterols, with great potential to be applied in functional food formulations.

  9. Further biochemical characterization of Mycobacterium leprae laminin-binding proteins

    Directory of Open Access Journals (Sweden)

    M.A.M. Marques

    2001-04-01

    Full Text Available It has been demonstrated that the alpha2 chain of laminin-2 present on the surface of Schwann cells is involved in the process of attachment of Mycobacterium leprae to these cells. Searching for M. leprae laminin-binding molecules, in a previous study we isolated and characterized the cationic proteins histone-like protein (Hlp and ribosomal proteins S4 and S5 as potential adhesins involved in M. leprae-Schwann cell interaction. Hlp was shown to bind alpha2-laminins and to greatly enhance the attachment of mycobacteria to ST88-14 Schwann cells. In the present study, we investigated the laminin-binding capacity of the ribosomal proteins S4 and S5. The genes coding for these proteins were PCR amplified and their recombinant products were shown to bind alpha2-laminins in overlay assays. However, when tested in ELISA-based assays and in adhesion assays with ST88-14 cells, in contrast to Hlp, S4 and S5 failed to bind laminin and act as adhesins. The laminin-binding property and adhesin capacity of two basic host-derived proteins were also tested, and only histones, but not cytochrome c, were able to increase bacterial attachment to ST88-14 cells. Our data suggest that the alanine/lysine-rich sequences shared by Hlp and eukaryotic H1 histones might be involved in the binding of these cationic proteins to laminin.

  10. Silver, gold and the corresponding core shell nanoparticles: synthesis and characterization

    International Nuclear Information System (INIS)

    Douglas, Fraser; Yanez, Ramon; Ros, Josep; Marin, Sergio; Escosura-Muniz, Alfredo de la; Alegret, Salvador; Merkoci, Arben

    2008-01-01

    Simple strategies for producing silver and gold nanoparticles (AgNP and AuNP) along with the corresponding core shell nanoparticles (Au-Ag and Ag-Au) by reduction of the metal salts AgBF 4 and HAuCl 4 by NaBH 4 in water will be presented. The morphologies of the obtained nanoparticles are determined by the order of addition of reactants. The obtained NPs, with sizes in the range 3-40 nm, are characterized by transmission electronic microscopy (TEM) and UV-Vis absorption spectroscopy, so as to evaluate their qualities. Moreover, a direct electrochemical detection protocol based on a cyclic voltammetry in water solution that involves the use of glassy carbon electrode is also applied to characterize the prepared NPs. The developed NPs and the related electroanalytical method seem to be with interest for future sensing and biosensing applications including DNA sensors and immunosensors.

  11. Controllable synthesis and characterization of novel copper-carbon core-shell structured nanoparticles

    International Nuclear Information System (INIS)

    Zhai, Jing; Tao, Xia; Pu, Yuan; Zeng, Xiao-Fei; Chen, Jian-Feng

    2011-01-01

    Highlights: → We reported a facile, green and cheap hydrothermal method to obtain novel copper-carbon core-shell nanoparticles. → The as-formed particles with controllable size and morphology are antioxidant. → The particles with organic-group-loaded surfaces and protective shells are expected to be applied in fields of medicine, electronics, sensors and lubricant. -- Abstract: A facile hydrothermal method was developed for preparing copper-carbon core-shell structured particles through a reaction at 160 o C in which glucose, copper sulfate pentahydrate and cetyltrimethylammonium bromide were used as starting materials. The original copper-carbon core-shell structured particles obtained were sized of 100-250 nm. The thickness of carbonaceous shells was controlled ranging from 25 to 100 nm by adjusting the hydrothermal duration time and the concentrations of glucose in the process. Products were characterized with transmission electron microscopy, X-ray diffraction, energy dispersive spectroscopy, Fourier transform infrared spectroscopy. Since no toxic materials were involved in the preparation, particles with stable carbonaceous framework and reactive surface also showed promising applications in medicine, electronics, sensors, lubricant, etc.

  12. Controllable synthesis and characterization of novel copper-carbon core-shell structured nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Zhai, Jing [Sin-China Nano Technology Center, Key Lab for Nanomaterials, Ministry of Education, Beijing University of Chemical Technology, Beijing 100029 (China); Research Center of the Ministry of Education for High Gravity Engineering and Technology, Beijing University of Chemical Technology, No. 15 Beisanhuan Dong Lu, Beijing 100029 (China); Tao, Xia; Pu, Yuan; Zeng, Xiao-Fei [Sin-China Nano Technology Center, Key Lab for Nanomaterials, Ministry of Education, Beijing University of Chemical Technology, Beijing 100029 (China); Chen, Jian-Feng, E-mail: chenjf@mail.buct.edu.cn [Research Center of the Ministry of Education for High Gravity Engineering and Technology, Beijing University of Chemical Technology, No. 15 Beisanhuan Dong Lu, Beijing 100029 (China)

    2011-06-15

    Highlights: {yields} We reported a facile, green and cheap hydrothermal method to obtain novel copper-carbon core-shell nanoparticles. {yields} The as-formed particles with controllable size and morphology are antioxidant. {yields} The particles with organic-group-loaded surfaces and protective shells are expected to be applied in fields of medicine, electronics, sensors and lubricant. -- Abstract: A facile hydrothermal method was developed for preparing copper-carbon core-shell structured particles through a reaction at 160 {sup o}C in which glucose, copper sulfate pentahydrate and cetyltrimethylammonium bromide were used as starting materials. The original copper-carbon core-shell structured particles obtained were sized of 100-250 nm. The thickness of carbonaceous shells was controlled ranging from 25 to 100 nm by adjusting the hydrothermal duration time and the concentrations of glucose in the process. Products were characterized with transmission electron microscopy, X-ray diffraction, energy dispersive spectroscopy, Fourier transform infrared spectroscopy. Since no toxic materials were involved in the preparation, particles with stable carbonaceous framework and reactive surface also showed promising applications in medicine, electronics, sensors, lubricant, etc.

  13. Characterization of single-core magnetite nanoparticles for magnetic imaging by SQUID relaxometry

    International Nuclear Information System (INIS)

    Adolphi, Natalie L; Huber, Dale L; Monson, Todd C; Provencio, Paula P; Bryant, Howard C; Fegan, Danielle L; Tessier, Trace E; Flynn, Edward R; Lim, JitKang; Majetich, Sara A; Trujillo, Jason E; Lovato, Debbie M; Butler, Kimberly S; Larson, Richard S; Hathaway, Helen J

    2010-01-01

    Optimizing the sensitivity of SQUID (superconducting quantum interference device) relaxometry for detecting cell-targeted magnetic nanoparticles for in vivo diagnostics requires nanoparticles with a narrow particle size distribution to ensure that the Neel relaxation times fall within the measurement timescale (50 ms-2 s, in this work). To determine the optimum particle size, single-core magnetite nanoparticles (with nominal average diameters 20, 25, 30 and 35 nm) were characterized by SQUID relaxometry, transmission electron microscopy, SQUID susceptometry, dynamic light scattering and zeta potential analysis. The SQUID relaxometry signal (detected magnetic moment/kg) from both the 25 nm and 30 nm particles was an improvement over previously studied multi-core particles. However, the detected moments were an order of magnitude lower than predicted based on a simple model that takes into account the measured size distributions (but neglects dipolar interactions and polydispersity of the anisotropy energy density), indicating that improved control of several different nanoparticle properties (size, shape and coating thickness) will be required to achieve the highest detection sensitivity. Antibody conjugation and cell incubation experiments show that single-core particles enable a higher detected moment per cell, but also demonstrate the need for improved surface treatments to mitigate aggregation and improve specificity.

  14. Characterization of single-core magnetite nanoparticles for magnetic imaging by SQUID relaxometry

    Energy Technology Data Exchange (ETDEWEB)

    Adolphi, Natalie L [Department of Biochemistry and Molecular Biology, University of New Mexico, Albuquerque, NM 87131 (United States); Huber, Dale L; Monson, Todd C; Provencio, Paula P [Sandia National Laboratories, P. O. Box 5800, Albuquerque, NM 87185 (United States); Bryant, Howard C; Fegan, Danielle L; Tessier, Trace E; Flynn, Edward R [Senior Scientific, LLC, 11109 Country Club NE, Albuquerque, NM 87111 (United States); Lim, JitKang; Majetich, Sara A [Department of Physics, Carnegie Mellon University, Pittsburgh, PA 15213 (United States); Trujillo, Jason E; Lovato, Debbie M; Butler, Kimberly S; Larson, Richard S [Department of Pathology, Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM 87131 (United States); Hathaway, Helen J, E-mail: NAdolphi@salud.unm.ed [Department of Cell Biology and Physiology, University of New Mexico, Albuquerque, NM 87131 (United States)

    2010-10-07

    Optimizing the sensitivity of SQUID (superconducting quantum interference device) relaxometry for detecting cell-targeted magnetic nanoparticles for in vivo diagnostics requires nanoparticles with a narrow particle size distribution to ensure that the Neel relaxation times fall within the measurement timescale (50 ms-2 s, in this work). To determine the optimum particle size, single-core magnetite nanoparticles (with nominal average diameters 20, 25, 30 and 35 nm) were characterized by SQUID relaxometry, transmission electron microscopy, SQUID susceptometry, dynamic light scattering and zeta potential analysis. The SQUID relaxometry signal (detected magnetic moment/kg) from both the 25 nm and 30 nm particles was an improvement over previously studied multi-core particles. However, the detected moments were an order of magnitude lower than predicted based on a simple model that takes into account the measured size distributions (but neglects dipolar interactions and polydispersity of the anisotropy energy density), indicating that improved control of several different nanoparticle properties (size, shape and coating thickness) will be required to achieve the highest detection sensitivity. Antibody conjugation and cell incubation experiments show that single-core particles enable a higher detected moment per cell, but also demonstrate the need for improved surface treatments to mitigate aggregation and improve specificity.

  15. Characterization of Proteins in Filtrate from Biodegradation of Crop Residue

    Science.gov (United States)

    Horton, Wileatha; Trotman, A. A.

    1997-01-01

    Biodegradation of plant biomass is a feasible path for transformation of crop residue and recycling of nutrients for crop growth. The need to model the effects of factors associated with recycling of plant biomass resulting from hydroponic sweet potato production has led to investigation of natural soil isolates with the capacity for starch hydrolysis. This study sought to use nondenaturing gel electrophoresis to characterize the proteins present in filtered effluent from bioreactors seeded with starch hydrolyzing bacterial culture used in the biodegradation of senesced sweet potato biomass. The study determined the relative molecular weight of proteins in sampled effluent and the protein banding pattern was characterized. The protein profiles of effluent were similar for samples taken from independent runs under similar conditions of starch hydrolysis. The method can be used as a quality control tool for confirmation of starch hydrolysis of crop biomass. In addition, this method will allow monitoring for presence of contaminants within the system-protein profiles indicative of new enzymes in the bioreactors.

  16. The Synthesis and Characterization of Gold-Core/LDH-Shell Nanoparticles

    Science.gov (United States)

    Rearick, Colton

    In recent years, the field of nanomedicine has progressed at an astonishing rate, particularly with respect to applications in cancer treatment and molecular imaging. Although organic systems have been the frontrunners, inorganic systems have also begun to show promise, especially those based upon silica and magnetic nanoparticles (NPs). Many of these systems are being designed for simultaneous therapeutic and diagnostic capabilities, thus coining the term, theranostics. A unique class of inorganic systems that shows great promise as theranostics is that of layered double hydroxides (LDH). By synthesis of a core/shell structures, e.g. a gold nanoparticle (NP) core and LDH shell, the multifunctional theranostic may be developed without a drastic increase in the structural complexity. To demonstrate initial proof-of-concept of a potential (inorganic) theranostic platform, a Au-core/LDH-shell nanovector has been synthesized and characterized. The LDH shell was heterogeneously nucleated and grown on the surface of silica coated gold NPs via a coprecipitation method. Polyethylene glycol (PEG) was introduced in the initial synthesis steps to improve crystallinity and colloidal stability. Additionally, during synthesis, fluorescein isothiocyanate (FITC) was intercalated into the interlayer spacing of the LDH. In contrast to the PEG stabilization, a post synthesis citric acid treatment was used as a method to control the size and short-term stability. The heterogeneous core-shell system was characterized with scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDX), dynamic light scattering (DLS), and powder x-ray diffraction (PXRD). A preliminary in vitro study carried out with the assistance of Dr. Kaushal Rege's group at Arizona State University was to demonstrate the endocytosis capability of homogeneously-grown LDH NPs. The DLS measurements of the core-shell NPs indicated an average particle size of 212nm. The PXRD analysis showed that PEG

  17. High-energy intermediates in protein unfolding characterized by thiol labeling under nativelike conditions.

    Science.gov (United States)

    Malhotra, Pooja; Udgaonkar, Jayant B

    2014-06-10

    A protein unfolding reaction usually appears to be so dominated by a large free energy barrier that identifying and characterizing high-energy intermediates and, hence, dissecting the unfolding reaction into multiple structural transitions have proven to be a challenge. In particular, it has been difficult to identify any detected high-energy intermediate with the dry (DMG) and wet (WMG) molten globules that have been implicated in the unfolding reactions of at least some proteins. In this study, a native-state thiol labeling methodology was used to identify high-energy intermediates, as well as to delineate the barriers to the disruption of side chain packing interactions and to site-specific solvent exposure in different regions of the small protein, single-chain monellin (MNEI). Labeling studies of four single-cysteine-containing variants of MNEI have identified three high-energy intermediates, populated to very low extents under nativelike conditions. A significant dispersion in the opening rates of the cysteine side chains has allowed multiple steps, leading to the loss of side chain packing, to be resolved temporally. A detailed structural analysis of the positions of the four cysteine residue positions, which are buried to different depths within the protein, has suggested a direct correlation with the structure of a DMG, detected in previous studies. It is observed that side chain packing within the core of the protein is maintained, while that at the surface is disrupted, in the DMG. The core of the protein becomes solvent-exposed only in a WMG populated after the rate-limiting step of unfolding at high denaturant concentrations.

  18. Geotechnical core and rock mass characterization for the UK radioactive waste repository design

    International Nuclear Information System (INIS)

    Rawlings, C.G.; Barton, N.; Loset, F.; Vik, G.; Bhasin, R.K.; Smallwood, A.; Davies, N.

    1996-01-01

    The NGI method of characterizing joints (using JRC, JCS and φ r ) and characterizing rock masses (using the Q-system) have been and are currently being used extensively in geotechnical consultancy projects. One such project recently completed for UK Nirex Ltd included the logging of 8 km of 100-mm-diameter drill core from boreholes up to 2km in depth. Preliminary rock reinforcement designs were derived from the Q-system statistics, which were logged in parallel with JRC, JCS and φ r . The data from the NGI method of characterizing joints and the Q-system for characterizing rock masses have also been used as the basis for UDEC-BB numerical modelling of the proposed cavern excavations for the disposal of solid, low- and intermediate-level radioactive wastes. The purpose of this numerical modelling was to investigate the stability of rock caverns and in particular the rock reinforcement requirements (giving predicted bolt loads and rock deformations), the extent of the disturbed zone (joint shearing and hydraulic aperture) with respect to cavern orientation, the effect of various pillar widths, and the effect of the cavern excavation sequence. (Author)

  19. Fabrication and characterization of optical sensors using metallic core-shell thin film nanoislands for ozone detection

    Science.gov (United States)

    Addanki, Satish; Nedumaran, D.

    2017-07-01

    Core-Shell nanostructures play a vital role in the sensor field owing to their performance improvements in sensing characteristics and well-established synthesis procedures. These nanostructures can be ingeniously tuned to achieve tailored properties for a particular application of interest. In this work, an Ag-Au core-shell thin film nanoislands with APTMS (3-Aminopropyl trimethoxysilane) and PVA (Polyvinyl alcohol) binding agents was modeled, synthesized and characterized. The simulation results were used to fabricate the sensor through chemical route. The results of this study confirmed that the APTMS based Ag-Au core-shell thin film nanoislands offered a better performance over the PVA based Ag-Au core-shell thin film nanoislands. Also, the APTMS based Ag-Au core-shell thin film nanoislands exhibited better sensitivity towards ozone sensing over the other types, viz., APTMS/PVA based Au-Ag core-shell and standalone Au/Ag thin film nanoislands.

  20. Characterization of pea (Pisum sativum) seed protein fractions.

    Science.gov (United States)

    Rubio, Luis A; Pérez, Alicia; Ruiz, Raquel; Guzmán, M Ángeles; Aranda-Olmedo, Isabel; Clemente, Alfonso

    2014-01-30

    Legume seed proteins have to be chemically characterized in order to properly link their nutritional effects with their chemical structure. Vicilin and albumin fractions devoid of cross-contamination, as assessed by mass peptide fingerprinting analysis, were obtained from defatted pea (Pisum sativum cv. Bilbo) meal. The extracted protein fractions contained 56.7-67.7 g non-starch polysaccharides kg⁻¹. The vicilin fraction was higher than legumins in arginine, isoleucine, leucine, phenylalanine and lysine. The most abundant amino acids in the albumin fraction were aspartic acid, glutamic acid, lysine and arginine, and the amounts of methionine were more than double than those in legumins and vicilins. The pea albumin fraction showed a clear enrichment of protease inhibitory activity when compared with the seed meal. In vitro digestibility values for pea proteins were 0.63 ±  0.04, 0.88 ±  0.04 and 0.41 ±  0.23 for legumins, vicilins and albumins respectively. Vicilin and albumin fractions devoid of cross-contamination with other proteins were obtained from pea seed meal. The vicilin fraction also contained low amounts of soluble non-starch polysaccharides and was enriched in isoleucine, leucine, phenylalanine and lysine. In vitro digestibility values for pea proteins were similar or even numerically higher than those for control proteins. © 2013 Society of Chemical Industry.

  1. Characterization of seed storage protein patterns of Heliotropium digynum

    Directory of Open Access Journals (Sweden)

    Mona Soliman Alwhibi

    2017-09-01

    Full Text Available Heliotropium digynum, is a shrub that has ecological importance. The height of the plant differs from one population to another and the difference in length of the inflorescence can be attributed to environmental factors, such as rainfall or type of soil and temperature. To date, no study has shed light on estimation in seed samples of H. digynum in Saudi Arabia. So, the aim is to evaluate and characterize the protein patterns of seed storage proteins of H. digynum to be used as fingerprint of this plant in Saudi Arabia. It is collected from different locations in the central region of Saudi Arabia and total protein extraction from plant was compared in SDS-PAGE. The genetic relationships among all cultivars were analyzed using UPGMA and NJ using Total Lab TL and in the same way using Jaccard Similarity Coefficient dendrogram using STATISTICA (ver.8 software. Results, our data show that amounts of protein are different, although they are of the same type or from the same geographical region. Amounts ranged between 22 and 1.5 mg/g of dry weight. Less amount of protein was obtained from the group of samples collected from Dir’iyah area, and the highest amount of protein was from the group of samples collected from Dyrab area in general.

  2. Characterization of seed storage protein patterns of Heliotropium digynum.

    Science.gov (United States)

    Alwhibi, Mona Soliman

    2017-09-01

    Heliotropium digynum , is a shrub that has ecological importance. The height of the plant differs from one population to another and the difference in length of the inflorescence can be attributed to environmental factors, such as rainfall or type of soil and temperature. To date, no study has shed light on estimation in seed samples of H. digynum in Saudi Arabia. So, the aim is to evaluate and characterize the protein patterns of seed storage proteins of H. digynum to be used as fingerprint of this plant in Saudi Arabia. It is collected from different locations in the central region of Saudi Arabia and total protein extraction from plant was compared in SDS-PAGE. The genetic relationships among all cultivars were analyzed using UPGMA and NJ using Total Lab TL and in the same way using Jaccard Similarity Coefficient dendrogram using STATISTICA (ver.8) software. Results, our data show that amounts of protein are different, although they are of the same type or from the same geographical region. Amounts ranged between 22 and 1.5 mg/g of dry weight. Less amount of protein was obtained from the group of samples collected from Dir'iyah area, and the highest amount of protein was from the group of samples collected from Dyrab area in general.

  3. Identification and characterization of Euphorbia nivulia latex proteins.

    Science.gov (United States)

    Badgujar, Shamkant B; Mahajan, Raghunath T

    2014-03-01

    The protein profile of latex of Euphorbia nivulia Buch.-Ham. is established. Three new proteins viz., Nivulian-I, II and III have been purified to homogeneity from the latex. The relative molecular masses of Nivulian-I, II and III are 31,486.985, 43,670.846 and 52,803.470 Da respectively. Nivulian-I is a simple type of protein while Nivulian-II and III are glycoproteins. Peptide mass fingerprint analysis revealed peptides of these proteins match with Tubulin alpha-1 chain of Eleusine indica, Maturase K of Banksia quercifolia and hypothetical protein of Zea mays respectively. Tryptic digestion profile of Nivulian-I, II and III, infer the exclusive nature of latex origin proteins and may be new and are additive molecules in the dictionaries of phytoproteins or botany. This is the first of its kind, regarding characterization and validation of Nivulian-I, II and III with respect to peptide sequencing. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Purification and characterization of Escherichia coli MreB protein.

    Science.gov (United States)

    Nurse, Pearl; Marians, Kenneth J

    2013-02-01

    The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μM.

  5. Purification and Characterization of Escherichia coli MreB Protein*

    Science.gov (United States)

    Nurse, Pearl; Marians, Kenneth J.

    2013-01-01

    The actin homolog MreB is required in rod-shaped bacteria for maintenance of cell shape and is intimately connected to the holoenzyme that synthesizes the peptidoglycan layer. The protein has been reported variously to exist in helical loops under the cell surface, to rotate, and to move in patches in both directions around the cell surface. Studies of the Escherichia coli protein in vitro have been hampered by its tendency to aggregate. Here we report the purification and characterization of native E. coli MreB. The protein requires ATP hydrolysis for polymerization, forms bundles with a left-hand twist that can be as long as 4 μm, forms sheets in the presence of calcium, and has a critical concentration for polymerization of 1.5 μm. PMID:23235161

  6. Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algae-Specific Variants and Post-Translational Modifications.

    Science.gov (United States)

    Khan, Aliyya; Eikani, Carlo K; Khan, Hana; Iavarone, Anthony T; Pesavento, James J

    2018-01-05

    The unicellular microalga Chlamydomonas reinhardtii has played an instrumental role in the development of many new fields (bioproducts, biofuels, etc.) as well as the advancement of basic science (photosynthetic apparati, flagellar function, etc.). Chlamydomonas' versatility ultimately derives from the genes encoded in its genome and the way that the expression of these genes is regulated, which is largely influenced by a family of DNA binding proteins called histones. We characterize C. reinhardtii core histones, both variants and their post-translational modifications, by chromatographic separation, followed by top-down mass spectrometry (TDMS). Because TDMS has not been previously used to study Chlamydomonas proteins, we show rampant artifactual protein oxidation using established nuclei purification and histone extraction methods. After addressing oxidation, both histones H3 and H4 are found to each have a single polypeptide sequence that is minimally acetylated and methylated. Surprisingly, we uncover a novel monomethylation at lysine 79 on histone H4 present on all observed molecules. Histone H2B and H2A are found to have two and three variants, respectively, and both are minimally modified. This study provides an updated assessment of the core histone proteins in the green alga C. reinhardtii by top-down mass spectrometry and lays the foundation for further investigation of these essential proteins.

  7. Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.

    Science.gov (United States)

    Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael; Zlotnick, Adam

    2018-01-29

    Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. © 2017, Schlicksup et al.

  8. Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

    Science.gov (United States)

    Schlicksup, Christopher John; Wang, Joseph Che-Yen; Francis, Samson; Venkatakrishnan, Balasubramanian; Turner, William W; VanNieuwenhze, Michael

    2018-01-01

    Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. PMID:29377794

  9. The H1 linker histones: multifunctional proteins beyond the nucleosomal core particle.

    Science.gov (United States)

    Hergeth, Sonja P; Schneider, Robert

    2015-11-01

    The linker histone H1 family members are a key component of chromatin and bind to the nucleosomal core particle around the DNA entry and exit sites. H1 can stabilize both nucleosome structure and higher-order chromatin architecture. In general, H1 molecules consist of a central globular domain with more flexible tail regions at both their N- and C-terminal ends. The existence of multiple H1 subtypes and a large variety of posttranslational modifications brings about a considerable degree of complexity and makes studying this protein family challenging. Here, we review recent progress in understanding the function of linker histones and their subtypes beyond their role as merely structural chromatin components. We summarize current findings on the role of H1 in heterochromatin formation, transcriptional regulation and embryogenesis with a focus on H1 subtypes and their specific modifications. © 2015 The Authors.

  10. TRF2 Protein Interacts with Core Histones to Stabilize Chromosome Ends.

    Science.gov (United States)

    Konishi, Akimitsu; Izumi, Takashi; Shimizu, Shigeomi

    2016-09-23

    Mammalian chromosome ends are protected by a specialized nucleoprotein complex called telomeres. Both shelterin, a telomere-specific multi-protein complex, and higher order telomeric chromatin structures combine to stabilize the chromosome ends. Here, we showed that TRF2, a component of shelterin, binds to core histones to protect chromosome ends from inappropriate DNA damage response and loss of telomeric DNA. The N-terminal Gly/Arg-rich domain (GAR domain) of TRF2 directly binds to the globular domain of core histones. The conserved arginine residues in the GAR domain of TRF2 are required for this interaction. A TRF2 mutant with these arginine residues substituted by alanine lost the ability to protect telomeres and induced rapid telomere shortening caused by the cleavage of a loop structure of the telomeric chromatin. These findings showed a previously unnoticed interaction between the shelterin complex and nucleosomal histones to stabilize the chromosome ends. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Neutron Environment Characterization of the Central Cavity in the Annular Core Research Reactor *

    Directory of Open Access Journals (Sweden)

    Parma Edward J.

    2016-01-01

    Full Text Available Characterization of the neutron environment in the central cavity of the Sandia National Laboratories' Annular Core Research Reactor (ACRR is important in order to provide experimenters with the most accurate spectral information and maintain a high degree of fidelity in performing reactor experiments. Characterization includes both modeling and experimental efforts. Building accurate neutronic models of the ACRR and the central cavity “bucket” environments that can be used by experimenters is important in planning and designing experiments, as well as assessing the experimental results and quantifying uncertainties. Neutron fluence characterizations of two bucket environments, LB44 and PLG, are presented. These two environments are used frequently and represent two extremes in the neutron spectrum. The LB44 bucket is designed to remove the thermal component of the neutron spectrum and significantly attenuate the gamma-ray fluence. The PLG bucket is designed to enhance the thermal component of the neutron spectrum and attenuate the gamma-ray fluence. The neutron characterization for each bucket was performed by irradiating 20 different activation foil types, some of which were cadmium covered, resulting in 37 different reactions at the peak axial flux location in each bucket. The dosimetry results were used in the LSL-M2 spectrum adjustment code with a 640-energy group MCNP-generated trial spectrum, self-shielding correction factors, the SNLRML or IRDFF dosimetry cross-section library, trial spectrum uncertainty, and trial covariance matrix, to generate a least-squares adjusted neutron spectrum, spectrum uncertainty, and covariance matrix. Both environment character-izations are well documented and the environments are available for use by experimenters.

  12. Controllable fabrication and characterization of biocompatible core-shell particles and hollow capsules as drug carrier

    Science.gov (United States)

    Hao, Lingyun; Gong, Xinglong; Xuan, Shouhu; Zhang, Hong; Gong, Xiuqing; Jiang, Wanquan; Chen, Zuyao

    2006-10-01

    SiO 2@CdSe core-shell particles were fabricated by controllable deposition CdSe nanoparticles on silica colloidal spheres. Step-wise coating process was tracked by the TEM and XRD measurements. In addition, SiO 2@CdSe/polypyrrole(PPy) multi-composite particles were synthesized based on the as-prepared SiO 2@CdSe particles by cationic polymerization. The direct electrochemistry of myoglobin (Mb) could be performed by immobilizing Mb on the surface of SiO 2@CdSe particles. Immobilized with Mb, SiO 2@CdSe/PPy-Mb also displayed good bioelectrochemical activity. It confirmed the good biocompatible property of the materials with protein. CdSe hollow capsules were further obtained as the removal of the cores of SiO 2@CdSe spheres. Hollow and porous character of CdSe sub-meter size capsules made them becoming hopeful candidates as drug carriers. Doxorubicin, a typical an antineoplastic drug, was introduced into the capsules. A good sustained drug release behavior of the loading capsules was discovered via performing a release test in the PBS buffer (pH 7.4) solution at 310 k. Furthermore, SiO 2@CdSe/PPy could be converted to various smart hollow capsules via selectively removal of their relevant components.

  13. Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein.

    Science.gov (United States)

    Lingappa, V R; Lingappa, J R; Prasad, R; Ebner, K E; Blobel, G

    1978-05-01

    mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized alpha-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat alpha-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of alpha-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communis lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature alpha-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by alpha-methyl mannoside, but not to R. communis lectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.

  14. Characterization of host proteins interacting with the lymphocytic choriomeningitis virus L protein.

    Science.gov (United States)

    Khamina, Kseniya; Lercher, Alexander; Caldera, Michael; Schliehe, Christopher; Vilagos, Bojan; Sahin, Mehmet; Kosack, Lindsay; Bhattacharya, Anannya; Májek, Peter; Stukalov, Alexey; Sacco, Roberto; James, Leo C; Pinschewer, Daniel D; Bennett, Keiryn L; Menche, Jörg; Bergthaler, Andreas

    2017-12-01

    RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.

  15. Protein corona between nanoparticles and bacterial proteins in activated sludge: Characterization and effect on nanoparticle aggregation.

    Science.gov (United States)

    Zhang, Peng; Xu, Xiao-Yan; Chen, You-Peng; Xiao, Meng-Qian; Feng, Bo; Tian, Kai-Xun; Chen, Yue-Hui; Dai, You-Zhi

    2018-02-01

    In this work, the protein coronas of activated sludge proteins on TiO 2 nanoparticles (TNPs) and ZnO nanoparticles (ZNPs) were characterized. The proteins with high affinity to TNPs and ZNPs were identified by shotgun proteomics, and their effects of on the distributions of TNPs and ZNPs in activated sludge were concluded. In addition, the effects of protein coronas on the aggregations of TNPs and ZNPs were evaluated. Thirty and nine proteins with high affinities to TNPs and ZNPs were identified, respectively. The proteomics and adsorption isotherms demonstrated that activated sludge had a higher affinity to TNPs than to ZNPs. The aggregation percentages of ZNPs at 35, 53, and 106 mg/L of proteins were 13%, 14%, and 18%, respectively, whereas those of TNPs were 21%, 30%, 41%, respectively. The proteins contributed to ZNPs aggregation by dissolved Zn ion-bridging, whereas the increasing protein concentrations enhanced the TNPs aggregation through macromolecule bridging flocculation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. A Case Study of German Language Core Journals for Characterizing Citation Patterns in the Social Sciences

    Directory of Open Access Journals (Sweden)

    Pei-Shan Chi

    2013-12-01

    Full Text Available Publication practices in the social sciences are characterized by the use of heterogeneous publication channels and a stronger national focus (Nederhof, 2006; Hicks & Wang, 2011. At the same time the use of bibliometric indicators in research evaluation promotes journal articles in international peer reviewed journals as the main style of publishing research results. The question emerges to which extent this changes publication practices in these disciplines. In our contribution we address this question and present results of a case study which investigates publication and referencing patterns of core German language journals in sociology and political science. Based on an explorative analysis of reference lists we describe patterns and changes of the parameters of the knowledge base of these journals. The analysis of the results in this study shows that with a total of 67% in the sociology and 76% in the political science the core German journals predominantly refer to non-journal publications. Besides, the share of non-source publications basically remains constant in the time period 2000-2009, and the share of references to source journals is the same in both disciplines. The difference between sociology and political science is: publications in the German language sociology journals have more references to monographs (46% than publications in the German language political science journals (38%, but these political science journals reference to other non-source publications (38% much more than sociology (21%.

  17. Ni3Si(Al)/a-SiOx core shell nanoparticles: characterization, shell formation, and stability

    Science.gov (United States)

    Pigozzi, G.; Mukherji, D.; Gilles, R.; Barbier, B.; Kostorz, G.

    2006-08-01

    We have used an electrochemical selective phase dissolution method to extract nanoprecipitates of the Ni3Si-type intermetallic phase from two-phase Ni-Si and Ni-Si-Al alloys by dissolving the matrix phase. The extracted nanoparticles are characterized by transmission electron microscopy, energy-dispersive x-ray spectrometry, x-ray powder diffraction, and electron powder diffraction. It is found that the Ni3Si-type nanoparticles have a core-shell structure. The core maintains the size, the shape, and the crystal structure of the precipitates that existed in the bulk alloys, while the shell is an amorphous phase, containing only Si and O (SiOx). The shell forms around the precipitates during the extraction process. After annealing the nanoparticles in nitrogen at 700 °C, the tridymite phase recrystallizes within the shell, which remains partially amorphous. In contrast, on annealing in air at 1000 °C, no changes in the composition or the structure of the nanoparticles occur. It is suggested that the shell forms after dealloying of the matrix phase, where Si atoms, the main constituents of the shell, migrate to the surface of the precipitates.

  18. Identification and characterization of the genes encoding the core histones and histone variants of Neurospora crassa.

    OpenAIRE

    Hays, Shan M; Swanson, Johanna; Selker, Eric U

    2002-01-01

    We have identified and characterized the complete complement of genes encoding the core histones of Neurospora crassa. In addition to the previously identified pair of genes that encode histones H3 and H4 (hH3 and hH4-1), we identified a second histone H4 gene (hH4-2), a divergently transcribed pair of genes that encode H2A and H2B (hH2A and hH2B), a homolog of the F/Z family of H2A variants (hH2Az), a homolog of the H3 variant CSE4 from Saccharomyces cerevisiae (hH3v), and a highly diverged ...

  19. Synthesis, characterization, and photocatalytic properties of core/shell mesoporous silica nanospheres supporting nanocrystalline titania

    International Nuclear Information System (INIS)

    Cendrowski, K.; Chen, X.; Zielinska, B.; Kalenczuk, R. J.; Rümmeli, M. H.; Büchner, B.; Klingeler, R.; Borowiak-Palen, E.

    2011-01-01

    The facile bulk synthesis of silica nanospheres makes them an attractive support for the transport of chemical compounds such as nanocrystalline titanium dioxide. In this contribution we present a promising route for the synthesis of mesoporous silica nanospheres (m-SiO 2 ) with diameter in range 200 nm, which are ideal supports for nanocrystalline titanium dioxide (TiO 2 ). The detailed microscopic and spectroscopic characterizations of core/shell structure (m-SiO 2 /TiO 2 ) were conducted. Moreover, the photocatalytic potential of the nanostructures was investigated via phenol decomposition and hydrogen generation. A clear enhancement of photoactivity in both reactions as compared to commercial TiO 2 -Degussa P25 catalyst is detected.

  20. Synthesis, characterization, and photocatalytic properties of core/shell mesoporous silica nanospheres supporting nanocrystalline titania

    Science.gov (United States)

    Cendrowski, K.; Chen, X.; Zielinska, B.; Kalenczuk, R. J.; Rümmeli, M. H.; Büchner, B.; Klingeler, R.; Borowiak-Palen, E.

    2011-11-01

    The facile bulk synthesis of silica nanospheres makes them an attractive support for the transport of chemical compounds such as nanocrystalline titanium dioxide. In this contribution we present a promising route for the synthesis of mesoporous silica nanospheres (m-SiO2) with diameter in range 200 nm, which are ideal supports for nanocrystalline titanium dioxide (TiO2). The detailed microscopic and spectroscopic characterizations of core/shell structure (m-SiO2/TiO2) were conducted. Moreover, the photocatalytic potential of the nanostructures was investigated via phenol decomposition and hydrogen generation. A clear enhancement of photoactivity in both reactions as compared to commercial TiO2-Degussa P25 catalyst is detected.

  1. Characterization of the Annular Core Research Reactor (ACRR Neutron Radiography System Imaging Plane

    Directory of Open Access Journals (Sweden)

    Kaiser Krista

    2016-01-01

    Full Text Available The Annular Core Research Reactor (ACRR at Sandia National Laboratories (SNL is an epithermal pool-type research reactor licensed up to a thermal power of 2.4 MW. The ACRR facility has a neutron radiography facility that is used for imaging a wide range of items including reactor fuel and neutron generators. The ACRR neutron radiography system has four apertures (65:1, 125:1, 250:1, and 500:1 available to experimenters. The neutron flux and spectrum as well as the gamma dose rate were characterized at the imaging plane for the ACRR's neutron radiography system for the 65:1, 125:1 and 250:1 apertures.

  2. Bayesian methods for the combination of core sampling data with historical models for tank characterization

    International Nuclear Information System (INIS)

    York, J.C.; Remund, K.M.; Chen, G.; Simpson, B.C.; Brown, T.M.

    1995-07-01

    A wide variety of information is available on the contents of the nuclear waste tanks at the Hanford site. This report describes an attempt to combine several sources of information using a Bayesian statistical approach. This methodology allows the combination of multiple disparate information sources. After each source of information is summarized in terms of a probability distribution function (pdf), Bayes' theorem is applied to combine them. This approach has been applied to characterizing tanks B-110, B-111, and B-201. These tanks were chosen for their simple waste matrices: B-110 and B-111 contain mostly 2C waste, and B-201 contains mostly 224 waste. Additionally,, the results of this analysis axe used to make predictions for tank T-111 (which contains both 2C and 224 waste). These predictions are compared to the estimates based on core samples from tank T-111

  3. "Hot cores" in proteins: Comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms

    Directory of Open Access Journals (Sweden)

    Bossa Francesco

    2008-02-01

    Full Text Available Abstract Background A wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. These include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. It has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. In this work, protein crystal structures from hyper/thermophilic organisms and their mesophilic homologs have been compared, in order to quantify the difference of apolar contact area and to assess the role played by the hydrophobic contacts in the stabilization of the protein core, at high temperatures. Results The construction of two datasets was carried out so as to satisfy several restrictive criteria, such as minimum redundancy, resolution and R-value thresholds and lack of any structural defect in the collected structures. This approach allowed to quantify with relatively high precision the apolar contact area between interacting residues, reducing the uncertainty due to the position of atoms in the crystal structures, the redundancy of data and the size of the dataset. To identify the common core regions of these proteins, the study was focused on segments that conserve a similar main chain conformation in the structures analyzed, excluding the intervening regions whose structure differs markedly. The results indicated that hyperthermophilic proteins underwent a significant increase of the hydrophobic contact area contributed by those residues composing the alpha-helices of the structurally conserved regions. Conclusion This study indicates the decreased flexibility of alpha-helices in proteins core as a major factor contributing to the enhanced termostability of a number of hyperthermophilic proteins. This effect, in turn, may be due to an increased number of buried methyl groups in

  4. Mechanisms and Effects on HBV Replication of the Interaction between HBV Core Protein and Cellular Filamin B.

    Science.gov (United States)

    Li, Yilin; Sun, Yishuang; Sun, Fuyun; Hua, Rong; Li, Chenlin; Chen, Lang; Guo, Deyin; Mu, Jingfang

    2018-03-28

    Hepatitis B virus (HBV) infection is one of the major problems that threatens global health. There have been many studies on HBV, but the relationship between HBV and host factors is largely unexplored and more studies are needed to clarify these interactions. Filamin B is an actin-binding protein that acts as a cytoskeleton protein, and it is involved in cell development and several signaling pathways. In this study, we showed that filamin B interacted with HBV core protein, and the interaction promoted HBV replication. The interaction between filamin B and core protein was observed in HEK 293T, Huh7 and HepG2 cell lines by co-immunoprecipitation and co-localization immnofluoresence. Overexpression of filamin B increased the levels of HBV total RNAs and pre-genome RNA (pgRNA), and improved the secretion level of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). In contrast, filamin B knockdown inhibited HBV replication, decreased the level of HBV total RNAs and pgRNA, and reduced the secretion level of HBsAg and HBeAg. In addition, we found that filamin B and core protein may interact with each other via four blocks of argentine residues at the C-terminus of core protein. In conclusion, we identify filamin B as a novel host factor that can interact with core protein to promote HBV replication in hepatocytes. Our study provides new insights into the relationship between HBV and host factors and may provide new strategies for the treatment of HBV infection.

  5. Cloning and sequencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer's disease: Identification as the microtubule-associated protein tau

    International Nuclear Information System (INIS)

    Goedert, M.; Wischik, C.M.; Crowther, R.A.; Walker, J.E.; Klug, A.

    1988-01-01

    Screening of cDNA libraries prepared from the frontal cortex of an Alzheimer's disease patient and from fetal human brain has led to isolation of the cDNA for a core protein of the paired helical filament of Alzheimer's disease. The partial amino acid sequence of this core protein was used to design synthetic oligonucleotide probes. The cDNA encodes a protein of 352 amino acids that contains a characteristic amino acid repeat in its carboxyl-terminal half. This protein is highly homologous to the sequence of the mouse microtubule-associated protein tau and thus constitutes the human equivalent of mouse tau. RNA blot analysis indicates the presence of two major transcripts, 6 and 2 kilobases long, with a wide distribution in normal human brain. Tau protein mRNAs were found in normal amounts in the frontal cortex from patients with Alzheimer's disease. The proof that at least part of tau protein forms a component of the paired helical filament core opens the way to understanding the mode of formation of paired helical filaments and thus, ultimately, the pathogenesis of Alzheimer's disease

  6. Characterization of an M-Cluster-Substituted Nitrogenase VFe Protein.

    Science.gov (United States)

    Rebelein, Johannes G; Lee, Chi Chung; Newcomb, Megan; Hu, Yilin; Ribbe, Markus W

    2018-03-13

    The Mo- and V-nitrogenases are two homologous members of the nitrogenase family that are distinguished mainly by the presence of different heterometals (Mo or V) at their respective cofactor sites (M- or V-cluster). However, the V-nitrogenase is ~600-fold more active than its Mo counterpart in reducing CO to hydrocarbons at ambient conditions. Here, we expressed an M-cluster-containing, hybrid V-nitrogenase in Azotobacter vinelandii and compared it to its native, V-cluster-containing counterpart in order to assess the impact of protein scaffold and cofactor species on the differential reactivities of Mo- and V-nitrogenases toward CO. Housed in the VFe protein component of V-nitrogenase, the M-cluster displayed electron paramagnetic resonance (EPR) features similar to those of the V-cluster and demonstrated an ~100-fold increase in hydrocarbon formation activity from CO reduction, suggesting a significant impact of protein environment on the overall CO-reducing activity of nitrogenase. On the other hand, the M-cluster was still ~6-fold less active than the V-cluster in the same protein scaffold, and it retained its inability to form detectable amounts of methane from CO reduction, illustrating a fine-tuning effect of the cofactor properties on this nitrogenase-catalyzed reaction. Together, these results provided important insights into the two major determinants for the enzymatic activity of CO reduction while establishing a useful framework for further elucidation of the essential catalytic elements for the CO reactivity of nitrogenase. IMPORTANCE This is the first report on the in vivo generation and in vitro characterization of an M-cluster-containing V-nitrogenase hybrid. The "normalization" of the protein scaffold to that of the V-nitrogenase permits a direct comparison between the cofactor species of the Mo- and V-nitrogenases (M- and V-clusters) in CO reduction, whereas the discrepancy between the protein scaffolds of the Mo- and V-nitrogenases (MoFe and VFe

  7. Purification and characterization of a thylakoid protein kinase

    International Nuclear Information System (INIS)

    Coughlan, S.J.; Hind, G.

    1986-01-01

    Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs) and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs

  8. Single Shell Tank Waste Characterization Project for Tank B-110, Core 9 - data package and PNL validation summary report

    International Nuclear Information System (INIS)

    Pool, K.N.; Jones, T.E.; McKinley, S.G.; Tingey, J.M.; Longaker, T.M.; Gibson, J.A.

    1990-01-01

    This Data Package contains results obtained by Pacific Northwest Laboratory (PNL) staff in the characterization and analyses of Core 9 segments taken from the Single-Shell Tank (SST) 110B. The characterization and analysis of Core 9 segments are outlined in the Waste Characterization Plan for Hanford Site Single-Shell Tanks and in the Pacific Northwest Laboratory (PNL) Single-Shell Tank Waste Characterization Support FY 89/90 Statement of Work (SOW), Rev. 1 dated March, 1990. Specific analyses for each sub-sample taken from a segment are delineated in Test Instructions prepared by the PNL Single-Shell Tank Waste Characterization Project Management Office (SST Project) in accordance with procedures contained in the SST Waste Characterization Procedure Compendium (PNL-MA-599). Analytical procedures used in the characterization activities are also included in PNL-MA-599. Core 9 included five segments although segment 1 did not have sufficient material for characterization. The five samplers were received from Westinghouse Hanford Company (WHC) on 11/21-22/89. Each segment was contained in a sampler and was enclosed in a shipping cask. The shipping cask was butted up to the 325-A hot cell and the sampler moved into the hot cell. The material in the sampler (i.e., the segment) was extruded from the sampler, limited physical characteristics assessed, and photographed. At this point samples were taken for particle size and volatile organic analyses. Each segment was then homogenized. Sub-samples were taken for required analyses as delineated in the appropriate Test Instruction. Table 1 includes sample numbers assigned to Core 9 segment materials being transferred from 325-A Hot Cell. Sample numbers 90-0298, 90-0299, 90-0302, and 90-0303 were included in Table 1 although no analyses were requested for these samples. Table 2 lists Core 9 sub-sample numbers per sample preparation method

  9. Identification and characterization of immunogenic proteins of Mycoplasma genitalium

    DEFF Research Database (Denmark)

    Svenstrup, Helle Friis; Jensen, J.S.; Gevaert, K.

    2006-01-01

    serum against M. genitalium G37, determine their identity by mass spectrometry, and develop an M. genitalium-specific enzyme-linked immunosorbent assay (ELISA) free from cross-reactivity with M. pneumoniae antibodies. Using recombinant fragments of the C-terminal part of MgPa (rMgPa), we developed....... genitalium strains were isolated (J. S. Jensen, H. T. Hansen, and K. Lind, J. Clin. Microbiol. 34:286-291, 1996). The objective of this study was to characterize immunogenic proteins of M. genitalium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting by using a hyperimmune rabbit...

  10. Characterization of tumour virus proteins. I. radioimmunoassay of the P27 protein of avian viruses

    International Nuclear Information System (INIS)

    Higuchi, T.

    1977-01-01

    The major structural protein of avian oncornaviruses, a core component of about 27000 daltons, has been measured by radioimmunoassay. The purified protein was labelled with 125 Iodine by chloramine-T method. The immune serum titer was defined as the highest serum dilution able to precipitate 50% of the labelled antigon present in the system. Standard competition curve was constructed in order to determine the equivalents of protein, in a system with limiting antibody concentration. In the experimental conditions used, 0.14 ng of AMV-P27 inhibited 50% of 125 I-AMV-P27 (1.0 ng) precipitation. The 125 I-AMV-P27 vs anti-AMV-P27 system was used to study the competition of normal cells, purified virus suspension, productive cells and supernatant fluids. Most of the chicken ombryo fibroblast showed expression of this viral component. The phenomena of cell transformation, the increase in total protein, and the expression of P27 were studied in rapid transformation of CEF by RSV-SR sub(A) [pt

  11. CD4+ Primary T Cells Expressing HCV-Core Protein Upregulate Foxp3 and IL-10, Suppressing CD4 and CD8 T Cells

    Science.gov (United States)

    Aguado, Enrique; Garcia-Cozar, Francisco

    2014-01-01

    Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127lowPD-1highTIM-3high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein. PMID:24465502

  12. CD4+ primary T cells expressing HCV-core protein upregulate Foxp3 and IL-10, suppressing CD4 and CD8 T cells.

    Directory of Open Access Journals (Sweden)

    Cecilia Fernandez-Ponce

    Full Text Available Adaptive T cell responses are critical for controlling HCV infection. While there is clinical evidence of a relevant role for regulatory T cells in chronic HCV-infected patients, based on their increased number and function; mechanisms underlying such a phenomena are still poorly understood. Accumulating evidence suggests that proteins from Hepatitis C virus can suppress host immune responses. We and others have shown that HCV is present in CD4+ lymphocytes from chronically infected patients and that HCV-core protein induces a state of unresponsiveness in the CD4+ tumor cell line Jurkat. Here we show that CD4+ primary T cells lentivirally transduced with HCV-core, not only acquire an anergic phenotype but also inhibit IL-2 production and proliferation of bystander CD4+ or CD8+ T cells in response to anti-CD3 plus anti-CD28 stimulation. Core-transduced CD4+ T cells show a phenotype characterized by an increased basal secretion of the regulatory cytokine IL-10, a decreased IFN-γ production upon stimulation, as well as expression of regulatory T cell markers, CTLA-4, and Foxp3. A significant induction of CD4+CD25+CD127(lowPD-1(highTIM-3(high regulatory T cells with an exhausted phenotype was also observed. Moreover, CCR7 expression decreased in HCV-core expressing CD4+ T cells explaining their sequestration in inflamed tissues such as the infected liver. This work provides a new perspective on de novo generation of regulatory CD4+ T cells in the periphery, induced by the expression of a single viral protein.

  13. Characterization of the Eimeria maxima sporozoite surface protein IMP1.

    Science.gov (United States)

    Jenkins, M C; Fetterer, R; Miska, K; Tuo, W; Kwok, O; Dubey, J P

    2015-07-30

    The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection. Published by Elsevier B.V.

  14. DISTRIBUTION OF GBM HEPARAN-SULFATE PROTEOGLYCAN CORE PROTEIN AND SIDE-CHAINS IN HUMAN GLOMERULAR-DISEASES

    NARCIS (Netherlands)

    VANDENBORN, J; VANDENHEUVEL, LPWJ; BAKKER, MAH; VEERKAMP, JH; ASSMANN, KJM; WEENING, JJ; BERDEN, JHM

    Using monoclonal antibodies (mAbs) recognizing either the core protein or the heparan sulfate (HS) side chain of human GBM heparan sulfate proteoglycan (HSPG), we investigated their glomerular distribution on cryostat sections of human kidney tissues. The study involved 95 biopsies comprising twelve

  15. Aberrant expression of mucin core proteins and o-linked glycans associated with progression of pancreatic cancer

    DEFF Research Database (Denmark)

    Remmers, Neeley; Anderson, Judy M; Linde, Erin M

    2013-01-01

    Mucin expression is a common feature of most adenocarcinomas and features prominently in current attempts to improve diagnosis and therapy for pancreatic cancer and other adenocarcinomas. We investigated the expression of a number of mucin core proteins and associated O-linked glycans expressed i...

  16. Characterization of tuyere-level core-drill coke samples from blast furnace operation

    Energy Technology Data Exchange (ETDEWEB)

    S. Dong; N. Paterson; S.G. Kazarian; D.R. Dugwell; R. Kandiyoti [Imperial College London, London (United Kingdom). Department of Chemical Engineering

    2007-12-15

    A suite of tuyere-level coke samples have been withdrawn from a working blast furnace during coal injection, using the core-drilling technique. The samples have been characterized by size exclusion chromatography (SEC), Fourier transform Raman spectroscopy (FT-RS), and X-ray powder diffraction (XRD) spectroscopy. The 1-methyl-2-pyrrolidinone (NMP) extracts of the cokes sampled from the 'bosh', the rear of the 'bird's nest', and the 'dead man' zones were found by SEC to contain heavy soot-like materials (ca. 10{sup 7}-10{sup 8} apparent mass units). In contrast, NMP extracts of cokes taken from the raceway and the front of the 'bird's nest' only contained a small amount of material of relatively lower apparent molecular mass (up to ca. 10{sup 5} u). Since the feed coke contained no materials extractable by the present method, the soot-like materials are thought to have formed during the reactions of volatile matter released from the injectant coal, probably via dehydrogenation and repolymerization of the tars. The Raman spectra of the NMP-extracted core-drilled coke samples showed variations reflecting their temperature histories. Area ratios of D-band to G-band decreased as the exposure temperature increased, while intensity ratios of D to G band and those of 2D to G bands increased with temperature. The graphitic (G), defect (D), and random (R) fractions of the carbon structure of the cokes were also derived from the Raman spectra. The R fractions decreased with increasing temperature, whereas G fractions increased, while the D fractions showed a more complex variation with temperature. These data appear to give clues regarding the graphitization mechanism of tuyere-level cokes in the blast furnace. 41 refs., 9 figs., 6 tabs.

  17. Characterization of core-drilled cokes in a working blast furnace

    Energy Technology Data Exchange (ETDEWEB)

    Shanning Dong; Nigel Paterson; Denis R. Dugwell; Rafael Kandiyoti [Imperial College London, London (United Kingdom). Dept. of Chemical Engineering

    2007-07-01

    A batch of tuyere-level core-drilled cokes, taken from a blast furnace working with coal injection has been characterized using a battery of analytical techniques. These included size exclusion chromatography (SEC), FT-Raman Spectroscopy (FT-RS) and X-ray Powder Diffraction (XRD). SEC tests on NMP-extracts of cokes taken from zones where temperatures were ca. 1500{sup o}C, showed the presence of heavy soot-like material (ca. 107-108 u apparent mass). By contrast, cokes in higher temperature zones (ca. 2000{sup o}C), only gave small amounts of extractable material with up to ca. 105 u apparent mass. The presence of soot-like material indicated the conversion-unfavoured locations at the tuyere-level. FT-Raman spectra of NMP-extracted cokes varied: the area ratios of D (at 1288-1295cm{sup -1}) to G (at ca. 1596cm{sup -1}) bands decreased as the exposure temperature increased. The random (r) fractions decreased with increasing exposure temperature, whereas, the graphitic (G) fractions increased whilst the defect (D) fraction showed a more complex variation with temperature. The latter is a likely indicator of graphitization of tuyere-level cokes in the blast furnace. The Raman spectral results were validated by XRD analyses of the demineralised and NMP-extracted cokes. Raceway coke possessed the largest crystalline dimensions and closest inter-layer spacing because it had encountered highest temperatures as well as iron catalysis. The combination of SEC and Raman spectrometry on core-drill samples has provided information relevant for maintaining stable operation in a blast-furnace operating with coal injection. 13 refs., 7 figs., 6 tabs.

  18. Characterization of combustion chamber products by core-level photoabsorption spectroscopy

    International Nuclear Information System (INIS)

    Kellar, S.A.; Huff, W.R.A.; Moler, E.J.

    1997-01-01

    The lubricating performance of motor oil is adversely affected by the carbon soot contamination that is a natural by-product of the combustion process. Particularly in diesel engines, open-quote blow-by close-quote is a problem that greatly decreases the longevity of the engine-lubricating oil. Motor oil manufacturers spend considerable resources developing new oil formulations that counteract the adverse affects of this combustion soot. At present, the only effective way to test new formulations is in a working engine. This process is obviously expensive and not especially efficient. In this ongoing work in collaboration with Chevron Research and Technology, the authors goal is to find a form of carbon that chemically resembles the soot created by the open-quote blow-by close-quote in a diesel engine. The chemically correct soot substitute can be used in bench tests to replace the expensive full motor testing for new formulations. The final testing would still be done in the test motors but only with promising candidates. To these ends, Near Edge X-ray Adsorption spectroscopy Extended Fine Structure (NEXAFS) is an attractive technique in that it has chemical specificity through the core-level binding energy and because it probes the chemically important unoccupied molecular orbitals of the material. Core-level photoabsorption has been used to characterize the empty electronic states of a wide variety of materials. Specifically, the near-edge region of the photoabsorption process has been used to determine the relative quantity of sp 2 and sp 3 bonding in carbon films. The samples were fine grained powders pressed into pellets. The C(1s) absorption spectra were collected from each sample by measuring the total electron yield from the sample as a function of photon energy. The absorption intensity was normalized to the incoming photon flux by measuring the photoyield from a fine gold mesh

  19. Bioinformatic analysis suggests that the Cypovirus 1 major core protein cistron harbours an overlapping gene

    Directory of Open Access Journals (Sweden)

    Atkins John F

    2008-05-01

    Full Text Available Abstract Members of the genus Cypovirus (family Reoviridae are common pathogens of insects. These viruses have linear dsRNA genomes divided into 10–11 segments, which have generally been assumed to be monocistronic. Here, bioinformatic evidence is presented for a short overlapping coding sequence (CDS in the cypovirus genome segment encoding the major core capsid protein VP1, overlapping the 5'-terminal region of the VP1 ORF in the +1 reading frame. In Cypovirus type 1 (CPV-1, a 62-codon AUG-initiated open reading frame (hereafter ORFX is present in all four available segment 1 sequences. The pattern of base variations across the sequence alignment indicates that ORFX is subject to functional constraints at the amino acid level (even when the constraints due to coding in the overlapping VP1 reading frame are taken into account; MLOGD software. In fact the translated ORFX shows greater amino acid conservation than the overlapping region of VP1. The genomic location of ORFX is consistent with translation via leaky scanning. A 62–64 codon AUG-initiated ORF is present in a corresponding location and reading frame in other available cypovirus sequences (2 CPV-14, 1 CPV-15 and an 87-codon ORFX homologue may also be present in Aedes pseudoscutellaris reovirus. The ORFX amino acid sequences are hydrophilic and basic, with between 12 and 16 Arg/Lys residues in each though, at 7.5–10.2 kDa, the putative ORFX product is too small to appear on typical published protein gels.

  20. Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

    Science.gov (United States)

    Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

    2016-01-01

    Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

  1. Identification of protein W, the elusive sixth subunit of the Rhodopseudomonas palustris reaction center-light harvesting 1 core complex.

    Science.gov (United States)

    Jackson, Philip J; Hitchcock, Andrew; Swainsbury, David J K; Qian, Pu; Martin, Elizabeth C; Farmer, David A; Dickman, Mark J; Canniffe, Daniel P; Hunter, C Neil

    2018-02-01

    The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc 1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  2. Human Adenovirus Infection Causes Cellular E3 Ubiquitin Ligase MKRN1 Degradation Involving the Viral Core Protein pVII.

    Science.gov (United States)

    Inturi, Raviteja; Mun, Kwangchol; Singethan, Katrin; Schreiner, Sabrina; Punga, Tanel

    2018-02-01

    Human adenoviruses (HAdVs) are common human pathogens encoding a highly abundant histone-like core protein, VII, which is involved in nuclear delivery and protection of viral DNA as well as in sequestering immune danger signals in infected cells. The molecular details of how protein VII acts as a multifunctional protein have remained to a large extent enigmatic. Here we report the identification of several cellular proteins interacting with the precursor pVII protein. We show that the cellular E3 ubiquitin ligase MKRN1 is a novel precursor pVII-interacting protein in HAdV-C5-infected cells. Surprisingly, the endogenous MKRN1 protein underwent proteasomal degradation during the late phase of HAdV-C5 infection in various human cell lines. MKRN1 protein degradation occurred independently of the HAdV E1B55K and E4orf6 proteins. We provide experimental evidence that the precursor pVII protein binding enhances MKRN1 self-ubiquitination, whereas the processed mature VII protein is deficient in this function. Based on these data, we propose that the pVII protein binding promotes MKRN1 self-ubiquitination, followed by proteasomal degradation of the MKRN1 protein, in HAdV-C5-infected cells. In addition, we show that measles virus and vesicular stomatitis virus infections reduce the MKRN1 protein accumulation in the recipient cells. Taken together, our results expand the functional repertoire of the HAdV-C5 precursor pVII protein in lytic virus infection and highlight MKRN1 as a potential common target during different virus infections. IMPORTANCE Human adenoviruses (HAdVs) are common pathogens causing a wide range of diseases. To achieve pathogenicity, HAdVs have to counteract a variety of host cell antiviral defense systems, which would otherwise hamper virus replication. In this study, we show that the HAdV-C5 histone-like core protein pVII binds to and promotes self-ubiquitination of a cellular E3 ubiquitin ligase named MKRN1. This mutual interaction between the pVII and

  3. On the characterization and software implementation of general protein lattice models.

    Directory of Open Access Journals (Sweden)

    Alessio Bechini

    Full Text Available models of proteins have been widely used as a practical means to computationally investigate general properties of the system. In lattice models any sterically feasible conformation is represented as a self-avoiding walk on a lattice, and residue types are limited in number. So far, only two- or three-dimensional lattices have been used. The inspection of the neighborhood of alpha carbons in the core of real proteins reveals that also lattices with higher coordination numbers, possibly in higher dimensional spaces, can be adopted. In this paper, a new general parametric lattice model for simplified protein conformations is proposed and investigated. It is shown how the supporting software can be consistently designed to let algorithms that operate on protein structures be implemented in a lattice-agnostic way. The necessary theoretical foundations are developed and organically presented, pinpointing the role of the concept of main directions in lattice-agnostic model handling. Subsequently, the model features across dimensions and lattice types are explored in tests performed on benchmark protein sequences, using a Python implementation. Simulations give insights on the use of square and triangular lattices in a range of dimensions. The trend of potential minimum for sequences of different lengths, varying the lattice dimension, is uncovered. Moreover, an extensive quantitative characterization of the usage of the so-called "move types" is reported for the first time. The proposed general framework for the development of lattice models is simple yet complete, and an object-oriented architecture can be proficiently employed for the supporting software, by designing ad-hoc classes. The proposed framework represents a new general viewpoint that potentially subsumes a number of solutions previously studied. The adoption of the described model pushes to look at protein structure issues from a more general and essential perspective, making

  4. Functional characterization of the Woronin body protein WscA of the pathogenic mold Aspergillus fumigatus.

    Science.gov (United States)

    Leonhardt, Yannik; Beck, Julia; Ebel, Frank

    2016-05-01

    Woronin bodies are fungal-specific organelles that seal damaged hyphal compartments and thereby contribute to the stress resistance and virulence of filamentous fungi. In this study, we have characterized the Aspergillus fumigatus Woronin body protein WscA. WscA is homologous to Neurospora crassa WSC, a protein that was shown to be important for biogenesis, segregation and positioning of Woronin bodies. WscA and WSC both belong to the Mpv17/PMP22 family of peroxisomal membrane proteins. An A. fumigatus ΔwscA mutant is unable to form Woronin bodies, and HexA, the protein that forms the crystal-like core of Woronin bodies, accumulates in large peroxisomes instead. The ΔwscA mutant showed no defect in segregation of HexA containing organelles, as has been reported for the corresponding N. crassa mutant. In the peroxisomes of the A. fumigatus mutant, HexA assembles into compact, donut-shaped structures. Experiments with GFP fusion proteins revealed that WscA function is highly sensitive to these modifications, in particular to an N-terminal fusion of GFP. In N. crassa, WSC was shown to be essentially required for Woronin body positioning, but the respective domain is not conserved in most other Pezizomycotina, including A. fumigatus. We have recently found evidence that HexA may have a direct role in WB positioning, since a HexA-GFP fusion protein, lacking a functional PTS1 motif, is efficiently recruited to the septal pore. In the current study we show that this targeting of HexA-GFP is independent of WscA. Copyright © 2016 Elsevier GmbH. All rights reserved.

  5. Interfacial characterization of ceramic core materials with veneering porcelain for all-ceramic bi-layered restorative systems.

    Science.gov (United States)

    Tagmatarchis, Alexander; Tripodakis, Aris-Petros; Filippatos, Gerasimos; Zinelis, Spiros; Eliades, George

    2014-01-01

    The aim of the study was to characterize the elemental distribution at the interface between all-ceramic core and veneering porcelain materials. Three groups of all-ceramic cores were selected: A) Glass-ceramics (Cergo, IPS Empress, IPS Empress 2, e-max Press, Finesse); B) Glass-infiltrated ceramics (Celay Alumina, Celay Zirconia) and C) Densely sintered ceramics (Cercon, Procera Alumina, ZirCAD, Noritake Zirconia). The cores were combined with compatible veneering porcelains and three flat square test specimens were produced for each system. The core-veneer interfaces were examined by scanning electron microscopy and energy dispersive x-ray microanalysis. The glass-ceramic systems showed interfacial zones reach in Si and O, with the presence of K, Ca, Al in core and Ca, Ce, Na, Mg or Al in veneer material, depending on the system tested. IPS Empress and IPS Empress 2 demonstrated distinct transitional phases at the core-veneer interface. In the glassinfiltrated systems, intermixing of core (Ce, La) with veneer (Na, Si) elements occurred, whereas an abrupt drop of the core-veneer elemental concentration was documented at the interfaces of all densely sintered ceramics. The results of the study provided no evidence of elemental interdiffusion at the core-veneer interfaces in densely sintered ceramics, which implies lack of primary chemical bonding. For the glass-containing systems (glassceramics and glass-infiltrated ceramics) interdiffusion of the glass-phase seems to play a critical role in establishing a primary bonding condition between ceramic core and veneering porcelain.

  6. Characterization of auxin-binding proteins from zucchini plasma membrane

    Science.gov (United States)

    Hicks, G. R.; Rice, M. S.; Lomax, T. L.

    1993-01-01

    We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may

  7. Structural and Functional Characterization of an Ancient Bacterial Transglutaminase Sheds Light on the Minimal Requirements for Protein Cross-Linking.

    Science.gov (United States)

    Fernandes, Catarina G; Plácido, Diana; Lousa, Diana; Brito, José A; Isidro, Anabela; Soares, Cláudio M; Pohl, Jan; Carrondo, Maria A; Archer, Margarida; Henriques, Adriano O

    2015-09-22

    Transglutaminases are best known for their ability to catalyze protein cross-linking reactions that impart chemical and physical resilience to cellular structures. Here, we report the crystal structure and characterization of Tgl, a transglutaminase from the bacterium Bacillus subtilis. Tgl is produced during sporulation and cross-links the surface of the highly resilient spore. Tgl-like proteins are found only in spore-forming bacteria of the Bacillus and Clostridia classes, indicating an ancient origin. Tgl is a single-domain protein, produced in active form, and the smallest transglutaminase characterized to date. We show that Tgl is structurally similar to bacterial cell wall endopeptidases and has an NlpC/P60 catalytic core, thought to represent the ancestral unit of the cysteine protease fold. We show that Tgl functions through a unique partially redundant catalytic dyad formed by Cys116 and Glu187 or Glu115. Strikingly, the catalytic Cys is insulated within a hydrophobic tunnel that traverses the molecule from side to side. The lack of similarity of Tgl to other transglutaminases together with its small size suggests that an NlpC/P60 catalytic core and insulation of the active site during catalysis may be essential requirements for protein cross-linking.

  8. Development of lycopene-loaded lipid-core nanocapsules: physicochemical characterization and stability study

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Priscilla Pereira dos [Universidade Federal do Rio Grande do Sul (UFRGS), Instituto de Ciência e Tecnologia de Alimentos (Brazil); Paese, Karina; Guterres, Silvia Stanisçuaski [Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia (Brazil); Pohlmann, Adriana Raffin [Universidade Federal do Rio Grande do Sul (UFRGS), Departamento de Química Orgânica, Instituto de Química (Brazil); Costa, Tania Hass [Universidade Federal do Rio Grande do Sul (UFRGS), Departamento de Química, Instituto de Química (Brazil); Jablonski, André [Universidade Federal do Rio Grande do Sul (UFRGS), Departamento de Engenharia de Minas (Brazil); Flôres, Simone Hickmann; Rios, Alessandro de Oliveira, E-mail: alessandro.rios@ufrgs.br [Universidade Federal do Rio Grande do Sul (UFRGS), Instituto de Ciência e Tecnologia de Alimentos (Brazil)

    2015-02-15

    The objective of this study was to develop lycopene-loaded lipid-core nanocapsules (Lyc-LNCs) by the interfacial deposition of preformed poly(ε-caprolactone) (PCL). Lyc extract (93.9 %) was obtained from tomatoes, and the organic phase was prepared with polymer (PCL), caprylic/capric triglycerides, sorbitan monostearate, and Lyc in a mixture of acetone and ethanol under magnetic stirring at 40 °C. The organic phase was injected into an aqueous phase containing polysorbate 80, and the suspension was concentrated under reduced pressure. The formulation with a Lyc concentration of 85 µg/mL was characterized in terms of size distribution, zeta potential, encapsulation efficiency, pH, viscosity, and color. The Lyc-LNC formulation presented stable values for the z-average (193 ± 4.7 nm) and zeta potential (−11.5 ± 0.40 mV). Despite the lower pH, Lyc content, and color change of the suspension, the nanocapsules showed satisfactory stability, presenting around 50 % Lyc content after 14 days of storage at room temperature (25 °C)

  9. Symmetrical N-acylsubstituted dihydrazones containing bithiophene core - Photophysical, electrochemical and thermal characterization

    Science.gov (United States)

    Jarczyk-Jedryka, Anna; Filapek, Michal; Malecki, Grzegorz; Kula, Slawomir; Janeczek, Henryk; Boharewicz, Bartosz; Iwan, Agnieszka; Schab-Balcerzak, Ewa

    2016-04-01

    Four symmetrical N-acylsubstituted dihydrazones containing bithiophene core were synthesized from condensation of 2,2‧-bithiophene-5,5‧-dicarboxyaldehyde with benzoic, isonicotinoyl, 2-thiophenic and 2-furoic hydrazide. The obtained compounds were characterized through the data from 1H nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), elemental analysis, UV-vis absorption spectroscopy, photoluminescence (PL), cyclic voltammetry (CV) and differential pulse voltammetry (DPV) measurements. Additionally, the electronic properties including orbital energies and resulting energy gaps were calculated by density functional theory (DFT). Their thermal behavior was investigated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). They were thermal sable up to 320 °C. The prepared N-acylsubstituted dihydrazones emitted light with λem in the range of 499-530 nm in solution, whereas, in solid state as blend with PMMA blue emission was observed. They undergo quasi-reversible and irreversible electrochemical reduction and oxidation processes, respectively. Additionally, the selected compounds were tested preliminary as component of active layer in organic photovoltaic cells. The highest value of power conversion efficiency, equal to 1.68% under simulated 100 mW/cm2 AM 1.5G irradiation was found for device with the architecture ITO/PEDOT:PSS/P3HT:PCBM:FBTH (1:2:2)/Al.

  10. Surface characterization of hydrophobic core-shell QDs using NMR techniques

    Science.gov (United States)

    Zhang, Chengqi; Zeng, Birong; Palui, Goutam; Mattoussi, Hedi

    2018-02-01

    Using a few solution phase NMR spectroscopy techniques, including 1H NMR and 31P NMR, we have characterized the organic shell on CdSe-ZnS core-shell quantum dots and tracked changes in its composition when the QD dispersions are subjected to varying degrees of purification. Combining solution phase NMR with diffusion ordered spectroscopy (DOSY), we were able to distinguish between freely diffusing ligands in the sample from those bound on the surfaces. Additionally, matrix assisted laser desorption ionization (MALDI) and FTIR measurements were used to provide complementary and supporting information on the organic ligand coating for these nanocrystals. We found that the organic shell is dominated by monomeric or oligomeric n-hexylphosphonic acid (HPA), along with small portion of 1-hexadecyl amine (HDA). The presence of TOP/TOPO (tri-n-octylphosphine / tri-noctylphosphine oxide) molecules is much smaller, even though large excess of TOP/TOPO were used during the QD growth. These results indicate that HPA (alkyl phosphonate) exhibits the strongest coordination affinity to ZnS-rich QD surfaces grown using the high temperature injection route.

  11. Development of lycopene-loaded lipid-core nanocapsules: physicochemical characterization and stability study

    International Nuclear Information System (INIS)

    Santos, Priscilla Pereira dos; Paese, Karina; Guterres, Silvia Stanisçuaski; Pohlmann, Adriana Raffin; Costa, Tania Hass; Jablonski, André; Flôres, Simone Hickmann; Rios, Alessandro de Oliveira

    2015-01-01

    The objective of this study was to develop lycopene-loaded lipid-core nanocapsules (Lyc-LNCs) by the interfacial deposition of preformed poly(ε-caprolactone) (PCL). Lyc extract (93.9 %) was obtained from tomatoes, and the organic phase was prepared with polymer (PCL), caprylic/capric triglycerides, sorbitan monostearate, and Lyc in a mixture of acetone and ethanol under magnetic stirring at 40 °C. The organic phase was injected into an aqueous phase containing polysorbate 80, and the suspension was concentrated under reduced pressure. The formulation with a Lyc concentration of 85 µg/mL was characterized in terms of size distribution, zeta potential, encapsulation efficiency, pH, viscosity, and color. The Lyc-LNC formulation presented stable values for the z-average (193 ± 4.7 nm) and zeta potential (−11.5 ± 0.40 mV). Despite the lower pH, Lyc content, and color change of the suspension, the nanocapsules showed satisfactory stability, presenting around 50 % Lyc content after 14 days of storage at room temperature (25 °C)

  12. Development of lycopene-loaded lipid-core nanocapsules: physicochemical characterization and stability study

    Science.gov (United States)

    dos Santos, Priscilla Pereira; Paese, Karina; Guterres, Silvia Stanisçuaski; Pohlmann, Adriana Raffin; Costa, Tania Hass; Jablonski, André; Flôres, Simone Hickmann; Rios, Alessandro de Oliveira

    2015-02-01

    The objective of this study was to develop lycopene-loaded lipid-core nanocapsules (Lyc-LNCs) by the interfacial deposition of preformed poly(ɛ-caprolactone) (PCL). Lyc extract (93.9 %) was obtained from tomatoes, and the organic phase was prepared with polymer (PCL), caprylic/capric triglycerides, sorbitan monostearate, and Lyc in a mixture of acetone and ethanol under magnetic stirring at 40 °C. The organic phase was injected into an aqueous phase containing polysorbate 80, and the suspension was concentrated under reduced pressure. The formulation with a Lyc concentration of 85 µg/mL was characterized in terms of size distribution, zeta potential, encapsulation efficiency, pH, viscosity, and color. The Lyc-LNC formulation presented stable values for the z-average (193 ± 4.7 nm) and zeta potential (-11.5 ± 0.40 mV). Despite the lower pH, Lyc content, and color change of the suspension, the nanocapsules showed satisfactory stability, presenting around 50 % Lyc content after 14 days of storage at room temperature (25 °C).

  13. Quantitative methods for structural characterization of proteins based on deep UV resonance Raman spectroscopy.

    Science.gov (United States)

    Shashilov, Victor A; Sikirzhytski, Vitali; Popova, Ludmila A; Lednev, Igor K

    2010-09-01

    Here we report on novel quantitative approaches for protein structural characterization using deep UV resonance Raman (DUVRR) spectroscopy. Specifically, we propose a new method combining hydrogen-deuterium (HD) exchange and Bayesian source separation for extracting the DUVRR signatures of various structural elements of aggregated proteins including the cross-beta core and unordered parts of amyloid fibrils. The proposed method is demonstrated using the set of DUVRR spectra of hen egg white lysozyme acquired at various stages of HD exchange. Prior information about the concentration matrix and the spectral features of the individual components was incorporated into the Bayesian equation to eliminate the ill-conditioning of the problem caused by 100% correlation of the concentration profiles of protonated and deuterated species. Secondary structure fractions obtained by partial least squares (PLS) and least squares support vector machines (LS-SVMs) were used as the initial guess for the Bayessian source separation. Advantages of the PLS and LS-SVMs methods over the classical least squares calibration (CLSC) are discussed and illustrated using the DUVRR data of the prion protein in its native and aggregated forms. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  14. Nonstructural protein 5A is incorporated into hepatitis C virus low-density particle through interaction with core protein and microtubules during intracellular transport.

    Directory of Open Access Journals (Sweden)

    Chao-Kuen Lai

    Full Text Available Nonstructural protein 5A (NS5A of hepatitis C virus (HCV serves dual functions in viral RNA replication and virus assembly. Here, we demonstrate that HCV replication complex along with NS5A and Core protein was transported to the lipid droplet (LD through microtubules, and NS5A-Core complexes were then transported from LD through early-to-late endosomes to the plasma membrane via microtubules. Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Furthermore, exosomal markers CD63 and CD81 were also detected in the low-density fractions, but not in the high-density fractions. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection.

  15. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

    Science.gov (United States)

    Yin, Yanting; de Waal, Parker W.; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H. Eric

    2017-01-01

    The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. PMID:28356352

  16. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

    Science.gov (United States)

    Yin, Yanting; de Waal, Parker W; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H Eric

    2017-06-16

    The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β 2 -adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Isolation and functional characterization of CE1 binding proteins

    Directory of Open Access Journals (Sweden)

    Yu Ji-hyun

    2010-12-01

    Full Text Available Abstract Background Abscisic acid (ABA is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE, has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1. Results To isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs. Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity. Conclusions Our results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or

  18. Isolation and functional characterization of CE1 binding proteins.

    Science.gov (United States)

    Lee, Sun-ji; Park, Ji Hye; Lee, Mi Hun; Yu, Ji-hyun; Kim, Soo Young

    2010-12-16

    Abscisic acid (ABA) is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE), has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE)" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1. To isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs). Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity. Our results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or abiotic stress response, but the physiological functions

  19. Suitability of magnetic single- and multi-core nanoparticles to detect protein binding with dynamic magnetic measurement techniques

    International Nuclear Information System (INIS)

    Remmer, Hilke; Dieckhoff, Jan; Schilling, Meinhard; Ludwig, Frank

    2015-01-01

    We investigated the binding of biotinylated proteins to various streptavidin functionalized magnetic nanoparticles with different dynamic magnetic measurement techniques to examine their potential for homogeneous bioassays. As particle systems, single-core nanoparticles with a nominal core diameter of 30 nm as well as multi-core nanoparticles with hydrodynamic sizes varying between nominally 60 nm and 100 nm were chosen. As experimental techniques, fluxgate magnetorelaxometry (MRX), complex ac susceptibility (ACS) and measurements of the phase lag between rotating field and sample magnetization are applied. MRX measurements are only suited for the detection of small analytes if the multivalency of functionalized nanoparticles and analytes causes cross-linking, thus forming larger aggregates. ACS measurements showed for all nanoparticle systems a shift of the imaginary part's maximum towards small frequencies. In rotating field measurements only the single-core nanoparticle systems with dominating Brownian mechanism exhibit an increase of the phase lag upon binding in the investigated frequency range. The coexistence of Brownian and Néel relaxation processes can cause a more complex phase lag change behavior, as demonstrated for multi-core nanoparticle systems. - Highlights: • Cealization of homogeneous magnetic bioassays using different magnetic techniques. • Comparison of single- and multi-core nanoparticle systems. • ac Susceptibility favorable for detection of small analytes. • Magnetorelaxometry favorable for detection of large analytes or cross-linking assays

  20. Purification, characterization and immunolocalization of porcine surfactant protein D

    DEFF Research Database (Denmark)

    Sørensen, C.M.; Nielsen, Ove Lilholm; Willis, A.

    2005-01-01

    in a dose and Ca2+-dependent manner with a saccharide specificity similar to rat and human SP-D. The purified protein was used for the production of a monoclonal anti-pSP-D antibody. The antibody reacted specifically with pSP-D in the reduced and unreduced state when analysed by Western blotting......Surfactant protein D (SP-D) is a collectin believed to play an important role in innate immunity. SP-D is characterized by having a collagen-like domain and a carbohydrate recognition domain (CRD), which has a specific Ca2+-dependent specificity for saccharides and thus the ability to bind complex...... glycoconjugates on micro-organisms. This paper describes the tissue immunolocalization of porcine SP-D (pSP-D) in normal slaughter pigs using a monoclonal antibody raised against purified pSP-D. Porcine SP-D was purified from porcine bronchoalveolar lavage (BAL) by maltose-agarose and immunoglobulin M affinity...

  1. Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization

    Directory of Open Access Journals (Sweden)

    Gao Fan

    2012-01-01

    Full Text Available Abstract Background To characterize the human humoral immune response against enterovirus 71 (EV71 infection and map human epitopes on the viral capsid proteins. Methods A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3 of BJ08 strain (genomic C4 were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl. Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel. Results A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3 were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15 was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71 were identified and mapped to VP1. Conclusion These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents.

  2. Decorin core protein (decoron) shape complements collagen fibril surface structure and mediates its binding.

    Science.gov (United States)

    Orgel, Joseph P R O; Eid, Aya; Antipova, Olga; Bella, Jordi; Scott, John E

    2009-09-15

    Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e(1) bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1) bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

  3. Decorin core protein (decoron shape complements collagen fibril surface structure and mediates its binding.

    Directory of Open Access Journals (Sweden)

    Joseph P R O Orgel

    2009-09-01

    Full Text Available Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM. With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein and binding sites in the d and e(1 bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e(1 bands. This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.

  4. Preparation, process optimization and characterization of core-shell polyurethane/chitosan nanofibers as a potential platform for bioactive scaffolds.

    Science.gov (United States)

    Maleknia, Laleh; Dilamian, Mandana; Pilehrood, Mohammad Kazemi; Sadeghi-Aliabadi, Hojjat; Hekmati, Amir Houshang

    2018-06-01

    In this paper, polyurethane (PU), chitosan (Cs)/polyethylene oxide (PEO), and core-shell PU/Cs nanofibers were produced at the optimal processing conditions using electrospinning technique. Several methods including SEM, TEM, FTIR, XRD, DSC, TGA and image analysis were utilized to characterize these nanofibrous structures. SEM images exhibited that the core-shell PU/Cs nanofibers were spun without any structural imperfections at the optimized processing conditions. TEM image confirmed the PU/Cs core-shell nanofibers were formed apparently. It that seems the inclusion of Cs/PEO to the shell, did not induce the significant variations in the crystallinity in the core-shell nanofibers. DSC analysis showed that the inclusion of Cs/PEO led to the glass temperature of the composition increased significantly compared to those of neat PU nanofibers. The thermal degradation of core-shell PU/Cs was similar to PU nanofibers degradation due to the higher PU concentration compared to other components. It was hypothesized that the core-shell PU/Cs nanofibers can be used as a potential platform for the bioactive scaffolds in tissue engineering. Further biological tests should be conducted to evaluate this platform as a three dimensional scaffold with the capabilities of releasing the bioactive molecules in a sustained manner.

  5. Hepatitis C virus core protein expression leads to biphasic regulation of the p21 cdk inhibitor and modulation of hepatocyte cell cycle

    International Nuclear Information System (INIS)

    Nguyen, Hau; Mudryj, Maria; Guadalupe, Moraima; Dandekar, Satya

    2003-01-01

    Hepatitis C virus (HCV) Core protein is implicated in viral pathogenesis by the modulation of hepatocyte gene expression and function. To determine the effect of Core protein on the cell-cycle control of hepatocytes, a HepG2 cell line containing a Flag-tagged Core under the control of an inducible promoter was generated. Initial Core protein expression included the presence of unprocessed (191 aa) and processed (173 aa) forms of the Core proteins with the processed form becoming dominant later. Expression of the 191 aa form of Core protein corresponded to an increase in the expression of the p21, a decrease in cdk2-dependent kinase activity, and a decrease in the percentage of cells in S-phase along with an accumulation of cells in the G 0 /G 1 phase of the cell cycle. As the processed form accumulated, the p21 levels started to decline, suggesting that Core protein regulates p21 expression in a biphasic manner. These findings implicate Core protein in potentially modulating hepatocyte cell cycle differentially in the early stages of infection through biphasic regulation of p21 cdk kinase inhibitor

  6. Characterization of spherical core–shell particles by static light scattering. Estimation of the core- and particle-size distributions

    International Nuclear Information System (INIS)

    Clementi, Luis A.; Vega, Jorge R.; Gugliotta, Luis M.; Quirantes, Arturo

    2012-01-01

    A numerical method is proposed for the characterization of core–shell spherical particles from static light scattering (SLS) measurements. The method is able to estimate the core size distribution (CSD) and the particle size distribution (PSD), through the following two-step procedure: (i) the estimation of the bivariate core–particle size distribution (C–PSD), by solving a linear ill-conditioned inverse problem through a generalized Tikhonov regularization strategy, and (ii) the calculation of the CSD and the PSD from the estimated C–PSD. First, the method was evaluated on the basis of several simulated examples, with polystyrene–poly(methyl methacrylate) core–shell particles of different CSDs and PSDs. Then, two samples of hematite–Yttrium basic carbonate core–shell particles were successfully characterized. In all analyzed examples, acceptable estimates of the PSD and the average diameter of the CSD were obtained. Based on the single-scattering Mie theory, the proposed method is an effective tool for characterizing core–shell colloidal particles larger than their Rayleigh limits without requiring any a-priori assumption on the shapes of the size distributions. Under such conditions, the PSDs can always be adequately estimated, while acceptable CSD estimates are obtained when the core/shell particles exhibit either a high optical contrast, or a moderate optical contrast but with a high ‘average core diameter’/‘average particle diameter’ ratio. -- Highlights: ► Particles with core–shell morphology are characterized by static light scattering. ► Core size distribution and particle size distribution are successfully estimated. ► Simulated and experimental examples are used to validate the numerical method. ► The positive effect of a large core/shell optical contrast is investigated. ► No a-priori assumption on the shapes of the size distributions is required.

  7. Characterization of Vadose Zone Sediment: Uncontaminated RCRA Borehole Core Samples and Composite Samples

    International Nuclear Information System (INIS)

    Serne, R. Jeffrey; Bjornstad, Bruce N.; Schaef, Herbert T.; Williams, Bruce A.; Lanigan, David C.; Horton, Duane G.; Clayton, Ray E.; Mitroshkov, Alexandre V.; Legore, Virginia L.; O'Hara, Matthew J.; Brown, Christopher F.; Parker, Kent E.; Kutnyakov, Igor V.; Serne, Jennifer N.; Last, George V.; Smith, Steven C.; Lindenmeier, Clark W.; Zachara, John M.; Burke, Deborah Sd.

    2001-01-01

    The overall goal of the of the Tank Farm Vadose Zone Project, led by CH2M HILL Hanford Group, Inc., is to define risks from past and future single-shell tank farm activities. To meet this goal, CH2M HILL Hanford Group, Inc. asked scientists from Pacific Northwest National Laboratory to perform detailed analyses on vadose zone sediment from within the S-SX Waste Management Area. This report is the first in a series of four reports to present the results of these analyses. Specifically, this report contains all the geologic, geochemical, and selected physical characterization data collected on vadose zone sediment recovered from RCRA borehole bore samples and composite samples. Intact cores from two RCRA boreholes (299-W22-48 and 299-W22-50) near the SX Tank Farm and four, large-quantity grab samples from outcrop sediment on and off the Hanford Site were sampled to better understand the fate of contaminants in the vadose zone beneath underground storage tanks at the Hanford Site. Borehole and outcrop samples analyzed for this report are located outside the tank farms, and therefore may be considered standard or background samples from which to compare contaminated sediments within the tank farms themselves. This report presents our interpretation of the physical, chemical, and mineralogical properties of the uncontaminated vadose zone sediments, and variations in the vertical distribution of these properties. The information presented in this report is intended to support preparation of the S-SX Field Investigation Report to be prepared by CH2M Hill Hanford Group, Inc. as well as future remediation actions at the S-SX Tank Farm

  8. Conformational Changes in the Hepatitis B Virus Core Protein Are Consistent with a Role for Allostery in Virus Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Packianathan, Charles; Katen, Sarah P.; Dann, III, Charles E.; Zlotnick, Adam (Indiana)

    2010-01-12

    In infected cells, virus components must be organized at the right place and time to ensure assembly of infectious virions. From a different perspective, assembly must be prevented until all components are available. Hypothetically, this can be achieved by allosterically controlling assembly. Consistent with this hypothesis, here we show that the structure of the hepatitis B virus (HBV) core protein dimer, which can spontaneously self-assemble, is incompatible with capsid assembly. Systematic differences between core protein dimer and capsid conformations demonstrate linkage between the intradimer interface and interdimer contact surface. These structures also provide explanations for the capsid-dimer selectivity of some antibodies and the activities of assembly effectors. Solution studies suggest that the assembly-inactive state is more accurately an ensemble of conformations. Simulations show that allostery supports controlled assembly and results in capsids that are resistant to dissociation. We propose that allostery, as demonstrated in HBV, is common to most self-assembling viruses.

  9. Characterization of compressive and short beam shear strength of bamboo opened cell foam core sandwich composites

    Energy Technology Data Exchange (ETDEWEB)

    Setyawan, Paryanto Dwi, E-mail: paryanto-ds@yahoo.com; Sugiman,; Saputra, Yudhi [Department of Mechanical Engineering, Faculty of Engineering, University of Mataram, Mataram, West Nusa Tenggara (Indonesia)

    2016-03-29

    The paper presents the compressive and the short beam shear strength of a sandwich composite with opened cell foam made of bamboo fiber as the core and plywood as the skins. The core thickness was varied from 10 mm to 40 mm keeping the volume fraction of fiber constant. Several test s were carried out including the core density, flatwise compressive and the short beam shear testing in three point bending. The results show that the density of bamboo opened cell foam is comparable with commercial plastic foam, such as polyurethane foam. The compressive strength tends to increase linearly with increasing the core thickness. The short beam shear failure load of the sandwich composite increases with the increase of core thickness, however on the contrary, the short beam shear strength which tends to sharply decrease from the thickness of 10 mm to 30 mm and then becomes flat.

  10. Characterization of Coated Sand Cores from Two Different Binder Systems for Grey Iron Castings

    DEFF Research Database (Denmark)

    Nwaogu, Ugochukwu Chibuzoh; Tiedje, Niels Skat; Poulsen, Thomas

    or veining and metal penetration defects. The use of refractory coatings on cores is fundamental to obtaining acceptable casting surface quality and is used on resin bonded cores in production foundries. In this study new sol gel-coated sand cores made from coldbox and furan binder systems were investigated......Expansion defects on the surface of the castings include sand burn-in, metal penetration and/or veining, finning or scab. Veining or finning and metal penetration are of interest. These defects are associated with silica sand and result from the penetration of liquid metal into cracks formed during...... differential expansion of the core during heating. The rapid expansion of silica sand up to 600 oC and especially at 573 oC, where the α – β phase transformation occurs, is the cause of stresses in the core system. These stresses cause crack formation and metal melt flows into these cracks causing finning...

  11. Fracture Characterization of PVC Foam Core Sandwich Specimen Using the DCB-UBM Test Method

    DEFF Research Database (Denmark)

    Saseendran, Vishnu; Berggreen, Christian; Carlsson, Leif A.

    coupled with experimental validation is paramount to determine the fracture resistance of the face/core interface. In this paper, the test-rig exploiting the double cantilever beam with uneven bending moments (DCB-UBM) concept is used to determine the fracture toughness of PVC foam core sandwich......Face/core debond failure in sandwich composites is a critical failure mode. Lack of cohesion between face and core will lead to loss of structural integrity. The estimation of interface fracture toughness especially at the face/core interface is extremely challenging, provided the dissimilarity...... composites. The DCB-UBM test enables fracture testing over a large range of mode-mixities as expressed by a phase angle (ψ) which is a measure of the amount of shear loading at the crack tip. A desired phase angle may be achieved by changing the moment-ratio (MR = Md/Ms)....

  12. Face/core mixed mode debond fracture toughness characterization using the modified TSD test method

    DEFF Research Database (Denmark)

    Berggreen, Christian; Quispitupa, Amilcar; Costache, Andrei

    2014-01-01

    The modified tilted sandwich debond (TSD) test method is used to examine face/core debond fracture toughness of sandwich specimens with glass/polyester face sheets and PVC H45 and H100 foam cores over a large range of mode-mixities. The modification was achieved by reinforcing the loaded face sheet....... The fracture process was inspected visually during and after testing. For specimens with H45 core the crack propagated in the core. For specimens with an H100 core, the crack propagated between the resin-rich layer and the face sheet. © The Author(s) 2013 Reprints and permissions: sagepub...... with a steel bar, and fracture testing of the test specimens was conducted over a range of tilt angles. The fracture toughness exhibited mode-mixity phase angle dependence, especially for mode II dominated loadings; although, the fracture toughness remained quite constant for mode I dominated crack loadings...

  13. Face/core interface fracture characterization of mixed mode bending sandwich specimens

    DEFF Research Database (Denmark)

    Quispitupa, Amilcar; Berggreen, Christian; Carlsson, L.A.

    2011-01-01

    and PVC H45, H100 and H250 foam core materials were evaluated. A methodology to perform precracking on fracture specimens in order to achieve a sharp and representative crack front is outlined. The mixed mode loading was controlled in the mixed mode bending (MMB) test rig by changing the loading......Debonding of the core from the face sheets is a critical failure mode in sandwich structures. This paper presents an experimental study on face/core debond fracture of foam core sandwich specimens under a wide range of mixed mode loading conditions. Sandwich beams with E‐glass fibre face sheets...... application point (lever arm distance). Finite element analysis was performed to determine the mode‐mixity at the crack tip. The results showed that the face/core interface fracture toughness increased with increased mode II loading. Post failure analysis of the fractured specimens revealed that the crack...

  14. Fabrication, characterization and screen printing of conductive ink based on carbon@Ag core-shell nanoparticles.

    Science.gov (United States)

    Wu, Wei; Yang, Shuanglei; Zhang, Shaofeng; Zhang, Hongbo; Jiang, Changzhong

    2014-08-01

    The large-scale synthesis and characterization of carbon-core/Ag-shell (C@Ag) nanoparticles by the successive reduction of silver ammonia are described. The resultant C@Ag nanoparticles had a mean core diameter of 360 nm and a controllable shell thickness from 10 to 40 nm by simple adjustments of repeat coating times. Various analysis techniques confirmed that the carbon cores were fully covered by Ag nanoshells. The results also show that C/Ag composite nanomaterials-based conductive inks, which can be easily produced on a large scale and possess outstanding electronic properties, have great potential for the convenient fabrication of flexible and low-cost carbon-based electronic devices and replace the traditional pure silver paste, by using a simple screen printing technique. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro.

    Science.gov (United States)

    Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

    2004-01-01

    The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.

  16. Rheological characterization of plasticized corn proteins for fused deposition modeling

    Science.gov (United States)

    Chaunier, Laurent; Dalgalarrondo, Michèle; Della Valle, Guy; Lourdin, Denis; Marion, Didier; Leroy, Eric

    2017-10-01

    Additive Manufacturing (AM) of tailored natural biopolymer-based objects by Fused Deposition Modeling (FDM) opens new perspectives for applications such as biomedical temporary devices, or pharmaceutical tablets. This exploits the biocompatibility, resorbability and edibility properties of biopolymers. When adequately plasticized, zeins, storage proteins from endosperm of maize kernels, displayed thermomechanical properties possibly matching FDM processing requirements at a convenient temperature Tprinting=130°C. Indeed, with 20% glycerol added (Tg=42°C), plasticized zeins present a high modulus, E'>1GPa, at ambient conditions, which drops below 0.6 MPa at the processing temperature T=130°C, before flowing in the molten state. The rheological characterization shows that the processing window is limited by a progressive increase of viscosity linked to proteins aggregation and crosslinking by S-S bonding between cysteine amino acid residues, which can lead to gelation. However, for short residence time typical of FDM, the viscosity of plasticized zeins is comparable to the one of standard polymers, like ABS or PLA in their FDM processing conditions: indeed, in presence of glycerol, the molten zeins show a shear-thinning behavior with |η*|≈3kPa.s at 1s-1, decreasing to |η*|≈0.3kPa.s at 100s-1, at 130°C. Moreover, zeins presenting both hydrophilic and hydrophobic domains, amphiphilic plasticizers can be used supplementary to tune their rheological behavior. With 20% oleic acid added to the previous composition, the viscosity is divided down to a ratio about 1/2 at 100s-1 at 130°C, below the value of a standard polymer as PLA at its printing temperature. These results show the possible enhancement of the printability of zein-based materials in the molten state, by combining polar and amphiphilic plasticizers.

  17. Core Data of Yeast Interacting Proteins Database (Original Version) - Yeast Interacting Proteins Database | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available y are in the reverse direction. *1 A comprehensive two-hybrid analysis to explore the yeast protein interact...s. 2000 Jan 1;28(1):73-6. *2 The yeast proteome database (YPD) and Caenorhabditis elegans proteome database (WormPD): comprehensive...000 Jan 1;28(1):73-6. *3 A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisia

  18. First identification and characterization of Borrobol-type tephra in the Greenland ice cores

    DEFF Research Database (Denmark)

    Cook, Eliza; Davies, Siwan M.; Guðmundsdóttir, Esther R.

    2018-01-01

    in the ice-cores or that it relates to just one of the ice-core events. A firm correlation cannot be established at present due to their strong geochemical similarities. The older tephra horizon, found within all three ice-cores and dated to 17326 ± 319 a b2k, can be correlated to a known layer within marine....... The older deposit is consistent with BT age estimates derived from Scottish sites, while the younger deposit overlaps with both BT and PT age estimates. We suggest that either the BT in Northern European terrestrial sequences represents an amalgamation of tephra from both of the GI-1e events identified...

  19. Face/core debond fatigue crack growth characterization using the sandwich mixed mode bending specimen

    DEFF Research Database (Denmark)

    Manca, Marcello; Quispitupa, Amilcar; Berggreen, Christian

    2012-01-01

    Face/core fatigue crack growth in foam-cored sandwich composites is examined using the mixed mode bending (MMB) test method. The mixed mode loading at the debond crack tip is controlled by changing the load application point in the MMB test fixture. Sandwich specimens were manufactured using H45...... and H100 PVC foam cores and E-glass/polyester face sheets. All specimens were pre-cracked in order to define a sharp crack front. The static debond fracture toughness for each material configuration was measured at different mode-mixity phase angles. Fatigue tests were performed at 80% of the static...

  20. Impairment of interferon regulatory factor-3 activation by hepatitis C virus core protein basic amino acid region 1.

    Science.gov (United States)

    Inoue, Kazuaki; Tsukiyama-Kohara, Kyoko; Matsuda, Chiho; Yoneyama, Mitsutoshi; Fujita, Takashi; Kuge, Shusuke; Yoshiba, Makoto; Kohara, Michinori

    2012-11-30

    Interferon regulatory factor-3 (IRF-3), a key transcriptional factor in the type I interferon system, is frequently impaired by hepatitis C virus (HCV), in order to establish persistent infection. However, the exact mechanism by which the virus establishes persistent infection has not been fully understood yet. The present study aimed to investigate the effects of various HCV proteins on IRF-3 activation, and elucidate the underlying mechanisms. To achieve this, full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into HepG2 cells. IFN-β induction, plaque formation, and IRF-3 dimerization were elicited by Newcastle disease virus (NDV) infection. The expressions of IRF-3 homodimer and its monomer, Ser386-phosphorylated IRF-3, and HCV core protein were detected by immunofluorescence and western blotting. IFN-β mRNA expression was quantified by real-time PCR (RT-PCR), and IRF-3 activity was measured by the levels of IRF-3 dimerization and phosphorylation, induced by NDV infection or polyriboinosinic:polyribocytidylic acid [poly(I:C)]. Switching of the expression of the complete HCV genome as well as the core proteins, E1, E2, and NS2, suppressed IFN-β mRNA levels and IRF-3 dimerization, induced by NDV infection. Our study revealed a crucial region of the HCV core protein, basic amino acid region 1 (BR1), to inhibit IRF-3 dimerization as well as its phosphorylation induced by NDV infection and poly (I:C), thus interfering with IRF-3 activation. Therefore, our study suggests that rescue of the IRF-3 pathway impairment may be an effective treatment for HCV infection. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Rotavirus NSP2 interferes with the core lattice protein VP2 in initiation of minus-strand synthesis

    International Nuclear Information System (INIS)

    Vende, Patrice; Tortorici, M. Alejandra; Taraporewala, Zenobia F.; Patton, John T.

    2003-01-01

    The rotavirus nonstructural protein NSP2 self-assembles into stable octameric structures that possess nonspecific affinity for single-stranded (ss)RNA and RNA-RNA helix-destabilizing and NTPase activities. Furthermore, NSP2 is a component of replication intermediates with replicase activity and plays a critical role in the packaging and replication of the segmented dsRNA genome of rotavirus. To better understand the function of the protein in genome replication, we examined the effect that purified recombinant NSP2 had on the synthesis of dsRNA by the open core replication system. The results showed that NSP2 inhibited the synthesis of dsRNA from viral mRNA in vitro, in a concentration-dependent manner. The inhibition was overcome by adding increasing amounts of viral mRNA or nonviral ssRNA to the system, indicating that the inhibition was mediated by the nonspecific RNA-binding activity of NSP2. Further analysis revealed that NSP2 interfered with the ability of the open core proteins, GTP, and viral mRNA to form the initiation complex for (-) strand synthesis. Additional experiments indicated that NSP2 did not perturb recognition of viral mRNA by the viral RNA polymerase VP1, but rather interfered with the function of VP2, a protein that is essential for (-) strand initiation and dsRNA synthesis and that forms the T = 1 lattice of the virion core. In contrast to initiation, NSP2 did not inhibit (-) strand elongation. Collectively, the findings provide evidence that the temporal order of interaction of RNA-binding proteins with viral mRNA is a crucial factor impacting the formation of replication intermediates

  2. Characterization of polyclonal antibodies against nonstructural protein 9 from the porcine reproductive and respiratory syndrome virus

    Directory of Open Access Journals (Sweden)

    Mengmeng ZHAO,Juanjuan QIAN,Jiexiong XIE,Tiantian CUI,Songling FENG,Guoqiang WANG,Ruining WANG,Guihong ZHANG

    2016-06-01

    Full Text Available Porcine reproductive and respiratory syndrome (PRRS is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. The nonstructural protein 9 gene, Nsp9, encoding the RNA-dependent RNA polymerase, is generally regarded as fairly conserved when compared to other viral proteins. Antibodies against Nsp9 will be of great importance for the diagnosis and treatment of the causal agent, PRRS virus. A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9. For this purpose, the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits. Antiserum was identified via an indirect ELISA, and then verified based on the ability to react with both naturally and artificially expressed Nsp9. Results of virus neutralization test showed that this antiserum could not neutralize the PRRSV. Nevertheless, this antiserum as a diagnostic core reagent should prove invaluable for further investigations into the mechanism of PRRS pathogenesis.

  3. Electrochemical characterization of core@shell CoFe{sub 2}O{sub 4}/Au composite

    Energy Technology Data Exchange (ETDEWEB)

    Carla, Francesco [' Ugo Schiff' , Universita degli Studi di Firenze, Dipartimento di Chimica (Italy); Campo, Giulio; Sangregorio, Claudio; Caneschi, Andrea; Julian Fernandez, Cesar de; Cabrera, Lourdes I., E-mail: lourisa_cabrera@yahoo.com [Universita degli Studi di Firenze, Laboratorio di Magnetismo Molecolare, INSTM, Dipartimento di Chimica (Italy)

    2013-08-15

    In this paper, we address the synthesis and characterization of the core@shell composite magneto-plasmonic cobalt ferrite-gold (Co-ferrite/Au) nanosystem. The synthesis Co-ferrite/Au nanocomposite is not obvious, hence it was of interest to generate it in a simple straightforward method. Co-ferrite/Au nanocomposite was generated by synthesizing first by thermal decomposition Co-ferrite nanoparticles (NPs). On a second step, ionic gold (Au{sup 3+}) was reduced at the surface of Co-ferrite NPs by ultrasound, to obtain the metallic Au shell. The characterization of the nanomaterial was achieved by microscopy, spectroscopy, and performing magnetic measurements. However, what is attractive about our work is the use of electrochemical techniques as analytical tools. The key technique was cyclic voltammetry, which provided information about the nature and structure of the nanocomposite, allowing us to confirm the core@shell structure.

  4. Protein-energy malnutrition induces an aberrant acute-phase response and modifies the circadian rhythm of core temperature.

    Science.gov (United States)

    Smith, Shari E; Ramos, Rafaela Andrade; Refinetti, Roberto; Farthing, Jonathan P; Paterson, Phyllis G

    2013-08-01

    Protein-energy malnutrition (PEM), present in 12%-19% of stroke patients upon hospital admission, appears to be a detrimental comorbidity factor that impairs functional outcome, but the mechanisms are not fully elucidated. Because ischemic brain injury is highly temperature-sensitive, the objectives of this study were to investigate whether PEM causes sustained changes in temperature that are associated with an inflammatory response. Activity levels were recorded as a possible explanation for the immediate elevation in temperature upon introduction to a low protein diet. Male, Sprague-Dawley rats (7 weeks old) were fed a control diet (18% protein) or a low protein diet (PEM, 2% protein) for either 7 or 28 days. Continuous core temperature recordings from bioelectrical sensor transmitters demonstrated a rapid increase in temperature amplitude, sustained over 28 days, in response to a low protein diet. Daily mean temperature rose transiently by day 2 (p = 0.01), falling to normal by day 4 (p = 0.08), after which mean temperature continually declined as malnutrition progressed. There were no alterations in activity mean (p = 0.3) or amplitude (p = 0.2) that were associated with the early rise in mean temperature. Increased serum alpha-2-macroglobulin (p protein diet had no effect on the signaling pathway of the pro-inflammatory transcription factor, NFκB, in the hippocampus. In conclusion, PEM induces an aberrant and sustained acute-phase response coupled with long-lasting effects on body temperature.

  5. Characterization of the Zebrafish Homolog of Zipper Interacting Protein Kinase

    Directory of Open Access Journals (Sweden)

    Brandon W. Carr

    2014-06-01

    Full Text Available Zipper-interacting protein kinase (ZIPK is a conserved vertebrate-specific regulator of actomyosin contractility in smooth muscle and non-muscle cells. Murine ZIPK has undergone an unusual divergence in sequence and regulation compared to other ZIPK orthologs. In humans, subcellular localization is controlled by phosphorylation of threonines 299 and 300. In contrast, ZIPK subcellular localization in mouse and rat is controlled by interaction with PAR-4. We carried out a comparative biochemical characterization of the regulation of the zebrafish ortholog of ZIPK. Like the human orthologs zebrafish ZIPK undergoes nucleocytoplasmic-shuttling and is abundant in the cytoplasm, unlike the primarily nuclear rat ZIPK. Rat ZIPK, but not human or zebrafish ZIPK, interacts with zebrafish PAR-4. Mutation of the conserved residues required for activation of the mammalian orthologs abrogated activity of the zebrafish ZIPK. In contrast to the human ortholog, mutation of threonine 299 and 300 in the zebrafish ZIPK has no effect on the activity or subcellular localization. Thus, we found that zebrafish ZIPK functions in a manner most similar to the human ZIPK and quite distinct from murine orthologs, yet the regulation of subcellular localization is not conserved.

  6. Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

    Science.gov (United States)

    Ali, Amjad; Nisar, Muhammad; Idrees, Muhammad; Rafique, Shazia; Iqbal, Muhammad

    2015-05-01

    Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the

  7. Synthesis and characterization of ZnO/TiO 2 composite core/shell ...

    Indian Academy of Sciences (India)

    Organic solar cells; ZnO/TiO2 core/shell; nanorod arrays; sol–gel. ... on indium tin oxide (ITO) substrate via a facile sol–gel dip-coating process. Effects of solution pH for ZnO, annealing temperature, growth time and temperature on the ... The optical and electrical properties of the bare TiO2 thin film and core/shell composite ...

  8. Preparation and Characterization of Silicone Liquid Core/Polymer Shell Microcapsules via Internal Phase Separation

    DEFF Research Database (Denmark)

    Gonzalez, Lidia; Kostrzewska, Malgorzata; Ma, Baoguang

    2014-01-01

    Microcapsules with a silicone liquid core surrounded by a polymeric shell were synthesisedthrough the controlled phase separation. The dispersed silicone phase consisted of the shellpolymer PMMA, a good solvent for the PMMA (dichloromethane, DCM) and a poor solvent(methylhydrosiloxane dimethylsil......Microcapsules with a silicone liquid core surrounded by a polymeric shell were synthesisedthrough the controlled phase separation. The dispersed silicone phase consisted of the shellpolymer PMMA, a good solvent for the PMMA (dichloromethane, DCM) and a poor solvent...

  9. The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion.

    Directory of Open Access Journals (Sweden)

    Loïc Etienne

    Full Text Available Hepatitis C virus (HCV assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis.

  10. Biochemical characterization of the small hydrophobic protein of avian metapneumovirus.

    Science.gov (United States)

    Deng, Qiji; Song, Minxun; Demers, Andrew; Weng, Yuejin; Lu, Wuxun; Wang, Dan; Kaushik, Radhey S; Yu, Qingzhong; Li, Feng

    2012-08-01

    Avian metapneumovirus (AMPV) is a paramyxovirus that has three membrane proteins (G, F, and SH). Among them, the SH protein is a small type II integral membrane protein that is incorporated into virions and is only present in certain paramyxoviruses. In the present study, we show that the AMPV SH protein is modified by N-linked glycans and can be released into the extracellular environment. Furthermore, we demonstrate that glycosylated AMPV SH proteins form homodimers through cysteine-mediated disulfide bonds, which has not been reported previously for SH proteins of paramyxoviruses. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Characterization of a translation inhibitory protein from Luffa aegyptiaca.

    Science.gov (United States)

    Ramakrishnan, S; Enghlid, J J; Bryant, H L; Xu, F J

    1989-04-28

    A protein with a molecular weight of about 30,000 was purified from the seeds of Luffa aegyptiaca. This protein inhibited cell free translation at pM concentrations. In spite of functional similarity to other ribosomal inhibitory proteins, the NH2-terminal analysis did not show any significant homology. Competitive inhibition studies indicate no immunological crossreactivity between the inhibitory protein from Luffa aegyptiaca, pokeweed antiviral protein (PAP) and recombinant ricin A chain. Chemical linkage of the protein to a monoclonal antibody reactive to transferrin receptor resulted in a highly cytotoxic conjugate.

  12. Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus.

    Science.gov (United States)

    Desai, Tanay M; Marin, Mariana; Sood, Chetan; Shi, Jiong; Nawaz, Fatima; Aiken, Christopher; Melikyan, Gregory B

    2015-10-29

    HIV-1 Vpr is recruited into virions during assembly and appears to remain associated with the viral core after the reverse transcription and uncoating steps of entry. This feature has prompted the use of fluorescently labeled Vpr to visualize viral particles and to follow trafficking of post-fusion HIV-1 cores in the cytoplasm. Here, we tracked single pseudovirus entry and fusion and observed that fluorescently tagged Vpr gradually dissociates from post-fusion viral cores over the course of several minutes and accumulates in the nucleus. Kinetics measurements showed that fluorescent Vpr released from the cores very rapidly entered the cell nucleus. More than 10,000 Vpr molecules can be delivered into the cell nucleus within 45 min of infection by HIV-1 particles pseudotyped with the avian sarcoma and leukosis virus envelope glycoprotein. The fraction of Vpr from cell-bound viruses that accumulated in the nucleus was proportional to the extent of virus-cell fusion and was fully blocked by viral fusion inhibitors. Entry of virus-derived Vpr into the nucleus occurred independently of envelope glycoproteins or target cells. Fluorescence correlation spectroscopy revealed two forms of nuclear Vpr-monomers and very large complexes, likely involving host factors. The kinetics of viral Vpr entering the nucleus after fusion was not affected by point mutations in the capsid protein that alter the stability of the viral core. The independence of Vpr shedding of capsid stability and its relatively rapid dissociation from post-fusion cores suggest that this process may precede capsid uncoating, which appears to occur on a slower time scale. Our results thus demonstrate that a bulk of fluorescently labeled Vpr incorporated into HIV-1 particles is released shortly after fusion. Future studies will address the question whether the quick and efficient nuclear delivery of Vpr derived from incoming viruses can regulate subsequent steps of HIV-1 infection.

  13. Purification, crystallization and preliminary X-ray diffraction study of human ribosomal protein L10 core domain

    International Nuclear Information System (INIS)

    Nishimura, Mitsuhiro; Kaminishi, Tatsuya; Kawazoe, Masahito; Shirouzu, Mikako; Takemoto, Chie; Yokoyama, Shigeyuki; Tanaka, Akiko; Sugano, Sumio; Yoshida, Takuya; Ohkubo, Tadayasu; Kobayashi, Yuji

    2007-01-01

    A truncated variant of human ribosomal protien L10 was prepared and crystallized. Diffraction data were collected to 2.5 Å resolution. Eukaryotic ribosomal protein L10 is an essential component of the large ribosomal subunit, which organizes the architecture of the aminoacyl-tRNA binding site. The human L10 protein is also called the QM protein and consists of 214 amino-acid residues. For crystallization, the L10 core domain (L10CD, Phe34–Glu182) was recombinantly expressed in Escherichia coli and purified to homogeneity. A hexagonal crystal of L10CD was obtained by the sitting-drop vapour-diffusion method. The L10CD crystal diffracted to 2.5 Å resolution and belongs to space group P3 1 21 or P3 2 21

  14. Preliminary stratigraphic and petrologic characterization of core samples from USW-G1, Yucca Mountain, Nevada

    International Nuclear Information System (INIS)

    Waters, A.C.; Carroll, P.R.

    1981-11-01

    Tuffs of the Nevada Test Site are currently under investigation to determine their potential for long-term storage of radioactive waste. As part of this program, hole USW-G1 was drilled to a depth of 6000 ft below the surface, in the central part of the Yucca Mountain area, Nevada Test Site, Nevada. Petrographic study of the USW-G1 core is presented in this report and shows the tuffs (which generally were variably welded ash flows) are partly recrystallized to a variety of secondary minerals. The important alteration products are zeolites (heulandite, clinoptilolite, mordenite and analcime), smectite clays with minor interstratified illite, albite, micas, potassium feldspar, and various forms of silica. Iijima's zeolite zones I through IV of burial metamorphism can be recognized in the core. Zeolites are first observed at about the 1300-ft depth, and the high-temperature boundary of zeolite stability in this core occurs at about 4350 ft. Analcime persists, either metastably or as a retrograde mineral, deeper in the core. The oxidation state of Fe-Ti oxide minerals, through most of the core, increases as the degree of welding decreases, but towards the bottom of the hole, reducing conditions generally prevail. Four stratigraphic units transected by the core may be potentially favorable sites for a waste repository. These four units, in order of increasing depth in the core, are (1) the lower cooling unit of the Topopah Spring Member, (2) cooling unit II of the Bullfrog Member, (3) the upper part of the Tram tuff, and (4) the Lithic-rich tuff

  15. Preliminary stratigraphic and petrologic characterization of core samples from USW-G1, Yucca Mountain, Nevada

    Energy Technology Data Exchange (ETDEWEB)

    Waters, A.C.; Carroll, P.R. (eds.)

    1981-11-01

    Tuffs of the Nevada Test Site are currently under investigation to determine their potential for long-term storage of radioactive waste. As part of this program, hole USW-G1 was drilled to a depth of 6000 ft below the surface, in the central part of the Yucca Mountain area, Nevada Test Site, Nevada. Petrographic study of the USW-G1 core is presented in this report and shows the tuffs (which generally were variably welded ash flows) are partly recrystallized to a variety of secondary minerals. The important alteration products are zeolites (heulandite, clinoptilolite, mordenite and analcime), smectite clays with minor interstratified illite, albite, micas, potassium feldspar, and various forms of silica. Iijima`s zeolite zones I through IV of burial metamorphism can be recognized in the core. Zeolites are first observed at about the 1300-ft depth, and the high-temperature boundary of zeolite stability in this core occurs at about 4350 ft. Analcime persists, either metastably or as a retrograde mineral, deeper in the core. The oxidation state of Fe-Ti oxide minerals, through most of the core, increases as the degree of welding decreases, but towards the bottom of the hole, reducing conditions generally prevail. Four stratigraphic units transected by the core may be potentially favorable sites for a waste repository. These four units, in order of increasing depth in the core, are (1) the lower cooling unit of the Topopah Spring Member, (2) cooling unit II of the Bullfrog Member, (3) the upper part of the Tram tuff, and (4) the Lithic-rich tuff.

  16. Static light scattering to characterize membrane proteins in detergent solution

    NARCIS (Netherlands)

    Slotboom, Dirk Jan; Duurkens, Ria H.; Olieman, Kees; Erkens, Guus B.

    2008-01-01

    Determination of the oligomeric state or the subunit stoichiometry of integral membrane proteins in detergent solution is notoriously difficult, because the amount of detergent (and lipid) associated with the proteins is usually not known. Only two classical methods (sedimentation equilibrium

  17. Synthesis and characterization of recombinant abductin-based proteins.

    Science.gov (United States)

    Su, Renay S-C; Renner, Julie N; Liu, Julie C

    2013-12-09

    Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II β-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.

  18. Electron spin resonance characterization of radical components in irradiated black pepper skin and core

    International Nuclear Information System (INIS)

    Yamaoki, Rumi; Kimura, Shojiro; Ohta, Masatoshi

    2011-01-01

    Characteristics of free radical components of irradiated black pepper fruit (skin) and the pepper seed (core) were analyzed using electron spin resonance. A weak signal near g=2.005 was observed in black pepper before irradiation. Complex spectra near g=2.005 with three lines (the skin) or seven lines (the core) were observed in irradiated black pepper (both end line width; ca. 6.8 mT). The spectral intensities decreased considerably at 30 days after irradiation, and continued to decrease steadily thereafter. The spectra simulated on the basis of the content and the stability of radical components derived from plant constituents, including fiber, starch, polyphenol, mono- and disaccharide, were in good agreement with the observed spectra. Analysis showed that the signal intensities derived from fiber in the skin for an absorbed dose were higher, and the rates of decrease were lower, than that in the core. In particular, the cellulose radical component in the skin was highly stable. - Highlights: → We identified the radical components in irradiated black pepper skin and core. → The ESR spectra near g=2.005 with 3-7 lines were emerged after irradiation. → Spectra simulated basing on the content and the stability of radical from the plant constituents. → Cellulose radical component in black pepper skin was highly stable. → Single signal near g=2.005 was the most stable in black pepper core.

  19. Synthesis and Characterization of Core-Shell Acrylate Based Latex and Study of Its Reactive Blends

    Directory of Open Access Journals (Sweden)

    Ying Nie

    2008-03-01

    Full Text Available Techniques in resin blending are simple and efficient method for improving the properties of polymers, and have been used widely in polymer modification field. However, polymer latex blends such as the combination of latexes, especially the latexes with water-soluble polymers, were rarely reported. Here, we report a core-shell composite latex synthesized using methyl methacrylate (MMA, butyl acrylate (BA, 2-ethylhexyl acrylate (EHA and glycidyl methacrylate (GMA as monomers and ammonium persulfate and sodium bisulfite redox system as the initiator. Two stages seeded semi-continuous emulsion polymerization were employed for constructing a core-shell structure with P(MMA-co-BA component as the core and P(EHA-co-GMA component as the shell. Results of Transmission Electron Microscopy (TEM and Dynamics Light Scattering (DLS tests confirmed that the particles obtained are indeed possessing a desired core-shell structural character. Stable reactive latex blends were prepared by adding the latex with waterborne melamine-formaldehyde resin (MF or urea-formaldehyde resin (UF. It was found that the glass transition temperature, the mechanical strength and the hygroscopic property of films cast from the latex blends present marked enhancements under higher thermal treatment temperature. It was revealed that the physical properties of chemically reactive latexes with core-shell structure could be altered via the change of crosslinking density both from the addition of crosslinkers and the thermal treatment.

  20. Global Structural Flexibility of Metalloproteins Regulates Reactivity of Transition Metal Ion in the Protein Core: An Experimental Study Using Thiol-subtilisin as a Model Protein.

    Science.gov (United States)

    Matsuo, Takashi; Kono, Takamasa; Shobu, Isamu; Ishida, Masaya; Gonda, Katsuya; Hirota, Shun

    2018-02-21

    The functions of metal-containing proteins (metalloproteins) are determined by the reactivities of transition metal ions at their active sites. Because protein macromolecular structures have several molecular degrees of freedom, global structural flexibility may also regulate the properties of metalloproteins. However, the influence of this factor has not been fully delineated in mechanistic studies of metalloproteins. Accordingly, we have investigated the relationship between global protein flexibility and the characteristics of a transition metal ion in the protein core using thiol-subtilisin (tSTL) with a Cys-coordinated Cu 2+ ion as a model system. Although tSTL has two Ca 2+ -binding sites, the Ca 2+ -binding status hardly affects its secondary structure. Nevertheless, guanidinium-induced denaturation and amide H/D exchange indicated the increase in the structural flexibility of tSTL by the removal of bound Ca 2+ ions. Electron paramagnetic resonance and absorption spectral changes have revealed that the protein flexibility determines the characteristics of a Cu 2+ ion in tSTL. Therefore, global protein flexibility should be recognized as an important factor that regulates the properties of metalloproteins. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Synthesis and characterization of fluorinated magnetic core-shell nanoparticles for inhibition of insulin amyloid fibril formation

    International Nuclear Information System (INIS)

    Skaat, Hadas; Margel, Shlomo; Belfort, Georges

    2009-01-01

    Maghemite (γ-Fe 2 O 3 ) magnetic nanoparticles of 15.0 ± 2.1 nm are formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Uniform magnetic γ-Fe 2 O 3 /poly (2,2,3,3,4,4,4-heptafluorobutyl acrylate) (γ-Fe 2 O 3 /PHFBA) core-shell nanoparticles are prepared by emulsion polymerization of the fluorinated monomer 2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA) in the presence of the maghemite nanoparticles. The kinetics of the insulin fibrillation process in the absence and in the presence of the γ-Fe 2 O 3 /PHFBA core-shell nanoparticles are elucidated. A significant direct slow transition from α-helix to β-sheets during insulin fibril formation is observed in the presence of the γ-Fe 2 O 3 /PHFBA nanoparticles. This is in contradiction to our previous manuscript, which illustrated that the γ-Fe 2 O 3 core nanoparticles do not affect the kinetics of the formation of the insulin fibrils, and to other previous publications that describe acceleration of the fibrillation process by using various types of nanoparticles. These core-shell nanoparticles may therefore be also useful for the inhibition of conformational changes of other amyloidogenic proteins that lead to neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, mad cow and prion diseases.

  2. Synthesis and characterization of multifunctional silica core-shell nanocomposites with magnetic and fluorescent functionalities

    International Nuclear Information System (INIS)

    Ma Zhiya; Dosev, Dosi; Nichkova, Mikaela; Dumas, Randy K.; Gee, Shirley J.; Hammock, Bruce D.; Liu Kai; Kennedy, Ian M.

    2009-01-01

    Multifunctional core-shell nanocomposites with a magnetic core and a silica shell doped with lanthanide chelate have been prepared by a simple method. First, citric acid-modified magnetite nanoparticles were synthesized by a chemical coprecipitation method. Then the magnetite nanoparticles were coated with silica shells doped with terbium (Tb 3+ ) complex by a modified Stoeber method based on hydrolyzing and condensation of tetraethyl orthosilicate (TEOS) and a silane precursor. These multifunctional nanocomposites are potentially useful in a variety of biological areas such as bio-imaging, bio-labeling and bioassays because they can be simultaneously manipulated with an external magnetic field and exhibit unique phosphorescence properties.

  3. Phosphine-free synthesis and characterization of type-II ZnSe/CdS core-shell quantum dots

    Science.gov (United States)

    Ghasemzadeh, Roghayyeh; Armanmehr, Mohammad Hasan; Abedi, Mohammad; Fateh, Davood Sadeghi; Bahreini, Zaker

    2018-01-01

    A phosphine-free route for synthesis of type-II ZnSe/CdS core-shell quantum dots, using green, low cost and environmentally friendly reagents and phosphine-free solvents such as 1-octadecene (ODE) and liquid paraffin has been reported. Hot-injection technique has been used for the synthesis of ZnSe core quantum dots. The CdS shell quantum dots prepared by reaction of CdO precursor and S powder in 1-octadecene (ODE). The ZnSe/CdS core-shell quantum dots were synthesized via successive ion layer adsorption and reaction (SILAR) technique. The characterization of produced quantum dots were performed by absorption and fluorescence spectroscopy, X-ray diffraction (XRD) and energy dispersive X-ray spectroscopy (EDX) and transmission electron microscopy (TEM). The results showed the formation of type-II ZnSe/CdS core-shell quantum dots with FWHM 32 nm and uniform size distribution.

  4. Alcohol Induced Hepatic Degeneration in a Hepatitis C Virus Core Protein Transgenic Mouse Model

    Directory of Open Access Journals (Sweden)

    Dong-Hyung Noh

    2014-03-01

    Full Text Available Hepatitis C virus (HCV has become a major public health issue. It is prevalent in most countries. HCV infection frequently begins without clinical symptoms, before progressing to persistent viremia, chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC in the majority of patients (70% to 80%. Alcohol is an independent cofactor that accelerates the development of HCC in chronic hepatitis C patients. The purpose of the current study was to evaluate ethanol-induced hepatic changes in HCV core-Tg mice and mutant core Tg mice. Wild type (NTG, core wild-Tg mice (TG-K, mutant core 116-Tg mice (TG-116 and mutant core 99-Tg mice (TG-99 were used in this investigation. All groups were given drinking water with 10% ethanol and 5% sucrose for 13 weeks. To observe liver morphological changes, we performed histopathological and immunohistochemical examinations. Histopathologically, NTG, TG-K and TG-116 mice showed moderate centrilobular necrosis, while severe centrilobular necrosis and hepatocyte dissociation were observed in TG-99 mice with increasing lymphocyte infiltration and piecemeal necrosis. In all groups, a small amount of collagen fiber was found, principally in portal areas. None of the mice were found to have myofibroblasts based on immunohistochemical staining specific for α-SMA. CYP2E1-positive cells were clearly detected in the centrilobular area in all groups. In the TG-99 mice, we also observed cells positive for CK8/18, TGF-β1 and phosphorylated (p-Smad2/3 and p21 around the necrotic hepatocytes in the centrilobular area (p < 0.01. Based on our data, alcohol intake induced piecemeal necrosis and hepatocyte dissociation in the TG-99 mice. These phenomena involved activation of the TGF-β1/p-Smad2/3/p21 signaling pathway in hepatocytes. Data from this study will be useful for elucidating the association between alcohol intake and HCV infection.

  5. Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein

    OpenAIRE

    Theo Luiz Ferraz de Souza; Sheila Maria Barbosa de Lima; Vanessa L. de Azevedo Braga; David S. Peabody; Davis Fernandes Ferreira; M. Lucia Bianconi; Andre Marco de Oliveira Gomes; Jerson Lima Silva; Andréa Cheble de Oliveira

    2016-01-01

    Background Hepatitis C virus (HCV) core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124) is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs) in vitro. The specific...

  6. Gold-Pluronic core-shell nanoparticles: synthesis, characterization and biological evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Simon, Timea; Boca, Sanda [Babes-Bolyai University, Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute on Bio-Nano-Sciences and Faculty of Physics (Romania); Biro, Dominic [Sapientia University, Department of Mechanical Engineering, Faculty of Technical and Human Sciences (Romania); Baldeck, Patrice [Universite Joseph Fourier and CNRS, Laboratoire Interdisciplinaire de Physique, UMR 5588, CNRS (France); Astilean, Simion, E-mail: simion.astilean@phys.ubbcluj.ro [Babes-Bolyai University, Nanobiophotonics and Laser Microspectroscopy Center, Interdisciplinary Research Institute on Bio-Nano-Sciences and Faculty of Physics (Romania)

    2013-04-15

    This study presents the synthesis of gold-Pluronic core-shell nanoparticles by a two-step method and investigates their biological impact on cancer cells, specifically nanoparticle internalization and cytotoxicity. Uniform, 9-10-nm-sized, hydrophobic gold nanoparticles were synthesized in organic phase by reducing gold salt with oleylamine, after which oleylamine-protected gold nanoparticles were phase-transferred into aqueous medium using Pluronic F127 block copolymer, resulting in gold-Pluronic core-shell nanoparticles with a mean hydrodynamic diameter of {approx}35 nm. The formation and phase-transfer of gold nanoparticles were analyzed by UV-Vis absorption spectroscopy, transmission electron microscopy, and dynamic light scattering. The obtained gold-Pluronic core-shell nanoparticles proved to be highly stable in salted solution. Cytotoxicity tests showed no modification of cellular viability in the presence of properly purified particles. Furthermore, dark-field cellular imaging demonstrated that gold-Pluronic nanoparticles were able to be efficiently uptaken by cells, being internalized through nonspecific endocytosis. The high stability, proven biocompatibility, and imaging properties of gold-Pluronic core-shell nanoparticles hold promise for relevant intracellular applications, with such a design providing the feasibility to combine all multiple functionalities in one nanoparticle for simultaneous detection and imaging.

  7. Three-dimensional vortex analysis and aeroacoustic source characterization of jet core breakdown

    NARCIS (Netherlands)

    Violato, D.; Scarano, F.

    2013-01-01

    The three-dimensional behavior of jet core breakdown is investigated with experiments conducted on a free water jet at Re = 5000 by time-resolved tomographic particle image velocimetry (TR-TOMO PIV). The investigated domain encompasses the range between 0 and 10 jet diameters. The characteristic

  8. Molecular Characterization of Methanogenic Communities in Core Sediments of the Dajiuhu Peatland, Central China

    Science.gov (United States)

    Wang, R.; Wang, H.

    2017-12-01

    Methane (CH4) is an important greenhouse gas with a global warming potential 22 times greater than carbon dioxide. Large amounts of CH4 can be produced and released by methanogenesis in peatland ecosystems, which make peatland ecosystems play an important role in mediating global climate change. Here we report the abundance and distribution of methanogenic communities and their correlation with physicochemical parameters along two sediment cores in the Dajiuhu Peatland via quantitative PCR, clone library construction of functional genes and statistical analysis. Uncultured Group and Fen Cluster were found to be the dominant methanogens at the upper part of the cores, and Rice and Related Rice Cluster became dominant in the bottom of the cores. Quantitative PCR showed that abundances of methanogenic communities ranged from 104 to 106 copies/ng DNA throughout the cores. Canonical Correlation Analysis (CCA) indicated that dissolved oxygen (DO) (P=0.046, F=1.4) was the main factor significantly controlling methanogenic communities. Our results enhance the understanding of the compositions and variations of methanogenic communities vertically and greatly help us to further investigate process of microbial methanogenesis in Dajiuhu Peatland.

  9. One-pot synthesis and characterization of rhodamine derivative-loaded magnetic core-shell nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Jin, E-mail: jzhang@eng.uwo.ca; Li Jiaxin [University of Western Ontario, Department of Chemical and Biochemical Engineering (Canada); Razavi, Fereidoon S. [Brock University, Department of Physics (Canada); Mumin, Abdul Md. [University of Western Ontario, Department of Chemical and Biochemical Engineering (Canada)

    2011-05-15

    A new method to produce elaborate nanostructure with magnetic and fluorescent properties in one entity is reported in this article. Magnetite (Fe{sub 3}O{sub 4}) coated with fluorescent silica (SiO{sub 2}) shell was produced through the one-pot reaction, in which one reactor was utilized to realize the synthesis of superparamagnetic core of Fe{sub 3}O{sub 4}, the formation of SiO{sub 2} coating through the condensation and polymerization of tetraethylorthosilicate (TEOS), and the encapsulation of tetramethyl rhodamine isothiocyanate-dextran (TRITC-dextran) within silica shell. Transmission electron microscopy (TEM), energy dispersive X-ray (EDX) analysis, and X-ray diffraction (XRD) were carried out to investigate the core-shell structure. The magnetic core of the core-shell nanoparticles is 60 {+-} 10 nm in diameter. The thickness of the fluorescent SiO{sub 2} shell is estimated at 15 {+-} 5 nm. In addition, the fluorescent signal of the SiO{sub 2} shell has been detected by the laser confocal scanning microscopy (LCSM) with emission wavelength ({lambda}{sub em}) at 566 nm. In addition, the magnetic properties of TRITC-dextran loaded silica-coating iron oxide nanoparticles (Fe{sub 3}O{sub 4}-SiO{sub 2} NPs) were studied. The hysteresis loop of the core-shell NPs measured at room temperature shows that the saturation magnetization (M{sub s}) is not reached even at the field of 70 kOe (7T). Meanwhile, the very low coercivity (H{sub c}) and remanent magnetization (M{sub r}) are 0.375 kOe and 6.6 emu/g, respectively, at room temperature. It indicates that the core-shell particles have the superparamagnetic properties. The measured blocking temperature (T{sub B}) of the TRITC-dextran loaded Fe{sub 3}O{sub 4}-SiO{sub 2} NPs is about 122.5 K. It is expected that the multifunctional core-shell nanoparticles can be used in bio-imaging.

  10. SCOWLP: a web-based database for detailed characterization and visualization of protein interfaces

    Directory of Open Access Journals (Sweden)

    Schroeder Michael

    2006-03-01

    Full Text Available Abstract Background Currently there is a strong need for methods that help to obtain an accurate description of protein interfaces in order to be able to understand the principles that govern molecular recognition and protein function. Many of the recent efforts to computationally identify and characterize protein networks extract protein interaction information at atomic resolution from the PDB. However, they pay none or little attention to small protein ligands and solvent. They are key components and mediators of protein interactions and fundamental for a complete description of protein interfaces. Interactome profiling requires the development of computational tools to extract and analyze protein-protein, protein-ligand and detailed solvent interaction information from the PDB in an automatic and comparative fashion. Adding this information to the existing one on protein-protein interactions will allow us to better understand protein interaction networks and protein function. Description SCOWLP (Structural Characterization Of Water, Ligands and Proteins is a user-friendly and publicly accessible web-based relational database for detailed characterization and visualization of the PDB protein interfaces. The SCOWLP database includes proteins, peptidic-ligands and interface water molecules as descriptors of protein interfaces. It contains currently 74,907 protein interfaces and 2,093,976 residue-residue interactions formed by 60,664 structural units (protein domains and peptidic-ligands and their interacting solvent. The SCOWLP web-server allows detailed structural analysis and comparisons of protein interfaces at atomic level by text query of PDB codes and/or by navigating a SCOP-based tree. It includes a visualization tool to interactively display the interfaces and label interacting residues and interface solvent by atomic physicochemical properties. SCOWLP is automatically updated with every SCOP release. Conclusion SCOWLP enriches

  11. Proteomic characterization of the human centrosome by protein correlation profiling

    DEFF Research Database (Denmark)

    Andersen, Jens S; Wilkinson, Christopher J; Mayor, Thibault

    2003-01-01

    chromosomes between dividing cells. Despite the importance of this organelle to cell biology and more than 100 years of study, many aspects of its function remain enigmatic and its structure and composition are still largely unknown. We performed a mass-spectrometry-based proteomic analysis of human...... centrosomes in the interphase of the cell cycle by quantitatively profiling hundreds of proteins across several centrifugation fractions. True centrosomal proteins were revealed by both correlation with already known centrosomal proteins and in vivo localization. We identified and validated 23 novel...... components and identified 41 likely candidates as well as the vast majority of the known centrosomal proteins in a large background of nonspecific proteins. Protein correlation profiling permits the analysis of any multiprotein complex that can be enriched by fractionation but not purified to homogeneity....

  12. Engineering and Characterization of a Superfolder Green Fluorescent Protein

    International Nuclear Information System (INIS)

    Pedelacq, J.; Cabantous, S.; Tran, T.; Terwilliger, T.; Waldo, G.

    2006-01-01

    Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP

  13. Characterization of core-shell GaAs/AlGaAs nanowire heterostructures using advanced electron microscopy

    International Nuclear Information System (INIS)

    Tambe, M J; Gradecak, S; Allard, L F

    2010-01-01

    To explore the unique properties of the nanoscale, advanced fabrication and characterization techniques are required. Specifically analyses in two orthogonal directions, plan-view and cross-section, were used to prove the core-shell morphology of GaAs/AlGaAs nanowires and determine their cross-section to be hexagonal. High-resolution transmission electron microscopy and high angle annular dark field scanning transmission electron microscopy confirmed the core-shell interface to be defect-free, coherent, and sharp ( 0.9 Ga 0.1 As uniformly along the length of the nanowire. These results demonstrate the power of electron microscopy to aid the development of semiconductor nanotechnology.

  14. Synthesis and characterization of silver-copper core-shell nanoparticles using polyol method for antimicrobial agent

    Science.gov (United States)

    Hikmah, N.; Idrus, N. F.; Jai, J.; Hadi, A.

    2016-06-01

    Silver and copper nanoparticles are well-known as the good antimicrobial agent. The nano-size of particles influences in enhancing the antimicrobial activity. This paper discusses the effect of molarity on the microstructure and morphology of silver-copper core-shell nanoparticles prepared by a polyol method. In this study, silver-copper nanoparticles are synthesized through the green approach of polyol method using ethylene glycol (EG) as green solvent and reductant, and polyoxyethylene-(80)-sorbitan monooleate (Tween 80) as a nontoxic stabilizer. The phase and morphology of silver-copper nanoparticles are characterized by X-ray diffraction (XRD) and Field emission scanning electron microscope (FESEM) and Transmission electron microscope (TEM). The results XRD confirm the pure crystalline of silver and copper nanoparticles with face-centered cubic (FCC) structure. FESEM and TEM analysis confirm the existence of Ag and Cu nanoparticles in core-shell shape.

  15. Purification and characterization of a soybean cell wall protein

    International Nuclear Information System (INIS)

    San Francisco, S.; Tierney, M.L.

    1989-01-01

    Plant cell wall composition is thought to reflect cellular responses to developmental and environmental signals. We have purified a 33 kDa protein from cell wall extracts of soybean seedlings which is most abundant in extracts from the hook region of the hypocotyl and is rich in proline and hydroxypyroline. In vivo 3 H-proline labelling of hypocotyl tissues indicates that the hook tissue is the predominant site for synthesis of this protein. In unwounded hook, label is incorporated into a 33 kDa protein, while in wounded hook this and additional proteins rich in proline are synthesized. Similarly treated cell wall extracts analyzed by Western blot analysis, using a polyclonal antibody raised against this 33kD protein, showed that the 33 kDa protein is most abundant in cell wall extracts from the hook region of unwounded seedlings and does not increase upon wounding. An immunologically related 35kD protein is also apparent in extracts from wounded hooks and appears to co-migrate with one of the labelled proteins extractable from this tissue. These data indicate that there are two related, proline-rich cell wall proteins in the hook region of soybean seedlings, one of which (33 kDa) is prominent during seedling development and another (35 kDa) which is wound inducible

  16. Preparation and characterization of core-shell electrodes for application in gel electrolyte-based dye-sensitized solar cells

    International Nuclear Information System (INIS)

    Avellaneda, Cesar O.; Goncalves, Agnaldo D.; Benedetti, Joao E.; Nogueira, Ana F.

    2010-01-01

    Core-shell electrodes based on TiO 2 covered with different oxides were prepared and characterized. These electrodes were applied in gel electrolyte-based dye-sensitized solar cells (DSSC). The TiO 2 electrodes were prepared from TiO 2 powder (P25 Degussa) and coated with thin layers of Al 2 O 3 , MgO, Nb 2 O 5 , and SrTiO 3 prepared by the sol-gel method. The core-shell electrodes were characterized by X-ray diffraction, scanning electron microscopy and atomic force microscopy measurements. J-V curves in the dark and under standard AM 1.5 conditions and photovoltage decay measurements under open-circuit conditions were carried out in order to evaluate the influence of the oxide layer on the charge recombination dynamics and on the device's performance. The results indicated an improvement in the conversion efficiency as a result of an increase in the open circuit voltage. The photovoltage decay curves under open-circuit conditions showed that the core-shell electrodes provide longer electron lifetime values compared to uncoated TiO 2 electrodes, corroborating with a minimization in the recombination losses at the nanoparticle surface/electrolyte interface. This is the first time that a study has been applied to DSSC based on gel polymer electrolyte. The optimum performance was achieved by solar cells based on TiO 2 /MgO core-shell electrodes: fill factor of ∼0.60, short-circuit current density J sc of 12 mA cm -2 , open-circuit voltage V oc of 0.78 V and overall energy conversion efficiency of ∼5% (under illumination of 100 mW cm -2 ).

  17. Characterization of structural proteins of hirame rhabdovirus, HRV

    Science.gov (United States)

    Nishizawa, Toyohiko; Yoshimizu, Mamoru; Winton, James; Ahne, Winfried; Kimura, Takahisa

    1991-01-01

    Structural proteins of hirame rhabdovirus (HRV) were analyzed by SDS-polyacrylarnide gel electrophoresis, western blotting, 2-dimensional gel electrophoresis, and Triton X-100 treatment. Purified HRV virions were composed of: polymerase (L), glycoprotein (G), nucleoprotein (N), and 2 matrix proteins (M1 and M2). Based upon their relative mobilities, the estimated molecular weights of the proteins were: L, 156 KDa; G, 68 KDa; N, 46.4 KDa; M1, 26.4 KDa; and M2, 19.9 KDa. The electrophorehc pattern formed by the structural proteins of HRV was clearly different from that formed by pike fry rhabdovirus, spring viremia of carp virus, eel virus of America, and eel virus European X which belong to the Vesiculovirus genus; however, it resembled the pattern formed by structural proteins of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) which are members of the Lyssavirus genus. Among HRV, IHNV, and VHSV, differences were observed in the relative mobilities of the G, N, M1, and M2 proteins. Western blot analysis revealed that the G. N, and M2 proteins of HRV shared antigenic determinants with IHNV and VHSV, but not with any of the 4 fish vesiculoviruses tested. Cross-reactions between the M1 proteins of HRV, IHNV, or VHSV were not detected in this assay. Two-dimensional gel electrophoresis was used to show that HRV differed from IHNV or VHSV in the isoelectric point (PI) of the M1 and M2 proteins. In this system, 2 forms of the M1 protein of HRV and IHNV were observed.These subspecies of M1 had the same relative mobility but different p1 values. Treatment of purified virions with 2% Triton X-100 in Tris buffer containing NaCl removed the G, M1, and M2 proteins of IHNV, but HRV virions were more stable under these conditions.

  18. Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

    Science.gov (United States)

    Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

    2014-12-11

    The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

  19. Multiple TPR motifs characterize the Fanconi anemia FANCG protein.

    NARCIS (Netherlands)

    Blom, E.; Vrugt, van der H.J.; Vries, de Y.; Winter, de J.P.; Arwert, F.; Joenje, H.

    2004-01-01

    The genome protection pathway that is defective in patients with Fanconi anemia (FA) is controlled by at least eight genes, including BRCA2. A key step in the pathway involves the monoubiquitylation of FANCD2, which critically depends on a multi-subunit nuclear 'core complex' of at least six FANC

  20. Geochemical and mineralogical characterization of the Eagle Ford Shale: Results from the USGS Gulf Coast #1 West Woodway core

    Science.gov (United States)

    Birdwell, Justin E.; Boehlke, Adam; Paxton, Stanley T.; Whidden, Katherine J.; Pearson, Ofori N.

    2017-01-01

    The Eagle Ford shale is a major continuous oil and gas resource play in southcentral Texas and a source for other oil accumulations in the East Texas Basin. As part of the U.S. Geological Survey’s (USGS) petroleum system assessment and research efforts, a coring program to obtain several immature, shallow cores from near the outcrop belt in central Texas has been undertaken. The first of these cores, USGS Gulf Coast #1 West Woodway, was collected near Waco, Texas, in September 2015 and has undergone extensive geochemical and mineralogical characterization using routine methods to ascertain variations in the lithologies and chemofacies present in the Eagle Ford at this locale. Approximately 270 ft of core was examined for this study, focusing on the Eagle Ford Group interval between the overlying Austin Chalk and underlying Buda Limestone (~20 ft of each). Based on previous work to identify the stratigraphy of the Eagle Ford Group in the Waco area and elsewhere (Liro et al., 1994; Robison, 1997; Ratcliffe et al., 2012; Boling and Dworkin, 2015; Fairbanks et al., 2016, and references therein), several lithological units were expected to be present, including the Pepper Shale (or Woodbine), the Lake Waco Formation (or Lower Eagle Ford, including the Bluebonnet, Cloice, and Bouldin or Flaggy Cloice members), and the South Bosque Member (Upper Eagle Ford). The results presented here indicate that there are three major chemofacies present in the cored interval, which are generally consistent with previous descriptions of the Eagle Ford Group in this area. The relatively high-resolution sampling (every two ft above the Buda, 432.8 ft depth, and below the Austin Chalk, 163.5 ft depth) provides great detail in terms of geochemical and mineralogical properties supplementing previous work on immature Eagle Ford Shale near the outcrop belt.

  1. Characterization of viral proteins of Oryctes baculovirus and comparison between two geographical isolates.

    Science.gov (United States)

    Mohan, K S; Gopinathan, K P

    1989-01-01

    Bacilliform Oryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infected Oryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reported Oryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

  2. A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

    DEFF Research Database (Denmark)

    Larsen, Martin; Skottrup, Peter; Enghild, Jan Johannes

    Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... graphite powder micro-columns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and would be ideal for proteomics studies and verification of correct glycosylation of recombinant glycoproteins....

  3. Enhancement of curcumin water dispersibility and antioxidant activity using core-shell protein-polysaccharide nanoparticles.

    Science.gov (United States)

    Huang, Xiaoxia; Huang, Xulin; Gong, Yushi; Xiao, Hang; McClements, David Julian; Hu, Kun

    2016-09-01

    Curcumin has strong antioxidant activity, but poor water-solubility and chemical stability, which limits its utilization as a nutraceutical in many applications. Previously, we developed a core-shell (zein-pectin) nanoparticle delivery system with high curcumin loading efficiency, high particle yield, and good water dispersibility. However, this system was unstable to aggregation around neutral pH and moderate ionic strengths due to weakening of electrostatic repulsion between nanoparticles. In the current study, we used a combination of alginate (high charge density) and pectin (low charge density) to form the shell around zein nanoparticles. Replacement of 30% of pectin with alginate greatly improved aggregation stability at pH 5 to 7 and at high ionic strengths (2000mM NaCl). Curcumin encapsulated within these core-shell nanoparticles exhibited higher antioxidant and radical scavenging activities than curcumin solubilized in ethanol solutions as determined by Fe (III) reducing power, 1, 1-Diphenyl-2-picrylhydrazyl free radical (DPPH·), and 2, 2'-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid radical cation (ABTS· + ) scavenging analysis. These core-shell nanoparticles may be useful for incorporating chemically unstable hydrophobic nutraceuticals such as curcumin into functional foods, dietary supplements, and pharmaceuticals. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Characterization of a cocaine binding protein in human placenta

    International Nuclear Information System (INIS)

    Ahmed, M.S.; Zhou, D.H.; Maulik, D.; Eldefrawi, M.E.

    1990-01-01

    [ 3 H]-Cocaine binding sites are identified in human placental villus tissue plasma membranes. These binding sites are associated with a protein and show saturable and specific binding of [ 3 H]-cocaine with a high affinity site of 170 fmole/mg protein. The binding is lost with pretreatment with trypsin or heat. The membrane bound protein is solubilized with the detergent 3-(3-cholamidopropyl)dimethyl-ammonio-1-propane sulphonate (CHAPS) with retention of its saturable and specific binding of [ 3 H]-cocaine. The detergent-protein complex migrates on a sepharose CL-6B gel chromatography column as a protein with an apparent molecular weight of 75,900. The protein has an S 20,w value of 5.1. The binding of this protein to norcocaine, pseudococaine, nomifensine, imipramine, desipramine, amphetamine and dopamine indicates that it shares some, but not all, the properties of the brain cocaine receptor. The physiologic significance of this protein in human placenta is currently unclear

  5. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    WINTEC

    GRAVY, Grand Average Hydropathy. structure of protein (3D coordinates data). The 3D structure of AFPs Q01758 and P05140 were gener- ated by homology modelling using Esypred34 server. The similar 3D structures (for the AFPs Q01758 and. P05140 sequences) in the Protein Data bank. (www.rscb.org) were identified ...

  6. Identification and characterization of stable membrane protein complexes

    NARCIS (Netherlands)

    Spelbrink, R.E.J.

    2007-01-01

    Many membrane proteins exist as oligomers. Such oligomers play an important role in a broad variety of cellular processes such as ion transport, energy transduction, osmosensing and cell wall synthesis. We developed an electrophoresis-based method of identifying oligomeric membrane proteins that are

  7. Characterization of chicken riboflavin carrier protein gene structure ...

    Indian Academy of Sciences (India)

    The chicken riboflavin carrier protein (RCP) is an estrogen induced egg yolk and white protein. Eggs from hens which have a splice mutation in RCP gene fail to hatch, indicating an absolute requirement of RCP for the transport of riboflavin to the oocyte. In order to understand the mechanism of regulation of this gene by ...

  8. Characterization of the "Escherichia Coli" Acyl Carrier Protein Phosphodiesterase

    Science.gov (United States)

    Thomas, Jacob

    2009-01-01

    Acyl carrier protein (ACP) is a small essential protein that functions as a carrier of the acyl intermediates of fatty acid synthesis. ACP requires the posttranslational attachment of a 4'phosphopantetheine functional group, derived from CoA, in order to perform its metabolic function. A Mn[superscript 2+] dependent enzymatic activity that removes…

  9. Biophysical characterization of membrane protein-small molecule interactions

    NARCIS (Netherlands)

    Chen, Dan

    2015-01-01

    Membrane proteins are account for up to two thirds of known druggable targets. Traditionally, new drugs against this class of proteins have been discovered through HTS. However, not all GPCRs are amenable to traditional screening methods. Recently, fragment-based drug discovery (FBDD) has emerged as

  10. Physicochemical characterization of fish protein adlayers with bacteria repelling properties

    DEFF Research Database (Denmark)

    Meyer, R. L.; Arpanaei, A.; Pillai, S.

    2013-01-01

    Materials coated with aqueous fish protein extracts can reduce bacterial adhesion, but the mechanism behind the observed effect is not fully understood. In this study we explore the physicochemical properties of fish muscle protein adlayers on four substrates: gold, stainless steel, polystyrene...

  11. Characterization of a Porous Carbon Material Functionalized with Cobalt-Oxide/Cobalt Core-Shell Nanoparticles for Lithium Ion Battery Electrodes

    KAUST Repository

    Anjum, Dalaver H.; Rasul, Shahid; Roldan-Gutierrez, Manuel A.; Da Costa, Pedro M. F. J.

    2016-01-01

    A nanoporous carbon (C) material, functionalized with Cobalt-Oxide/Cobalt (CoO/Co) core-shell nanoparticles (NPs), was structurally and chemically characterized with transmission electron microcopy (TEM) while its electrochemical response

  12. Simulant - water experiments to characterize the debris bed formed in severe core melt accidents

    International Nuclear Information System (INIS)

    Mathai, Amala M.; Anandan, J.; Sharma, Anil Kumar; Murthy, S.S.; Malarvizhi, B.; Lydia, G.; Das, Sanjay Kumar; Nashine, B.K.; Selvaraj, P.

    2015-01-01

    Molten Fuel Coolant Interaction (WO) and debris bed configuration on the core catcher plate assumes importance in assessing the Post Accident Heat Removal (PARR) of a heat generating debris bed. The key factors affecting the coolability of the debris bed are the bed porosity, morphology of the fragmented particles, degree of spreading/heaping of the debris on the core catcher and the fraction of lump formed. Experiments are conducted to understand the fragmentation kinetics and subsequent debris bed formation of molten woods metal in water at interface temperatures near the spontaneous nucleation temperature of water. Morphology of the debris particles is investigated to understand the fragmentation mechanisms involved. The spreading behavior of the debris on the catcher plate and the particle size distribution are presented for 5 kg and 10 kg melt inventories. Porosity of the undisturbed bed on the catcher plate is evaluated using a LASER sensor technique. (author)

  13. Performance Characterization of Multi-threaded Graph Processing Applications on Intel Many-Integrated-Core Architecture

    OpenAIRE

    Liu, Xu; Chen, Langshi; Firoz, Jesun S.; Qiu, Judy; Jiang, Lei

    2017-01-01

    Intel Xeon Phi many-integrated-core (MIC) architectures usher in a new era of terascale integration. Among emerging killer applications, parallel graph processing has been a critical technique to analyze connected data. In this paper, we empirically evaluate various computing platforms including an Intel Xeon E5 CPU, a Nvidia Geforce GTX1070 GPU and an Xeon Phi 7210 processor codenamed Knights Landing (KNL) in the domain of parallel graph processing. We show that the KNL gains encouraging per...

  14. Gold nanorod@iron oxide core-shell heterostructures: synthesis, characterization, and photocatalytic performance.

    Science.gov (United States)

    Li, Yue; Zhao, Junwei; You, Wenlong; Cheng, Danhong; Ni, Weihai

    2017-03-17

    Iron oxides are directly coated on the surface of cetyl-trimethylammonium bromide (CTAB)-capped gold nanorods (AuNRs) in aqueous solutions at room temperature, which results in AuNR@Fe 2 O 3 , AuNR@Fe 3 O 4 , and AuNR@Fe 2 O 3 @Fe 3 O 4 core-shell heterostructures. The iron oxide shells are uniform, smooth, with characteristic porous structure, and their thickness can be readily tuned. The shell formation is highly dependent on the reaction parameters including pH and CTAB concentration. The Fe 2 O 3 shell is amorphous and exhibits nearly zero remanence and coercivity, while the Fe 3 O 4 shell is ferromagnetic with a low saturation magnetization of about 0.5 emu g -1 due to its low crystallinity and the porous structure. At elevated temperatures achieved by plasmonic heating of the Au core, the Fe 2 O 3 shell transforms from amorphous to γ-Fe 2 O 3 and α-Fe 2 O 3 phases, while the Fe 3 O 4 phase disappears because of the oxidation of Fe 2+ . A 1.4-fold increase of photocatalytic performance is observed due to the plasmonic resonance provided by the Au core. The photocatalytic efficiency of Fe 3 O 4 is about 1.7-fold higher than Fe 2 O 3 as more surface defects are present on the Fe 3 O 4 shell, promoting the adsorption and activation of reagents on the surface during the catalytic reactions. This approach can be readily extended to other nanostructures including Au spherical nanoparticles and nanostars. These highly uniform and multifunctional core-shell heterostructures can be of great potential in a variety of energy, magnetic, and environment applications.

  15. Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7

    DEFF Research Database (Denmark)

    Galli, Andrea; Scheel, Troels K H; Prentoe, Jannick C

    2013-01-01

    Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (c......LDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain...... JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A...

  16. Characterization of core-shell nanoparticles by small angle neutron scattering

    Energy Technology Data Exchange (ETDEWEB)

    Strunz, P. [Nuclear Physics Institute (NPI), Rez (Czech Republic); Research Centre Rez, Rez (Czech Republic); Mukherji, D. [TU Braunschweig, IfW, Braunschweig (Germany); Pigozzi, G. [ETH Zuerich, Laboratory for Nanometallurgy, Zuerich (Switzerland); Gilles, R. [TU Muenchen, ZWE FRM-II, Garching (Germany); Geue, T. [PSI and ETH Zuerich, Laboratory for Neutron Scattering, Villigen PSI (Switzerland); Pranzas, K. [GKSS Research Centre, Institute of Materials Research, Geesthacht (Germany)

    2007-08-15

    The Ni{sub 3}Si-type nanoparticles dispersed in a mixture of H{sub 2}O/D{sub 2}O were characterised by SANS using the contrast variation method. The existence of a core-shell structure in the nanoparticles with a Ni{sub 3}Si(Al) core and amorphous SiO{sub x} shell is confirmed by the SANS measurements. The nanoparticles were produced by extracting precipitates from a bulk Ni-13.3Si-2Al (at. %) alloy using electrochemical phase separation technique and were pre-characterised by X-ray diffraction and transmission electron microscopy. By comparing the precipitate morphology in the Ni-Si-Al alloy with the extracted nanoparticles in the SANS measurements, it is clearly established that the precipitates shape and size are unaffected by the extraction process and that the amorphous shell forms on top of the particle core. However, the present measurement could not confirm or exclude the presence of H atoms in the shell structure. (orig.)

  17. Synthesis and characterization of core-shell Fe3O4-gold-chitosan nanostructure

    Directory of Open Access Journals (Sweden)

    Salehizadeh Hossein

    2012-01-01

    Full Text Available Abstract Background Fe3O4-gold-chitosan core-shell nanostructure can be used in biotechnological and biomedical applications such as magnetic bioseparation, water and wastewater treatment, biodetection and bioimaging, drug delivery, and cancer treatment. Results Magnetite nanoparticles with an average size of 9.8 nm in diameter were synthesized using the chemical co-precipitation method. A gold-coated Fe3O4 monotonous core-shell nanostructure was produced with an average size of 15 nm in diameter by glucose reduction of Au3+ which is then stabilized with a chitosan cross linked by formaldehyde. The results of analyses with X-ray diffraction (XRD, Fourier Transformed Infrared Spectroscopy (FTIR, Transmission Electron Microscopy (TEM, and Atomic Force Microscopy (AFM indicated that the nanoparticles were regularly shaped, and agglomerate-free, with a narrow size distribution. Conclusions A rapid, mild method for synthesizing Fe3O4-gold nanoparticles using chitosan was investigated. A magnetic core-shell-chitosan nanocomposite, including both the supermagnetic properties of iron oxide and the optical characteristics of colloidal gold nanoparticles, was synthesized.

  18. Piecewise linear approximation: application to control rod step counting in a nuclear reactor core and image contours characterization

    International Nuclear Information System (INIS)

    Kaoutar, M.

    1986-09-01

    After a survey of main algorithms for piecewise linear approximation, a new method is suggested. It consists of two stages: a sequential detection stage and an optimization stage, which derives from general dynamic clustering principle. It is applied to control rod step counting in a nuclear reactor core and images contours characterization. Another version of our method is presented. Its originality cames from the variability of the line segments number during iterations. A comparative study is made by comparing the results of the proposed method with of another methods already existing thereby it attests the efficiency and reliability of our method [fr

  19. Pokemon siRNA Delivery Mediated by RGD-Modified HBV Core Protein Suppressed the Growth of Hepatocellular Carcinoma.

    Science.gov (United States)

    Kong, Jing; Liu, Xiaoping; Jia, Jianbo; Wu, Jinsheng; Wu, Ning; Chen, Jun; Fang, Fang

    2015-10-01

    Hepatocellular carcinoma (HCC) is a deadly human malignant tumor that is among the most common cancers in the world, especially in Asia. Hepatitis B virus (HBV) infection has been well established as a high risk factor for hepatic malignance. Studies have shown that Pokemon is a master oncogene for HCC growth, suggesting it as an ideal therapeutic target. However, efficient delivery system is still lacking for Pokemon targeting treatment. In this study, we used core proteins of HBV, which is modified with RGD peptides, to construct a biomimetic vector for the delivery of Pokemon siRNAs (namely, RGD-HBc-Pokemon siRNA). Quantitative PCR and Western blot assays revealed that RGD-HBc-Pokemon siRNA possessed the highest efficiency of Pokemon suppression in HCC cells. In vitro experiments further indicated that RGD-HBc-Pokemon-siRNA exerted a higher tumor suppressor activity on HCC cell lines, evidenced by reduced proliferation and attenuated invasiveness, than Pokemon-siRNA or RGD-HBc alone. Finally, animal studies demonstrated that RGD-HBc-Pokemon siRNA suppressed the growth of HCC xenografts in mice by a greater extent than Pokemon-siRNA or RGD-HBc alone. Based on the above results, Pokemon siRNA delivery mediated by RGD-modified HBV core protein was shown to be an effective strategy of HCC gene therapy.

  20. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

    Science.gov (United States)

    Milbradt, Jens; Sonntag, Eric; Wagner, Sabrina; Strojan, Hanife; Wangen, Christina; Lenac Rovis, Tihana; Lisnic, Berislav; Jonjic, Stipan; Sticht, Heinrich; Britt, William J; Schlötzer-Schrehardt, Ursula; Marschall, Manfred

    2018-01-13

    The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  1. Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97

    Directory of Open Access Journals (Sweden)

    Jens Milbradt

    2018-01-01

    Full Text Available The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

  2. Hepatitis C virus core protein targets 4E-BP1 expression and phosphorylation and potentiates Myc-induced liver carcinogenesis in transgenic mice.

    Science.gov (United States)

    Abdallah, Cosette; Lejamtel, Charlène; Benzoubir, Nassima; Battaglia, Serena; Sidahmed-Adrar, Nazha; Desterke, Christophe; Lemasson, Matthieu; Rosenberg, Arielle R; Samuel, Didier; Bréchot, Christian; Pflieger, Delphine; Le Naour, François; Bourgeade, Marie-Françoise

    2017-08-22

    Hepatitis C virus (HCV) is a leading cause of liver diseases including the development of hepatocellular carcinoma (HCC). Particularly, core protein has been involved in HCV-related liver pathologies. However, the impact of HCV core on signaling pathways supporting the genesis of HCC remains largely elusive. To decipher the host cell signaling pathways involved in the oncogenic potential of HCV core, a global quantitative phosphoproteomic approach was carried out. This study shed light on novel differentially phosphorylated proteins, in particular several components involved in translation. Among the eukaryotic initiation factors that govern the translational machinery, 4E-BP1 represents a master regulator of protein synthesis that is associated with the development and progression of cancers due to its ability to increase protein expression of oncogenic pathways. Enhanced levels of 4E-BP1 in non-modified and phosphorylated forms were validated in human hepatoma cells and in mouse primary hepatocytes expressing HCV core, in the livers of HCV core transgenic mice as well as in HCV-infected human primary hepatocytes. The contribution of HCV core in carcinogenesis and the status of 4E-BP1 expression and phosphorylation were studied in HCV core/Myc double transgenic mice. HCV core increased the levels of 4E-BP1 expression and phosphorylation and significantly accelerated the onset of Myc-induced tumorigenesis in these double transgenic mice. These results reveal a novel function of HCV core in liver carcinogenesis potentiation. They position 4E-BP1 as a tumor-specific target of HCV core and support the involvement of the 4E-BP1/eIF4E axis in hepatocarcinogenesis.

  3. Characterization of a Fiber-Coupled 36-Core 3-Mode Photonic Lantern Spatial Multiplexer

    DEFF Research Database (Denmark)

    Rommel, Simon; Mendinueta, José Manuel Delgado; Klaus, Werner

    2017-01-01

    A fiber-coupled 108-port photonic lantern spatial-MUX is characterized with a spatially-diverse optical vector network analyzer. Insertion loss, mode-dependent losses, and time response are measured, showing significant mode mixing at a fiber splice.......A fiber-coupled 108-port photonic lantern spatial-MUX is characterized with a spatially-diverse optical vector network analyzer. Insertion loss, mode-dependent losses, and time response are measured, showing significant mode mixing at a fiber splice....

  4. Identification of odorant binding proteins and chemosensory proteins in Microplitis mediator as well as functional characterization of chemosensory protein 3.

    Directory of Open Access Journals (Sweden)

    Yong Peng

    Full Text Available Odorant binding proteins (OBPs and chemosensory proteins (CSPs play important roles in transporting semiochemicals through the sensillar lymph to olfactory receptors in insect antennae. In the present study, twenty OBPs and three CSPs were identified from the antennal transcriptome of Microplitis mediator. Ten OBPs (MmedOBP11-20 and two CSPs (MmedCSP2-3 were newly identified. The expression patterns of these new genes in olfactory and non-olfactory tissues were investigated by real-time quantitative PCR (qPCR measurement. The results indicated that MmedOBP14, MmedOBP18, MmedCSP2 and MmedCSP3 were primarily expressed in antennae suggesting potential olfactory roles in M. mediator. However, other genes including MmedOBP11-13, 15-17, 19-20 appeared to be expressed at higher levels in body parts than in antennae. Focusing on the functional characterization of MmedCSP3, immunocytochemistry and fluorescent competitive binding assays were conducted indoors. It was found that MmedCSP3 was specifically located in the sensillum lymph of olfactory sensilla basiconca type 2. The recombinant MmedCSP3 could bind several types of host insects odors and plant volatiles. Interestingly, three sex pheromone components of Noctuidae insects, cis-11-hexadecenyl aldehyde (Z11-16: Ald, cis-11-hexadecanol (Z11-16: OH, and trans-11-tetradecenyl acetate (E11-14: Ac, showed high binding affinities (Ki = 17.24-18.77 μM. The MmedCSP3 may be involved in locating host insects. Our data provide a base for further investigating the physiological roles of OBPs and CSPs in M. mediator, and extend the function of MmedCSP3 in chemoreception of M. mediator.

  5. Cloning and characterization of an insecticidal crystal protein gene ...

    Indian Academy of Sciences (India)

    Unknown

    The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene from Bacillus .... diet or by topical application on food substrates as .... has very high similarity (99.74%) at DNA level with.

  6. In silico characterization of antifreeze proteins using computational ...

    Indian Academy of Sciences (India)

    WINTEC

    structure. SOSUI server predicts one transmembrane region in winter flounder fish and atlantic cod and ..... result in a better interaction with water. The secon- ... structure of antifreeze protein P05140 (using PDB template 2AFP_A). The 10 ...

  7. Characterization of DNA-binding proteins from pea mitochondria

    DEFF Research Database (Denmark)

    Hatzack, F.A.; Dombrowski, S.; Brennicke, A.

    1998-01-01

    We studied transcription initiation in the mitochondria of higher plants, with particular respect to promoter structures. Conserved elements of these promoters have been successfully identified by in vitro transcription systems in different species, whereas the involved protein components are still...

  8. Preparation, characterization and infrared emissivity study of helical polyurethane-SiO2 core-shell composite

    International Nuclear Information System (INIS)

    Wang Zhiqiang; Zhou Yuming; Yao Qingzhao; Sun Yanqing

    2009-01-01

    Helical polyurethane-SiO 2 (HPU-SiO 2 ) core-shell composite was prepared after surface modification of SiO 2 nanoparticles. HPU-SiO 2 was characterized by Fourier-transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), ultraviolet (UV) spectroscopy, X-ray diffraction (XRD) and transmission electron microscopy (TEM). The results indicate that the helical polyurethane has been successfully grafted onto the surfaces of the modified SiO 2 . HPU-SiO 2 composite exhibits clearly core-shell structure. The ultraviolet absorption and crystallizability of HPU-SiO 2 are changed due to the shell of helical polyurethane, which possesses regular single-handed conformation and inter-chain hydrogen bonds. The infrared emissivity of HPU-SiO 2 was also investigated. The result indicates that the interfacial interactions between organic shell and inorganic core induce the infrared emissivity value being reduced from 0.781 for SiO 2 to 0.503 for HPU-SiO 2 .

  9. Synthesis and Characterization of Ti-Phenyl at SiO2 Core-Shell Nanoparticles Catalyst

    International Nuclear Information System (INIS)

    Syamsi Aini; Jon Efendi; Syamsi Aini; Jon Efendi

    2012-01-01

    This study highlights the potential use of Ti-Phenyl at SiO 2 core-shell nanoparticles as heterogeneous catalysis in oxidation reaction. The Ti-Phenyl at SiO 2 was synthesized by reduction of TiCl 4 and diazonium salt with sodium borohydride to produce phenyl titanium nanoparticles (Ti-Phenyl), followed by the silica shell coating using tetraethyl orthosilicate (TEOS). The Ti-Phenyl at SiO 2 nanoparticles were characterized by Fourier transform infrared (FTIR) spectrometer, diffuse reflectance (DR) UV-visible spectrometer, thermogravimetric analyzer (TGA), X-ray diffraction (XRD) spectrometer, field emission scanning electron microscope (FESEM) and transmission electron microscope (TEM). The core-shell size of Ti-Phenyl at SiO 2 was in the range of 40 to 100 nm with its core composed with an agglomeration of Ti-Phenyl. The Ti-Phenyl at SiO 2 was active as a catalyst in the liquid phase epoxidation of 1-octene with aqueous hydrogen peroxide as an oxidant. (author)

  10. Sensitivity of physics parameters for establishment of a burned CANDU full-core model for decommissioning waste characterization

    International Nuclear Information System (INIS)

    Cho, Dong-Keun; Sun, Gwang-Min; Choi, Jongwon; Hwang, Dong-Hyun; Hwang, Tae-Won; Yang, Ho-Yeon; Park, Dong-Hwan

    2011-01-01

    The sensitivity of parameters related with reactor physics on the source terms of decommissioning wastes from a CANDU reactor was investigated in order to find a viable, simplified burned core model of a Monte Carlo simulation for decommissioning waste characterization. First, a sensitivity study was performed for the level of nuclide consideration in an irradiated fuel and implicit geometry modeling, the effects of side structural components of the core, and structural supporters for reactive devices. The overall effects for computation memory, calculation time, and accuracy were then investigated with a full-core model. From the results, it was revealed that the level of nuclide consideration and geometry homogenization are not important factors when the ratio of macroscopic neutron absorption cross section (MNAC) relative to a total value exceeded 0.95. The most important factor affecting the neutron flux of the pressure tube was shown to be the structural supporters for reactivity devices, showing an 10% difference. Finally, it was concluded that a bundle-average homogeneous model considering a MNAC of 0.95, which is the simplest model in this study, could be a viable approximate model, with about 25% lower computation memory, 40% faster simulation time, and reasonable engineering accuracy compared with a model with an explicit geometry employing an MNAC of 0.99. (author)

  11. Synthesis, characterization and nitrite ion sensing performance of reclaimable composite samples through a core-shell structure

    Science.gov (United States)

    Cui, Xiao; Yuqing, Zhao; Cui, Jiantao; Zheng, Qian; Bo, Wang

    2018-02-01

    The following paper reported and discussed a nitrite ion optical sensing platform based on a core-shell structure, using superamagnetic nanoparticles as the core, a silica molecular sieve MCM-41 as the shell and two rhodamine derivatives as probe, respectively. This superamagnetic core made this sensing platform reclaimable after finishing nitrite ion sensing procedure. This sensing platform was carefully characterized by means of electron microscopy images, porous structure analysis, magnetic response, IR spectra and thermal stability analysis. Detailed analysis suggested that the emission of these composite samples was quenchable by nitrite ion, showing emission turn off effect. A static sensing mechanism based on an additive reaction between chemosensors and nitrite ion was proposed. These composite samples followed Demas quenching equation against different nitrite ion concentrations. Limit of detection value was obtained as low as 0.4 μM. It was found that, after being quenched by nitrite ion, these composite samples could be reclaimed and recovered by sulphamic acid, confirming their recyclability.

  12. Identification, characterization and mycotoxigenic ability of Alternaria spp. causing core rot of apple fruit in Greece.

    Science.gov (United States)

    Ntasiou, Panagiota; Myresiotis, Charalampos; Konstantinou, Sotiris; Papadopoulou-Mourkidou, Euphemia; Karaoglanidis, George S

    2015-03-16

    Alternaria core rot is a major postharvest disease of apple fruit in several countries of the world, including Greece. The study was conducted aiming to identify the disease causal agents at species level, investigate the aggressiveness of Alternaria spp. isolates and the susceptibility of different apple varieties and determine the mycotoxigenic potential of Alternaria spp. isolates from apple fruit. Seventy-five Alternaria spp. isolates obtained from apple fruit showing core rot symptoms were identified as either Alternaria tenuissima or Alternaria arborescens at frequencies of 89.3 and 11.7%, respectively, based on the sequence of endopolygalacturonase (EndoPG) gene. Artificial inoculations of fruit of 4 different varieties (Fuji, Golden Delicious, Granny Smith and Red Delicious) and incubation at two different temperatures (2 and 25°C) showed that fruit of Fuji variety were the most susceptible and fruit of Golden Delicious the most resistant to both pathogens. In addition, the production of 3 mycotoxins, alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) was investigated in 30 isolates of both species. Mycotoxin determination was conducted both in vitro, on artificial nutrient medium and in vivo on artificially inoculated apple fruit, using a high performance liquid chromatography with diode array detector (HPLC-DAD). The results showed that most of the isolates of both species were able to produce all the 3 metabolites both in vivo and in vitro. On apple fruit A. tenuissima isolates produced more AOH than A. arborescens isolates, whereas the latter produced more TEN than the former. Such results indicate that Alternaria core rot represents a major threat of apple fruit production not only due to quantitative yield losses but also for qualitative deterioration of apple by-products. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Extraction and characterization of proteins from banana (Musa Sapientum L) flower and evaluation of antimicrobial activities.

    Science.gov (United States)

    Sitthiya, Kewalee; Devkota, Lavaraj; Sadiq, Muhammad Bilal; Anal, Anil Kumar

    2018-02-01

    Ultrasonic assisted alkaline extraction of protein from banana flower was optimized using response surface methodology. The extracted proteins were characterized by Fourier transform infrared spectroscopy and molecular weight distribution was determined by gel electrophoresis. The maximum protein yield of 252.25 mg/g was obtained under optimized extraction conditions: temperature 50 °C, 30 min extraction time and 1 M NaOH concentration. The alkaline extraction produced a significantly high protein yield compared to enzymatic extraction of banana flower. Chemical finger printing of proteins showed the presence of tyrosine, tryptophan and amide bonds in extracted protein. Alkaline and pepsin assisted extracted banana flower proteins showed characteristic bands at 40 and 10 kDA, respectively. The extracted proteins showed antibacterial effects against both gram positive and gram negative bacteria. The high protein content and antimicrobial activity indicate the potential applications of banana flower in the food and feed industry.

  14. Characterization of interfacial waves and pressure drop in horizontal oil-water core-annular flows

    Science.gov (United States)

    Tripathi, Sumit; Tabor, Rico F.; Singh, Ramesh; Bhattacharya, Amitabh

    2017-08-01

    We study the transportation of highly viscous furnace-oil in a horizontal pipe as core-annular flow (CAF) using experiments. Pressure drop and high-speed images of the fully developed CAF are recorded for a wide range of flow rate combinations. The height profiles (with respect to the centerline of the pipe) of the upper and lower interfaces of the core are obtained using a high-speed camera and image analysis. Time series of the interface height are used to calculate the average holdup of the oil phase, speed of the interface, and the power spectra of the interface profile. We find that the ratio of the effective velocity of the annular fluid to the core velocity, α , shows a large scatter. Using the average value of this ratio (α =0.74 ) yields a good estimate of the measured holdup for the whole range of flow rate ratios, mainly due to the low sensitivity of the holdup ratio to the velocity ratio. Dimensional analysis implies that, if the thickness of the annular fluid is much smaller than the pipe radius, then, for the given range of parameters in our experiments, the non-dimensional interface shape, as well as the non-dimensional wall shear stress, can depend only on the shear Reynolds number and the velocity ratio. Our experimental data show that, for both lower and upper interfaces, the normalized power spectrum of the interface height has a strong dependence on the shear Reynolds number. Specifically, for low shear Reynolds numbers, interfacial modes with large wavelengths dominate, while, for large shear Reynolds numbers, interfacial modes with small wavelengths dominate. Normalized variance of the interface height is higher at lower shear Reynolds numbers and tends to a constant with increasing shear Reynolds number. Surprisingly, our experimental data also show that the effective wall shear stress is, to a large extent, proportional to the square of the core velocity. Using the implied scalings for the holdup ratio and wall shear stress, we can derive

  15. Synthesis and characterization of fluorinated magnetic core-shell nanoparticles for inhibition of insulin amyloid fibril formation

    Energy Technology Data Exchange (ETDEWEB)

    Skaat, Hadas; Margel, Shlomo [Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900 (Israel); Belfort, Georges [Howard P Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180 (United States)], E-mail: ch348@mail.biu.ac.il, E-mail: belfog@rpi.edu, E-mail: Shlomo.margel@mail.biu.ac.il

    2009-06-03

    Maghemite ({gamma}-Fe{sub 2}O{sub 3}) magnetic nanoparticles of 15.0 {+-} 2.1 nm are formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Uniform magnetic {gamma}-Fe{sub 2}O{sub 3}/poly (2,2,3,3,4,4,4-heptafluorobutyl acrylate) ({gamma}-Fe{sub 2}O{sub 3}/PHFBA) core-shell nanoparticles are prepared by emulsion polymerization of the fluorinated monomer 2,2,3,3,4,4,4-heptafluorobutyl acrylate (HFBA) in the presence of the maghemite nanoparticles. The kinetics of the insulin fibrillation process in the absence and in the presence of the {gamma}-Fe{sub 2}O{sub 3}/PHFBA core-shell nanoparticles are elucidated. A significant direct slow transition from {alpha}-helix to {beta}-sheets during insulin fibril formation is observed in the presence of the {gamma}-Fe{sub 2}O{sub 3}/PHFBA nanoparticles. This is in contradiction to our previous manuscript, which illustrated that the {gamma}-Fe{sub 2}O{sub 3} core nanoparticles do not affect the kinetics of the formation of the insulin fibrils, and to other previous publications that describe acceleration of the fibrillation process by using various types of nanoparticles. These core-shell nanoparticles may therefore be also useful for the inhibition of conformational changes of other amyloidogenic proteins that lead to neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's, mad cow and prion diseases.

  16. Characterization of cap binding proteins associated with the nucleus

    International Nuclear Information System (INIS)

    Patzelt, E.

    1986-04-01

    Eucaryotic mRNAs a carry 7-methylguanosine triphosphate residue (called cap structure) at their 5' terminus. The cap plays an important role in RNA recognition. Cap binding proteins (CBP) of HeLa cells were identified by photoaffinity labelling using the cap analogue γ-( 32 P)-(4-(benzoyl-phenyl)methylamido)-7-methylguanosine-5'-triphosphate (BP-m 7 GTP). Photoreaction of this cap analogue with HeLa cell initiation factors resulted in specific labelling of two polypeptides of Msub(r) 37000 and 26000. The latter was also labelled in crude initiation factors prepared from reticulocytes and is identical to the cap binding protein CBP I previously identified. These cap binding proteins were also affinity labelled in poliovirus infected cell extracts. Photoaffinity reaction with BP-m 7 GTP of whole HeLa cell homogenate showed three additional polypeptides with Msub(r) 120000, 89000 and 80000. These cap binding proteins were found to be associated with the nucleus and are therefore referred to as nuclear cap binding proteins, i.e. NCBP 1, NCBP 2 and NCBP 3. They were also present in splicing extracts. Photoaffinity labelling in these nuclear extracts was differentially inhibited by various cap analogues and capped mRNAs. Affinity chromatography on immobilized globin mRNA led to a partial separation of the three nuclear cap binding proteins. Chromatography on m 7 GTP-Sepharose resulted in a specific binding of NCBP 3. The different behaviour of the cap binding proteins suggests that they are functionally distinct and that they might be involved in different processes requiring cap recognition. (Author)

  17. Analytical methods to characterize heterogeneous raw material for thermal spray process: cored wire Inconel 625

    Science.gov (United States)

    Lindner, T.; Bonebeau, S.; Drehmann, R.; Grund, T.; Pawlowski, L.; Lampke, T.

    2016-03-01

    In wire arc spraying, the raw material needs to exhibit sufficient formability and ductility in order to be processed. By using an electrically conductive, metallic sheath, it is also possible to handle non-conductive and/or brittle materials such as ceramics. In comparison to massive wire, a cored wire has a heterogeneous material distribution. Due to this fact and the complex thermodynamic processes during wire arc spraying, it is very difficult to predict the resulting chemical composition in the coating with sufficient accuracy. An Inconel 625 cored wire was used to investigate this issue. In a comparative study, the analytical results of the raw material were compared to arc sprayed coatings and droplets, which were remelted in an arc furnace under argon atmosphere. Energy-dispersive X-ray spectroscopy (EDX) and X-ray fluorescence (XRF) analysis were used to determine the chemical composition. The phase determination was performed by X-ray diffraction (XRD). The results were related to the manufacturer specifications and evaluated in respect to differences in the chemical composition. The comparison between the feedstock powder, the remelted droplets and the thermally sprayed coatings allows to evaluate the influence of the processing methods on the resulting chemical and phase composition.

  18. Identification and preliminary characterization of protein-cysteine farnesyltransferase

    International Nuclear Information System (INIS)

    Manne, V.; Roberts, D.; Tobin, A.; O'Rourke, E.; Barbacid, M.; De Virgilio, M.; Meyers, C.; Ahmed, N.; Kurz, B.; Resh, M.; Kung, Hsiang-Fu

    1990-01-01

    Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2 Valendash19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an onocogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, the authors describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway

  19. Characterization of a DUF820 family protein Alr3200 of the ...

    Indian Academy of Sciences (India)

    2016-10-14

    Oct 14, 2016 ... Supplementary materials pertaining to this article are available on the ... paper deals with the characterization of Alr3200 protein .... Studies are currently underway to ... 2002) methods also matched well with the predicted.

  20. Identification and characterization of cytosolic Hansenula polymorpha proteins belonging to the Hsp70 protein family

    NARCIS (Netherlands)

    Titorenko, Vladimir I.; Evers, Melchior E.; Diesel, Andre; Samyn, Bart; Beeumen, Josef van; Roggenkamp, Rainer; Kiel, Jan A.K.W.; Klei, Ida J. van der; Veenhuis, Marten

    We have isolated two members of the Hsp70 protein family from the yeast Hansenula polymorpha using affinity chromatography. Both proteins were located in the cytoplasm. One of these, designated Hsp72, was inducible in nature (e.g. by heat shock). The second protein (designated Hsc74) was

  1. Characterization of known protein complexes using k-connectivity and other topological measures

    Science.gov (United States)

    Gallagher, Suzanne R; Goldberg, Debra S

    2015-01-01

    Many protein complexes are densely packed, so proteins within complexes often interact with several other proteins in the complex. Steric constraints prevent most proteins from simultaneously binding more than a handful of other proteins, regardless of the number of proteins in the complex. Because of this, as complex size increases, several measures of the complex decrease within protein-protein interaction networks. However, k-connectivity, the number of vertices or edges that need to be removed in order to disconnect a graph, may be consistently high for protein complexes. The property of k-connectivity has been little used previously in the investigation of protein-protein interactions. To understand the discriminative power of k-connectivity and other topological measures for identifying unknown protein complexes, we characterized these properties in known Saccharomyces cerevisiae protein complexes in networks generated both from highly accurate X-ray crystallography experiments which give an accurate model of each complex, and also as the complexes appear in high-throughput yeast 2-hybrid studies in which new complexes may be discovered. We also computed these properties for appropriate random subgraphs.We found that clustering coefficient, mutual clustering coefficient, and k-connectivity are better indicators of known protein complexes than edge density, degree, or betweenness. This suggests new directions for future protein complex-finding algorithms. PMID:26913183

  2. Characterization of binding of N'-nitrosonornicotine to protein

    International Nuclear Information System (INIS)

    Hughes, M.F.

    1986-01-01

    The NADPH-dependent activation of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN) to a reactive intermediate which binds covalently to protein was assessed using male Sprague-Dawley rat liver and lung microsomes. The NADPH-dependent covalent binding of [ 14 C]NNN to liver and lung microsomes was linear with time up to 90 and 45 min, respectively and was also linear with protein concentrations up to 3.0 and 2.0 mg/ml, respectively. The apparent K/sub m/ and V/sub max/ of the NADPH-dependent binding to liver microsomes were determined from the initial velocities. Addition of the thiols glutathione, cystein, N-acetylcysteine or 2-mercapthoethanol significantly decreased the non-NADPH-dependent binding to liver microsomal protein, but did not affect the NADPH-dependent binding. Glutathione was required in order to observe any NADPH-dependent binding to lung microsomal protein. In lung microsomes, SKF-525A significantly decreased the NADPH-dependent binding by 79%. Replacement of an air atmosphere with N 2 or CO:O 2 (8:2) significantly decreased the NADPH-dependent binding of [ 14 C]NNN to liver microsomal protein by 40% or 27% respectively. Extensive covalent binding of [ 14 C]NNN to liver and muscle microsomal protein occurred in the absence of an NADPH-generating system, in the presence of 50% methanol and also to bovine serum albumin, indicating a nonenzymatic reaction. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN, but also suggest additional mechanisms of activation

  3. AFM characterization of protein net formation on a fibrous medium

    Directory of Open Access Journals (Sweden)

    Assis O.B.G.

    2000-01-01

    Full Text Available Lysozyme protein net is set on a glass fiber support using the self-assembly technique. Enzymatic film formation is followed by surface imaging via atomic force microscopy (AFM. Change in roughness as a function of deposition time is used as an indirect indicator of film formation. The objective was to form a protein film that would have no effect on the permeability of the medium, aiming at its application as a bioactive membrane or reactor suitable for bacteria and chemical interactions in aqueous media.

  4. Infrared Spectroscopy for Rapid Characterization of Drill Core and Cutting Mineralogy

    Science.gov (United States)

    Calvin, W. M.; Kratt, C.; Kruse, F. A.

    2009-12-01

    Water geochemistry can vary with depth and location within a geothermal reservoir, owing to natural factors such as changing rock type, gas content, fluid source and temperature. The interaction of these variable fluids with the host rock will cause well known changes in alteration mineral assemblages that are commonly factored into the exploration of hydrothermal systems for economic metals, but are less utilized with regard to mapping borehole geology for geothermal energy production. Chemistry of geothermal fluids and rock alteration products can impact production factors such as pipeline corrosion and scaling and early studies explored the use of both silica and chlorites as geothermometers. Infrared spectroscopy is particularly good at identifying a wide variety of alteration minerals, especially in discrimination among clay minerals, with no sample preparation. The technique has been extensively used in the remote identification of materials, but is not commonly used on drill core or chips. We have performed several promising pilot studies that suggest the power of the technique to sample continuously and provide mineral logs akin to geophysical ones. We have surveyed a variety of samples, including drill chip boards, boxed core, and drill cuttings from envelopes, sample bottles and chip trays. This work has demonstrated that core and drill chips can be rapidly surveyed, acquiring spectra every few to tens of cm of section, or the vertical resolution of the chip tray (typically 10 feet). Depending on the sample type we can acquire spectral data over thousands of feet depth at high vertical resolution in a fraction of the time that is needed for traditional analytical methods such as XRD or TEM with better accuracy than traditional geologic drill or chip logging that uses visual inspection alone. We have successfully identified layered silicates such as illite, kaolinite, montmorillonite chlorite and prehnite, zeolites, opal, calcite, jarosite and iron oxides

  5. 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.

  6. Development of a core set of SSR markers for the characterization of Gossypium germplasm

    Science.gov (United States)

    Molecular markers such as simple sequence repeats (SSR) are a useful tool for characterizing genetic diversity of Gossypium germplasm collections. Genetic profiles by DNA fingerprinting of cotton accessions can only be compared among different collections if a common set of molecular markers are us...

  7. Computational and biological characterization of fusion proteins of two insecticidal proteins for control of insect pests.

    Science.gov (United States)

    Javaid, Shaista; Naz, Sehrish; Amin, Imran; Jander, Georg; Ul-Haq, Zaheer; Mansoor, Shahid

    2018-03-19

    Sucking pests pose a serious agricultural challenge, as available transgenic technologies such as Bacillus thuringiensis crystal toxins (Bt) are not effective against them. One approach is to produce fusion protein toxins for the control of these pests. Two protein toxins, Hvt (ω-atracotoxin from Hadronyche versuta) and onion leaf lectin, were translationally fused to evaluate the negative effects of fusion proteins on Phenacoccus solenopsis (mealybug), a phloem-feeding insect pest. Hvt was cloned both N-terminally (HL) and then C-terminally (LH) in the fusion protein constructs, which were expressed transiently in Nicotiana tabacum using a Potato Virus X (PVX) vector. The HL fusion protein was found to be more effective against P. solenopsis, with an 83% mortality rate, as compared to the LH protein, which caused 65% mortality. Hvt and lectin alone caused 42% and 45%, respectively, under the same conditions. Computational studies of both fusion proteins showed that the HL protein is more stable than the LH protein. Together, these results demonstrate that translational fusion of two insecticidal proteins improved the insecticidal activity relative to each protein individually and could be expressed in transgenic plants for effective control of sucking pests.

  8. Synthesis and characterization of FePt/Au core-shell nanoparticles

    International Nuclear Information System (INIS)

    Presa, P. de la; Multigner, M.; Morales, M.P.; Rueda, T.; Fernandez-Pinel, E.; Hernando, A.

    2007-01-01

    In this work, the structural and magnetic properties of the gold-coated FePt nanoparticles synthesized from high-temperature solution phase are presented. The amount of gold was optimized to obtain most of the FePt particles coated. The particle diameter increases from 4 to 10 nm as observed by TEM. The magnetic properties are largely affected by the coating. At low temperature, the coercive field Hc of the coated nanoparticles decreases about three times respect to the uncoated and the blocking temperature reduces to the half. The changes of the magnetic behavior are discussed in terms of the effect of the gold atoms at the FePt core surface

  9. The duck hepatitis B virus polymerase and core proteins accumulate in different patterns from their common mRNA

    International Nuclear Information System (INIS)

    Yao Ermei; Schaller, Heinz; Tavis, John E.

    2003-01-01

    Hepadnaviral reverse transcription occurs in capsids in which the core (C) protein surrounds the reverse transcriptase (P) and pregenomic RNA (pgRNA). We analyzed the accumulation patterns of duck hepatitis B virus P, C, and pgRNA in transfected LMH cells, infected primary duck hepatocytes (PDH), and infected duck liver. In all three systems, P accumulated over time in a different pattern compared with C, despite translation of both proteins from the pgRNA. Although the accumulation patterns of the proteins varied between the systems, in each case P became detectable at the same time or earlier than C and the ratio of P relative to C dropped with time. These accumulation patterns were consistent with the translation rates and half-lives of P and C. Comparing the translation rates of P and C with the pgRNA level over time revealed that translation of P and C was negatively regulated in LMH cells. These data provide a framework for comparing replication studies performed in LMH cells, PDHs and ducks

  10. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

    International Nuclear Information System (INIS)

    Simonetti, Angelita; Marzi, Stefano; Fabbretti, Attilio; Hazemann, Isabelle; Jenner, Lasse; Urzhumtsev, Alexandre; Gualerzi, Claudio O.; Klaholz, Bruno P.

    2013-01-01

    The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue

  11. Structure of the protein core of translation initiation factor 2 in apo, GTP-bound and GDP-bound forms

    Energy Technology Data Exchange (ETDEWEB)

    Simonetti, Angelita [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Marzi, Stefano [Architecture et Réactivité de l’ARN, UPR 9002 CNRS, IBMC (Institute of Molecular and Cellular Biology), 15 Rue R. Descartes, 67084 Strasbourg, France, Université de Strasbourg, 67000 Strasbourg (France); Fabbretti, Attilio [University of Camerino, 62032 Camerino (Monaco) (Italy); Hazemann, Isabelle; Jenner, Lasse [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale -INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Urzhumtsev, Alexandre [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France); Université de Lorraine, 54506 Vandoeuvre-lès-Nancy (France); Gualerzi, Claudio O. [University of Camerino, 62032 Camerino (Monaco) (Italy); Klaholz, Bruno P., E-mail: klaholz@igbmc.fr [IGBMC (Institute of Genetics and of Molecular and Cellular Biology), Centre National de la Recherche Scientifique (CNRS) UMR 7104/Institut National de la Santé de la Recherche Médicale - INSERM U964/Université de Strasbourg, 1 Rue Laurent Fries, 67404 Illkirch (France)

    2013-06-01

    The crystal structures of the eubacterial translation initiation factor 2 in apo form and with bound GDP and GTP reveal conformational changes upon nucleotide binding and hydrolysis, notably of the catalytically important histidine in the switch II region. Translation initiation factor 2 (IF2) is involved in the early steps of bacterial protein synthesis. It promotes the stabilization of the initiator tRNA on the 30S initiation complex (IC) and triggers GTP hydrolysis upon ribosomal subunit joining. While the structure of an archaeal homologue (a/eIF5B) is known, there are significant sequence and functional differences in eubacterial IF2, while the trimeric eukaryotic IF2 is completely unrelated. Here, the crystal structure of the apo IF2 protein core from Thermus thermophilus has been determined by MAD phasing and the structures of GTP and GDP complexes were also obtained. The IF2–GTP complex was trapped by soaking with GTP in the cryoprotectant. The structures revealed conformational changes of the protein upon nucleotide binding, in particular in the P-loop region, which extend to the functionally relevant switch II region. The latter carries a catalytically important and conserved histidine residue which is observed in different conformations in the GTP and GDP complexes. Overall, this work provides the first crystal structure of a eubacterial IF2 and suggests that activation of GTP hydrolysis may occur by a conformational repositioning of the histidine residue.

  12. Highly temperature responsive core-shell magnetic particles: synthesis, characterization and colloidal properties.

    Science.gov (United States)

    Rahman, Md Mahbubor; Chehimi, Mohamed M; Fessi, Hatem; Elaissari, Abdelhamid

    2011-08-15

    Temperature responsive magnetic polymer submicron particles were prepared by two step seed emulsion polymerization process. First, magnetic seed polymer particles were obtained by emulsion polymerization of styrene using potassium persulfate (KPS) as an initiator and divinylbenzne (DVB) as a cross-linker in the presence of oil-in-water magnetic emulsion (organic ferrofluid droplets). Thereafter, DVB cross-linked magnetic polymer particles were used as seed in the precipitation polymerization of N-isopropylacrylamide (NIPAM) to induce thermosensitive PNIPAM shell onto the hydrophobic polymer surface of the cross-linked magnetic polymer particles. To impart cationic functional groups in the thermosensitive PNIPAM backbone, the functional monomer aminoethylmethacrylate hydrochloride (AEMH) was used to polymerize with NIPAM while N,N'-methylenebisacrylamide (MBA) and 2, 2'-azobis (2-methylpropionamidine) dihydrochloride (V-50) were used as a cross-linker and as an initiator respectively. The effect of seed to monomer (w/w) ratio along with seed nature on the final particle morphology was investigated. Dynamic light scattering (DLS) results demonstrated particles swelling at below volume phase transition temperature (VPTT) and deswelling above the VPTT. The perfect core (magnetic) shell (polymer) structure of the particles prepared was confirmed by Transmission Electron Microscopy (TEM). The chemical composition of the particles were determined by thermogravimetric analysis (TGA). The effect of temperature, pH, ionic strength on the colloidal properties such as size and zeta potential of the micron sized thermo-sensitive magnetic particles were also studied. In addition, a short mechanistic discussion on the formation of core-shell morphology of magnetic polymer particles has also been discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. Characterization of Indian and exotic quality protein maize (QPM ...

    African Journals Online (AJOL)

    Polymorphism analysis and genetic diversity of normal maize and quality protein maize (QPM) inbreds among locally well adapted germplasm is a prerequisite for hybrid maize breeding program. The diversity analyses of 48 maize accessions including Indian and exotic germplasm using 75 simple sequence repeat (SSR) ...

  14. In silico Characterization of Plant and Microbial Antifreeze Proteins

    Directory of Open Access Journals (Sweden)

    Abdul Mohin Sajib

    2012-12-01

    Full Text Available Antifreeze proteins (AFPs are class of proteins that protect organisms from the damage caused by freezing through their ability to inhibit ice growth and effectively lower the temperature at which water freezes. In this study, a total of 25 antifreeze proteins were selected from four different sources (plant, bacteria and fungus where they represent distinct physicochemical and structural features. Several Physico-chemical properties such as grand average hydropathy (GRAVY, aliphatic index (AI, extinction coefficient (EC, isolelectric point (pI, and instability index (II were computed. S-S bridges and secondary structures were analyzed using CYS_REC and SOPMA programs respectively. The three dimensional structure of Antifreeze proteins is predicted by using three homology modelling server Geno3D, Swiss-model and CPHmodels. These models were evaluated with PROCHECK, What If, and ProSA programs. Model visualization and analysis was done with Pymol. These structures will provide a good foundation for functional analysis of experimentally derived crystal structures.

  15. Cloning and characterization of an insecticidal crystal protein gene ...

    Indian Academy of Sciences (India)

    A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed in E. coli. The recombinant protein produced 92% mortality in first-instar larvae of Spodoptera litura and 86% inhibition of adult emergence in Phthorimaea operculella, but showed very low toxicity against ...

  16. Characterization of Pseudomonas aeruginosa Chitinase, a Gradually Secreted Protein

    NARCIS (Netherlands)

    Folders, J. (Jindra); Algra, J. (Jon); Roelofs, M.S. (Marc); Loon, L.C. van; Tommassen, J.P.M.; Bitter, Wilbert

    2001-01-01

    The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino

  17. PROTEIN & SENSORY ANALYSIS TO CHARACTERIZE MEXICAN CHIHUAHUA CHEESES

    Science.gov (United States)

    It has been established that native microflora in raw milk cheeses, including Queso Chihuahua, a Mexican cheese variety, contributes to the development of unique flavors through degradation of milk proteins resulting in the release of free amino acids and short peptides that influence the taste and ...

  18. Characterization of cottonseed protein isolate as a paper additive

    Science.gov (United States)

    There is current interest in using agro-based biopolymers in industrial applications. Because cottonseed protein is abundantly available, it would be useful to explore its feasibility as a polymeric additive and possible substitute for petroleum-based materials. In this work we studied cottonseed ...

  19. Non-conventional approaches to food processing in CELSS, 1. Algal proteins: Characterization and process optimization

    Science.gov (United States)

    Nakhost, Z.; Karel, M.; Krukonis, V. J.

    1987-01-01

    Protein isolate obtained from green algae cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine make algal protein isolate a high quality component of closed ecological life support system diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical carbon dioxide resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

  20. Non-conventional approaches to food processing in CELSS. I - Algal proteins: Characterization and process optimization

    Science.gov (United States)

    Nakhost, Z.; Karel, M.; Krukonis, V. J.

    1987-01-01

    Protein isolate obtained from green algae (Scenedesmus obliquus) cultivated under controlled conditions was characterized. Molecular weight determination of fractionated algal proteins using SDS-polyacrylamide gel electrophoresis revealed a wide spectrum of molecular weights ranging from 15,000 to 220,000. Isoelectric points of dissociated proteins were in the range of 3.95 to 6.20. Amino acid composition of protein isolate compared favorably with FAO standards. High content of essential amino acids leucine, valine, phenylalanine and lysine makes algal protein isolate a high quality component of CELSS diets. To optimize the removal of algal lipids and pigments supercritical carbon dioxide extraction (with and without ethanol as a co-solvent) was used. Addition of ethanol to supercritical CO2 resulted in more efficient removal of algal lipids and produced protein isolate with a good yield and protein recovery. The protein isolate extracted by the above mixture had an improved water solubility.

  1. Methods for validating the presence of and characterizing proteins deposited onto an array

    Science.gov (United States)

    Schabacker, Daniel S.

    2010-09-21

    A method of determining if proteins have been transferred from liquid-phase protein fractions to an array comprising staining the array with a total protein stain and imaging the array, optionally comparing the staining with a standard curve generated by staining known amounts of a known protein on the same or a similar array; a method of characterizing proteins transferred from liquid-phase protein fractions to an array including staining the array with a post-translational modification-specific (PTM-specific) stain and imaging the array and, optionally, after staining the array with a PTM-specific stain and imaging the array, washing the array, re-staining the array with a total protein stain, imaging the array, and comparing the imaging with the PTM-specific stain with the imaging with the total protein stain; stained arrays; and images of stained arrays.

  2. Molecular characterization of the porcine surfactant, pulmonary-associated protein C gene

    DEFF Research Database (Denmark)

    Cirera, S.; Nygård, A.B.; Jensen, H.E.

    2006-01-01

    The surfactant, pulmonary-associated protein C (SFTPC) is a peptide secreted by the alveolar type II pneumocytes of the lung. We have characterized the porcine SFTPC gene at genomic, transcriptional, and protein levels. The porcine SFTPC is a single-copy gene on pig chromosome 14. Two transcripts...

  3. Functional characterization of the vaccinia virus I5 protein

    Directory of Open Access Journals (Sweden)

    Stanitsa Eleni S

    2008-12-01

    Full Text Available The I5L gene is one of ~90 genes that are conserved throughout the chordopoxvirus family, and hence are presumed to play vital roles in the poxvirus life cycle. Previous work had indicated that the VP13 protein, a component of the virion membrane, was encoded by the I5L gene, but no additional studies had been reported. Using a recombinant virus that encodes an I5 protein fused to a V5 epitope tag at the endogenous locus (vI5V5, we show here that the I5 protein is expressed as a post-replicative gene and that the ~9 kDa protein does not appear to be phosphorylated in vivo. I5 does not appear to traffic to any cellular organelle, but ultrastructural and biochemical analyses indicate that I5 is associated with the membranous components of assembling and mature virions. Intact virions can be labeled with anti-V5 antibody as assessed by immunoelectron microscopy, indicating that the C' terminus of the protein is exposed on the virion surface. Using a recombinant virus which encodes only a TET-regulated copy of the I5V5 gene (vΔindI5V5, or one in which the I5 locus has been deleted (vΔI5, we also show that I5 is dispensable for replication in tissue culture. Neither plaque size nor the viral yield produced in BSC40 cells or primary human fibroblasts are affected by the absence of I5 expression.

  4. Hepatitis C virus core protein regulates p300/CBP co-activation function. Possible role in the regulation of NF-AT1 transcriptional activity

    International Nuclear Information System (INIS)

    Gomez-Gonzalo, Marta; Benedicto, Ignacio; Carretero, Marta; Lara-Pezzi, Enrique; Maldonado-Rodriguez, Alejandra; Moreno-Otero, Ricardo; Lai, Michael M.C.; Lopez-Cabrera, Manuel

    2004-01-01

    Hepatitis C virus (HCV) core is a viral structural protein; it also participates in some cellular processes, including transcriptional regulation. However, the mechanisms of core-mediated transcriptional regulation remain poorly understood. Oncogenic virus proteins often target p300/CBP, a known co-activator of a wide variety of transcription factors, to regulate the expression of cellular and viral genes. Here we demonstrate, for the first time, that HCV core protein interacts with p300/CBP and enhances both its acetyl-transferase and transcriptional activities. In addition, we demonstrate that nuclear core protein activates the NH 2 -terminal transcription activation domain (TAD) of NF-AT1 in a p300/CBP-dependent manner. We propose a model in which core protein regulates the co-activation function of p300/CBP and activates NF-AT1, and probably other p300/CBP-regulated transcription factors, by a novel mechanism involving the regulation of the acetylation state of histones and/or components of the transcriptional machinery

  5. Tandem fusion of hepatitis B core antigen allows assembly of virus-like particles in bacteria and plants with enhanced capacity to accommodate foreign proteins.

    Directory of Open Access Journals (Sweden)

    Hadrien Peyret

    Full Text Available The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.

  6. Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules

    Science.gov (United States)

    Killian, C. E.; Wilt, F. H.

    1996-01-01

    In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.

  7. Identification and characterization of proteins involved in nuclear organization using Drosophila GFP protein trap lines.

    Directory of Open Access Journals (Sweden)

    Margaret Rohrbaugh

    Full Text Available Strains from a collection of Drosophila GFP protein trap lines express GFP in the normal tissues where the endogenous protein is present. This collection can be used to screen for proteins distributed in the nucleus in a non-uniform pattern.We analyzed four lines that show peripheral or punctate nuclear staining. One of these lines affects an uncharacterized gene named CG11138. The CG11138 protein shows a punctate distribution in the nuclear periphery similar to that of Drosophila insulator proteins but does not co-localize with known insulators. Interestingly, mutations in Lamin proteins result in alterations in CG11138 localization, suggesting that this protein may be a novel component of the nuclear lamina. A second line affects the Decondensation factor 31 (Df31 gene, which encodes a protein with a unique nuclear distribution that appears to segment the nucleus into four different compartments. The X-chromosome of males is confined to one of these compartments. We also find that Drosophila Nucleoplasmin (dNlp is present in regions of active transcription. Heat shock leads to loss of dNlp from previously transcribed regions of polytene chromosome without redistribution to the heat shock genes. Analysis of Stonewall (Stwl, a protein previously found to be necessary for the maintenance of germline stem cells, shows that Stwl is present in a punctate pattern in the nucleus that partially overlaps with that of known insulator proteins. Finally we show that Stwl, dNlp, and Df31 form part of a highly interactive network. The properties of other components of this network may help understand the role of these proteins in nuclear biology.These results establish screening of GFP protein trap alleles as a strategy to identify factors with novel cellular functions. Information gained from the analysis of CG11138 Stwl, dNlp, and Df31 sets the stage for future studies of these proteins.

  8. Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

    Science.gov (United States)

    2016-08-01

    RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI 1. INTRODUCTION 1.1 Background Vaccinia virus (VACV) is the active component of the...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in...PURIFICATION AND CHARACTERIZATION OF RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI ECBC-TR-1370

  9. Characterization of the Fault Core and Damage Zone of the Borrego Fault, 2010 M7.2 Rupture

    Science.gov (United States)

    Dorsey, M. T.; Rockwell, T. K.; Girty, G.; Ostermeijer, G.; Mitchell, T. M.; Fletcher, J. M.

    2017-12-01

    We collected a continuous sample of the fault core and 23 samples of the damage zone out to 52 m across the rupture trace of the 2010 M7.2 El Mayor-Cucapa earthquake to characterize the physical damage and chemical transformations associated with this active seismic source. In addition to quantifying fracture intensity from macroscopic analysis, we cut a continuous thin section through the fault core and from various samples in the damage zone, and ran each sample for XRD analyses for clay mineralogy, XRF for bulk geochemical analyses, and bulk and grain density from which porosity and volumetric strain were derived. The parent rock is a hydrothermally-altered biotite tonalite, with biotite partially altered to chlorite. The presence of epidote with chlorite suggests that these rocks were subjected to relatively high temperatures of 300-400° C. Adjacent to the outermost damage zone is a chaotic breccia zone with distinct chemical and physical characteristics, indicating possible connection to an ancestral fault to the southwest. The damage zone consists of an outer zone of protocataclasite, which grades inward towards mesocataclasite with seams of ultracataclasite. The fault core is anomalous in that it is largely composed of a sliver of marble that has been translated along the fault, so direct comparison with the damage zone is impaired. From collected data, we observe that chloritization increases into the breccia and damage zones, as does the presence of illite. Porosity reaches maximum values in the damage zone adjacent to the core, and closely follows trends in fracture intensity. Statistically significant gains in Mg, Na, K, Mn, and total bulk mass occurred within the inner damage zone, with losses of Ca and P mass, which led to the formation of chlorite and albite. The outer damage zone displays gains in Mg and Na mass with losses in Ca and P mass. The breccia zone shows gains in mass of Mg and Mn and loss in total bulk mass. A gain in LOI in both the

  10. Protein characterization of protein bodies from cotyledons of Mucuna pruriens (L.) DC.

    Science.gov (United States)

    Bellani, Lorenza; Giglioni, Stefania; Muccifora, Simonetta

    2013-03-01

    Seeds of Mucuna pruriens (L.) DC. (Fabaceae) were analyzed for protein composition of protein bodies isolated from cotyledons. Protein bodies were successfully separated by Lympholyte and those of dry seeds, observed by scanning electron microscope, were elliptical or spherical in shape with a diameter of 5-12 μm. Protein content in dry seed protein bodies was 10.6 mg/g dry weight. Globulin was the largest protein fraction isolated (62.5 %), followed by albumin (18.3 %), glutelin (15.8 %) and prolamin (3.4 %). The prolamin fraction and high glutelin content are uncommon in legumes. SDS-PAGE of albumins, globulins, prolamins and glutelins provided different band numbers and molecular weights under reducing and non reducing conditions and suggested that the albumin fraction is rich in disulphide bonds.

  11. Docking-based modeling of protein-protein interfaces for extensive structural and functional characterization of missense mutations.

    Science.gov (United States)

    Barradas-Bautista, Didier; Fernández-Recio, Juan

    2017-01-01

    Next-generation sequencing (NGS) technologies are providing genomic information for an increasing number of healthy individuals and patient populations. In the context of the large amount of generated genomic data that is being generated, understanding the effect of disease-related mutations at molecular level can contribute to close the gap between genotype and phenotype and thus improve prevention, diagnosis or treatment of a pathological condition. In order to fully characterize the effect of a pathological mutation and have useful information for prediction purposes, it is important first to identify whether the mutation is located at a protein-binding interface, and second to understand the effect on the binding affinity of the affected interaction/s. Computational methods, such as protein docking are currently used to complement experimental efforts and could help to build the human structural interactome. Here we have extended the original pyDockNIP method to predict the location of disease-associated nsSNPs at protein-protein interfaces, when there is no available structure for the protein-protein complex. We have applied this approach to the pathological interaction networks of six diseases with low structural data on PPIs. This approach can almost double the number of nsSNPs that can be characterized and identify edgetic effects in many nsSNPs that were previously unknown. This can help to annotate and interpret genomic data from large-scale population studies, and to achieve a better understanding of disease at molecular level.

  12. Docking-based modeling of protein-protein interfaces for extensive structural and functional characterization of missense mutations.

    Directory of Open Access Journals (Sweden)

    Didier Barradas-Bautista

    Full Text Available Next-generation sequencing (NGS technologies are providing genomic information for an increasing number of healthy individuals and patient populations. In the context of the large amount of generated genomic data that is being generated, understanding the effect of disease-related mutations at molecular level can contribute to close the gap between genotype and phenotype and thus improve prevention, diagnosis or treatment of a pathological condition. In order to fully characterize the effect of a pathological mutation and have useful information for prediction purposes, it is important first to identify whether the mutation is located at a protein-binding interface, and second to understand the effect on the binding affinity of the affected interaction/s. Computational methods, such as protein docking are currently used to complement experimental efforts and could help to build the human structural interactome. Here we have extended the original pyDockNIP method to predict the location of disease-associated nsSNPs at protein-protein interfaces, when there is no available structure for the protein-protein complex. We have applied this approach to the pathological interaction networks of six diseases with low structural data on PPIs. This approach can almost double the number of nsSNPs that can be characterized and identify edgetic effects in many nsSNPs that were previously unknown. This can help to annotate and interpret genomic data from large-scale population studies, and to achieve a better understanding of disease at molecular level.

  13. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Shi Ruina [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Huang Yong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Wang Dan [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Zhao Meiping [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China); Li Yuanzong [College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China)]. E-mail: yzli@pku.edu.cn

    2006-09-25

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10{sup -8} to 2.0 x 10{sup -6} M with a detection limit of 1.6 x 10{sup -8} M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications.

  14. Expression and characterization of insulin growth factor-I-enhanced green fluorescent protein fused protein as a tracer for immunoassay

    International Nuclear Information System (INIS)

    Shi Ruina; Huang Yong; Wang Dan; Zhao Meiping; Li Yuanzong

    2006-01-01

    The insulin-like growth factor-I (IGF-I) is an important polypeptide hormone under investigation for body metabolism study and for doping detection. Here, we describe for the first time the expression of a recombinant fusion protein of IGF-I and the enhanced green fluorescent protein (EGFP). The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and homogeneous structure. The fused protein (EGFP-IGF-I) was expressed as a soluble protein in cytoplasm of Escherichia coli and its fluorescence and immunoreaction properties were thoroughly characterized. Finally, we demonstrated the utility of the EGFP-IGF-I fusion protein for the fluorescence immunoassay of IGF-1. The linear range of the assay is 1.6 x 10 -8 to 2.0 x 10 -6 M with a detection limit of 1.6 x 10 -8 M. To our knowledge, this is the first time that EGFP has been used as a quantitative label in a fusion protein to develop a quantitative assay for IGF-I. Furthermore, the use of genetically engineered fusion proteins, which combine peptide hormones with fluorescent protein, can lead to a new labeling approach to a number of bioanalytical applications

  15. Molecular Characterization and Analysis of a Novel Protein Disulfide Isomerase-Like Protein of Eimeria tenella

    OpenAIRE

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDI...

  16. Study the Characterization of Spectral Absorbance on Irradiated Milk Protein

    Science.gov (United States)

    Fohely, F.; Suardi, N.

    2018-04-01

    The milk has been adopted as a structural nature food for a long era since it is containing most of the growth factors, protective agents, and enzymes needed for the body. a few attempts have been conducted to treat the dairy products especially raw milk by the means of ionizing radiation. as its production has been an expanding industry for many years due to the high demands from the consumers worldwide, there is still some doubt about preserving these products by irradiation. In this work, a preliminary effort to describe the influences of ionizing radiation on raw milk’s protein will be devoted to measuring the spectral absorbance of the total protein (after subjected to varied radiation doses) by UV-VIS-NIR spectroscopy analysis. The absorbance spectrum then analyzed based on absorbance spectra of organic compounds. A comparison is made between the effects of different radiation doses to estimate the influence in milk’s structure.

  17. Identification and characterization of N-glycosylated proteins using proteomics

    DEFF Research Database (Denmark)

    Selby, David S; Larsen, Martin R; Calvano, Cosima Damiana

    2008-01-01

    and analysis of glycoproteins and glycopeptides. Combinations of affinity-enrichment techniques, chemical and biochemical protocols, and advanced mass spectrometry facilitate detailed glycoprotein analysis in proteomics, from fundamental biological studies to biomarker discovery in biomedicine....... is a complex task and is currently achieved by mass spectrometry-based methods that enable identification of glycoproteins and localization, classification, and analysis of individual glycan structures on proteins. In this chapter we briefly introduce a range of analytical technologies for recovery...

  18. Immunochemical characterization of the brain glutamate binding protein

    International Nuclear Information System (INIS)

    Roy, S.

    1986-01-01

    A glutamate binding protein (GBP) was purified from bovine and rat brain to near homogeneity. Polyclonal antibodies were raised against this protein. An enzyme-linked-immunosorbent-assay was used to quantify and determine the specificity of the antibody response. The antibodies were shown to strongly react with bovine brain GBP and the analogous protein from rat brain. The antibodies did not show any crossreactivity with the glutamate metabolizing enzymes, glutamate dehydrogenase, glutamine synthetase and glutamyl transpeptidase, however it crossreacted moderately with glutamate decarboxylase. The antibodies were also used to define the possible physiologic activity of GBP in synaptic membranes. The antibodies were shown: (i) to inhibit the excitatory amino-acid stimulation of thiocyanate (SCN)flux, (ii) had no effect on transport of L-Glutamic acid across the synaptic membrane, and (iii) had no effect on the depolarization-induced release of L-glutamate. When the anti-GBP antibodies were used to localize and quantify the GBP distribution in various subcellular fractions and in brain tissue samples, it was found that the hippocampus had the highest immunoreactivity followed by the cerebral cortex, cerebellar cortex and caudate-putamen. The distribution of immunoreactivity in the subcellular fraction were as follows: synaptic membranes > crude mitochondrial fraction > homogenate > myelin. In conclusion these studies suggest that: (a) the rat brain GBP and the bovine brain GBP are immunologically homologous protein, (b) there are no structural similarities between the GBP and the glutamate metabolizing enzymes with the exception of glutamate decarboxylase and (c) the subcellular and regional distribution of the GBP immunoreactivity followed a similar pattern as observed for L-[ 3 H]-binding

  19. Characterization and Prediction of Protein Phosphorylation Hotspots in Arabidopsis thaliana.

    Science.gov (United States)

    Christian, Jan-Ole; Braginets, Rostyslav; Schulze, Waltraud X; Walther, Dirk

    2012-01-01

    The regulation of protein function by modulating the surface charge status via sequence-locally enriched phosphorylation sites (P-sites) in so called phosphorylation "hotspots" has gained increased attention in recent years. We set out to identify P-hotspots in the model plant Arabidopsis thaliana. We analyzed the spacing of experimentally detected P-sites within peptide-covered regions along Arabidopsis protein sequences as available from the PhosPhAt database. Confirming earlier reports (Schweiger and Linial, 2010), we found that, indeed, P-sites tend to cluster and that distributions between serine and threonine P-sites to their respected closest next P-site differ significantly from those for tyrosine P-sites. The ability to predict P-hotspots by applying available computational P-site prediction programs that focus on identifying single P-sites was observed to be severely compromised by the inevitable interference of nearby P-sites. We devised a new approach, named HotSPotter, for the prediction of phosphorylation hotspots. HotSPotter is based primarily on local amino acid compositional preferences rather than sequence position-specific motifs and uses support vector machines as the underlying classification engine. HotSPotter correctly identified experimentally determined phosphorylation hotspots in A. thaliana with high accuracy. Applied to the Arabidopsis proteome, HotSPotter-predicted 13,677 candidate P-hotspots in 9,599 proteins corresponding to 7,847 unique genes. Hotspot containing proteins are involved predominantly in signaling processes confirming the surmised modulating role of hotspots in signaling and interaction events. Our study provides new bioinformatics means to identify phosphorylation hotspots and lays the basis for further investigating novel candidate P-hotspots. All phosphorylation hotspot annotations and predictions have been made available as part of the PhosPhAt database at http://phosphat.mpimp-golm.mpg.de.

  20. Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

    Science.gov (United States)

    Folders, J; Algra, J; Roelofs, M S; van Loon, L C; Tommassen, J; Bitter, W

    2001-12-01

    The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.

  1. Identification and Characterization of Perinucleolar Compartment-Associated Protein

    National Research Council Canada - National Science Library

    Leary, Daniel

    2002-01-01

    .... hSof1, like fibrillarin, localizes to both the nucleolus and nucleoplasm. However, unlike fibrillarin, hSof1 is also in the granular component of nucleoli and responds differently to the inhibition of the transcription of pre- rRNA. In addition, hSof1 -GFP also exhibits a higher nuclear mobility than fibrillarin-GFP and is a nucleocytoplasmic shuttling protein.

  2. Role of Decorin Core Protein in Collagen Organisation in Congenital Stromal Corneal Dystrophy (CSCD.

    Directory of Open Access Journals (Sweden)

    Christina S Kamma-Lorger

    Full Text Available The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD. In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT in the decorin (DCN gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS, to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.

  3. Characterization of the human GARP (Golgi associated retrograde protein) complex

    International Nuclear Information System (INIS)

    Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank

    2005-01-01

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells

  4. Design and characterization of protein-quercetin bioactive nanoparticles

    Directory of Open Access Journals (Sweden)

    Leng Xiaojing

    2011-05-01

    Full Text Available Abstract Background The synthesis of bioactive nanoparticles with precise molecular level control is a major challenge in bionanotechnology. Understanding the nature of the interactions between the active components and transport biomaterials is thus essential for the rational formulation of bio-nanocarriers. The current study presents a single molecule of bovine serum albumin (BSA, lysozyme (Lys, or myoglobin (Mb used to load hydrophobic drugs such as quercetin (Q and other flavonoids. Results Induced by dimethyl sulfoxide (DMSO, BSA, Lys, and Mb formed spherical nanocarriers with sizes less than 70 nm. After loading Q, the size was further reduced by 30%. The adsorption of Q on protein is mainly hydrophobic, and is related to the synergy of Trp residues with the molecular environment of the proteins. Seven Q molecules could be entrapped by one Lys molecule, 9 by one Mb, and 11 by one BSA. The controlled releasing measurements indicate that these bioactive nanoparticles have long-term antioxidant protection effects on the activity of Q in both acidic and neutral conditions. The antioxidant activity evaluation indicates that the activity of Q is not hindered by the formation of protein nanoparticles. Other flavonoids, such as kaempferol and rutin, were also investigated. Conclusions BSA exhibits the most remarkable abilities of loading, controlled release, and antioxidant protection of active drugs, indicating that such type of bionanoparticles is very promising in the field of bionanotechnology.

  5. Characterization of a Lactococcus lactis promoter for heterologous protein production

    Directory of Open Access Journals (Sweden)

    Christian E. Ogaugwu

    2018-03-01

    Full Text Available Constitutively active promoter elements for heterologous protein production in Lactococcus lactis are scarce. Here, the promoter of the PTS-IIC gene cluster from L. lactis NZ3900 is described. This promoter was cloned upstream of an enhanced green fluorescent protein, GFPmut3a, and transformed into L. lactis. Transformants produced up to 13.5 μg of GFPmut3a per milliliter of log phase cells. Addition of cellobiose further increased the production of GFPmut3a by up to two-fold when compared to glucose. Analysis of mutations at two specific positions in the PTS-IIC promoter showed that a ‘T’ to ‘G’ mutation within the −35 element resulted in constitutive expression in glucose, while a ‘C’ at nucleotide 7 in the putative cre site enhanced promoter activity in cellobiose. Finally, this PTS-IIC promoter is capable of mediating protein expression in Bacillus subtilis and Escherichia coli Nissle 1917, suggesting the potential for future biotechnological applications of this element and its derivatives.

  6. Characterization and Expression of Genes Encoding Three Small Heat Shock Proteins in Sesamia inferens (Lepidoptera: Noctuidae)

    OpenAIRE

    Sun, Meng; Lu, Ming-Xing; Tang, Xiao-Tian; Du, Yu-Zhou

    2014-01-01

    The pink stem borer, Sesamia inferens (Walker), is a major pest of rice and is endemic in China and other parts of Asia. Small heat shock proteins (sHSPs) encompass a diverse, widespread class of stress proteins that have not been characterized in S. inferens. In the present study, we isolated and characterized three S. inferens genes that encode members of the α-crystallin/sHSP family, namely, Sihsp21.4, Sihsp20.6, and Sihsp19.6. The three cDNAs encoded proteins of 187, 183 and 174 amino a...

  7. The diverse functions of the hepatitis B core/capsid protein (HBc) in the viral life cycle: Implications for the development of HBc-targeting antivirals.

    Science.gov (United States)

    Diab, Ahmed; Foca, Adrien; Zoulim, Fabien; Durantel, David; Andrisani, Ourania

    2018-01-01

    Virally encoded proteins have evolved to perform multiple functions, and the core protein (HBc) of the hepatitis B virus (HBV) is a perfect example. While HBc is the structural component of the viral nucleocapsid, additional novel functions for the nucleus-localized HBc have recently been described. These results extend for HBc, beyond its structural role, a regulatory function in the viral life cycle and potentially a role in pathogenesis. In this article, we review the diverse roles of HBc in HBV replication and pathogenesis, emphasizing how the unique structure of this protein is key to its various functions. We focus in particular on recent advances in understanding the significance of HBc phosphorylations, its interaction with host proteins and the role of HBc in regulating the transcription of host genes. We also briefly allude to the emerging niche for new direct-acting antivirals targeting HBc, known as Core (protein) Allosteric Modulators (CAMs). Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Miguel A Martín-Acebes

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

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

    Science.gov (United States)

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

    2013-01-01

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

  10. The dense core vesicle protein IA-2, but not IA-2β, is required for active avoidance learning.

    Science.gov (United States)

    Carmona, G N; Nishimura, T; Schindler, C W; Panlilio, L V; Notkins, A L

    2014-06-06

    The islet-antigens IA-2 and IA-2β are major autoantigens in type-1 diabetes and transmembrane proteins in dense core vesicles (DCV). Recently we showed that deletion of both IA-2 and IA-2β alters the secretion of hormones and neurotransmitters and impairs behavior and learning. The present study was designed to evaluate the contribution to learning of each of these genes by using single knockout (SKO) and double knockout (DKO) mice in an active avoidance test. After 5 days of training, wild-type (WT) mice showed 60-70% active avoidance responses, whereas the DKO mice showed only 10-15% active avoidance responses. The degree of active avoidance responses in the IA-2 SKO mice was similar to that of the DKO mice, but in contrast, the IA-2β SKO mice behaved like WT mice showing 60-70% active avoidance responses. Molecular studies revealed a marked decrease in the phosphorylation of the cAMP response element-binding protein (CREB) and Ca(2+)/calmodulin-dependent protein kinase II (CAMKII) in the striatum and hippocampus of the IA-2 SKO and DKO mice, but not in the IA-2β SKO mice. To evaluate the role of CREB and CAMKII in the SKO and DKO mice, GBR-12909, which selectively blocks the dopamine uptake transporter and increases CREB and CAMKII phosphorylation, was administered. GBR-12909 restored the phosphorylation of CREB and CAMKII and increased active avoidance learning in the DKO and IA-2 SKO to near the normal levels found in the WT and IA-2β SKO mice. We conclude that in the absence of the DCV protein IA-2, active avoidance learning is impaired. Published by Elsevier Ltd.

  11. Fabrication and characterization of porous-core honeycomb bandgap THz fibers

    DEFF Research Database (Denmark)

    Bao, Hualong; Nielsen, Kristian; Rasmussen, Henrik K.

    We have fabricated a porous-core honeycomb fiber in the cyclic olefin copolymer (COC) Topas® by drill-draw technology [1]. A cross-sectional image of the fabricated fiber is shown in the left Panel of Fig. 1. Simulation of the electromagnetic properties of the fiber shows two wide bandgaps within......-TDS system (Picometrix T-Ray 4000). The reference pulse before coupling into the fiber is shown in Fig. 1(a) and the time trace of the THz pulse after propagation through a 5-cm long segment of fiber is shown in Fig. 1(b) (blue curve). After adding some water on the outside of the fiber surface......, the transmitted pulse experiences less pronounced oscillations at times later than 20 ps ( red curve in Fig. 1(b)). Figs. 1(c) and (d) show the short-time Fourier transforms of the two time-domain traces in Fig. 1(b), overlaid with the calculated group delay in the two bandgaps (black squares). The frequencies...

  12. Qualification and characterization of electronics of the fast neutron Hodoscope detectors using neutrons from CABRI core

    Science.gov (United States)

    Mirotta, S.; Guillot, J.; Chevalier, V.; Biard, B.

    2018-01-01

    The study of Reactivity Initiated Accidents (RIA) is important to determine up to which limits nuclear fuels can withstand such accidents without clad failure. The CABRI International Program (CIP), conducted by IRSN under an OECD/NEA agreement, has been launched to perform representative RIA Integral Effect Tests (IET) on real irradiated fuel rods in prototypical Pressurized Water Reactors (PWR) conditions. For this purpose, the CABRI experimental pulse reactor, operated by CEA in Cadarache, France, has been strongly renovated, and equipped with a pressurized water loop. The behavior of the test rod, located in that loop in the center of the driver core, is followed in real time during the power transients thanks to the hodoscope, a unique online fuel motion monitoring system, and one of the major distinctive features of CABRI. The hodoscope measures the fast neutrons emitted by the tested rod during the power pulse with a complete set of 153 Fission Chambers and 153 Proton Recoil Counters. During the CABRI facility renovation, the electronic chain of these detectors has been upgraded. In this paper, the performance of the new system is presented describing gain calibration methodology in order to get maximal Signal/Noise ratio for amplification modules, threshold tuning methodology for the discrimination modules (old and new ones), and linear detectors response limit versus different reactor powers for the whole electronic chain.

  13. Fabrication and characterization of flaky core-shell particles by magnetron sputtering silver onto diatomite

    Science.gov (United States)

    Wang, Yuanyuan; Zhang, Deyuan; Cai, Jun

    2016-02-01

    Diatomite has delicate porous structures and various shapes, making them ideal templates for microscopic core-shell particles fabrication. In this study, a new process of magnetron sputtering assisted with photoresist positioning was proposed to fabricate lightweight silver coated porous diatomite with superior coating quality and performance. The diatomite has been treated with different sputtering time to investigate the silver film growing process on the surface. The morphologies, constituents, phase structures and surface roughness of the silver coated diatomite were analyzed with SEM, EDS, XRD and AFM respectively. The results showed that the optimized magnetron sputtering time was 8-16 min, under which the diatomite templates were successfully coated with uniform silver film, which exhibits face centered cubic (fcc) structure, and the initial porous structures were kept. Moreover, this silver coating has lower surface roughness (RMS 4.513 ± 0.2 nm) than that obtained by electroless plating (RMS 15.692 ± 0.5 nm). And the infrared emissivity of coatings made with magnetron sputtering and electroless plating silver coated diatomite can reach to the lowest value of 0.528 and 0.716 respectively.

  14. Characterization of BIV Env core: Implication for mechanism of BIV-mediated cell fusion

    International Nuclear Information System (INIS)

    Li Shu; Zhu Jieqing; Peng Yu; Cui Shanshan; Wang Chunping; Gao, George F.; Tien Po

    2005-01-01

    Entry of lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), requires folding of two heptad repeat regions (HR1 and HR2) of gp41 into a trimer-of-hairpins, which subsequently brings virus and cell membrane into fusion. This motif is a generalized feature of viral fusion proteins and has been exploited in generating antiviral fusion agents. In the present paper, we report structural characters of Env protein from another lentivirus, bovine immunodeficiency virus (BIV), which contributes to a good animal model of HIV. BIV HR1 and HR2 regions are predicted by two different programs and expressed separately or conjointly in Escherichia coli. Biochemical and biophysical analyses show that the predicted HRs of BIV Env can form a stable trimer-of-hairpins or six-helix bundle just like that formed by feline immunodeficiency virus Env. Cell fusion assay demonstrates that the HR2 peptide of BIV can efficiently inhibit the virus-mediated cell fusion

  15. The SNARE protein vti1a functions in dense-core vesicle biogenesis

    DEFF Research Database (Denmark)

    Walter, Alexander M; Kurps, Julia; de Wit, Heidi

    2014-01-01

    overlapping with syntaxin-6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca(2+)-channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin-2 content. In contrast, release kinetics and Ca(2+)-sensitivity remain unchanged, indicating that the final fusion......The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans-Golgi network, partially...... reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long-term re-expression of vti1a (days...

  16. [Identification and characterization of proteins from human bronchial secretion (author's transl)].

    Science.gov (United States)

    Laine, A; Hayem, A

    1976-03-01

    An analysis of bronchial mucus proteins was carried out by crossed immunoelectrophoresis. Before electrophoretic migration, sputum was treated with Ecteola-cellulose, which retains acid mucins. The proteins were then extracted by a phosphate/saline buffer pH 7.5. Crossed immunoelectrophoresis of the "bronchial extracts" was carried out with an anti-human serum: fifteen proteins were detected. Among them, IgA and protease inhibitiors play an important role in bronchial pathology. Bronchial extracts were also studied with immune serums against milk proteins, whole saliva and proteins of bronchial mucus. Bronchotransferrin, amylase and two esterases were characterized. Four other proteins were also detected with immune serums against bronchial mucus-proteins: their biological role is still unknown.

  17. Synthesis and characterization of aligned ZnO/BeO core/shell nanocable arrays on glass substrate

    Directory of Open Access Journals (Sweden)

    Zhou Minjie

    2011-01-01

    Full Text Available Abstract By sequential hydrothermal growth of ZnO nanowire arrays and thermal evaporation of Be, large-scale vertically aligned ZnO/BeO core/shell nanocable arrays on glass substrate have been successfully synthesized without further heat treatment. Detailed characterizations on the sample morphologies, compositions, and microstructures were systematically carried out, which results disclose the growth behaviors of the ZnO/BeO nanocable. Furthermore, incorporation of BeO shell onto ZnO core resulted in distinct improvement of optical properties of ZnO nanowire, i.e., significant enhancement of near band edge (NBE emission as well as effective suppression of defects emission in ZnO. In particular, the NBE emission of nanocable sample shows a noticeable blue-shift compared with that of pristine ZnO nanowire, which characteristics most likely originate from Be alloying into ZnO. Consequently, the integration of ZnO and BeO into nanoscale heterostructure could bring up new opportunities in developing ZnO-based device for application in deep ultraviolet region. PACS 61.46.K; 78.67.Uh; 81.07.Gf.

  18. Ni(3)Si(Al)/a-SiO(x) core-shell nanoparticles: characterization, shell formation, and stability.

    Science.gov (United States)

    Pigozzi, G; Mukherji, D; Gilles, R; Barbier, B; Kostorz, G

    2006-08-28

    We have used an electrochemical selective phase dissolution method to extract nanoprecipitates of the Ni(3)Si-type intermetallic phase from two-phase Ni-Si and Ni-Si-Al alloys by dissolving the matrix phase. The extracted nanoparticles are characterized by transmission electron microscopy, energy-dispersive x-ray spectrometry, x-ray powder diffraction, and electron powder diffraction. It is found that the Ni(3)Si-type nanoparticles have a core-shell structure. The core maintains the size, the shape, and the crystal structure of the precipitates that existed in the bulk alloys, while the shell is an amorphous phase, containing only Si and O (SiO(x)). The shell forms around the precipitates during the extraction process. After annealing the nanoparticles in nitrogen at 700 °C, the tridymite phase recrystallizes within the shell, which remains partially amorphous. In contrast, on annealing in air at 1000 °C, no changes in the composition or the structure of the nanoparticles occur. It is suggested that the shell forms after dealloying of the matrix phase, where Si atoms, the main constituents of the shell, migrate to the surface of the precipitates.

  19. Synthesis and characterization of core-shell gold nanoparticles with poly(vinyl pyrrolidone) from a new precursor salt

    Science.gov (United States)

    Behera, M.; Ram, S.

    2013-02-01

    In this article, we report a facile one-step chemical synthesis of gold (Au) nanoparticles (GNPs) from a new precursor salt i.e., gold hydroxide in the presence of poly(vinyl pyrrolidone) (PVP) polymer. The non-aqueous dispersion of GNPs was comprehensively characterized by UV-Visible, FTIR, zeta potential, and transmission electron microscope (TEM). A strong surface plasmon resonance band at 529 nm in the UV-Visible spectrum confirms the formation of GNPs in the Au colloid. The FTIR spectroscopic results showed that PVP molecules get chemisorbed onto the surface of GNP via O-atom of carbonyl group. A negative zeta potential of (-)16 mV reveals accumulation of nonbonding electrons of O-atom of carbonyl group of PVP molecules on the nanosurface of GNP. TEM images demonstrate a core-shell nanostructure with an Au-crystalline core covered by a thin amorphous PVP-shell. PVP-capped GNPs could be a potential candidate for bio-sensing, catalysis, and other applications.

  20. Synthesis and characterization of Fe3O4–TiO2 core-shell nanoparticles

    International Nuclear Information System (INIS)

    Stefan, M.; Pana, O.; Leostean, C.; Silipas, D.; Bele, C.; Senila, M.; Gautron, E.

    2014-01-01

    Composite core-shell nanoparticles may have morpho-structural, magnetic, and optical (photoluminescence (PL)) properties different from each of the components considered separately. The properties of Fe 3 O 4 –TiO 2 nanoparticles can be controlled by adjusting the titania amount (shell thinness). Core–shell nanoparticles were prepared by seed mediated growth of semiconductor (TiO 2 ) through a modified sol-gel process onto preformed magnetite (Fe 3 O 4 ) cores resulted from the co-precipitation method. The structure and morphology of samples were characterized by X-ray diffraction, transmission electron microscopy (TEM), and high resolution-TEM respectively. X-ray photoelectron spectroscopy was correlated with ICP-AES. Magnetic measurements, optical absorption spectra, as well as PL spectroscopy indicate the presence of a charge/spin transfer from the conduction band of magnetite into the band gap of titania nanocrystals. The process modifies both Fe 3 O 4 and TiO 2 magnetic and optical properties, respectively.

  1. Characterization of welding of AISI 304l stainless steel similar to the core encircling of a BWR reactor

    International Nuclear Information System (INIS)

    Gachuz M, M.E.; Palacios P, F.; Robles P, E.F.

    2003-01-01

    Plates of austenitic stainless steel AISI 304l of 0.0381 m thickness were welded by means of the SMAW process according to that recommended in the Section 9 of the ASME Code, so that it was reproduced the welding process used to assemble the encircling of the core of a BWR/5 reactor similar to that of the Laguna Verde Nucleo electric plant, there being generated the necessary documentation for the qualification of the one welding procedure and of the welder. They were characterized so much the one base metal, as the welding cord by means of metallographic techniques, scanning electron microscopy, X-ray diffraction, mechanical essays and fracture mechanics. From the obtained results it highlights the presence of an area affected by the heat of up to 1.5 mm of wide and a value of fracture tenacity (J IC ) to ambient temperature for the base metal of 528 KJ/m 2 , which is diminished by the presence of the welding and by the increment in the temperature of the one essay. Also it was carried out an fractographic analysis of the fracture zone generated by the tenacity essays, what evidence a ductile fracture. The experimental values of resistance and tenacity are important for the study of the structural integrity of the encircling one of the core. (Author)

  2. Combining proteomic tools to characterize the protein fraction of llama (Lama glama) milk.

    Science.gov (United States)

    Saadaoui, Besma; Bianchi, Leonardo; Henry, Céline; Miranda, Guy; Martin, Patrice; Cebo, Christelle

    2014-05-01

    Llamas belong to the Camelidae family along with camels. While dromedary camel milk has been broadly characterized, data on llama milk proteins are scarce. The objective of this study was thus to investigate the protein composition of llama milk. Skimmed llama milk proteins were first characterized by a 2D separation technique coupling RP-HPLC in the first dimension with SDS-PAGE in the second dimension (RP-HPLC/SDS-PAGE). Llama milk proteins, namely caseins (αs1 -, αs2 -, β-, and κ-caseins), α-lactalbumin, lactoferrin, and serum albumin, were identified using PMF. Llama milk proteins were also characterized by online LC-ESI-MS analysis. This approach allowed attributing precise molecular masses for most of the previously MS-identified llama milk proteins. Interestingly, α-lactalbumin exhibits distinct chromatographic behaviors between llama and dromedary camel milk. De novo sequencing of the llama α-lactalbumin protein by LC coupled with MS/MS (LC-MS/MS) showed the occurrence of two amino acid substitutions (R62L/I and K89L/I) that partly explained the higher hydrophobicity of llama α-lactalbumin compared with its dromedary counterpart. Taken together, these results provide for the first time a thorough description of the protein fraction of Lama glama milk. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. An in vivo characterization of colostrum protein uptake in porcine gut during early lactation

    DEFF Research Database (Denmark)

    Danielsen, Marianne; Pedersen, Lene Juul; Bendixen, Emøke

    2011-01-01

    Understanding the bioactive roles of colostrum proteins has gained much attention, and in particular, their potential use in human and veterinary medicine has been extensively studied. However, studies of bioactivity have mainly been conducted in vitro, but it has not yet been well characterized...... at the individual protein level which colostrum components are internalized by the intestinal tissue of the neonate. The aim of this study was to characterize the in vivo processing of porcine colostrum in the gastrointestinal tract, and describe which of the potential bioactive proteins can be observed...... in the small intestinal tissue, and therefore may be functionally important. Using 2D-LC-MS/MS analysis we mapped the proteins in porcine colostrum. The colostrum proteins were then traced in the stomach content, as well as in the small intestinal tissue of 5 piglets suckled for 24 h. For comparison, we also...

  4. Synthesis and characterization of an MRI Gd-based probe designed to target the translocator protein

    International Nuclear Information System (INIS)

    Cerutti, Erika; Aime, Silvio; Damont, Annelaure; Dolle, Frederic; Baroni, Simona

    2013-01-01

    DPA-713 is the lead compound of a recently reported pyrazolo[1,5-a]pyrimidine acetamide series, targeting the translocator protein (TSPO 18 kDa), and as such, this structure, as well as closely related derivatives, have been already successfully used as positron emission tomography radioligands. On the basis of the pharmacological core of this ligands series, a new magnetic resonance imaging probe, coded DPA-C6-(Gd)DOTAMA was designed and successfully synthesized in six steps and 13% overall yield from DPA-713. The Gd-DOTA monoamide cage (DOTA = 1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetraacetic acid) represents the magnetic resonance imaging reporter, which is spaced from the phenyl-pyrazolo[1,5-a]pyrimidine acetamide moiety (DPA-713 motif) by a six carbon-atom chain. DPA-C6-(Gd)DOTAMA relaxometric characterization showed the typical behavior of a small-sized molecule (relaxivity value: 6.02 mM -1 s -1 at 20 MHz). The good hydrophilicity of the metal chelate makes DPAC6-(Gd)DOTAMA soluble in water, affecting thus its biodistribution with respect to the parent lipophilic DPA-713 molecule. For this reason, it was deemed of interest to load the probe to a large carrier in order to increase its residence lifetime in blood. Whereas DPA-C6-(Gd)DOTAMA binds to serum albumin with a low affinity constant, it can be entrapped into liposomes (both in the membrane and in the inner aqueous cavity). The stability of the supramolecular adduct formed by the Gd-complex and liposomes was assessed by a competition test with albumin. (authors)

  5. Characterization of the neutronic fields obtained by means of neutron traps inside the nuclear reactor core IPEN/MB-01

    International Nuclear Information System (INIS)

    Mura, Luiz Ernesto Credidio

    2011-01-01

    This paper presents the results of the neutron flux values obtained from the deployment of a Flux Trap of neutrons in the reactor core IPEN/MB-01. We analyzed several configurations of Flux Traps deployed in the reactor core IPEN/MB-01 in order to get elected to Flux Trap configuration more efficient. To characterize the neutron spectrum were irradiated in the center of the Flux Trap activation detectors of different materials (Au, Sc, In, Ti, Ni). The respective gamma spectroscopy of these elements after irradiation with and without cadmium cover, provided the experimental values of the nuclear reaction rates (saturation activity) by the target nuclei and their uncertainties used as input to the code SANDBP who calculated the energy spectrum of neutrons in the center of the 'Flux-Trap' in 50 energy groups, using the input spectra calculated at the irradiation position (center of the 'Flux Trap') by codes for Reactor Physics. The results found an increase in the thermal neutron flux in the center of the Flux Trap configuration 203 for the standard configuration (default) of about 350% without having the need to increase the reactor power. We also made comparisons between the spectra obtained by SANDBP deployed, compared to those calculated by MCNP-4C code and XSDRNPM. The spatial characterization of the thermal neutron flux is made with activation foils in the form of an infinitely dilute bulk alloy of 1% Au and 99% Al in some internal points of the configuration 203 (axially to Flux Trap a nd adjacent radial) and the results showed a significant increase in the magnitude of their values when compared to standard rectangular configuration. (author)

  6. A scalable double-barcode sequencing platform for characterization of dynamic protein-protein interactions.

    Science.gov (United States)

    Schlecht, Ulrich; Liu, Zhimin; Blundell, Jamie R; St Onge, Robert P; Levy, Sasha F

    2017-05-25

    Several large-scale efforts have systematically catalogued protein-protein interactions (PPIs) of a cell in a single environment. However, little is known about how the protein interactome changes across environmental perturbations. Current technologies, which assay one PPI at a time, are too low throughput to make it practical to study protein interactome dynamics. Here, we develop a highly parallel protein-protein interaction sequencing (PPiSeq) platform that uses a novel double barcoding system in conjunction with the dihydrofolate reductase protein-fragment complementation assay in Saccharomyces cerevisiae. PPiSeq detects PPIs at a rate that is on par with current assays and, in contrast with current methods, quantitatively scores PPIs with enough accuracy and sensitivity to detect changes across environments. Both PPI scoring and the bulk of strain construction can be performed with cell pools, making the assay scalable and easily reproduced across environments. PPiSeq is therefore a powerful new tool for large-scale investigations of dynamic PPIs.

  7. Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

    International Nuclear Information System (INIS)

    Vázquez-Iglesias, Lorena; Lostalé-Seijo, Irene; Martínez-Costas, José; Benavente, Javier

    2012-01-01

    A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

  8. Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

    Energy Technology Data Exchange (ETDEWEB)

    Vazquez-Iglesias, Lorena; Lostale-Seijo, Irene; Martinez-Costas, Jose [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain); Benavente, Javier, E-mail: franciscojavier.benavente@usc.es [Departamento de Bioquimica y Biologia Molecular, Facultad de Farmacia, y Centro Singular de Investigacion en Quimica Biologica y Materiales Moleculares (CIQUS), Universidad de Santiago de Compostela, 15782-Santiago de Compostela (Spain)

    2012-10-25

    A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.

  9. Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor.

    Directory of Open Access Journals (Sweden)

    Alessandro Pandini

    Full Text Available Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM domains (amino-terminal (FliGN, middle (FliGM and FliGC as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6. FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM

  10. Characterization of ferritin core on redox reactions as a nanocomposite for electron transfer

    International Nuclear Information System (INIS)

    Shin, Kwang Min; Watt, Richard K.; Watt, Gerald D.; Choi, Sang H.; Kim, Hyug-Han; Kim, Sun I.; Kim, Seon Jeong

    2010-01-01

    The kinetics of the change in mass related to the release from and deposition onto the cavities of a ferritin in the SWCNT nanocomposite by electrochemical redox reactions, and the effects of the SWCNT on the kinetics of the variation in mass of the ferritin nanocomposite were characterized using an electrochemical quartz crystal microbalance. The change in mass of reconstituted ferritin in the SWCNT nanocomposite shows reversible variation and stability of the ferritin/SWCNT nanocomposite on redox reactions was confirmed by using a coreless apoferritin and a Fe 2+ chelating agent. The ferritin/SWCNT nanocomposite is a good candidate for applications based on electron transfer, such as biosensor, biobatteries and electrodes for biofuel cell.

  11. Synthesis and characterization of magnetic and non-magnetic core-shell polyepoxide micrometer-sized particles of narrow size distribution.

    Science.gov (United States)

    Omer-Mizrahi, Melany; Margel, Shlomo

    2009-01-15

    Core polystyrene microspheres of narrow size distribution were prepared by dispersion polymerization of styrene in a mixture of ethanol and 2-methoxy ethanol. Uniform polyglycidyl methacrylate/polystyrene core-shell micrometer-sized particles were prepared by emulsion polymerization at 73 degrees C of glycidyl methacrylate in the presence of the core polystyrene microspheres. Core-shell particles with different properties (size, surface morphology and composition) have been prepared by changing various parameters belonging to the above seeded emulsion polymerization process, e.g., volumes of the monomer glycidyl methacrylate and the crosslinker monomer ethylene glycol dimethacrylate. Magnetic Fe(3)O(4)/polyglycidyl methacrylate/polystyrene micrometer-sized particles were prepared by coating the former core-shell particles with magnetite nanoparticles via a nucleation and growth mechanism. Characterization of the various particles has been accomplished by routine methods such as light microscopy, SEM, FTIR, BET and magnetic measurements.

  12. Structural Insight into the Core of CAD, the Multifunctional Protein Leading De Novo Pyrimidine Biosynthesis.

    Science.gov (United States)

    Moreno-Morcillo, María; Grande-García, Araceli; Ruiz-Ramos, Alba; Del Caño-Ochoa, Francisco; Boskovic, Jasminka; Ramón-Maiques, Santiago

    2017-06-06

    CAD, the multifunctional protein initiating and controlling de novo biosynthesis of pyrimidines in animals, self-assembles into ∼1.5 MDa hexamers. The structures of the dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains of human CAD have been previously determined, but we lack information on how these domains associate and interact with the rest of CAD forming a multienzymatic unit. Here, we prove that a construct covering human DHO and ATC oligomerizes as a dimer of trimers and that this arrangement is conserved in CAD-like from fungi, which holds an inactive DHO-like domain. The crystal structures of the ATC trimer and DHO-like dimer from the fungus Chaetomium thermophilum confirm the similarity with the human CAD homologs. These results demonstrate that, despite being inactive, the fungal DHO-like domain has a conserved structural function. We propose a model that sets the DHO and ATC complex as the central element in the architecture of CAD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Characterization of immunogenic proteins of Cysticercus tenuicollis of goats

    Directory of Open Access Journals (Sweden)

    A. Goswami

    2013-10-01

    Full Text Available Aim: To identify immunodominant proteins of cystic fluid antigens and whole cyst lysate antigens of Cysticercus tenuicollis, the larval stage of the canine tapeworm Taenia hydatigena. Materials and Methods: Three numbers of cysts of C. tenuicollis were collected from the mesentry of small intestine of goats after slaughter. C. tenuicollis cysts of each sample were washed thoroughly with PBS (pH 7.4. Two types of antigens i.e. cystic fluid antigens and whole cyst lysate antigens were prepared from each sample. Polypeptide profiles of cystic fluid antigens and whole cyst lysate antigens preparations of C. tunicollis were analysed by Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE containing 10% gel. C. tenuicollis proteins resolved by SDS-PAGE were electrophoretically transferred from the gel to a nitrocellulose membrane and probed by western blotting using natural immune sera obtained from infected goats. C. tenuicollis proteins that were resolved by SDS-PAGE and reactive by immune sera of goat was estimated by a Soft ware programme named KODAK 1D Image Analysis Software. Results: A total of eight major polypeptides of molecular weight (Mr 149.4 kDa, 92.9 kDa, 74.2 kDa, 63.5 kDa, 36.2 kDa, 23.9 kDa, 15.7 kDa and 9.6 kDa were resolved by SDS-PAGE with minor variations. Out of these, three polypeptides of Mr 36.2 kDa, 23.9 kDa and 9.6 kDa were recognized as immunodominant polypeptides after western blotting. Both cystic fluid antigens and whole cyst lysate antigens resolved same Mr polypeptides by SDS-PAGE and identified same immunodominant polypeptides after western blotting. The immunodominant polipeptides of Mr 23.9 kDa and 9.6 kDa identified for the first time from both cystic fluid antigens and whole cyst lysate antigens prepared from Cysticercus tenuicollis. Conclusion: Eight polypeptides were resolved from cystic fluid antigens and whole cyst lysate antigens of Cysticercus tenuicollis by SDS-PAGe of which polypeptides of

  14. Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

    DEFF Research Database (Denmark)

    Nesgaard, Lise W.; Hoffmann, Søren Vrønning; Andersen, Christian Beyschau

    2008-01-01

    Circular dichroism using synchrotron radiation (SRCD) can extend the spectral range down to approximately 130 nm for dry proteins, potentially providing new structural information. Using a selection of dried model proteins, including alpha-helical, beta-sheet, and mixed-structure proteins, we...... with previously published theoretical calculations related to pi-orbital transitions. We also show that drying does not lead to large changes in the secondary structure and does not induce orientational artifacts. In combination with principal component analysis, our SRCD data allow us to distinguish between two...... different types of protein fibrils, highlighting that bona fide fibrils formed by lysozyme are structurally more similar to the nonclassical fibrillar aggregates formed by the SerADan peptide than with the amyloid formed by alpha-synuclein. Thus, despite the lack of direct structural conclusions...

  15. Purification and Characterization of Antioxidant Peptide from Sunflower Protein Hydrolysate

    Directory of Open Access Journals (Sweden)

    Xi-Qun Zheng

    2010-01-01

    Full Text Available Sunflower proteins were hydrolyzed with Flavourzyme for the production of antioxidant peptide. DEAE-Sepharose Fast Flow, Sephadex G-25 gel filtration chromatography and reversed-phase HPLC were consecutively employed to purify a novel sunflower antioxidant peptide, and the ability to inhibit the autoxidation of pyrogallol was expressed as the antioxidative activity of the peptide. The amino acid sequence was identified as Ala-Cys-Ala-His-Asp-Lys-Val by a Q-Tof2 mass spectrometer. This novel peptide exhibited a high antioxidative activity of 79.42 U/mL, which is expected to protect against oxidative damage in living systems in relation to aging and carcinogenesis. Higher antioxidative activities were presumed mainly due to the presence of hydrophobic amino acids in its sequence.

  16. Characterization of a MAPKK-like protein kinase TOPK

    International Nuclear Information System (INIS)

    Matsumoto, Suguru; Abe, Yasuhito; Fujibuchi, Taketsugu; Takeuchi, Takashi; Kito, Katsumi; Ueda, Norifumi; Shigemoto, Kazuhiro; Gyo, Kiyofumi

    2004-01-01

    A MAPKK-like protein kinase TOPK expresses in a wide range of proliferating cells and tissues such as cancer cells and testis. However, details of this kinase are still uncovered. We investigated the intracellular distribution of TOPK and its association with cdk1/cyclin B and microtubules. In interphase cells, TOPK expresses in cytosol and nucleus without any significant association with microtubule networks. During mitosis, TOPK-Thr-9 was phosphorylated by cdk1/cyclin B and TOPK significantly associates with mitotic spindles. When TOPK expression was suppressed, formation of spindle midzone was thinned and dimmed and cytokinesis was disturbed. We propose that TOPK plays a role in the formation of spindle midzone and in cytokinesis

  17. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    Science.gov (United States)

    Han, Hongyu; Dong, Hui; Zhu, Shunhai; Zhao, Qiping; Jiang, Lianlian; Wang, Yange; Li, Liujia; Wu, Youlin; Huang, Bing

    2014-01-01

    Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results

  18. Molecular characterization and analysis of a novel protein disulfide isomerase-like protein of Eimeria tenella.

    Directory of Open Access Journals (Sweden)

    Hongyu Han

    Full Text Available Protein disulfide isomerase (PDI and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE according to the expressed sequence tag (EST. The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC. BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells

  19. Further characterization of protein kinase C in mouse mast cells

    International Nuclear Information System (INIS)

    White, J.R.; Ishizaka, T.

    1986-01-01

    Bridging of cell-bound IgE antibody molecules on colony stimulating factor dependent mouse mast cell line (PT-18) cells by multivalent antigen induces the mobilization and uptake of Ca 2+ monitored by Quin-2 and the production of diacylglycerol. Exposure of the sensitized cells to antigen also induces a substantial increase in protein kinase C (PKC) activity in the plasma membrane (340 units to 1375 units: 1 unit = 1 pmol of 32 P incorporated into Histone H-1/min/10 7 cells), within 30 seconds. There is also an increase in 3 H phorbol-12, 13-dibutyrate ( 3 H-PDB) binding which parallels the increase in PKC activity both in kinetics and antigen dose dependency. Determination of K/sub m/ and V/sub max/ for PKC revealed no difference between the cytosolic and membranous forms of PKC. Partial purification of PKC from the membrane of sensitized mast cells which had been labeled with 32 P and stimulated with DNP-HSA revealed a protein of 80-84,000 molecular weight, which migrated on polyacrylamide gel electrophoresis just above an authentic standard of PKC purified from rat brain. Treatment of the PKC from mouse mast cell membrane with alkaline phosphatase resulted in a reduction of phosphorylating activity and bindability of 3 H-PDB. In conclusion, the authors speculate that activation of mouse mast cells by cross-linking IgE results in the phosphorylation of a silent-pool of PKC converting it from an inactive state to an activated form

  20. Characterization of Carbohydrate Active Enzymes Involved in Arabinogalactan Protein Metabolism

    DEFF Research Database (Denmark)

    Knoch, Eva

    and tissues, their functions and synthesis are still poorly understood. The aim of the research presented in the thesis was to characterize carbohydrate active enzymes involved in AGP biosynthesis and modification to gain insights into the biosynthesis of the glycoproteins in plants. Candidate...... glycosyltransferases and glycoside hydrolases were selected based on co-expression profiles from a transcriptomics analysis. Reverse genetics approach on a novel glucuronosyltransferase involved in AGP biosynthesis has revealed that the enzyme activity is required for normal cell elongation in etiolated seedlings....... The enzymatic activity of a hydrolase from GH family 17 was investigated, without successful determination of the activity. Members of hydrolase family 43 appeared to be localized in the Golgi-apparatus, which is also the compartment for glycan biosynthesis. The localization of these glycoside hydrolases...

  1. Characterization of Vadose Zone Sediment: Uncontaminated RCRA Borehole Core Samples and Composite Samples

    International Nuclear Information System (INIS)

    Serne, R. Jeffrey; Bjornstad, Bruce N.; Schaef, Herbert T.; Williams, Bruce A.; Lanigan, David C.; Horton, Duane G.; Clayton, Ray E.; Mitroshkov, Alexandre V.; Legore, Virginia L.; O'Hara, Matthew J.; Brown, Christopher F.; Parker, Kent E.; Kutnyakov, Igor V.; Serne, Jennifer N.; Last, George V.; Smith, Steven C.; Lindenmeier, Clark W.; Zachara, John M.; Burke, Deborah S.

    2008-01-01

    This report was revised in September 2008 to remove acid-extractable sodium data from Tables 4.14, 4.16, 5.20, 5.22, 5.43, and 5.45. The sodium data was removed due to potential contamination introduced during the acid extraction process. The rest of the text remains unchanged from the original report issued in February 2002. The overall goal of the of the Tank Farm Vadose Zone Project, led by CH2M HILL Hanford Group, Inc., is to define risks from past and future single-shell tank farm activities. To meet this goal, CH2M HILL Hanford Group, Inc. asked scientists from Pacific Northwest National Laboratory to perform detailed analyses on vadose zone sediment from within the S-SX Waste Management Area. This report is one in a series of four reports to present the results of these analyses. Specifically, this report contains all the geologic, geochemical, and selected physical characterization data collected on vadose zone sediment recovered from Resource Conservation and Recovery Act (RCRA) borehole bore samples and composite samples

  2. Characterization of Vadose Zone Sediment: Uncontaminated RCRA Borehole Core Samples and Composite Samples

    Energy Technology Data Exchange (ETDEWEB)

    Serne, R. Jeffrey; Bjornstad, Bruce N.; Schaef, Herbert T.; Williams, Bruce A.; Lanigan, David C.; Horton, Duane G.; Clayton, Ray E.; Mitroshkov, Alexandre V.; Legore, Virginia L.; O' Hara, Matthew J.; Brown, Christopher F.; Parker, Kent E.; Kutnyakov, Igor V.; Serne, Jennifer N.; Last, George V.; Smith, Steven C.; Lindenmeier, Clark W.; Zachara, John M.; Burke, Deborah S.

    2008-09-11

    This report was revised in September 2008 to remove acid-extractable sodium data from Tables 4.14, 4.16, 5.20, 5.22, 5.43, and 5.45. The sodium data was removed due to potential contamination introduced during the acid extraction process. The rest of the text remains unchanged from the original report issued in February 2002. The overall goal of the of the Tank Farm Vadose Zone Project, led by CH2M HILL Hanford Group, Inc., is to define risks from past and future single-shell tank farm activities. To meet this goal, CH2M HILL Hanford Group, Inc. asked scientists from Pacific Northwest National Laboratory to perform detailed analyses on vadose zone sediment from within the S-SX Waste Management Area. This report is one in a series of four reports to present the results of these analyses. Specifically, this report contains all the geologic, geochemical, and selected physical characterization data collected on vadose zone sediment recovered from Resource Conservation and Recovery Act (RCRA) borehole bore samples and composite samples.

  3. HCV core protein-induced down-regulation of microRNA-152 promoted aberrant proliferation by regulating Wnt1 in HepG2 cells.

    Directory of Open Access Journals (Sweden)

    Shifeng Huang

    Full Text Available Hepatitis C virus (HCV has been reported to regulate cellular microRNAs (miRNAs. The HCV core protein is considered to be a potential oncoprotein in HCV-related hepatocellular carcinoma (HCV-HCC, but HCV core-regulated miRNAs are largely unknown. Our preliminary experiments revealed significant down-regulation of microRNA-152 (miR-152 by HCV core protein in HepG2 cells. Through target gene prediction softwares, Wnt1 was predicted to be a potential target of miR-152. The present study was initiated to investigate whether miR-152 is aberrantly regulated by the HCV core protein, and involved in the regulation of the aberrant proliferation of HCV-HCC cells.MiR-152 levels were examined by stem-loop real-time RT-PCR (SLqRT-PCR. Cell proliferation was analyzed by MTT and colony formation assay. Cell cycle analysis was performed by flow cytometry. Luciferase reporter assay was conducted to confirm miRNA-target association. Wnt1 expression was determined by real-time qPCR and Western blotting.HCV core protein significantly suppressed miR-152 expression, and led to significant Wnt1 up-regulation with a concomitant aberrantly promoted proliferation. Moreover, we validated that miR-152 inhibition promoted, while miR-152 mimics inhibited cell proliferation. Using, qRT-PCR and western blot, Wnt1 was demonstrated to be regulated by miR-152. Luciferase activity assay showed that while miR-152 mimics significantly reduced the luciferase activity by 83.76% (P<0.0001, miR-152 inhibitor showed no effect on luciferase reporter. Most notably, salvage expression of miR-152 after Ad-HCV core infection for 24 h almost totally reversed the proliferation-promoting effect of the HCV core protein, and meanwhile, reduced the expression of both Wnt1 mRNA and protein to basal levels.These findings provide important evidence that the reduced miR-152 expression by HCV core protein can indirectly lose an inhibitory effect on Wnt1, which might, at least partially lead to cell

  4. Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins.

    Science.gov (United States)

    Lakbub, Jude C; Shipman, Joshua T; Desaire, Heather

    2018-04-01

    Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass

  5. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    Directory of Open Access Journals (Sweden)

    Sanjukta Chakrabarti

    2016-06-01

    Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

  6. Characterization of white grub (Melolonthidae; Coleoptera in salak plantation based on morphology and protein banding pattern

    Directory of Open Access Journals (Sweden)

    SUGIYARTO

    2009-07-01

    Full Text Available Maryati KT, Sugiyarto. 2010. Characterization of white grub (Melolonthidae; Coleoptera in salak plantation based on morphology and protein banding pattern. Nusantara Bioscience 1: 72-77. This research aims to find out the white grub (Melolonthidae; Coleoptera variability based on the morphological characteristic and protein banding pattern found in ”salak pondoh” farm in Regencies of Sleman, Yogyakarta and Magelang, Central Java. Each area has five sampling points. Morphological analysis on white grub was conducted using descriptive method and analysis on protein banding pattern was conducted using qualitative analysis based on the presence or absent of band pattern on the gel, and qualitatively based on the relative mobility value (Rf of protein. The result indicated that the white grub in Sleman and Magelang, based on morphology characteristic is only one species, namely Holothricia sp. Based on the protein banding pattern, the white grub sample have differences of protein band number and protein molecular weight. Key words: Salacca zalacca, white grub, morphology, protein banding pattern.Abstrak. Maryati KT, Sugiyarto. 2010. Karakterisasi lundi putih (Melolonthidae: Coleoptera pada pertanaman salak berdasarkan ciri morfologi dan pola pita protein. Nusantara Bioscience 1: 72-77. Penelitian ini bertujuan untuk mengetahui keanekaragaman lundi putih (Melolonthidae; Coleoptera berdasarkan ciri morfologi dan pola pita protein yang ditemukan di lahan pertanaman salak pondoh di Kabupaten Sleman, Yogyakarta dan Kabupaten Magelang, Jawa Tengah. Pada masing-masing wilayah diambil lima titik sampling. Analisis morfologi lundi putih digunakan metode deskriptif, dan analisis pola pita protein digunakan analisis kualitatif berdasarkan muncul tidaknya pola pita pada gel, dan secara kuantitatif berdasarkan nilai mobilitas relatif protein (RF. Hasil penelitian menunjukkan bahwa sampel lundi putih di Kabupaten Sleman dan Magelang, berdasar karakter

  7. Characterization of Mediator Complex and its Associated Proteins from Rice.

    Science.gov (United States)

    Samanta, Subhasis; Thakur, Jitendra Kumar

    2017-01-01

    The Mediator complex is a multi-protein complex that acts as a molecular bridge conveying transcriptional messages from the cis element-bound transcription factor to the RNA Polymerase II machinery. It is found in all eukaryotes including members of the plant kingdom. Increasing number of reports from plants regarding different Mediator subunits involved in a multitude of processes spanning from plant development to environmental interactions have firmly established it as a central hub of plant regulatory networks. Routine isolation of Mediator complex in a particular species is a necessity because of many reasons. First, composition of the Mediator complex varies from species to species. Second, the composition of the Mediator complex in a particular species is not static under all developmental and environmental conditions. Besides this, at times, Mediator complex is used in in vitro transcription systems. Rice, a staple food crop of the world, is used as a model monocot crop. Realizing the need of a reliable protocol for the isolation of Mediator complex from plants, we describe here the isolation of Mediator complex from rice.

  8. Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-β Precursor Protein Transgenic Mice

    Science.gov (United States)

    Liu, Peng; Reichl, John H.; Rao, Eshaan R.; McNellis, Brittany M.; Huang, Eric S.; Hemmy, Laura S.; Forster, Colleen L.; Kuskowski, Michael A.; Borchelt, David R.; Vassar, Robert; Ashe, Karen H.; Zahs, Kathleen R.

    2016-01-01

    There exist several dozen lines of transgenic mice that express human amyloid-β precursor protein (AβPP) with Alzheimer’s disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ~4.5 times that of 21-month Tg2576 mice and ~15 times that of 21–24-month rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort. PMID:28059792

  9. Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-β Protein Precursor Transgenic Mice.

    Science.gov (United States)

    Liu, Peng; Reichl, John H; Rao, Eshaan R; McNellis, Brittany M; Huang, Eric S; Hemmy, Laura S; Forster, Colleen L; Kuskowski, Michael A; Borchelt, David R; Vassar, Robert; Ashe, Karen H; Zahs, Kathleen R

    2017-01-01

    There exist several dozen lines of transgenic mice that express human amyloid-β protein precursor (AβPP) with Alzheimer's disease (AD)-linked mutations. AβPP transgenic mouse lines differ in the types and amounts of Aβ that they generate and in their spatiotemporal patterns of expression of Aβ assemblies, providing a toolkit to study Aβ amyloidosis and the influence of Aβ aggregation on brain function. More complete quantitative descriptions of the types of Aβ assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aβ toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AβPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ∼4.5 times that of 21-month-old Tg2576 mice and ∼15 times that of 21-24-month-old rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort.

  10. Characterization of hapten-protein conjugates: antibody generation and immunoassay development for chlorophenoxyacetic acid pesticides.

    Science.gov (United States)

    Boro, Robin C; Singh, K Vikas; Suri, C Raman

    2009-01-01

    The generation of specific and sensitive antibodies against small molecules is greatly dependent upon the characteristics of the hapten-protein conjugates. In this study, we report a new fluorescence-based method for the characterization of hapten-protein conjugates. The method is based on an effect promoted by hapten-protein conjugation density upon the fluorescence intensity of the intrinsic tryptophan chromophore molecules of the protein. The proposed methodology is applied to quantify the hapten-protein conjugation density for two different chlorophenoxyacetic acid pesticides, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4-dichlorophenoxybutyric acid (2,4-DB), coupled to carrier protein. Highly sensitive anti-2,4-D and anti-2,4-DB antibodies were obtained using these well-characterized hapten-protein conjugates. The generated antibodies were used in an immunoassay format demonstrating inhibitory concentration (IC50) values equal to 30 and 7 ng/mL for 2,4-D and 2,4-DB, respectively. Linearity was observed in the concentration range between 0.1-500 nglmL with LODs around 4 and 3 ng/mL for 2,4-D and 2,4-DB, respectively, in standard water samples. The proposed method was successfully applied for the determination of the extent of hapten-protein conjugation to produce specific antibodies for immunoassay development against pesticides.

  11. Characterization and antifungal properties of wheat nonspecific lipid transfer proteins.

    Science.gov (United States)

    Sun, Jin-Yue; Gaudet, Denis A; Lu, Zhen-Xiang; Frick, Michele; Puchalski, Byron; Laroche, André

    2008-03-01

    This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZalpha and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 microM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.

  12. Functional characterization of antibodies against Neisseria gonorrhoeae opacity protein loops.

    Directory of Open Access Journals (Sweden)

    Jessica G Cole

    2009-12-01

    Full Text Available The development of a gonorrhea vaccine is challenged by the lack of correlates of protection. The antigenically variable neisserial opacity (Opa proteins are expressed during infection and have a semivariable (SV and highly conserved (4L loop that could be targeted in a vaccine. Here we compared antibodies to linear (Ab(linear and cyclic (Ab(cyclic peptides that correspond to the SV and 4L loops and selected hypervariable (HV(2 loops for surface-binding and protective activity in vitro and in vivo.Ab(SV cyclic bound a greater number of different Opa variants than Ab(SV linear, including variants that differed by seven amino acids. Antibodies to the 4L peptide did not bind Opa-expressing bacteria. Ab(SV (cyclic and Ab(HV2 (cyclic, but not Ab(SV (linear or Ab(HV2 linear agglutinated homologous Opa variants, and Ab(HV2BD (cyclic but not Ab(HV2BD (linear blocked the association of OpaB variants with human endocervical cells. Only Ab(HV2BD (linear were bactericidal against the serum resistant parent strain. Consistent with host restrictions in the complement cascade, the bactericidal activity of Ab(HV2BD (linear was increased 8-fold when rabbit complement was used. None of the antibodies was protective when administered vaginally to mice. Antibody duration in the vagina was short-lived, however, with <50% of the antibodies recovered 3 hrs post-administration.We conclude that an SV loop-specific cyclic peptide can be used to induce antibodies that recognize a broad spectrum of antigenically distinct Opa variants and have agglutination abilities. HV(2 loop-specific cyclic peptides elicited antibodies with agglutination and adherence blocking abilities. The use of human complement when testing the bactericidal activity of vaccine-induced antibodies against serum resistant gonococci is also important.

  13. Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima.

    Science.gov (United States)

    Clark, A M; Jacobsen, K R; Bostwick, D E; Dannenhoffer, J M; Skaggs, M I; Thompson, G A

    1997-07-01

    Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lactin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lactin, further suggesting an interaction between these phloem-specific proteins.

  14. Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

    Science.gov (United States)

    Brito-Argáez, Ligia; Tamayo-Sansores, José A; Madera-Piña, Dianeli; García-Villalobos, Francisco J; Moo-Puc, Rosa E; Kú-González, Ángela; Villanueva, Marco A; Islas-Flores, Ignacio

    2016-12-01

    The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an ∼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Characterizing Mobile/Less-Mobile Porosity and Solute Exchange in Dual-Domain Media Using Tracer Experiments and Electrical Measurements in a Hassler-Type Core Holder

    Science.gov (United States)

    Falzone, S.; Slater, L. D.; Day-Lewis, F. D.; Parker, B. L.; Keating, K.; Robinson, J.

    2017-12-01

    Mass transfer is the process by which solute is retained in less-mobile porosity domains, and later released into the mobile porosity domain. This process is often responsible for the slow arrival and gradual release of contaminants and solute tracers. Recent studies have outlined methods using dual-domain mass transfer (DDMT) models for characterizing this phenomenon. These models use the non-linear relationship of bulk (σb) and fluid (σf) conductivity, collected from electrical methods during tracer experiments, to characterize the less-mobile/mobile porosity ratio (β) and the mass-transfer rate coefficient (α). DDMT models use the hysteretic σb-σf relationship observed while solute tracers are injected and then flushed from a sample media. Due to limitations in observing the hysteretic σb-σf relationship, this method has not been used to characterize low permeability samples. We have developed an experimental method for testing porous rock cores that allows us to develop a fundamental understanding of contaminant storage and release in consolidated rock. We test the approach on cores from sedimentary rock sites where mass transfer is expected to occur between hydraulically connected fractures and the adjacent low permeability rock matrix. Our method uses a Hassler-type core holder, designed to apply confining pressure around the outside of a sample core, which hydraulically isolates the sample core, allowing water to be injected into it at increased pressures. The experimental apparatus was also designed to measure σb with spectral induced polarization (SIP) measurements, and σf from a sampling port located at the center of the core. Cores were initially saturated with a solution with high electrical conductivity ( 80000 μS/cm). DI water was then injected into the cores at elevated pressures (>60 psi) and the saturating solution was flushed from the cores, in order to generate flow rates fast enough to capture the non-linear σb-σf relationship

  16. An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice

    International Nuclear Information System (INIS)

    Geldmacher, Astrid; Skrastina, Dace; Petrovskis, Ivars; Borisova, Galina; Berriman, John A.; Roseman, Alan M.; Crowther, R. Anthony; Fischer, Jan; Musema, Shamil; Gelderblom, Hans R.; Lundkvist, Aake; Renhofa, Regina; Ose, Velta; Krueger, Detlev H.; Pumpens, Paul; Ulrich, Rainer

    2004-01-01

    Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains

  17. Liver cancer-derived hepatitis C virus core proteins shift TGF-beta responses from tumor suppression to epithelial-mesenchymal transition.

    Directory of Open Access Journals (Sweden)

    Serena Battaglia

    Full Text Available BACKGROUND: Chronic hepatitis C virus (HCV infection and associated liver cirrhosis represent a major risk factor for hepatocellular carcinoma (HCC development. TGF-beta is an important driver of liver fibrogenesis and cancer; however, its actual impact in human cancer progression is still poorly known. The aim of this study was to investigate the role of HCC-derived HCV core natural variants on cancer progression through their impact on TGF-beta signaling. PRINCIPAL FINDINGS: We provide evidence that HCC-derived core protein expression in primary human or mouse hepatocyte alleviates TGF-beta responses in terms or growth inhibition or apoptosis. Instead, in these hepatocytes TGF-beta was still able to induce an epithelial to mesenchymal transition (EMT, a process that contributes to the promotion of cell invasion and metastasis. Moreover, we demonstrate that different thresholds of Smad3 activation dictate the TGF-beta responses in hepatic cells and that HCV core protein, by decreasing Smad3 activation, may switch TGF-beta growth inhibitory effects to tumor promoting responses. CONCLUSION/SIGNIFICANCE: Our data illustrate the capacity of hepatocytes to develop EMT and plasticity under TGF-beta, emphasize the role of HCV core protein in the dynamic of these effects and provide evidence for a paradigm whereby a viral protein implicated in oncogenesis is capable to shift TGF-beta responses from cytostatic effects to EMT development.

  18. Room-temperature synthesis of core-shell structured magnetic covalent organic frameworks for efficient enrichment of peptides and simultaneous exclusion of proteins.

    Science.gov (United States)

    Lin, Guo; Gao, Chaohong; Zheng, Qiong; Lei, Zhixian; Geng, Huijuan; Lin, Zian; Yang, Huanghao; Cai, Zongwei

    2017-03-28

    Core-shell structured magnetic covalent organic frameworks (Fe 3 O 4 @COFs) were synthesized via a facile approach at room temperature. Combining the advantages of high porosity, magnetic responsiveness, chemical stability and selectivity, Fe 3 O 4 @COFs can serve as an ideal absorbent for the highly efficient enrichment of peptides and the simultaneous exclusion of proteins from complex biological samples.

  19. Characterization of Mammalian Selenoprotein O: A Redox-Active Mitochondrial Protein

    OpenAIRE

    Han, Seong-Jeong; Lee, Byung Cheon; Yim, Sun Hee; Gladyshev, Vadim N.; Lee, Seung-Rock

    2014-01-01

    Selenoproteins exhibit diverse biological functions, most of which are associated with redox control. However, the functions of approximately half of mammalian selenoproteins are not known. One such protein is Selenoprotein O (SelO), the largest mammalian selenoprotein with orthologs found in a wide range of organisms, including bacteria and yeast. Here, we report characterization of mammalian SelO. Expression of this protein could be verified in HEK 293T cells by metabolic labeling of cells ...

  20. In-Depth Characterization of Protein Disulfide Bonds by Online Liquid Chromatography-Electrochemistry-Mass Spectrometry

    Science.gov (United States)

    Switzar, Linda; Nicolardi, Simone; Rutten, Julie W.; Oberstein, Saskia A. J. Lesnik; Aartsma-Rus, Annemieke; van der Burgt, Yuri E. M.

    2016-01-01

    Disulfide bonds are an important class of protein post-translational modifications, yet this structurally crucial modification type is commonly overlooked in mass spectrometry (MS)-based proteomics approaches. Recently, the benefits of online electrochemistry-assisted reduction of protein S-S bonds prior to MS analysis were exemplified by successful characterization of disulfide bonds in peptides and small proteins. In the current study, we have combined liquid chromatography (LC) with electrochemistry (EC) and mass analysis by Fourier transform ion cyclotron resonance (FTICR) MS in an online LC-EC-MS platform to characterize protein disulfide bonds in a bottom-up proteomics workflow. A key advantage of a LC-based strategy is the use of the retention time in identifying both intra- and interpeptide disulfide bonds. This is demonstrated by performing two sequential analyses of a certain protein digest, once without and once with electrochemical reduction. In this way, the "parent" disulfide-linked peptide detected in the first run has a retention time-based correlation with the EC-reduced peptides detected in the second run, thus simplifying disulfide bond mapping. Using this platform, both inter- and intra-disulfide-linked peptides were characterized in two different proteins, ß-lactoglobulin and ribonuclease B. In order to prevent disulfide reshuffling during the digestion process, proteins were digested at a relatively low pH, using (a combination of) the high specificity proteases trypsin and Glu-C. With this approach, disulfide bonds in ß-lactoglobulin and ribonuclease B were comprehensively identified and localized, showing that online LC-EC-MS is a useful tool for the characterization of protein disulfide bonds.

  1. Structural characterization and comparative analysis of human and piscine cartilage acidic protein (CRTAC1/CRTAC2)

    OpenAIRE

    Guerreiro, Marta Lúcia Amaro

    2014-01-01

    Dissertação de mestrado, Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014 CRTAC (Cartilage Acidic Protein) firstly identified as a chondrocyte marker in humans and implicated in a number of diseases. This ancient protein is present from prokaryotes to vertebrates and the teleost are the only group that contain duplicates (CRTAC1/CRTAC2). The structure of CRTACs is poorly characterized and was the starting point of the present study. To establi...

  2. Characterization of mitosis-specific phosphorylation of tumor-associated microtubule-associated protein

    OpenAIRE

    Hong, Kyung Uk; Kim, Hyun-Jun; Bae, Chang-Dae; Park, Joobae

    2009-01-01

    Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton associated protein 2 (CKAP2), has been recently shown to be involved in the assembly and maintenance of mitotic spindle and also plays an essential role in maintaining the fidelity of chromosome segregation during mitosis. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis, and characterized the mechanism and functional importance of phosphorylation at one o...

  3. Characterization of Particles in Protein Solutions: Reaching the Limits of Current Technologies

    OpenAIRE

    Demeule, Barth?lemy; Messick, Steven; Shire, Steven J.; Liu, Jun

    2010-01-01

    Recent publications have emphasized the lack of characterization methods available for protein particles in a size range comprised between 0.1 and 10??m and the potential risk of immunogenicity associated with such particles. In the present paper, we have investigated the performance of light obscuration, flow microscopy, and Coulter counter instruments for particle counting and sizing in protein formulations. We focused on particles 2?10??m in diameter and studied the effect of silicon oil d...

  4. Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis.

    Science.gov (United States)

    Lee, Jin Ju; Lim, Jeong Ju; Kim, Dae Geun; Simborio, Hannah Leah; Kim, Dong Hyeok; Reyes, Alisha Wehdnesday Bernardo; Min, WonGi; Lee, Hu Jang; Kim, Dong Hee; Chang, Hong Hee; Kim, Suk

    2014-09-01

    In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Characterization of atmospheric trace elements in the Puruogangri ice core: a preliminary account of Tibetan Plateau environmental and contamination histories

    Science.gov (United States)

    Beaudon, E.; Gabrielli, P.; Sierra Hernandez, R.; Wegner, A.; Thompson, L. G.

    2014-12-01

    Asia is facing enormous challenges including large-scale environmental changes, rapid population growth and industrialization. The inherent generated pollution contributes to half of all Earth's anthropogenic trace metals emissions that, when deposited to glaciers of the surrounding mountains of the Third Pole region, leave a characteristic chemical fingerprint. Records of past atmospheric deposition preserved in snow and ice from Third Pole glaciers provide unique insights into changes of the chemical composition of the atmosphere and into the nature and intensity of the regional atmospheric circulation systems. The determination of the elemental composition of aeolian dust stored in Himalayan and Tibetan Plateau glaciers can help to qualify the potential contamination of glacial meltwater as a part of the greater fresh Asian water source. The 215 m long Puruogangri ice core retrieved in 2000 at 6500 m a.s.l. in central Tibetan Plateau (Western Tanggula Shan, China) provides one of the first multi-millennium-long environmental archives (spanning the last 7000 years and annually resolved for the last 400 years) obtained from the Tibetan Plateau region. The Puruogangri's area is climatologically of particular interest because of its location at the boundary between the monsoon (wet) and the westerly (dry) dominated atmospheric circulation. The major objective of this study is to determine the concentration of trace and ultra-trace elements in the Puruogangri ice core between 1600 and 2000 AD in order to characterize the atmospheric aerosols entrapped in the ice. Particular attention is given to assess the amount of trace elements originating from anthropogenic sources during both the pre-industrial and industrial periods. The distinction between the anthropogenic contribution and the crustal background may rely on the precise decoupling of the dry and wet seasons signals, the former being largely influenced by dust contribution.

  6. {sup 210}Pb geochronology and chemical characterization of sediment cores from lakes of the Parana river alluvial plain

    Energy Technology Data Exchange (ETDEWEB)

    Teixeira, L.F.L.; Damatto, S.R.; Scapin, M.A. [IPEN - Instituto de Pesquisas Energeticas e Nucleares (Brazil); Remor, M.B.; Sampaio, S.C. [UNIOESTE - Universidade Estadual do Oeste do Parana (Brazil)

    2014-07-01

    The flood plain of the upper Parana River is located among the lakes formed by the Brazilian hydroelectric plants being the last part of the Parana river, in Brazil, where there is an ecosystem with interaction river-flood plain. This flood plain has considerable habitat variability, with great diversity of terrestrial and aquatic species, and the floods are the main factor that regulates the operation of this ecosystem. The seasonality of the flood pulses is mainly influenced by the El Nino phenomenon, which increases precipitation in the drainage basin of the flood plain of the upper Parana River. Because of its unique characteristics this ecosystem is the subject of intense study since 1980, mainly from the ecological point of view. Therefore, two sediment cores were collected in the ponds formed by the floods, Patos pond and Garcas pond, in order to characterize the sediment chemically and evaluate a possible historic contamination. The trace element concentrations As, Ba, Br, Ce, Co, Cr, Cs, Eu, Hf, La, Lu, Nd, Rb, Sb, Sc, Sm, Ta, Tb, Th, U, Yb and Zn (mg.kg{sup -1}) and the major elements Si, Al, Fe, Ti, K, Ca, Mg, P, V, Mn, and Na (%) were determined in the sediment cores dated by {sup 210}Pb method, using instrumental neutron activation analysis, X-ray fluorescence and gross beta counting, respectively. The results obtained for the elements Ce, Cr, Cs, La, Nd, Sc, Sm and Th are higher than the values of Upper Continental Crust for both ponds. The sedimentation rates obtained for Garca pond, 0.77 cm.y{sup -1}, and Patos pond, 0.62 cm.y{sup -1} are in agreement with studies performed in sedimentary environments similar to the present work, such as Brazilian wetland Pantanal. The enrichment factor and the geo-accumulation index were used to assess the presence of anthropogenic sources of pollution. Document available in abstract form only. (authors)

  7. Characterization of the ternary Usher syndrome SANS/ush2a/whirlin protein complex.

    Science.gov (United States)

    Sorusch, Nasrin; Bauß, Katharina; Plutniok, Janet; Samanta, Ananya; Knapp, Barbara; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe

    2017-03-15

    The Usher syndrome (USH) is the most common form of inherited deaf-blindness, accompanied by vestibular dysfunction. Due to the heterogeneous manifestation of the clinical symptoms, three USH types (USH1-3) and additional atypical forms are distinguished. USH1 and USH2 proteins have been shown to function together in multiprotein networks in photoreceptor cells and hair cells. Mutations in USH proteins are considered to disrupt distinct USH protein networks and finally lead to the development of USH.To get novel insights into the molecular pathomechanisms underlying USH, we further characterize the periciliary USH protein network in photoreceptor cells. We show the direct interaction between the scaffold protein SANS (USH1G) and the transmembrane adhesion protein ush2a and that both assemble into a ternary USH1/USH2 complex together with the PDZ-domain protein whirlin (USH2D) via mutual interactions. Immunohistochemistry and proximity ligation assays demonstrate co-localization of complex partners and complex formation, respectively, in the periciliary region, the inner segment and at the synapses of rodent and human photoreceptor cells. Protein-protein interaction assays and co-expression of complex partners reveal that pathogenic mutations in USH1G severely affect formation of the SANS/ush2a/whirlin complex. Translational read-through drug treatment, targeting the c.728C > A (p.S243X) nonsense mutation, restored SANS scaffold function. We conclude that USH1 and USH2 proteins function together in higher order protein complexes. The maintenance of USH1/USH2 protein complexes depends on multiple USH1/USH2 protein interactions, which are disrupted by pathogenic mutations in USH1G protein SANS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. Characterization of Protein-Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy.

    Science.gov (United States)

    Koshari, Stijn H S; Ross, Jean L; Nayak, Purnendu K; Zarraga, Isidro E; Rajagopal, Karthikan; Wagner, Norman J; Lenhoff, Abraham M

    2017-02-06

    Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.

  9. Characterization of a baculovirus nuclear localization signal domain in the late expression factor 3 protein

    International Nuclear Information System (INIS)

    Au, Victoria; Yu Mei; Carstens, Eric B.

    2009-01-01

    The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) single-stranded DNA binding protein LEF-3 is a multi-functional protein that is required to transport the helicase protein P143 into the nucleus of infected cells where they function to replicate viral DNA. The N-terminal 56 amino acid region of LEF-3 is required for nuclear transport. In this report, we analyzed the effect of site-specific mutagenesis of LEF-3 on its intracellular distribution. Fluorescence microscopy of expression plasmid-transfected cells demonstrated that the residues 28 to 32 formed the core nuclear localization signal, but other adjacent positively-charged residues augmented these sequences. Comparison with other group I Alphabaculoviruses suggested that this core region functionally duplicated residues including 18 and 19. This was demonstrated by the loss of nuclear localization when the equivalent residues (18 to 20) in Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) LEF-3 were mutated. The AcMNPV LEF-3 nuclear localization domain was also shown to drive nuclear transport in mammalian cells indicating that the protein nuclear import systems in insect and mammalian cells are conserved. We also demonstrated by mutagenesis that two conserved cysteine residues located at 82 and 106 were not essential for nuclear localization or for interaction with P143. However, by using a modified construct of P143 that localized on its own to the nucleus, we demonstrated that a functional nuclear localization domain on LEF-3 was required for interaction between LEF-3 and P143

  10. Functional characterization of JMJD2A, a histone deacetylase- and retinoblastoma-binding protein.

    Science.gov (United States)

    Gray, Steven G; Iglesias, Antonio H; Lizcano, Fernando; Villanueva, Raul; Camelo, Sandra; Jingu, Hisaka; Teh, Bin T; Koibuchi, Noriyuki; Chin, William W; Kokkotou, Efi; Dangond, Fernando

    2005-08-05

    To effectively direct targeted repression, the class I histone deacetylases (HDACs) associate with many important regulatory proteins. In this paper we describe the molecular characterization of a member of the Jumonji domain 2 (JMJD2) family of proteins, and demonstrate its binding to both class I HDACs and the retinoblastoma protein (pRb). JMJD2 proteins are characterized by the presence of two leukemia-associated protein/plant homeodomain (LAP/PHD) zinc fingers, one JmjN, one JmjC (containing an internal retinoblastoma-binding protein 2 (RBBP2)-like sequence), and two Tudor domains. The first member of this group, JMJD2A, is widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA was found in several cell types, including human T-cell lymphotropic virus 1 (HTLV-1)-infected cell lines. JMJD2A and JMJD2B exhibit cell type-specific responses to the HDAC inhibitor trichostatin A. We show that the JMJD2A protein associates in vivo with pRb and class I HDACs, and mediates repression of E2F-regulated promoters. In HTLV-1 virus-infected cells, we find that JMJD2A binds to the viral Tax protein. Antibodies to JMJD2A recognize the native protein but also a half-sized protein fragment, the latter up-regulated in THP-1 cells during the G(2)/M phase of the cell cycle. The ability of JMJD2A to associate with pRb and HDACs and potentiate pRb-mediated repression of E2F-regulated promoters implies an important role for this protein in cell proliferation and oncogenesis.

  11. KARAKTERISASI BIJI DAN PROTEIN KORO KOMAK (Lablab purpureus (L Sweet SEBAGAI SUMBER PROTEIN [Characterization of Hyacinth Bean (Lablab purpureus (L. Sweet Seed and Its Protein

    Directory of Open Access Journals (Sweden)

    Andrew S R

    2006-06-01

    Full Text Available This research aimed to characterize the physiochemical properties of hyacinth beans as new protein source. The result of research showed that hyacinth beans are oval shaped and orange and yellow coloured. The edible part of hyacinth beans is 83.2 ± 1.1 % of dry seed; in which the carbohydrate is 67.9 ± 1.1 %; protein: 17.1 ± 1.5 % and fat: 1.1 ± 0.4 %. According to their solubility, the protein fractions were found as albumin: 18.22 %; globuli : 55.15 % and glutelin : 26.13 %, whereas prolamin was not detected. Further analyis showed that, the globulin is consisted of globulin 7S (3.50% and globulin 11S (0.67 %. The hyacinth beans are potential to be used for protein source.

  12. A complete dosimetry experimental program in support to the core characterization and to the power calibration of the CABRI reactor. A complete dosimetry experimental program in support of the core characterization and of the power calibration of the CABRI reactor

    Energy Technology Data Exchange (ETDEWEB)

    Rodiac, F.; Hudelot, JP.; Lecerf, J.; Garnier, Y.; Ritter, G. [CEA, DEN, CAD/DER/SRES/LPRE, Cadarache, F-13108 Saint-Paul-lez-Durance, (France); Gueton, O.; Colombier, AC. [CEA, DEN, CAD/DER/SPRC/LPN, Cadarache, F-13108 Saint-Paul-lez-Durance, (France); Domergue, C. [CEA, DEN, CAD/DER/SPEx/LDCI, Cadarache, F-13108 Saint-Paul-lez-Durance, (France)

    2015-07-01

    CABRI is an experimental pulse reactor operated by CEA at the Cadarache research center. Since 1978 the experimental programs have aimed at studying the fuel behavior under Reactivity Initiated Accident (RIA) conditions. Since 2003, it has been refurbished in order to be able to provide RIA and LOCA (Loss Of Coolant Accident) experiments in prototypical PWR conditions (155 bar, 300 deg. C). This project is part of a broader scope including an overall facility refurbishment and a safety review. The global modification is conducted by the CEA project team. It is funded by IRSN, which is conducting the CIP experimental program, in the framework of the OECD/NEA project CIP. It is financed in the framework of an international collaboration. During the reactor restart, commissioning tests are realized for all equipment, systems and circuits of the reactor. In particular neutronics and power commissioning tests will be performed respectively in 2015 and 2016. This paper focuses on the design of a complete and original dosimetry program that was built in support to the CABRI core characterization and to the power calibration. Each one of the above experimental goals will be fully described, as well as the target uncertainties and the forecasted experimental techniques and data treatment. (authors)

  13. Synthesis, characterization of Ag-Au core-shell bimetal nanoparticles and its application for electrocatalytic oxidation/sensing of L-methionine

    Energy Technology Data Exchange (ETDEWEB)

    Murugavelu, M.; Karthikeyan, B., E-mail: bkarthi_au@yahoo.com

    2017-01-01

    The Ag-Au core-shell bimetal nanoparticles (BNPs) was prepared using chemical reduction method. The prepared Ag-Au core-shell BNPs were characterized by UV–Visible (UV–Vis) spectroscopy, field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HR-TEM), X-ray diffraction (XRD), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) pattern. These results showed the Ag-Au BNPs exhibited core-shell shape. The Ag-Au core-shell BNPs was examined towards electrocatalytic oxidation of L-methionine (L-Met) by cyclic voltammetry (CV), linear sweep voltammetry (LSV) and chronoamperometry. According to the results, L-Met is determined with detection limit of 30 μM. Interference studies in biological buffer was also studied. - Highlights: • The Ag-Au core-shell BNPs are synthesized and characterized • Ag-Au core-shell BNPs modified (Ag-Au/GCE) has been examined for L-methionine oxidation/sensing by using electrochemical method. • The Ag-Au/GCE exhibited good performance for the detection of L-methionine.

  14. In vitro assembly into virus-like particles is an intrinsic quality of Pichia pastoris derived HCV core protein

    International Nuclear Information System (INIS)

    Acosta-Rivero, Nelson; Rodriguez, Armando; Musacchio, Alexis; Falcon, Viviana; Suarez, Viana M.; Martinez, Gillian; Guerra, Ivis; Paz-Lago, Dalila; Morera, Yanelys; Rosa, Maria C. de la; Morales-Grillo, Juan; Duenas-Carrera, Santiago

    2004-01-01

    Different variants of hepatitis C virus core protein (HCcAg) have proved to self-assemble in vitro into virus-like particles (VLPs). However, difficulties in obtaining purified mature HCcAg have limited these studies. In this study, a high degree of monomeric HCcAg purification was accomplished using chromatographic procedures under denaturing conditions. Size exclusion chromatography and sucrose density gradient centrifugation of renatured HCcAg (in the absence of structured RNA) under reducing conditions suggested that it assembled into empty capsids. The electron microscopy analysis of renatured HCcAg showed the presence of spherical VLPs with irregular shapes and an average diameter of 35 nm. Data indicated that HCcAg monomers assembled in vitro into VLPs in the absence of structured RNA, suggesting that recombinant HCcAg used in this work contains all the information necessary for the assembly process. However, they also suggest that some cellular factors might be required for the proper in vitro assembly of capsids

  15. Characterization of particulate drug delivery systems for oral delivery of Peptide and protein drugs.

    Science.gov (United States)

    Christophersen, Philip Carsten; Fano, Mathias; Saaby, Lasse; Yang, Mingshi; Nielsen, Hanne Mørck; Mu, Huiling

    2015-01-01

    Oral drug delivery is a preferred route because of good patient compliance. However, most peptide/ protein drugs are delivered via parenteral routes because of the absorption barriers in the gastrointestinal (GI) tract such as enzymatic degradation by proteases and low permeability acrossthe biological membranes. To overcome these barriers, different formulation strategies for oral delivery of biomacromolecules have been proposed, including lipid based formulations and polymer-based particulate drug delivery systems (DDS). The aim of this review is to summarize the existing knowledge about oral delivery of peptide/protein drugs and to provide an overview of formulationand characterization strategies. For a better understanding of the challenges in oral delivery of peptide/protein drugs, the composition of GI fluids and the digestion processes of different kinds of excipients in the GI tract are summarized. Additionally, the paper provides an overview of recent studies on characterization of solid drug carriers for peptide/protein drugs, drug distribution in particles, drug release and stability in simulated GI fluids, as well as the absorption of peptide/protein drugs in cell-based models. The use of biorelevant media when applicable can increase the knowledge about the quality of DDS for oral protein delivery. Hopefully, the knowledge provided in this review will aid the establishment of improved biorelevant models capable of forecasting the performance of particulate DDS for oral peptide/protein delivery.

  16. Preparation and characterization of alginate microspheres for sustained protein delivery within tissue scaffolds

    International Nuclear Information System (INIS)

    Zhai Peng; Chen, X B; Schreyer, David J

    2013-01-01

    Tissue engineering scaffolds are designed not only to provide structural support for the repair of damaged tissue, but can also serve the function of bioactive protein delivery. Here we present a study on the preparation and characterization of protein-loaded microspheres, either alone or incorporated into mock tissue scaffolds, for sustained protein delivery. Alginate microspheres were prepared by a novel, small-scale water-in-oil emulsion technique and loaded with fluorescently labeled immunoglobulin G (IgG). Microsphere size appears to be influenced by the magnitude and distribution of force generated by mechanical stirring during emulsion. Protein release studies show that sustained IgG release from microspheres could be achieved and that application of a secondary coating of chitosan could further slow the rate of protein release. Preservation of bioactivity of released IgG protein was confirmed using an immunohistochemical assay. When IgG-loaded microspheres were incorporated into mock scaffolds, initial protein release was diminished and the overall time course of release was extended. The present study demonstrates that protein-loaded microspheres can be prepared with a controlled release profile and preserved biological activity, and can be incorporated into scaffolds to achieve sustained and prolonged protein delivery in a tissue engineering application. (paper)

  17. Construction and characterization of a pure protein hydrogel for drug delivery application.

    Science.gov (United States)

    Xu, Xu; Xu, ZhaoKang; Yang, XiaoFeng; He, YanHao; Lin, Rong

    2017-02-01

    Injectable hydrogels have a variety of applications, including regenerative medicine, tissue engineering and controlled drug delivery. In this paper, we reported on a pure protein hydrogel based on tetrameric recombinant proteins for the potential drug delivery application. This protein hydrogel was formed instantly by simply mixing two recombinant proteins (ULD-TIP1 and ULD-GGGWRESAI) through the specific protein-peptide interaction. The protein hydrogel was characterized by rheology and scanning electron microscopy (SEM). In vitro cytotoxicity test indicated that the developed protein hydrogel had no apparent cytotoxicity against L-929 cells and HCEC cells after 48h incubation. The formed protein hydrogels was gradually degraded after incubation in phosphate buffered solution (PBS, pH=7.4) for a period of 144h study, as indicated by in vitro degradation test. Encapsulation of model drug (sodium diclofenac; DIC) were achieved by simple mixing of drugs with hydrogelator and the entrapped drugs was almost completely released from hydrogels within 24h via a diffusion manner. As a conclusion, the simple and mild preparation procedure and good biocompatibility of protein hydrogel would render its good promising candidate for drug delivery applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Characterization of the antigenicity of Cpl1, a surface protein of Cryptococcus neoformans var. neoformans.

    Science.gov (United States)

    Cai, Jian-Piao; Liu, Ling-Li; To, Kelvin K W; Lau, Candy C Y; Woo, Patrick C Y; Lau, Susanna K P; Guo, Yong-Hui; Ngan, Antonio H Y; Che, Xiao-Yan; Yuen, Kwok-Yung

    2015-01-01

    Cryptococcus neoformans var. neoformans is an important fungal pathogen. The capsule is a well established virulence factor and a target site for diagnostic tests. The CPL1 gene is required for capsular formation and virulence. The protein product Cpl1 has been proposed to be a secreted protein, but the characteristics of this protein have not been reported. Here we sought to characterize Cpl1. Phylogenetic analysis showed that the Cpl1 of C. neoformans var. neoformans and the Cpl1 orthologs identified in C. neoformans var. grubii and C. gattii formed a distinct cluster among related fungi; while the putative ortholog found in Trichosporon asahii was distantly related to the Cryptococcus cluster. We expressed Cpl1 abundantly as a secreted His-tagged protein in Pichia pastoris. The protein was used to immunize guinea pigs and rabbits for high titer mono-specific polyclonal antibody that was shown to be highly specific against the cell wall of C. neoformans var. neoformans and did not cross react with C. gattii, T. asahii, Aspergillus spp., Candida spp. and Penicillium spp. Using the anti-Cpl1 antibody, we detected Cpl1 protein in the fresh culture supernatant of C. neoformans var. neoformans and we showed by immunostaining that the Cpl1 protein was located on the surface. The Cpl1 protein is a specific surface protein of C. neoformans var. neoformans. © 2015 by The Mycological Society of America.

  19. Systematic Proteomic Approach to Characterize the Impacts of Chemical Interactions on Protein and Cytotoxicity Responses to Metal Mixture Exposures

    Science.gov (United States)

    Chemical interactions have posed a big challenge in toxicity characterization and human health risk assessment of environmental mixtures. To characterize the impacts of chemical interactions on protein and cytotoxicity responses to environmental mixtures, we established a systems...

  20. Prediction and characterization of protein-protein interaction networks in swine

    Directory of Open Access Journals (Sweden)

    Wang Fen

    2012-01-01

    Full Text Available Abstract Background Studying the large-scale protein-protein interaction (PPI network is important in understanding biological processes. The current research presents the first PPI map of swine, which aims to give new insights into understanding their biological processes. Results We used three methods, Interolog-based prediction of porcine PPI network, domain-motif interactions from structural topology-based prediction of porcine PPI network and motif-motif interactions from structural topology-based prediction of porcine PPI network, to predict porcine protein interactions among 25,767 porcine proteins. We predicted 20,213, 331,484, and 218,705 porcine PPIs respectively, merged the three results into 567,441 PPIs, constructed four PPI networks, and analyzed the topological properties of the porcine PPI networks. Our predictions were validated with Pfam domain annotations and GO annotations. Averages of 70, 10,495, and 863 interactions were related to the Pfam domain-interacting pairs in iPfam database. For comparison, randomized networks were generated, and averages of only 4.24, 66.79, and 44.26 interactions were associated with Pfam domain-interacting pairs in iPfam database. In GO annotations, we found 52.68%, 75.54%, 27.20% of the predicted PPIs sharing GO terms respectively. However, the number of PPI pairs sharing GO terms in the 10,000 randomized networks reached 52.68%, 75.54%, 27.20% is 0. Finally, we determined the accuracy and precision of the methods. The methods yielded accuracies of 0.92, 0.53, and 0.50 at precisions of about 0.93, 0.74, and 0.75, respectively. Conclusion The results reveal that the predicted PPI networks are considerably reliable. The present research is an important pioneering work on protein function research. The porcine PPI data set, the confidence score of each interaction and a list of related data are available at (http://pppid.biositemap.com/.

  1. Experimental characterization of cement-bentonite interaction using core infiltration techniques coupled with 4D X-ray tomography

    International Nuclear Information System (INIS)

    Dolder, F.; Maeder, U.; Jenni, A.

    2012-01-01

    Document available in extended abstract form only. Deep geological disposal of radioactive waste foresee cementitious materials as reinforcement of tunnels, backfill, or as waste matrix. Bentonite, or sand-bentonite mixtures, are proposed as backfill material enclosing spent fuel drums, or as drift seals. The emplacement of cementitious material next to clay material generates an enormous chemical gradient in pore water, which drives diffusive transport. Laboratory studies and reactive transport modeling predict significant mineral alteration on both sides of interfaces, mainly resulting in a decrease of porosity in bentonite. The goal of the project is to characterize and quantify the cement/bentonite skin effects spatially and temporally. The expected analytical data set will be amenable to interpretation by reactive transport modeling. The project focuses on the advective-diffusive transport domain, resolved at intermediate spatial scales. The experiments are carried out in a newly developed X-ray transparent core infiltration apparatus, constructed from carbon fiber compounds and suitable for confining and infiltration pressures up to 80 bars. This setup allows to performing X-ray computed tomography scans (CT) periodically without interrupting the running core infiltration experiment. The experiment is performed on cylindrical bentonite samples (50 by 50 mm) compacted and saturated in-house with a dedicated device (Fig. 1, top). A sample is mounted in the core infiltration apparatus (Fig. 1, bottom) and subjected to a confining pressure in order to simulate underground repository conditions proposed by NAGRA (Swiss National Cooperative for the Disposal of Radioactive Waste). The infiltration of high-pH cement pore-fluids into the bentonite plug is expected to alter the mineral assemblage over time as an advancing reaction front. The related change in phase densities, porosity and bulk density is tracked over time recording periodically non-destructive CT scans

  2. Novel synthesis and characterization of Ag@TiO2 core shell nanostructure for non-enzymatic glucose sensor

    Science.gov (United States)

    T, Dayakar; Venkateswara Rao, K.; Vinodkumar, M.; Bikshalu, K.; Chakradhar, B.; Ramachandra Rao, K.

    2018-03-01

    Ag@TiO2 core-shell nano composite (ATCSNC) were synthesized by using Ocimum tenuiflorum leaves extract through a simple one-step hydrothermal route for Non-enzymatic glucose sensing material. The prepared NCs were characterized and found high crystallinity, red shift absorbance, interface-bonding parameters, rough surface and network like microstructure through XRD, Uv-vis, FTIR, SEM, and TEM. The prepared ATCSNC have been used for fabrication of glassy carbon electrode (GCE) and the same was applied to test its electro catalytic activity of glucose in 0.1 M NaOH. The promising results were recorded for ATCSNC/GCE with a high sensitivity (1968.72 μAm M-1cm-2), wide linear range (1 μM-8.1 mM), good response time (3 s), and excellent low detection limit (0.19 μM, S/N = 3). Furthermore, the designed sensor exhibits admirable stability and reproducibility, as well as attractive achievability for real sample analysis. As such, the proposed ATCSNC could be highly beneficial in the development of sustainable and eco-friendly glucose sensing devices.

  3. Application of TZERO calibrated modulated temperature differential scanning calorimetry to characterize model protein formulations.

    Science.gov (United States)

    Badkar, Aniket; Yohannes, Paulos; Banga, Ajay

    2006-02-17

    The objective of this study was to evaluate the feasibility of using T(ZERO) modulated temperature differential scanning calorimetry (MDSC) as a novel technique to characterize protein solutions using lysozyme as a model protein and IgG as a model monoclonal antibody. MDSC involves the application of modulated heating program, along with the standard heating program that enables the separation of overlapping thermal transitions. Although characterization of unfolding transitions for protein solutions requires the application of high sensitive DSC, separation of overlapping transitions like aggregation and other exothermic events may be possible only by use of MDSC. A newer T(ZERO) calibrated MDSC model from TA instruments that has improved sensitivity than previous models was used. MDSC analysis showed total, reversing and non-reversing heat flow signals. Total heat flow signals showed a combination of melting endotherms and overlapping exothermic events. Under the operating conditions used, the melting endotherms were seen in reversing heat flow signal while the exothermic events were seen in non-reversing heat flow signal. This enabled the separation of overlapping thermal transitions, improved data analysis and decreased baseline noise. MDSC was used here for characterization of lysozyme solutions, but its feasibility for characterizing therapeutic protein solutions needs further assessment.

  4. In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Musumeci, Lucia; Tautz, Lutz

    2005-01-01

    Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains...

  5. Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs)

    NARCIS (Netherlands)

    Bastiaan-Net, S.; Chanput, W.; Hertz, A.; Zwittink, R.D.; Mes, J.J.; Wichers, H.J.

    2013-01-01

    In this study two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in

  6. GeLCMS for in-depth protein characterization and advanced analysis of proteomes

    DEFF Research Database (Denmark)

    Lundby, Alicia; Olsen, Jesper V

    2011-01-01

    In recent years the array of mass spectrometry (MS) applications to address questions in molecular and cellular biology has greatly expanded and continues to grow. Modern mass spectrometers allow for identification, characterization, as well as quantification of protein compositions and their mod...

  7. Salt-bridge networks within globular and disordered proteins: characterizing trends for designable interactions.

    Science.gov (United States)

    Basu, Sankar; Mukharjee, Debasish

    2017-07-01

    There has been considerable debate about the contribution of salt bridges to the stabilization of protein folds, in spite of their participation in crucial protein functions. Salt bridges appear to contribute to the activity-stability trade-off within proteins by bringing high-entropy charged amino acids into close contacts during the course of their functions. The current study analyzes the modes of association of salt bridges (in terms of networks) within globular proteins and at protein-protein interfaces. While the most common and trivial type of salt bridge is the isolated salt bridge, bifurcated salt bridge appears to be a distinct salt-bridge motif having a special topology and geometry. Bifurcated salt bridges are found ubiquitously in proteins and interprotein complexes. Interesting and attractive examples presenting different modes of interaction are highlighted. Bifurcated salt bridges appear to function as molecular clips that are used to stitch together large surface contours at interacting protein interfaces. The present work also emphasizes the key role of salt-bridge-mediated interactions in the partial folding of proteins containing long stretches of disordered regions. Salt-bridge-mediated interactions seem to be pivotal to the promotion of "disorder-to-order" transitions in small disordered protein fragments and their stabilization upon binding. The results obtained in this work should help to guide efforts to elucidate the modus operandi of these partially disordered proteins, and to conceptualize how these proteins manage to maintain the required amount of disorder even in their bound forms. This work could also potentially facilitate explorations of geometrically specific designable salt bridges through the characterization of composite salt-bridge networks. Graphical abstract ᅟ.

  8. Characterization of Periplasmic Protein BP26 Epitopes of Brucella melitensis Reacting with Murine Monoclonal and Sheep Antibodies

    Science.gov (United States)

    Wu, Jingbo; Zhang, Hui; Wang, Yuanzhi; Qiao, Jun; Chen, Chuangfu; Gao, Goege F.; Allain, Jean-Pierre; Li, Chengyao

    2012-01-01

    More than 35,000 new cases of human brucellosis were reported in 2010 by the Chinese Center for Disease Control and Prevention. An attenuated B. melitensis vaccine M5-90 is currently used for vaccination of sheep and goats in China. In the study, a periplasmic protein BP26 from M5-90 was characterized for its epitope reactivity with mouse monoclonal and sheep antibodies. A total of 29 monoclonal antibodies (mAbs) against recombinant BP26 (rBP26) were produced, which were tested for reactivity with a panel of BP26 peptides, three truncated rBP26 and native BP26 containing membrane protein extracts (NMP) of B. melitensis M5-90 in ELISA and Western-Blot. The linear, semi-conformational and conformational epitopes from native BP26 were identified. Two linear epitopes recognized by mAbs were revealed by 28 of 16mer overlapping peptides, which were accurately mapped as the core motif of amino acid residues 93DRDLQTGGI101 (position 93 to 101) or residues 104QPIYVYPD111, respectively. The reactivity of linear epitope peptides, rBP26 and NMP was tested with 137 sheep sera by ELISAs, of which the two linear epitopes had 65–70% reactivity and NMP 90% consistent with the results of a combination of two standard serological tests. The results were helpful for evaluating the reactivity of BP26 antigen in M5-90. PMID:22457830

  9. Genetic characterization of early maturing maize hybrids (Zea mays L. obtained by protein and RAPD markers

    Directory of Open Access Journals (Sweden)

    Bauer Iva

    2005-01-01

    Full Text Available Knowledge of maize germplasm genetic diversity is important for planning breeding programmes, germplasm conservation per se etc. Genetic variability of maize hybrids grown in the fields is also very important because genetic uniformity implies risks of genetic vulnerability to stress factors and can cause great losts in yield. Early maturing maize hybrids are characterized by shorter vegetation period and they are grown in areas with shorter vegetation season. Because of different climatic conditions in these areas lines and hybrids are developed with different features in respect to drought resistance and disease resistance. The objective of our study was to characterize set of early maturing maize hybrids with protein and RAPD markers and to compare this clasification with their pedigree information. RAPD markers gave significantly higher rate of polymorphism than protein markers. Better corelation was found among pedigree information and protein markers.

  10. Isolation and characterization of gelatin-binding proteins from goat seminal plasma

    Directory of Open Access Journals (Sweden)

    Lazure Claude

    2003-04-01

    Full Text Available Abstract A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation and destabilization (capacitation process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80°C was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins. Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in

  11. Characterization and possible function of glyceraldehyde-3-phosphate dehydrogenase-spermatogenic protein GAPDHS in mammalian sperm.

    Science.gov (United States)

    Margaryan, Hasmik; Dorosh, Andriy; Capkova, Jana; Manaskova-Postlerova, Pavla; Philimonenko, Anatoly; Hozak, Pavel; Peknicova, Jana

    2015-03-08

    Sperm proteins are important for the sperm cell function in fertilization. Some of them are involved in the binding of sperm to the egg. We characterized the acrosomal sperm protein detected by a monoclonal antibody (MoAb) (Hs-8) that was prepared in our laboratory by immunization of BALB/c mice with human ejaculated sperms and we tested the possible role of this protein in the binding assay. Indirect immunofluorescence and immunogold labelling, gel electrophoresis, Western blotting and protein sequencing were used for Hs-8 antigen characterization. Functional analysis of GAPDHS from the sperm acrosome was performed in the boar model using sperm/zona pellucida binding assay. Monoclonal antibody Hs-8 is an anti-human sperm antibody that cross-reacts with the Hs-8-related protein in spermatozoa of other mammalian species (boar, mouse). In the immunofluorescence test, Hs-8 antibody recognized the protein localized in the acrosomal part of the sperm head and in the principal piece of the sperm flagellum. In immunoblotting test, MoAb Hs-8 labelled a protein of 45 kDa in the extract of human sperm. Sequence analysis identified protein Hs-8 as GAPDHS (glyceraldehyde 3-phosphate dehydrohenase-spermatogenic). For this reason, commercial mouse anti-GAPDHS MoAb was applied in control tests. Both antibodies showed similar staining patterns in immunofluorescence tests, in electron microscopy and in immunoblot analysis. Moreover, both Hs-8 and anti-GAPDHS antibodies blocked sperm/zona pellucida binding. GAPDHS is a sperm-specific glycolytic enzyme involved in energy production during spermatogenesis and sperm motility; its role in the sperm head is unknown. In this study, we identified the antigen with Hs8 antibody and confirmed its localization in the apical part of the sperm head in addition to the principal piece of the flagellum. In an indirect binding assay, we confirmed the potential role of GAPDHS as a binding protein that is involved in the secondary sperm

  12. C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich 167RRRSQSPRR175 Domain Is Critical for HBV Replication

    Science.gov (United States)

    Kim, Taeyeung; Shin, Bo-Hye; Park, Gil-Soon; Park, Sun; Chwae, Yong-Joon; Shin, Ho-Joon; Kim, Kyongmin

    2012-01-01

    To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221–262 amino acids of DHBV C protein, in place of 146–185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221–241 and 251–262 amino acids of DHBV C, in place of HBV C 146–166 and 176–185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242–250 of DHBV C (242RAGSPLPRS 250) introduced in place of 167–175 of HBV C (167RRRSQSPRR 175) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich 167RRRSQSPRR175 domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important. PMID:22911745

  13. Highly efficient enrichment of low-abundance intact proteins by core-shell structured Fe3O4-chitosan@graphene composites.

    Science.gov (United States)

    Zhang, Peng; Fang, Xiaoni; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

    2017-11-01

    In proteomics research, the screening and monitoring of disease biomarkers is still a major challenge, mainly due to their low concentration in biological samples. However, the universal enrichment of intact proteins has not been further studied. In this work, we developed a Fe 3 O 4 -chitosan@graphene (Fe 3 O 4 -CS@G) core-shell composite to enrich low-abundance proteins from biological samples. Fe 3 O 4 -CS@G composite holds chitosan layer decorated Fe 3 O 4 core, which improves the hydrophilicity of materials greatly. Meanwhile, the graphene nanosheets shell formed via electrostatic assembly endows the composite with huge surface area (178m 2 /g). The good water dispersibility ensures the sufficient contact opportunities between graphene composites and proteins, and the large surface area provides enough adsorption sites for the enrichment of proteins. Using Fe 3 O 4 -CS@G, four standard proteins Cyt-c, BSA, Myo and OVA were enriched with better adsorption capacity and recovery rate, compared with previously reported magnetic graphene composites. Additionally, the mechanism of compared to" is corrected into "compared with". proteins adsorption on Fe 3 O 4 -CS@G was further studied, which indicates that hydrophobic and electrostatic interaction work together to facilitate the universal and efficient enrichment of proteins. Human plasma sample was employed to further evaluate the enrichment performance of Fe 3 O 4 -CS@G. Eventually, 123 proteins were identified from one of SAX fractions of human plasma, which is much better than commercial Sep-pak C18 enrichment column (39 proteins). All these outstanding performances suggest that Fe 3 O 4 -CS@G is an ideal platform for the enrichment of low-abundance intact proteins and thus holds great potential to facilitate the identification of biomarkers from biological samples in proteomics research. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    KAUST Repository

    Ooi, Amanda Siok Lee

    2016-07-19

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

  15. Experimental strategies for the identification and characterization of adhesive proteins in animals: a review

    Science.gov (United States)

    Hennebert, Elise; Maldonado, Barbara; Ladurner, Peter; Flammang, Patrick; Santos, Romana

    2015-01-01

    Adhesive secretions occur in both aquatic and terrestrial animals, in which they perform diverse functions. Biological adhesives can therefore be remarkably complex and involve a large range of components with different functions and interactions. However, being mainly protein based, biological adhesives can be characterized by classical molecular methods. This review compiles experimental strategies that were successfully used to identify, characterize and obtain the full-length sequence of adhesive proteins from nine biological models: echinoderms, barnacles, tubeworms, mussels, sticklebacks, slugs, velvet worms, spiders and ticks. A brief description and practical examples are given for a variety of tools used to study adhesive molecules at different levels from genes to secreted proteins. In most studies, proteins, extracted from secreted materials or from adhesive organs, are analysed for the presence of post-translational modifications and submitted to peptide sequencing. The peptide sequences are then used directly for a BLAST search in genomic or transcriptomic databases, or to design degenerate primers to perform RT-PCR, both allowing the recovery of the sequence of the cDNA coding for the investigated protein. These sequences can then be used for functional validation and recombinant production. In recent years, the dual proteomic and transcriptomic approach has emerged as the best way leading to the identification of novel adhesive proteins and retrieval of their complete sequences. PMID:25657842

  16. A Guide to Transient Expression of Membrane Proteins in HEK-293 Cells for Functional Characterization

    KAUST Repository

    Ooi, Amanda Siok Lee; Wong, Aloysius Tze; Esau, Luke; Lemtiri-Chlieh, Fouad; Gehring, Christoph A

    2016-01-01

    The human embryonic kidney 293 (HEK-293) cells are commonly used as host for the heterologous expression of membrane proteins not least because they have a high transfection efficiency and faithfully translate and process proteins. In addition, their cell size, morphology and division rate, and low expression of native channels are traits that are particularly attractive for current-voltage measurements. Nevertheless, the heterologous expression of complex membrane proteins such as receptors and ion channels for biological characterization and in particular for single-cell applications such as electrophysiology remains a challenge. Expression of functional proteins depends largely on careful step-by-step optimization that includes the design of expression vectors with suitable identification tags, as well as the selection of transfection methods and detection parameters appropriate for the application. Here, we use the heterologous expression of a plant potassium channel, the Arabidopsis thaliana guard cell outward-rectifying K+ channel, AtGORK (At5G37500) in HEK-293 cells as an example, to evaluate commonly used transfection reagents and fluorescent detection methods, and provide a detailed methodology for optimized transient transfection and expression of membrane proteins for in vivo studies in general and for single-cell applications in particular. This optimized protocol will facilitate the physiological and cellular characterization of complex membrane proteins.

  17. Molecular Characterization of LRB7 Gene and a Water Channel Protein TIP2 in Chorispora bungeana

    Directory of Open Access Journals (Sweden)

    Ming Li

    2016-01-01

    Full Text Available Background. Water channel proteins, also called aquaporins, are integral membrane proteins from major intrinsic protein (MIP family and involved in several pathways including not only water transport but also cell signaling, reproduction, and photosynthesis. The full cDNA and protein sequences of aquaporin in Chorispora bungeana Fisch. & C.A. Mey (C. bungeana are still unknown. Results. In this study, PCR and rapid amplification of cDNA ends approaches were used to clone the full cDNA of LRB7 (GenBank accession number: EU636988 of C. bungeana. Sequence analysis indicated that it was 1235 bp, which had two introns and encoded a protein of 250 amino acids. Structure analysis revealed that the protein had two conserved NPA motifs, one of which is MIP signature sequence (SGxHxNPAVT, six membrane helix regions, and additional membrane-embedded domains. Phylogenetic analysis suggested that the protein was from TIP2 subgroup. Surprisingly, semiquantitative RT-PCR experiment and western blot analysis showed that LRB7 and TIP2 were only detectable in roots, unlike Arabidopsis and Raphanus. Connecting with our previous studies, LRB7 was supported to associate with chilling-tolerance in C. bungeana. Conclusion. This is the first time to characterize the full sequences of LRB7 gene and water channel protein in C. bungeana. Our findings contribute to understanding the water transports in plants under low temperatures.

  18. In Silico Characterization and Structural Modeling of Dermacentor andersoni p36 Immunosuppressive Protein

    Directory of Open Access Journals (Sweden)

    Martin Omulindi Oyugi

    2018-01-01

    Full Text Available Ticks cause approximately $17–19 billion economic losses to the livestock industry globally. Development of recombinant antitick vaccine is greatly hindered by insufficient knowledge and understanding of proteins expressed by ticks. Ticks secrete immunosuppressant proteins that modulate the host’s immune system during blood feeding; these molecules could be a target for antivector vaccine development. Recombinant p36, a 36 kDa immunosuppressor from the saliva of female Dermacentor andersoni, suppresses T-lymphocytes proliferation in vitro. To identify potential unique structural and dynamic properties responsible for the immunosuppressive function of p36 proteins, this study utilized bioinformatic tool to characterize and model structure of D. andersoni p36 protein. Evaluation of p36 protein family as suitable vaccine antigens predicted a p36 homolog in Rhipicephalus appendiculatus, the tick vector of East Coast fever, with an antigenicity score of 0.7701 that compares well with that of Bm86 (0.7681, the protein antigen that constitute commercial tick vaccine Tickgard™. Ab initio modeling of the D. andersoni p36 protein yielded a 3D structure that predicted conserved antigenic region, which has potential of binding immunomodulating ligands including glycerol and lactose, found located within exposed loop, suggesting a likely role in immunosuppressive function of tick p36 proteins. Laboratory confirmation of these preliminary results is necessary in future studies.

  19. Light assisted drying (LAD) for protein stabilization: optical characterization of samples

    Science.gov (United States)

    Young, Madison A.; McKinnon, Madison E.; Elliott, Gloria D.; Trammell, Susan R.

    2018-02-01

    Light-Assisted Drying (LAD) is a novel biopreservation technique which allows proteins to be immobilized in a dry, amorphous solid at room temperature. Indicator proteins are used in a variety of diagnostic assays ranging from highthroughput 96-well plates to new microfluidic devices. A challenge in the development of protein-based assays is preserving the structure of the protein during production and storage of the assay, as the structure of the protein is responsible for its functional activity. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. An inexpensive, simple processing method that enables supra-zero temperature storage of proteins used in assays is needed. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is optically characterize samples processed with LAD. We use polarized light imaging (PLI) to look at crystallization kinetics of samples and determine optimal humidity. PLI shows a 62.5% chance of crystallization during LAD processing and negligible crystallization during low RH storage.

  20. The nuclear core of sun: theoretical perspectives and characterization of the scientific performances of the GOLF / SOHO experiment detector

    International Nuclear Information System (INIS)

    Dzitko, H.

    1995-01-01

    The subject of this thesis is the nuclear core of the sun. The first part is theoretical and concerns neutrino flux predictions. A precise description of the solar plasma is necessary to predict boron, beryllium and CNO cycle neutrinos. We treat here the nuclear reaction rates. They are mainly determined by the cross sections and the enhancement factors due to plasma particles, the co-called screening factors. We have discussed the various possible formalisms that could be used in stellar evolution and performed direct calculations of screened cross sections. We concluded that the screening prescriptions which have been used so far in stellar evolution should be replaced by the Mitler formalism. Next, we examine the cross section uncertainties and we show that it is possible to get a better agreement between theory and experiment. Discrepancies between the gallium experiments and the calculations suggest that we should go beyond the classical solar model. This has motivated our study on possible magnetic fields deeply buried in the solar core. We discuss here the influence of a magnetic pressure perturbation on solar evolution. In the experimental part of this work, we deal with the GOLF experiment, one of the three helio-seismological experiments on board the space probe SOHO. The purpose of this instrument is the study of the global oscillation modes in the frequency range 10 -7 to 6 10 -3 Hz with a sensitivity for frequencies higher than 2 10 -4 Hz of about 1 mm/s over 20 days of continuous integration at counting rates of 12 10 6 cs/s. One part of this work was devoted to the precise characterization of the photomultipliers and their associated electronics in order to select them according to their intrinsic performances. This step was followed by long duration tests of three weeks simulating as well as possible the flight conditions. We show that the detection chain effectively meets the stability requirements of around 10 -7 by velocity measurement. We also

  1. Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

    Science.gov (United States)

    Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi

    2016-01-01

    Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Continuous production of core-shell protein nanoparticles by antisolvent precipitation using dual-channel microfluidization: Caseinate-coated zein nanoparticles.

    Science.gov (United States)

    Ebert, Sandra; Koo, Charmaine K W; Weiss, Jochen; McClements, David Julian

    2017-02-01

    Antisolvent precipitation is commonly used to fabricate protein nanoparticles using a simple batch method that involves injecting a protein-solvent mixture into an antisolvent. In this study, the potential of producing core-shell protein nanoparticles by antisolvent precipitation using a continuous dual-channel microfluidization method was investigated. The solvent phase (zein in ethanol) and antisolvent phase (casein in water) were made to impinge on each other at high velocity, which generates intense shear, turbulent, and cavitation forces that ensure thorough mixing and breakup of the phases. Relatively small core-shell protein nanoparticles (dnanoparticles went from positive at low pH to negative at high pH, with a point of zero charge around pH5. Electron microscopy indicated that the protein particles formed had a roughly spherical shape. The results suggest that the dual-channel microfluidizer could be used to continuously form protein nanoparticles by antisolvent precipitation. Nevertheless, when the microfluidization method was compared with the simple batch method the size of the particles produced under similar conditions were fairly similar. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Mesitylene-Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Ribeiro, Orquidea; Du, Yang

    2016-01-01

    Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents...

  4. Characterization and functional analyses of the human G protein-coupled receptor kinase 4 gene promoter.

    Science.gov (United States)

    Hasenkamp, Sandra; Telgmann, Ralph; Staessen, Jan A; Hagedorn, Claudia; Dördelmann, Corinna; Bek, Martin; Brand-Herrmann, Stefan-Martin; Brand, Eva

    2008-10-01

    The G protein-coupled receptor kinase 4 is involved in renal sodium handling and blood pressure regulation. Missense variants have already been tested functionally and are associated with hypertension, but no data on promoter analyses are yet available. We scanned 94 hypertensive white subjects for genetic variation and performed promoter reporter gene analyses in HEK293T, COS7, and SaOs-2 cells. Transient transfections with various full lengths and wild-type deletion constructs revealed that 1851 bp of the flanking region and 275 bp of the 5'-untranslated region were sufficient for transcriptional activities and composed a powerful cis-active element in the distal 293 bp. The -1702T and +2T alleles resulted in drastic general reductions of promoter function, whereas an activity increasing effect of +268C was cell type specific. Electrophoretic mobility-shift assay, supershift, and cotransfection analyses of transcription factor binding sites predicted in silico (Alibaba2.1/Transfac7) resulted in allele-specific binding patterns of nuclear proteins and identified the participation of CCAAT/enhancer-binding protein transcription factor family members. The G protein-coupled receptor kinase 4 core promoter resides in the first 1851 bp upstream of its transcription start site. The 4 identified genetic variants within this region exert allele-specific impact on both cell type- and stimulation-dependent transcription and may affect the expression balance of renal G protein-coupled receptor kinase 4.

  5. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

    Science.gov (United States)

    Stoyanov, B; Volinia, S; Hanck, T; Rubio, I; Loubtchenkov, M; Malek, D; Stoyanova, S; Vanhaesebroeck, B; Dhand, R; Nürnberg, B

    1995-08-04

    Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

  6. A centrifugation-based physicochemical characterization method for the interaction between proteins and nanoparticles

    Science.gov (United States)

    Bekdemir, Ahmet; Stellacci, Francesco

    2016-10-01

    Nanomedicine requires in-depth knowledge of nanoparticle-protein interactions. These interactions are studied with methods limited to large or fluorescently labelled nanoparticles as they rely on scattering or fluorescence-correlation signals. Here, we have developed a method based on analytical ultracentrifugation (AUC) as an absorbance-based, label-free tool to determine dissociation constants (KD), stoichiometry (Nmax), and Hill coefficient (n), for the association of bovine serum albumin (BSA) with gold nanoparticles. Absorption at 520 nm in AUC renders the measurements insensitive to unbound and aggregated proteins. Measurements remain accurate and do not become more challenging for small (sub-10 nm) nanoparticles. In AUC, frictional ratio analysis allows for the qualitative assessment of the shape of the analyte. Data suggests that small-nanoparticles/protein complexes significantly deviate from a spherical shape even at maximum coverage. We believe that this method could become one of the established approaches for the characterization of the interaction of (small) nanoparticles with proteins.

  7. 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.

  8. Recombinant expression, purification, and characterization of an acyl-CoA binding protein from Aspergillus oryzae.

    Science.gov (United States)

    Hao, Qing; Liu, Xiaoguang; Zhao, Guozhong; Jiang, Lu; Li, Ming; Zeng, Bin

    2016-03-01

    To characterize biochemically the lipid metabolism-regulating acyl-CoA binding protein (ACBP) from the industrially-important fungus Aspergillus oryzae. A full-length cDNA encoding a candidate ACBP from A. oryzae (AoACBP) was cloned and expressed in Escherichia coli as a maltose-binding protein (MBP) fusion protein. The MBP-AoACBP protein was purified by an amylose resin chromatography column. SDS-PAGE showed that MBP-AoACBP has an estimated molecular weight of 82 kDa. Microscale thermophoresis binding assay showed that the recombinant AoACBP displayed much greater affinity for palmitoyl-CoA (K d = 80 nM) than for myristoyl-CoA (K d = 510 nM), thus demonstrating the preference of AoACBP for long-chain acyl-CoA. The data support the identification of AoACBP as a long-chain ACBP in A. oryzae.

  9. Biophysical characterization of the olfactomedin domain of myocilin, an extracellular matrix protein implicated in inherited forms of glaucoma.

    Directory of Open Access Journals (Sweden)

    Susan D Orwig

    Full Text Available Myocilin is an eye protein found in the trabecular extracellular matrix (TEM, within the anatomic region that controls fluid flow. Variants of myocilin, localized to its olfactomedin (OLF domain, have been linked to inherited forms of glaucoma, a disease associated with elevated intraocular pressure. OLF domains have also been implicated in psychiatric diseases and cancers by their involvement in signaling, neuronal growth, and development. However, molecular characterization of OLFs has been hampered by challenges in recombinant expression, a hurdle we have recently overcome for the myocilin OLF domain (myoc-OLF. Here, we report the first detailed solution biophysical characterization of myoc-OLF to gain insight into its structure and function. Myoc-OLF is stable in the presence of glycosaminoglycans, as well as in a wide pH range in buffers with functional groups reminiscent of such glycosaminoglycans. Circular dichroism (CD reveals significant β-sheet and β-turn secondary structure. Unexpectedly, the CD signature is reminiscent of α-chymotrypsin as well as another ocular protein family, the βγ-crystallins. At neutral pH, intrinsic tryptophan fluorescence and CD melts indicate a highly cooperative transition with a melting temperature of ∼55 °C. Limited proteolysis combined with mass spectrometry reveals that the compact core structural domain of OLF consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. The data presented here inform new testable hypotheses for interactions with specific TEM components, and will assist in design of therapeutic agents for myocilin glaucoma.

  10. Characterization of SLCO5A1/OATP5A1, a solute carrier transport protein with non-classical function.

    Directory of Open Access Journals (Sweden)

    Katrin Sebastian

    Full Text Available Organic anion transporting polypeptides (OATP/SLCO have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20 and genes implicated in developmental processes (e.g. TGM2. A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration.

  11. Hepatitis C virus core protein induces dysfunction of liver sinusoidal endothelial cell by down-regulation of silent information regulator 1.

    Science.gov (United States)

    Sun, Li-Jie; Yu, Jian-Wu; Shi, Yu-Guang; Zhang, Xiao-Yu; Shu, Meng-Ni; Chen, Mo-Yang

    2018-05-01

    Hepatic fibrosis is a frequent feature of chronic hepatitis C virus (HCV) infection. Some evidence has suggested the potential role of silent information regulator 1 (SIRT1) in organ fibrosis. The aim of this study was to investigate the effect of HCV core protein on expression of SIRT1 of liver sinusoidal endothelial cell (LSEC) and function of LSEC. LSECs were co-cultured with HepG2 cells or HepG2 cells expressing HCV core protein and LSECs cultured alone were used as controls. After co-culture, the activity and expression levels of mRNA and protein of SIRT1 in LSEC were detected by a SIRT1 fluorometric assay kit, real time-PCR (RT-PCR), Western blot, respectively. The levels of adiponectin receptor 2 (AdipoR2), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were measured by Western blot. Cluster of differentiation 31 (CD31), CD14, and von Willebrand factor (vWf) of LSECs was performed by flow cytometry. The level of reactive oxygen species (ROS) was assayed. Malondialdehyde (MDA), superoxide dismutase (SOD), adiponectin, nitric oxide (NO), and endothelin-1 (ET-1) levels in the co-culture supernatant were measured. The co-culture supernatant was then used to cultivate LX-2 cells. The levels of α-smooth muscle actin (ASMA) and transforming growth factor-β1 (TGF-β1) protein in LX-2 cells were measured by Western blot. Compared with LSEC co-cultured with HepG2 cells group, in LSEC co-cultured with HepG2-core cells group, the activity and expression level of mRNA and protein of SIRT1 reduced; the level of adiponectin reduced and the expression level of AdipoR2 protein decreased; ROS levels increased; the expression level of eNOS, VEGF protein decreased; and the expression level of CD14 decreased; the expression level of vWf and CD31 increased; NO and SOD levels decreased; whereas ET-1 and MDA levels increased; the levels of ASMA and TGF-β1 protein in LX-2 cells increased. SIRT1 activator improved the above-mentioned changes

  12. New insights into micro/nanoscale combined probes (nanoAuger, μXPS) to characterize Ag/Au@SiO2 core-shell assemblies

    Science.gov (United States)

    Ledeuil, J. B.; Uhart, A.; Soulé, S.; Allouche, J.; Dupin, J. C.; Martinez, H.

    2014-09-01

    This work has examined the elemental distribution and local morphology at the nanoscale of core@shell Ag/Au@SiO2 particles. The characterization of such complex metal/insulator materials becomes more efficient when using an initial cross-section method of preparation of the core@shell nanoparticles (ion milling cross polisher). The originality of this route of preparation allows one to obtain undamaged, well-defined and planar layers of cross-cut nano-objects. Once combined with high-resolution techniques of characterization (XPS, Auger and SEM), the process appears as a powerful way to minimize charging effects and enhance the outcoming electron signal (potentially affected by the topography of the material) during analysis. SEM experiments have unambiguously revealed the hollow-morphology of the metal core, while Auger spectroscopy observations showed chemical heterogeneity within the particles (as silver and gold are randomly found in the core ring). To our knowledge, this is the first time that Auger nano probe spectroscopy has been used and successfully optimized for the study of some complex metal/inorganic interfaces at such a high degree of resolution (~12 nm). Complementarily, XPS Au 4f and Ag 3d peaks were finally detected attesting the possibility of access to the whole chemistry of such nanostructured assemblies.This work has examined the elemental distribution and local morphology at the nanoscale of core@shell Ag/Au@SiO2 particles. The characterization of such complex metal/insulator materials becomes more efficient when using an initial cross-section method of preparation of the core@shell