Uses of phage display in agriculture: a review of food-related protein-protein interactions discovered by biopanning over diverse baits.

Science.gov (United States)

Kushwaha, Rekha; Payne, Christina M; Downie, A Bruce

2013-01-01

This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s) contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions.

Differential activation of G-proteins by μ-opioid receptor agonists

Science.gov (United States)

Saidak, Zuzana; Blake-Palmer, Katherine; Hay, Debbie L; Northup, John K; Glass, Michelle

2006-01-01

We investigated the ability of the activated μ-opioid receptor (MOR) to differentiate between myristoylated Gαi1 and GαoA type Gα proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each Gα protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The Gα subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified Gα protein by CB1 cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[35S]GTPγS exchange was then compared for Gαi1 and GαoA. Activation of MOR by DAMGO produced a high-affinity saturable interaction for GαoA (Km=20±1 nM) but a low-affinity interaction with Gαi1 (Km=116±12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal Gα activation among the agonists evaluated. Endomorphins 1 and 2, methadone and β-endorphin activated both Gα to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between Gαi1 and GαoA. Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two Gα. Differences in maximal activity and potency, for Gαi1 versus GαoA, are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects. PMID:16415903

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

OpenAIRE

Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice...

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.

Science.gov (United States)

Ananphongmanee, Vorawit; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Boonchird, Chuenchit

2015-01-01

Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral

Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV in Shrimp.

Directory of Open Access Journals (Sweden)

Vorawit Ananphongmanee

Full Text Available Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7 and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1 promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7 and partial VP28 (pVP28 were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against

Dietary protein considerations to support active aging.

Science.gov (United States)

Wall, Benjamin T; Cermak, Naomi M; van Loon, Luc J C

2014-11-01

Given our rapidly aging world-wide population, the loss of skeletal muscle mass with healthy aging (sarcopenia) represents an important societal and public health concern. Maintaining or adopting an active lifestyle alleviates age-related muscle loss to a certain extent. Over time, even small losses of muscle tissue can hinder the ability to maintain an active lifestyle and, as such, contribute to the development of frailty and metabolic disease. Considerable research focus has addressed the application of dietary protein supplementation to support exercise-induced gains in muscle mass in younger individuals. In contrast, the role of dietary protein in supporting the maintenance (or gain) of skeletal muscle mass in active older persons has received less attention. Older individuals display a blunted muscle protein synthetic response to dietary protein ingestion. However, this reduced anabolic response can largely be overcome when physical activity is performed in close temporal proximity to protein consumption. Moreover, recent evidence has helped elucidate the optimal type and amount of dietary protein that should be ingested by the older adult throughout the day in order to maximize the skeletal muscle adaptive response to physical activity. Evidence demonstrates that when these principles are adhered to, muscle maintenance or hypertrophy over prolonged periods can be further augmented in active older persons. The present review outlines the current understanding of the role that dietary protein occupies in the lifestyle of active older adults as a means to increase skeletal muscle mass, strength and function, and thus support healthier aging.

A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

Science.gov (United States)

Donnini, Sandra; Finetti, Federica; Francese, Simona; Boscaro, Francesca; Dani, Francesca R; Maset, Fabio; Frasson, Roberta; Palmieri, Michele; Pazzagli, Mario; De Filippis, Vincenzo; Garaci, Enrico; Ziche, Marina

2011-12-01

Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

Novel Biosensor of Membrane Protein Proximity Based on Fluorogen Activated Proteins.

Science.gov (United States)

Vasilev, Kalin V; Gallo, Eugenio; Shank, Nathaniel; Jarvik, Jonathan W

2016-01-01

We describe a novel biosensor system for reporting proximity between cell surface proteins in live cultured cells. The biosensor takes advantage of recently developed fluorogen-activating proteins (FAPs) that display fluorescence only when bound to otherwise-nonfluorescent fluorogen molecules. To demonstrate feasibility for the approach, two recombinant rapamycin-binding proteins were expressed as single-pass plasma membrane proteins in HeLa cells; one of the proteins (scAvd- FRB) carried an extracellular avidin tag; the other (HL1-TO1-FKBP) carried an extracellular FAP. Cells were incubated with a membrane-impermeable bivalent ligand (biotin-PEG2000-DIR) consisting of biotin joined to a dimethyl-indole red (DIR) fluorogen by a polyethylene glycol linker, thus tethering the fluorogen to the scAvd-FRB fusion protein. Addition of rapamycin, which promotes FKBP-FRB dimerization and thereby brings the FAP in close proximity to the tethered fluorogen, led to a significant increase in DIR fluorescence. We call the new proximity assay TEFLA, for tethered fluorogen assay.

Adenovirus Particles that Display the Plasmodium falciparum Circumsporozoite Protein NANP Repeat Induce Sporozoite-Neutralizing Antibodies in Mice

Science.gov (United States)

Palma, Christopher; Overstreet, Michael G.; Guedon, Jean-Marc; Hoiczyk, Egbert; Ward, Cameron; Karen, Kasey A.; Zavala, Fidel; Ketner, Gary

2011-01-01

Adenovirus particles can be engineered to display exogenous peptides on their surfaces by modification of viral capsid proteins, and particles that display pathogen-derived peptides can induce protective immunity. We constructed viable recombinant adenoviruses that display B-cell epitopes from the Plasmodium falciparum circumsporozoite protein (PfCSP) in the major adenovirus capsid protein, hexon. Recombinants induced high-titer antibodies against CSP when injected intraperitoneally into mice. Serum obtained from immunized mice recognized both recombinant PfCSP protein and P. falciparum sporozoites, and neutralized P. falciparum sporozoites in vitro. Replicating adenovirus vaccines have provided economical protection against adenovirus disease for over three decades. The recombinants described here may provide a path to an affordable malaria vaccine in the developing world. PMID:21199707

Phosphodiesterase inhibitor KMUP-3 displays cardioprotection via protein kinase G and increases cardiac output via G-protein-coupled receptor agonist activity and Ca2+ sensitization

Directory of Open Access Journals (Sweden)

Chung-Pin Liu

2016-02-01

Full Text Available KMUP-3 (7-{2-[4-(4-nitrobenzene piperazinyl]ethyl}-1, 3-dimethylxanthine displays cardioprotection and increases cardiac output, and is suggested to increase cardiac performance and improve myocardial infarction. To determine whether KMUP-3 improves outcomes in hypoperfused myocardium by inducing Ca2+ sensitization to oppose protein kinase (PKG-mediated Ca2+ blockade, we measured left ventricular systolic blood pressure, maximal rates of pressure development, mean arterial pressure and heart rate in rats, and measured contractility and expression of PKs/RhoA/Rho kinase (ROCKII in beating guinea pig left atria. Hemodynamic changes induced by KMUP-3 (0.5–3.0 mg/kg, intravenously were inhibited by Y27632 [(R-(+-trans-4-1-aminoethyl-N-(4-Pyridyl cyclohexane carboxamide] and ketanserin (1 mg/kg, intravenously. In electrically stimulated left guinea pig atria, positive inotropy induced by KMUP-3 (0.1–100μM was inhibited by the endothelial NO synthase (eNOS inhibitors N-nitro-l-arginine methyl ester (L-NAME and 7-nitroindazole, cyclic AMP antagonist SQ22536 [9-(terahydro-2-furanyl-9H-purin-6-amine], soluble guanylyl cyclase (sGC antagonist ODQ (1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one, RhoA inhibitor C3 exoenzyme, β-blocker propranolol, 5-hydroxytryptamine 2A antagonist ketanserin, ROCK inhibitor Y27632 and KMUP-1 (7-{2-[4-(2-chlorobenzene piperazinyl]ethyl}-1, 3-dimethylxanthine at 10μM. Western blotting assays indicated that KMUP-3 (0.1–10μM increased PKA, RhoA/ROCKII, and PKC translocation and CIP-17 (an endogenous 17-kDa inhibitory protein activation. In spontaneous right atria, KMUP-3 induced negative chronotropy that was blunted by 7-nitroindazole and atropine. In neonatal myocytes, L-NAME inhibited KMUP-3-induced eNOS phosphorylation and RhoA/ROCK activation. In H9c2 cells, Y-27632 (50μM and PKG antagonist KT5823 [2,3,9,10,11,12-hexahydro-10R- methoxy-2,9-dimethyl-1-oxo-9S,12R-epoxy-1H-diindolo(1,2,3-fg:3′,2′,1�

Yeast cell surface display for lipase whole cell catalyst and its applications

Energy Technology Data Exchange (ETDEWEB)

Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

2014-08-01

The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chain length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.

Identification of Novel Immunogenic Proteins of Neisseria gonorrhoeae by Phage Display.

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Daniel O Connor

Full Text Available Neisseria gonorrhoeae is one of the most prevalent sexually transmitted diseases worldwide with more than 100 million new infections per year. A lack of intense research over the last decades and increasing resistances to the recommended antibiotics call for a better understanding of gonococcal infection, fast diagnostics and therapeutic measures against N. gonorrhoeae. Therefore, the aim of this work was to identify novel immunogenic proteins as a first step to advance those unresolved problems. For the identification of immunogenic proteins, pHORF oligopeptide phage display libraries of the entire N. gonorrhoeae genome were constructed. Several immunogenic oligopeptides were identified using polyclonal rabbit antibodies against N. gonorrhoeae. Corresponding full-length proteins of the identified oligopeptides were expressed and their immunogenic character was verified by ELISA. The immunogenic character of six proteins was identified for the first time. Additional 13 proteins were verified as immunogenic proteins in N. gonorrhoeae.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

DEFF Research Database (Denmark)

Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

2013-01-01

knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

Chaperone activity of human small heat shock protein-GST fusion proteins.

Science.gov (United States)

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST

MOLECULAR DESIGNER: an interactive program for the display of protein structure on the IBM-PC.

Science.gov (United States)

Hannon, G J; Jentoft, J E

1985-09-01

A BASIC interactive graphics program has been developed for the IBM-PC which utilizes the graphics capabilities of that computer to display and manipulate protein structure from coordinates. Structures may be generated from typed files, or from Brookhaven National Laboratories' Protein Data Bank data tapes. Once displayed, images may be rotated, translated and expanded to any desired size. Figures may be viewed as ball-and-stick or space-filling models. Calculated multiple-point perspective may also be added to the display. Docking manipulations are possible since more than a single figure may be displayed and manipulated simultaneously. Further, stereo images and red/blue three-dimensional images may be generated using the accompanying DESIPLOT program and an HP-7475A plotter. A version of the program is also currently available for the Apple Macintosh. Full implementation on the Macintosh requires 512 K and at least one disk drive. Otherwise this version is essentially identical to the IBM-PC version described herein.

Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold

Science.gov (United States)

Li, Yizhou; De Luca, Roberto; Cazzamalli, Samuele; Pretto, Francesca; Bajic, Davor; Scheuermann, Jörg; Neri, Dario

2018-03-01

In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

Yeast arming systems: pros and cons of different protein anchors and other elements required for display.

Science.gov (United States)

Andreu, Cecilia; Del Olmo, Marcel Lí

2018-03-01

Yeast display is a powerful strategy that consists in exposing peptides or proteins of interest on the cell surface of this microorganism. Ever since initial experiments with this methodology were carried out, its scope has extended and many applications have been successfully developed in different science and technology fields. Several yeast display systems have been designed, which all involve introducting into yeast cells the gene fusions that contain the coding regions of a signal peptide, an anchor protein, to properly attach the target to the cell surface, and the protein of interest to be exposed, all of which are controlled by a strong promoter. In this work, we report the description of such elements for the alternative systems introduced by focusing particularly on anchor proteins. The comparisons made between them are included whenever possible, and the main advantages and inconveniences of each one are discussed. Despite the huge number of publications on yeast surface display and the revisions published to date, this topic has not yet been widely considered. Finally, given the growing interest in developing systems for non-Saccharomyces yeasts, the main strategies reported for some are also summarized.

Baculovirus display of functional antibody Fab fragments.

Science.gov (United States)

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses.

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Stefan W Metz

2016-10-01

Full Text Available Dengue virus (DENV is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE, purified and adsorbed to poly (lactic-co-glycolic acid (PLGA nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.

A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity.

Science.gov (United States)

Wendel, Sofie; Fischer, Emil C; Martínez, Virginia; Seppälä, Susanna; Nørholm, Morten H H

2016-05-03

Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay for evaluating and developing surface display systems is missing. Using a single domain antibody (also called nanobody) with high affinity for green fluorescent protein (GFP), we constructed a system that allows for fast, fluorescence-based detection of displayed proteins. The outer membrane hybrid protein LppOmpA and the autotransporter C-IgAP exposed the nanobody on the surface of Escherichia coli with very different efficiency. Both anchors were capable of functionally displaying the enzyme Chitinase A as a fusion with the nanobody, and this considerably increased expression levels compared to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency. We have developed an inexpensive and easy read-out assay for surface display using nanobody:GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies on the mechanism of protein transport to the surface of living cells, as well as the optimisation of applications in industrial biotech.

Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi.

Science.gov (United States)

Meireles, D A; Domingos, R M; Gaiarsa, J W; Ragnoni, E G; Bannitz-Fernandes, R; da Silva Neto, J F; de Souza, R F; Netto, L E S

2017-08-01

Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species). Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr), the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i) the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols), but not by β-mercaptoethanol or GSH (monothiols), even in large excess; (ii) MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH) over hydrogen peroxide; (iii) MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13)×10 8 M -1 s -1 ). Both Cys 87 and Cys 154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pK a value of the Cys p residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria. Copyright © 2017. Published by Elsevier B.V.

Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi

Directory of Open Access Journals (Sweden)

D.A. Meireles

2017-08-01

Full Text Available Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species. Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr, the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols, but not by β-mercaptoethanol or GSH (monothiols, even in large excess; (ii MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH over hydrogen peroxide; (iii MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13×108 M−1 s−1. Both Cys87 and Cys154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pKa value of the Cysp residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria. Keywords: Ohr/OsmC, Thiol-dependent peroxidases, Phylogeny

A novel active heads-up display for driver assistance.

Science.gov (United States)

Doshi, Anup; Cheng, Shinko Yuanhsien; Trivedi, Mohan Manubhai

2009-02-01

In this paper, we introduce a novel laser-based wide-area heads-up windshield display which is capable of actively interfacing with a human as part of a driver assistance system. The dynamic active display (DAD) is a unique prototype interface that presents safety-critical visual icons to the driver in a manner that minimizes the deviation of his or her gaze direction without adding to unnecessary visual clutter. As part of an automotive safety system, the DAD presents alerts in the field of view of the driver only if necessary, which is based upon the state and pose of the driver, vehicle, and environment. This paper examines the effectiveness of DAD through a comprehensive comparative experimental evaluation of a speed compliance driver assistance system, which is implemented on a vehicular test bed. Three different types of display protocols for assisting a driver to comply with speed limits are tested on actual roadways, and these are compared with a conventional dashboard display. Given the inclination, drivers who are given an overspeed warning alert reduced the time required to slow down to the speed limit by 38% (p system.

CLINICAL SURFACES - Activity-Based Computing for Distributed Multi-Display Environments in Hospitals

Science.gov (United States)

Bardram, Jakob E.; Bunde-Pedersen, Jonathan; Doryab, Afsaneh; Sørensen, Steffen

A multi-display environment (MDE) is made up of co-located and networked personal and public devices that form an integrated workspace enabling co-located group work. Traditionally, MDEs have, however, mainly been designed to support a single “smart room”, and have had little sense of the tasks and activities that the MDE is being used for. This paper presents a novel approach to support activity-based computing in distributed MDEs, where displays are physically distributed across a large building. CLINICAL SURFACES was designed for clinical work in hospitals, and enables context-sensitive retrieval and browsing of patient data on public displays. We present the design and implementation of CLINICAL SURFACES, and report from an evaluation of the system at a large hospital. The evaluation shows that using distributed public displays to support activity-based computing inside a hospital is very useful for clinical work, and that the apparent contradiction between maintaining privacy of medical data in a public display environment can be mitigated by the use of CLINICAL SURFACES.

Mitochondria-localized phospholipase A2, AoPlaA, in Aspergillus oryzae displays phosphatidylethanolamine-specific activity and is involved in the maintenance of mitochondrial phospholipid composition.

Science.gov (United States)

Kotani, Shohei; Izawa, Sho; Komai, Noriyuki; Takayanagi, Ayumi; Arioka, Manabu

2016-11-01

In mammals, cytosolic phospholipases A 2 (cPLA 2 s) play important physiological roles by releasing arachidonic acid, a precursor for bioactive lipid mediators, from the biological membranes. In contrast, fungal cPLA 2 -like proteins are much less characterized and their roles have remained elusive. AoPlaA is a cPLA 2 -like protein in the filamentous fungus Aspergillus oryzae which, unlike mammalian cPLA 2 , localizes to mitochondria. In this study, we investigated the biochemical and physiological functions of AoPlaA. Recombinant AoPlaA produced in E. coli displayed Ca 2+ -independent lipolytic activity. Mass spectrometry analysis demonstrated that AoPlaA displayed PLA 2 activity to phosphatidylethanolamine (PE), but not to other phospholipids, and generated 1-acylated lysoPE. Catalytic site mutants of AoPlaA displayed almost no or largely reduced activity to PE. Consistent with PE-specific activity of AoPlaA, AoplaA-overexpressing strain showed decreased PE content in the mitochondrial fraction. In contrast, AoplaA-disruption strain displayed increased content of cardiolipin. AoplaA-overexpressing strain, but not its counterparts overexpressing the catalytic site mutants, exhibited retarded growth at low temperature, possibly because of the impairment of the mitochondrial function caused by excess degradation of PE. These results suggest that AoPlaA is a novel PE-specific PLA 2 that plays a regulatory role in the maintenance of mitochondrial phospholipid composition. Copyright © 2016 Elsevier Inc. All rights reserved.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

Science.gov (United States)

Plamont, Marie-Aude; Billon-Denis, Emmanuelle; Maurin, Sylvie; Gauron, Carole; Pimenta, Frederico M; Specht, Christian G; Shi, Jian; Quérard, Jérôme; Pan, Buyan; Rossignol, Julien; Moncoq, Karine; Morellet, Nelly; Volovitch, Michel; Lescop, Ewen; Chen, Yong; Triller, Antoine; Vriz, Sophie; Le Saux, Thomas; Jullien, Ludovic; Gautier, Arnaud

2016-01-19

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

The E5 Proteins

OpenAIRE

DiMaio, Daniel; Petti, Lisa

2013-01-01

The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating ...

Chemical Synthesis and In Vitro Evaluation of a Phage Display-Derived Peptide Active against Infectious Salmon Anemia Virus.

Science.gov (United States)

Ojeda, Nicolás; Cárdenas, Constanza; Guzmán, Fanny; Marshall, Sergio H

2016-04-01

Infectious salmon anemia virus (ISAV) is the etiological agent of the disease by the same name and causes major losses in the salmon industry worldwide. Epizootic ISAV outbreaks have occurred in Norway and, to a lesser degree, in Canada. In 2007, an ISAV outbreak in Chile destroyed most of the seasonal production and endangered the entire Chilean salmon industry. None of the existing prophylactic approaches have demonstrated efficacy in providing absolute protection from or even a palliative effect on ISAV proliferation. Sanitary control measures for ISAV, based on molecular epidemiology data, have proven insufficient, mainly due to high salmon culture densities and a constant presence of a nonpathogenic strain of the virus. This report describes an alternative treatment approach based on interfering peptides selected from a phage display library. The screening of a phage display heptapeptide library resulted in the selection of a novel peptide with significant in vitro antiviral activity against ISAV. This peptide specifically interacted with the viral hemagglutinin-esterase protein, thereby impairing virus binding, with plaque reduction assays showing a significant reduction in viral yields. The identified peptide acts at micromolar concentrations against at least two different pathogenic strains of the virus, without detectable cytotoxic effects on the tested fish cells. Therefore, antiviral peptides represent a novel alternative for controlling ISAV and, potentially, other fish pathogens. Identifying novel methods for the efficient control of infectious diseases is imperative for the future of global aquaculture. The present study used a phage display heptapeptide library to identify a peptide with interfering activity against a key protein of the infectious salmon anemia virus (ISAV). A piscine orthomyxovirus, ISAV is a continuous threat to the commercial sustainability of cultured salmon production worldwide. The complex epidemiological strategy of this

Transposon display supports transpositional activity of P elements in ...

Indian Academy of Sciences (India)

. Abstract. Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances ...

Engineering bacterial surface displayed human norovirus capsid proteins: A novel system to explore interaction between norovirus and ligands

Directory of Open Access Journals (Sweden)

Mengya eNiu

2015-12-01

Full Text Available Human noroviruses (HuNoVs are major contributors to acute nonbacterial gastroenteritis outbreaks. Many aspects of HuNoVs are poorly understood due to both the current inability to culture HuNoVs, and the lack of efficient small animal models. Surrogates for HuNoVs, such as recombinant viral like particles (VLPs expressed in eukaryotic system or P particles expressed in prokaryotic system, have been used for studies in immunology and interaction between the virus and its receptors. However, it is difficult to use VLPs or P particles to collect or isolate potential ligands binding to these recombinant capsid proteins. In this study, a new strategy was used to collect HuNoVs binding ligands through the use of ice nucleation protein (INP to display recombinant capsid proteins of HuNoVs on bacterial surfaces. The viral protein-ligand complex could be easily separated by a low speed centrifugation step. This system was also used to explore interaction between recombinant capsid proteins of HuNoVs and their receptors. In this system, the VP1 capsid encoding gene (ORF2 and the protruding domain (P domain encoding gene (3’ terminal fragment of ORF2 of HuNoVs GI.1 and GII.4 were fused with 5’ terminal fragment of ice nucleation protein encoding gene (inaQn. The results demonstrated that the recombinant VP1 and P domains of HuNoVs were expressed and anchored on the surface of Escherichia coli BL21 cells after the bacteria were transformed with the corresponding plasmids. Both cell surface displayed VP1 and P domains could be recognized by HuNoVs specific antibodies and interact with the viral histo-blood group antigens receptors. In both cases, displayed P domains had better binding abilities than VP1. This new strategy of using displayed HuNoVs capsid proteins on the bacterial surface could be utilized to separate HuNoVs binding components from complex samples, to investigate interaction between the virus and its receptors, as well as to develop an

HOXA1 and TALE proteins display cross-regulatory interactions and form a combinatorial binding code on HOXA1 targets.

Science.gov (United States)

De Kumar, Bony; Parker, Hugo J; Paulson, Ariel; Parrish, Mark E; Pushel, Irina; Singh, Narendra Pratap; Zhang, Ying; Slaughter, Brian D; Unruh, Jay R; Florens, Laurence; Zeitlinger, Julia; Krumlauf, Robb

2017-09-01

Hoxa1 has diverse functional roles in differentiation and development. We identify and characterize properties of regions bound by HOXA1 on a genome-wide basis in differentiating mouse ES cells. HOXA1-bound regions are enriched for clusters of consensus binding motifs for HOX, PBX, and MEIS, and many display co-occupancy of PBX and MEIS. PBX and MEIS are members of the TALE family and genome-wide analysis of multiple TALE members (PBX, MEIS, TGIF, PREP1, and PREP2) shows that nearly all HOXA1 targets display occupancy of one or more TALE members. The combinatorial binding patterns of TALE proteins define distinct classes of HOXA1 targets, which may create functional diversity. Transgenic reporter assays in zebrafish confirm enhancer activities for many HOXA1-bound regions and the importance of HOX-PBX and TGIF motifs for their regulation. Proteomic analyses show that HOXA1 physically interacts on chromatin with PBX, MEIS, and PREP family members, but not with TGIF, suggesting that TGIF may have an independent input into HOXA1-bound regions. Therefore, TALE proteins appear to represent a wide repertoire of HOX cofactors, which may coregulate enhancers through distinct mechanisms. We also discover extensive auto- and cross-regulatory interactions among the Hoxa1 and TALE genes, indicating that the specificity of HOXA1 during development may be regulated though a complex cross-regulatory network of HOXA1 and TALE proteins. This study provides new insight into a regulatory network involving combinatorial interactions between HOXA1 and TALE proteins. © 2017 De Kumar et al.; Published by Cold Spring Harbor Laboratory Press.

The Pseudomonas aeruginosa catabolite repression control protein Crc is devoid of RNA binding activity.

Science.gov (United States)

Milojevic, Tetyana; Grishkovskaya, Irina; Sonnleitner, Elisabeth; Djinovic-Carugo, Kristina; Bläsi, Udo

2013-01-01

The Crc protein has been shown to mediate catabolite repression control in Pseudomonas, leading to a preferential assimilation of carbon sources. It has been suggested that Crc acts as a translational repressor of mRNAs, encoding functions involved in uptake and breakdown of different carbon sources. Moreover, the regulatory RNA CrcZ, the level of which is increased in the presence of less preferred carbon sources, was suggested to bind to and sequester Crc, resulting in a relief of catabolite repression. Here, we determined the crystal structure of Pseudomonas aeruginosa Crc, a member of apurinic/apyrimidinic (AP) endonuclease family, at 1.8 Å. Although Crc displays high sequence similarity with its orthologs, there are amino acid alterations in the area corresponding to the active site in AP proteins. Unlike typical AP endonuclease family proteins, Crc has a reduced overall positive charge and the conserved positively charged amino-acid residues of the DNA-binding surface of AP proteins are partially substituted by negatively charged, polar and hydrophobic residues. Crc protein purified to homogeneity from P. aeruginosa did neither display DNase activity, nor did it bind to previously identified RNA substrates. Rather, the RNA chaperone Hfq was identified as a contaminant in His-tagged Crc preparations purified by one step Ni-affinity chromatography from Escherichia coli, and was shown to account for the RNA binding activity observed with the His-Crc preparations. Taken together, these data challenge a role of Crc as a direct translational repressor in carbon catabolite repression in P. aeruginosa.

A nanobody:GFP bacterial platform that enables functional enzyme display and easy quantification of display capacity

DEFF Research Database (Denmark)

Wendel, Sofie; Christian Fischer, Emil; Martinez, Virginia

2016-01-01

Background: Bacterial surface display is an attractive technique for the production of cell-anchored, functional proteins and engineering of whole-cell catalysts. Although various outer membrane proteins have been used for surface display, an easy and versatile high-throughput-compatible assay...... to displaying the nanobody alone. We used flow cytometry to analyse display capability on single-cell versus population level and found that the signal peptide of the anchor has great effect on display efficiency.Conclusions: We have developed an inexpensive and easy read-out assay for surface display using...... nanobody: GFP interactions. The assay is compatible with the most common fluorescence detection methods, including multi-well plate whole-cell fluorescence detection, SDS-PAGE in-gel fluorescence, microscopy and flow cytometry. We anticipate that the platform will facilitate future in-depth studies...

European display scene

Science.gov (United States)

Bartlett, Christopher T.

2000-08-01

The manufacture of Flat Panel Displays (FPDs) is dominated by Far Eastern sources, particularly in Active Matrix Liquid Crystal Displays (AMLCD) and Plasma. The United States has a very powerful capability in micro-displays. It is not well known that Europe has a very active research capability which has lead to many innovations in display technology. In addition there is a capability in display manufacturing of organic technologies as well as the licensed build of Japanese or Korean designs. Finally, Europe has a display systems capability in military products which is world class.

Radioresistant Spodoptera frugiperda 9 insect cells display excessive resistance to 'endoplasmic reticulum' stress and calcium disturbances via pre-emptive activation of unfolded protein response pathway

International Nuclear Information System (INIS)

Guleria, Ayushi; Chandna, Sudhir

2016-01-01

Endoplasmic Reticulum (ER) performs multiple cellular functions such as proper folding of newly synthesized proteins and calcium homeostasis. ER stress triggers unfolded protein response (UPR) that attempts to restore normal ER function and resists damage-induced cell death. Lepidopteran Sf9 insect cells (derived from Spodoptera frugiperda) display 100-200 times higher radioresistance than mammalian cells. We have earlier reported that gamma-radiation doses <1000 Gy fail to trigger increase in cytosolic calcium in Sf9 cells, indicating resilience to calcium/ ER disturbances

Tsetse salivary gland proteins 1 and 2 are high affinity nucleic acid binding proteins with residual nuclease activity.

Directory of Open Access Journals (Sweden)

Guy Caljon

Full Text Available Analysis of the tsetse fly salivary gland EST database revealed the presence of a highly enriched cluster of putative endonuclease genes, including tsal1 and tsal2. Tsal proteins are the major components of tsetse fly (G. morsitans morsitans saliva where they are present as monomers as well as high molecular weight complexes with other saliva proteins. We demonstrate that the recombinant tsetse salivary gland proteins 1&2 (Tsal1&2 display DNA/RNA non-specific, high affinity nucleic acid binding with K(D values in the low nanomolar range and a non-exclusive preference for duplex. These Tsal proteins exert only a residual nuclease activity with a preference for dsDNA in a broad pH range. Knockdown of Tsal expression by in vivo RNA interference in the tsetse fly revealed a partially impaired blood digestion phenotype as evidenced by higher gut nucleic acid, hematin and protein contents.

Mining the active proteome of Arabidopsis thaliana

Directory of Open Access Journals (Sweden)

Renier A. L. Van Der Hoorn

2011-11-01

Full Text Available Assigning functions to the >30.000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied Activity-based Protein Profiling (ABPP. ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed of activities of 76 Arabidopsis proteins. These proteins represent over ten different protein classes that contain over 250 Arabidopsis proteins, including cysteine- serine- and metallo-proteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click-chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed novel protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities e.g. of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry and proteomics.

Peptides of the constant region of antibodies display fungicidal activity.

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Luciano Polonelli

Full Text Available Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA of antibodies (Fc-peptides exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents.

Peptides of the Constant Region of Antibodies Display Fungicidal Activity

Science.gov (United States)

Polonelli, Luciano; Ciociola, Tecla; Magliani, Walter; Zanello, Pier Paolo; D'Adda, Tiziana; Galati, Serena; De Bernardis, Flavia; Arancia, Silvia; Gabrielli, Elena; Pericolini, Eva; Vecchiarelli, Anna; Arruda, Denise C.; Pinto, Marcia R.; Travassos, Luiz R.; Pertinhez, Thelma A.; Spisni, Alberto; Conti, Stefania

2012-01-01

Synthetic peptides with sequences identical to fragments of the constant region of different classes (IgG, IgM, IgA) of antibodies (Fc-peptides) exerted a fungicidal activity in vitro against pathogenic yeasts, such as Candida albicans, Candida glabrata, Cryptococcus neoformans, and Malassezia furfur, including caspofungin and triazole resistant strains. Alanine-substituted derivatives of fungicidal Fc-peptides, tested to evaluate the critical role of each residue, displayed unaltered, increased or decreased candidacidal activity in vitro. An Fc-peptide, included in all human IgGs, displayed a therapeutic effect against experimental mucosal and systemic candidiasis in mouse models. It is intriguing to hypothesize that some Fc-peptides may influence the antifungal immune response and constitute the basis for devising new antifungal agents. PMID:22470523

Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6

International Nuclear Information System (INIS)

Heidenreich, K.A.; Toledo, S.P.

1989-01-01

As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and [gamma-32P]ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons

Development of oral cancer vaccine using recombinant Bifidobacterium displaying Wilms' tumor 1 protein.

Science.gov (United States)

Kitagawa, Koichi; Oda, Tsugumi; Saito, Hiroki; Araki, Ayame; Gonoi, Reina; Shigemura, Katsumi; Hashii, Yoshiko; Katayama, Takane; Fujisawa, Masato; Shirakawa, Toshiro

2017-06-01

Several types of vaccine-delivering tumor-associated antigens (TAAs) have been developed in basic and clinical research. Wilms' tumor 1 (WT1), identified as a gene responsible for pediatric renal neoplasm, is one of the most promising TAA for cancer immunotherapy. Peptide and dendritic cell-based WT1 cancer vaccines showed some therapeutic efficacy in clinical and pre-clinical studies but as yet no oral WT1 vaccine can be administrated in a simple and easy way. In the present study, we constructed a novel oral cancer vaccine using a recombinant Bifidobacterium longum displaying WT1 protein. B. longum 420 was orally administered into mice inoculated with WT1-expressing tumor cells for 4 weeks to examine anti-tumor effects. To analyze the WT1-specific cellular immune responses to oral B. longum 420, mice splenocytes were isolated and cytokine production and cytotoxic activities were determined. Oral administrations of B. longum 420 significantly inhibited WT1-expressing tumor growth and prolonged survival in mice. Immunohistochemical study and immunological assays revealed that B. longum 420 substantially induced tumor infiltration of CD4 + T and CD8 + T cells, systemic WT1-specific cytokine production, and cytotoxic activity mediated by WT1-epitope specific cytotoxic T lymphocytes, with no apparent adverse effects. Our novel oral cancer vaccine safely induced WT1-specific cellular immunity via activation of the gut mucosal immune system and achieved therapeutic efficacy with several practical advantages over existing non-oral vaccines.

Antibody complementarity-determining regions (CDRs can display differential antimicrobial, antiviral and antitumor activities.

Directory of Open Access Journals (Sweden)

Luciano Polonelli

Full Text Available BACKGROUND: Complementarity-determining regions (CDRs are immunoglobulin (Ig hypervariable domains that determine specific antibody (Ab binding. We have shown that synthetic CDR-related peptides and many decapeptides spanning the variable region of a recombinant yeast killer toxin-like antiidiotypic Ab are candidacidal in vitro. An alanine-substituted decapeptide from the variable region of this Ab displayed increased cytotoxicity in vitro and/or therapeutic effects in vivo against various bacteria, fungi, protozoa and viruses. The possibility that isolated CDRs, represented by short synthetic peptides, may display antimicrobial, antiviral and antitumor activities irrespective of Ab specificity for a given antigen is addressed here. METHODOLOGY/PRINCIPAL FINDINGS: CDR-based synthetic peptides of murine and human monoclonal Abs directed to: a a protein epitope of Candida albicans cell wall stress mannoprotein; b a synthetic peptide containing well-characterized B-cell and T-cell epitopes; c a carbohydrate blood group A substance, showed differential inhibitory activities in vitro, ex vivo and/or in vivo against C. albicans, HIV-1 and B16F10-Nex2 melanoma cells, conceivably involving different mechanisms of action. Antitumor activities involved peptide-induced caspase-dependent apoptosis. Engineered peptides, obtained by alanine substitution of Ig CDR sequences, and used as surrogates of natural point mutations, showed further differential increased/unaltered/decreased antimicrobial, antiviral and/or antitumor activities. The inhibitory effects observed were largely independent of the specificity of the native Ab and involved chiefly germline encoded CDR1 and CDR2 of light and heavy chains. CONCLUSIONS/SIGNIFICANCE: The high frequency of bioactive peptides based on CDRs suggests that Ig molecules are sources of an unlimited number of sequences potentially active against infectious agents and tumor cells. The easy production and low cost of small

The making of the minibody: an engineered beta-protein for the display of conformationally constrained peptides.

Science.gov (United States)

Tramontano, A; Bianchi, E; Venturini, S; Martin, F; Pessi, A; Sollazzo, M

1994-03-01

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost the drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue beta-protein aimed at retaining some desirable features of immunoglobulin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of the main chain conformation of the same regions, based on the 'canonical structures' model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical synthesis and expression in E. coli and characterized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low solubility. We were able to overcome this problem both by further mutagenesis of the framework and by addition of a solubilizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.

[Three-dimensional display simulation of lung surgery using "active shutter glasses"].

Science.gov (United States)

Onuki, Takamasa; Kanzaki, Masato; Sakamoto, Kei; Kikkawa, Takuma; Isaka, Tamami; Shimizu, Toshihide; Oyama, Kunihiro; Murasugi, Masahide

2011-08-01

We have reported preoperative 3-dimensional (3D) simulation of thoracoscopic lung surgery using self-made software and internet shareware of 3D-modeler. Using "active shutter glasses", we have tried the "3D display simulation" of lung surgery. 3D display was more effective to grasp clear 3D interrelation between the bronchii and pulmonary vascular system than those in images of currently in use with the same information volume.

Bacterial surface-displayed GII.4 human norovirus capsid proteins bound to surface of Romaine lettuce through HBGA-like molecules

Science.gov (United States)

Human Noroviruses (HuNoVs) are the main cause of nonbacterial gastroenteritis. Contaminated produce is a main vehicle for dissemination of HuNoVs. In this study, we used an ice nucleation protein (INP) mediated surface display system to present the protruding domain of GII.4 HuNoV capsid protein (G...

Blocking peptides against HBV: PreS1 protein selected from a phage display library

International Nuclear Information System (INIS)

Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui; Xiao, Gengfu

2011-01-01

Highlights: → Successfully selected specific PreS1-interacting peptides by using phage displayed library. → Alignment of the positive phage clones revealed a consensus PreS1 binding motif. → A highly enriched peptide named P7 had a strong binding ability for PreS1. → P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX n HX m HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

Blocking peptides against HBV: PreS1 protein selected from a phage display library

Energy Technology Data Exchange (ETDEWEB)

Wang, Wei; Liu, Yang; Zu, Xiangyang; Jin, Rui [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Xiao, Gengfu, E-mail: xiaogf@wh.iov.cn [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)

2011-09-09

Highlights: {yields} Successfully selected specific PreS1-interacting peptides by using phage displayed library. {yields} Alignment of the positive phage clones revealed a consensus PreS1 binding motif. {yields} A highly enriched peptide named P7 had a strong binding ability for PreS1. {yields} P7 could block PreS1 attachment. -- Abstract: The PreS1 protein is present on the outermost part of the hepatitis B virus (HBV) surface and has been shown to have a pivotal function in viral infectivity and assembly. The development of reagents with high affinity and specificity for PreS1 is of great significance for early diagnosis and treatment of HBV infection. A phage display library of dodecapeptide was screened for interactions with purified PreS1 protein. Alignment of the positive phage clones revealed a putative consensus PreS1 binding motif of HX{sub n}HX{sub m}HP/R. Moreover, a peptide named P7 (KHMHWHPPALNT) was highly enriched and occurred with a surprisingly high frequency of 72%. A thermodynamic study revealed that P7 has a higher binding affinity to PreS1 than the other peptides. Furthermore, P7 was able to abrogate the binding of HBV virions to the PreS1 antibody, suggesting that P7 covers key functional sites on the native PreS1 protein. This newly isolated peptide may, therefore, be a new therapeutic candidate for the treatment of HBV. The consensus motif could be modified to deliver imaging, diagnostic, and therapeutic agents to tissues affected by HBV.

Fluorogen-activating proteins: beyond classical fluorescent proteins

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Shengnan Xu

2018-05-01

Full Text Available Fluorescence imaging is a powerful technique for the real-time noninvasive monitoring of protein dynamics. Recently, fluorogen activating proteins (FAPs/fluorogen probes for protein imaging were developed. Unlike the traditional fluorescent proteins (FPs, FAPs do not fluoresce unless bound to their specific small-molecule fluorogens. When using FAPs/fluorogen probes, a washing step is not required for the removal of free probes from the cells, thus allowing rapid and specific detection of proteins in living cells with high signal-to-noise ratio. Furthermore, with different fluorogens, living cell multi-color proteins labeling system was developed. In this review, we describe about the discovery of FAPs, the design strategy of FAP fluorogens, the application of the FAP technology and the advances of FAP technology in protein labeling systems. KEY WORDS: Fluorogen activating proteins, Fluorogens, Genetically encoded sensors, Fluorescence imaging, Molecular imaging

Effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg meat during display storage.

Science.gov (United States)

Kim, Hyun-Wook; Kim, Ji-Han; Yan, Feifei; Cheng, Heng-Wei; Brad Kim, Yuan H

2017-12-01

The present study aimed to evaluate the effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg during display storage. Two hundred and forty, 1-day-old male chicks (5 birds per pen) were subjected to four treatments in a 2 (thermoneutral condition at 21 °C and cyclic heat stress at 32-21-32 °C for 10 h day -1 ) × 2 (regular diet with 0 or 0.25 g kg -1 Bacillus subtilis) factorial design. Chickens were harvested at day 46, and pairs of whole legs were collected at 1 day postmortem. The chicken legs were deboned, ground, tray-packaged with oxygen-permeable film, and displayed for 3 days. Heat stress and probiotic supplementation had no impact on pH, water-holding capacity, color, protein functionality, lipid lipolysis and lipid/protein oxidation stability (P > 0.05). Display storage increased the pH and lipid oxidation of ground chicken legs (P chicken leg meat. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

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Han Lanlan

2011-10-01

Full Text Available Abstract Background Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB. In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine. Results Multiple sequence alignment revealed that the C-terminal region (LcsB of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP or beta-galactosidase (Gal was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor. Conclusion The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological

Functional display of proteins, mutant proteins, fragments of proteins and peptides on the surface of filamentous (bacterio) phages: A review

NARCIS (Netherlands)

Pannekoek, H.; van Meijer, M.; Gaardsvoll, H.; van Zonneveld, A. J.

1995-01-01

Cytoplasmic expression of complex eukaryotic proteins inEscherichia coli usually yields inactive protein preparations. In some cases, (part) of the biological activity can be recovered by rather inefficient denaturation-renaturation procedures. Recently, novel concepts have been developed for the

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

Science.gov (United States)

Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

2016-01-13

Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Ribosome display: next-generation display technologies for production of antibodies in vitro.

Science.gov (United States)

He, Mingyue; Khan, Farid

2005-06-01

Antibodies represent an important and growing class of biologic research reagents and biopharmaceutical products. They can be used as therapeutics in a variety of diseases. With the rapid expansion of proteomic studies and biomarker discovery, there is a need for the generation of highly specific binding reagents to study the vast number of proteins encoded by the genome. Display technologies provide powerful tools for obtaining antibodies. Aside from the preservation of natural antibody repertoires, they are capable of exploiting diversity by DNA recombination to create very large libraries for selection of novel molecules. In contrast to in vivo immunization processes, display technologies allow selection of antibodies under in vitro-defined selection condition(s), resulting in enrichment of antibodies with desired properties from large populations. In addition, in vitro selection enables the isolation of antibodies against difficult antigens including self-antigens, and this can be applied to the generation of human antibodies against human targets. Display technologies can also be combined with DNA mutagenesis for antibody evolution in vitro. Some methods are amenable to automation, permitting high-throughput generation of antibodies. Ribosome display is considered as representative of the next generation of display technologies since it overcomes the limitations of cell-based display methods by using a cell-free system, offering advantages of screening larger libraries and continuously expanding new diversity during selection. Production of display-derived antibodies can be achieved by choosing one of a variety of prokaryotic and eukaryotic cell-based expression systems. In the near future, cell-free protein synthesis may be developed as an alternative for large-scale generation of antibodies.

Conserved TRAM Domain Functions as an Archaeal Cold Shock Protein via RNA Chaperone Activity

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Bo Zhang

2017-08-01

Full Text Available Cold shock proteins (Csps enable organisms to acclimate to and survive in cold environments and the bacterial CspA family exerts the cold protection via its RNA chaperone activity. However, most Archaea do not contain orthologs to the bacterial csp. TRAM, a conserved domain among RNA modification proteins ubiquitously distributed in organisms, occurs as an individual protein in most archaeal phyla and has a structural similarity to Csp proteins, yet its biological functions remain unknown. Through physiological and biochemical studies on four TRAM proteins from a cold adaptive archaeon Methanolobus psychrophilus R15, this work demonstrated that TRAM is an archaeal Csp and exhibits RNA chaperone activity. Three TRAM encoding genes (Mpsy_0643, Mpsy_3043, and Mpsy_3066 exhibited remarkable cold-shock induced transcription and were preferentially translated at lower temperature (18°C, while the fourth (Mpsy_2002 was constitutively expressed. They were all able to complement the cspABGE mutant of Escherichia coli BX04 that does not grow in cold temperatures and showed transcriptional antitermination. TRAM3066 (gene product of Mpsy_3066 and TRAM2002 (gene product of Mpsy_2002 displayed sequence-non-specific RNA but not DNA binding activity, and TRAM3066 assisted RNases in degradation of structured RNA, thus validating the RNA chaperone activity of TRAMs. Given the chaperone activity, TRAM is predicted to function beyond a Csp.

Eukaryotic ribosome display with in situ DNA recovery.

Science.gov (United States)

He, Mingyue; Edwards, Bryan M; Kastelic, Damjana; Taussig, Michael J

2012-01-01

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Actively addressed single pixel full-colour plasmonic display

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Franklin, Daniel; Frank, Russell; Wu, Shin-Tson; Chanda, Debashis

2017-05-01

Dynamic, colour-changing surfaces have many applications including displays, wearables and active camouflage. Plasmonic nanostructures can fill this role by having the advantages of ultra-small pixels, high reflectivity and post-fabrication tuning through control of the surrounding media. However, previous reports of post-fabrication tuning have yet to cover a full red-green-blue (RGB) colour basis set with a single nanostructure of singular dimensions. Here, we report a method which greatly advances this tuning and demonstrates a liquid crystal-plasmonic system that covers the full RGB colour basis set, only as a function of voltage. This is accomplished through a surface morphology-induced, polarization-dependent plasmonic resonance and a combination of bulk and surface liquid crystal effects that manifest at different voltages. We further demonstrate the system's compatibility with existing LCD technology by integrating it with a commercially available thin-film-transistor array. The imprinted surface interfaces readily with computers to display images as well as video.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein*

Science.gov (United States)

Townsend, Philip D.; Rodgers, Thomas L.; Glover, Laura C.; Korhonen, Heidi J.; Richards, Shane A.; Colwell, Lucy J.; Pohl, Ehmke; Wilson, Mark R.; Hodgson, David R. W.; McLeish, Tom C. B.; Cann, Martin J.

2015-01-01

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. PMID:26187469

Construction and Selection of Affilin® Phage Display Libraries.

Science.gov (United States)

Settele, Florian; Zwarg, Madlen; Fiedler, Sebastian; Koscheinz, Daniel; Bosse-Doenecke, Eva

2018-01-01

Affilin ® molecules represent a new class of so-called scaffold proteins. The concept of scaffold proteins is to use stable and versatile protein structures which can be endowed with de novo binding properties and specificities by introducing mutations in surface exposed amino acid residues. Complex variations and combinations are generated by genetic methods of randomization resulting in large cDNA libraries. The selection for candidates binding to a desired target can be executed by display methods, especially the very robust and flexible phage display. Here, we describe the construction of ubiquitin based Affilin ® phage display libraries and their use in biopanning experiments for the identification of novel protein ligands.

The Murine Factor H-Related Protein FHR-B Promotes Complement Activation

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Marcell Cserhalmi

2017-09-01

Full Text Available Factor H-related (FHR proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH. While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3, and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

Investigation of the Maillard Reaction between Polysaccharides and Proteins from Longan Pulp and the Improvement in Activities.

Science.gov (United States)

Han, Miao-Miao; Yi, Yang; Wang, Hong-Xun; Huang, Fei

2017-06-05

The purpose of this study was to investigate the Maillard reaction between polysaccharides and proteins from longan pulp and the effects of reaction on their in vitro activities. The polysaccharide-protein mixtures of fresh longan pulp (LPPMs) were co-prepared by an alkali extraction-acid precipitation method. They were then dry-heated under controlled conditions for monitoring the characterization of the Maillard reaction by the measurement of the free amino group content, ultraviolet-visible spectrum, Fourier transform infrared spectrum and molecular weight distribution. All the physicochemical analyses indicated the development of the Maillard reaction between polysaccharides and proteins. The in vitro activity evaluation indicated that the Maillard reaction could effectively enhance the antioxidant, antitumor and immunostimulating activities of LPPMs. The enhancement of 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and ferric reducing antioxidant power displayed both a positive correlation with the reaction time ( p Maillard-type intermacromolecular interaction is suggested to be an effective and controllable method for improving the functional activities of polysaccharides and proteins from longan pulp.

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

Science.gov (United States)

Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

2014-07-03

The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may

Heterologous surface display on lactic acid bacteria: non-GMO alternative?

Science.gov (United States)

Zadravec, Petra; Štrukelj, Borut; Berlec, Aleš

2015-01-01

Lactic acid bacteria (LAB) are food-grade hosts for surface display with potential applications in food and therapy. Alternative approaches to surface display on LAB would avoid the use of recombinant DNA technology and genetically-modified organism (GMO)-related regulatory requirements. Non-covalent surface display of proteins can be achieved by fusing them to various cell-wall binding domains, of which the Lysine motif domain (LysM) is particularly well studied. Fusion proteins have been isolated from recombinant bacteria or from their growth medium and displayed on unmodified bacteria, enabling heterologous surface display. This was demonstrated on non-viable cells devoid of protein content, termed bacteria-like particles, and on various species of genus Lactobacillus. Of the latter, Lactobacillus salivarius ATCC 11741 was recently shown to be particularly amenable for LysM-mediated display. Possible regulatory implications of heterologous surface display are discussed, particularly those relevant for the European Union.

A plasma coagulation assay for an activated protein C-independent anticoagulant activity of protein S

NARCIS (Netherlands)

van Wijnen, M.; van 't Veer, C.; Meijers, J. C.; Bertina, R. M.; Bouma, B. N.

1998-01-01

To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in

Sulfated glycopeptide nanostructures for multipotent protein activation

Energy Technology Data Exchange (ETDEWEB)

Lee, Sungsoo S.; Fyrner, Timmy; Chen, Feng; Álvarez, Zaida; Sleep, Eduard; Chun, Danielle S.; Weiner, Joseph A.; Cook, Ralph W.; Freshman, Ryan D.; Schallmo, Michael S.; Katchko, Karina M.; Schneider, Andrew D.; Smith, Justin T.; Yun, Chawon; Singh, Gurmit; Hashmi, Sohaib Z.; McClendon, Mark T.; Yu, Zhilin; Stock, Stuart R.; Hsu, Wellington K.; Hsu, Erin L.; Stupp , Samuel I. (NWU)

2017-06-19

Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with different polysaccharide-binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signalling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than that required in the animal model. These highly bioactive nanostructures may enable many therapies in the future involving proteins.

The N-terminal, polybasic region is critical for prion protein neuroprotective activity.

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Jessie A Turnbaugh

Full Text Available Several lines of evidence suggest that the normal form of the prion protein, PrP(C, exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrP(C to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (Δ32-134, called F35. To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (Δ23-31, Δ23-111, and Δ23-134 to rescue the phenotype of Tg(F35 mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although Δ23-31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrP(C neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

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Wieczorek Andrew S

2010-09-01

Full Text Available Abstract Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA. Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase, and were

G-Protein Coupled Receptors: Surface Display and Biosensor Technology

Science.gov (United States)

McMurchie, Edward; Leifert, Wayne

Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein.

Science.gov (United States)

Townsend, Philip D; Rodgers, Thomas L; Glover, Laura C; Korhonen, Heidi J; Richards, Shane A; Colwell, Lucy J; Pohl, Ehmke; Wilson, Mark R; Hodgson, David R W; McLeish, Tom C B; Cann, Martin J

2015-09-04

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S

NARCIS (Netherlands)

van Wijnen, M.; Stam, J. G.; van't Veer, C.; Meijers, J. C.; Reitsma, P. H.; Bertina, R. M.; Bouma, B. N.

1996-01-01

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in

The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

Directory of Open Access Journals (Sweden)

Yi-Chieh Lin

Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

To 'display' or not to 'display'- that is the peptide

CSIR Research Space (South Africa)

Crampton, Michael C

2008-11-01

Full Text Available eukaryotic and prokaryotic systems but has mainly focused around phages (Etz et al, 2001), yeast (Kondo and Ueda, 2004) and bacteria (Lee et al 2003). The central variable domain of the FliC protein is dispensable and can be used for the insertion and display...

The establishment of Saccharomyces boulardii surface display system using a single expression vector.

Science.gov (United States)

Wang, Tiantian; Sun, Hui; Zhang, Jie; Liu, Qing; Wang, Longjiang; Chen, Peipei; Wang, Fangkun; Li, Hongmei; Xiao, Yihong; Zhao, Xiaomin

2014-03-01

In the present study, an a-agglutinin-based Saccharomyces boulardii surface display system was successfully established using a single expression vector. Based on the two protein co-expression vector pSP-G1 built by Partow et al., a S. boulardii surface display vector-pSDSb containing all the display elements was constructed. The display results of heterologous proteins were confirmed by successfully displaying enhanced green fluorescent protein (EGFP) and chicken Eimeria tenella Microneme-2 proteins (EtMic2) on the S. boulardii cell surface. The DNA sequence of AGA1 gene from S. boulardii (SbAGA1) was determined and used as the cell wall anchor partner. This is the first time heterologous proteins have been displayed on the cell surface of S. boulardii. Because S. boulardii is probiotic and eukaryotic, its surface display system would be very valuable, particularly in the development of a live vaccine against various pathogenic organisms especially eukaryotic pathogens such as protistan parasites. Copyright © 2013 Elsevier Inc. All rights reserved.

Identification of novel 2-benzoxazolinone derivatives with specific inhibitory activity against the HIV-1 nucleocapsid protein.

Science.gov (United States)

Gamba, Elia; Mori, Mattia; Kovalenko, Lesia; Giannini, Alessia; Sosic, Alice; Saladini, Francesco; Fabris, Dan; Mély, Yves; Gatto, Barbara; Botta, Maurizio

2018-02-10

In this report, we present a new benzoxazole derivative endowed with inhibitory activity against the HIV-1 nucleocapsid protein (NC). NC is a 55-residue basic protein with nucleic acid chaperone properties, which has emerged as a novel and potential pharmacological target against HIV-1. In the pursuit of novel NC-inhibitor chemotypes, we performed virtual screening and in vitro biological evaluation of a large library of chemical entities. We found that compounds sharing a benzoxazolinone moiety displayed putative inhibitory properties, which we further investigated by considering a series of chemical analogues. This approach provided valuable information on the structure-activity relationships of these compounds and, in the process, demonstrated that their anti-NC activity could be finely tuned by the addition of specific substituents to the initial benzoxazolinone scaffold. This study represents the starting point for the possible development of a new class of antiretroviral agents targeting the HIV-1 NC protein. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

investigating acid production by Streptococcus mutans with a surface-displayed pH-sensitive green fluorescent protein.

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Lihong Guo

Full Text Available Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87 was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.

Plastic Substrate Active Matrix Displays Final Report CRADA No. TC-2011-00

Energy Technology Data Exchange (ETDEWEB)

Bernhardt, A. F. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Smith, P. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

2017-09-28

This project was a collaborative effort between the University of California, Lawrence Livermore National Laboratory (LLNL) and FlexICs, Inc. to develop thin film transistor (TFT) electronics for active matrix displays.

Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

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D. Olivera-Severo

2006-07-01

Full Text Available Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

Versatile microbial surface-display for environmental remediation and biofuels production

Energy Technology Data Exchange (ETDEWEB)

Wu, Cindy H.; Mulchandani, Ashok; Chen, wilfred

2008-02-14

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.

JTEC panel on display technologies in Japan

Science.gov (United States)

Tannas, Lawrence E., Jr.; Glenn, William E.; Credelle, Thomas; Doane, J. William; Firester, Arthur H.; Thompson, Malcolm

1992-01-01

This report is one in a series of reports that describes research and development efforts in Japan in the area of display technologies. The following are included in this report: flat panel displays (technical findings, liquid crystal display development and production, large flat panel displays (FPD's), electroluminescent displays and plasma panels, infrastructure in Japan's FPD industry, market and projected sales, and new a-Si active matrix liquid crystal display (AMLCD) factory); materials for flat panel displays (liquid crystal materials, and light-emissive display materials); manufacturing and infrastructure of active matrix liquid crystal displays (manufacturing logistics and equipment); passive matrix liquid crystal displays (LCD basics, twisted nematics LCD's, supertwisted nematic LCD's, ferroelectric LCD's, and a comparison of passive matrix LCD technology); active matrix technology (basic active matrix technology, investment environment, amorphous silicon, polysilicon, and commercial products and prototypes); and projection displays (comparison of Japanese and U.S. display research, and technical evaluation of work).

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus

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Kyle K. Biggar

2015-04-01

Full Text Available Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK family members to primate torpor were compared in six organs of control (aroused versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs, c-jun NH2-terminal kinases (JNKs, MAPK kinase (MEK, and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27. The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor.

Intracellular route and biological activity of exogenously delivered Rep proteins from the adeno-associated virus type 2

International Nuclear Information System (INIS)

Awedikian, Rafi; Francois, Achille; Guilbaud, Mickael; Moullier, Philippe; Salvetti, Anna

2005-01-01

The two large Rep proteins, Rep78 and Rep68, from the adeno-associated virus type 2 (AAV-2) are required for AAV-2 DNA replication, site-specific integration, and for the regulation of viral gene expression. The study of their activities is dependent on the ability to deliver these proteins to the cells in a time and dose-dependent manner. We evaluated the ability of a protein transduction domain (PTD) derived from the human immunodeficiency virus 1 (HIV-1) TAT protein to drive the cellular internalization of exogenously delivered PTD-fused Rep68 proteins. This analysis unexpectedly revealed that recombinant Rep68 alone, in the absence of any PTD, could be endocytosed by the cells. Rep68 as the chimeric TAT-Rep68 proteins were internalized through endocytosis in clathrin-coated vesicles and retained in late endosomes/lysosomes with no detectable nuclear localization. In the presence of adenovirus, the Rep proteins could translocate into the nucleus where they displayed a biological activity. These findings support recent reports on the mechanism of entry of TAT-fused proteins and also revealed a new property of Rep68

Gas Vesicle Nanoparticles for Antigen Display

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Shiladitya DasSarma

2015-09-01

Full Text Available Microorganisms like the halophilic archaeon Halobacterium sp. NRC-1 produce gas-filled buoyant organelles, which are easily purified as protein nanoparticles (called gas vesicles or GVNPs. GVNPs are non-toxic, exceptionally stable, bioengineerable, and self-adjuvanting. A large gene cluster encoding more than a dozen proteins has been implicated in their biogenesis. One protein, GvpC, found on the exterior surface of the nanoparticles, can accommodate insertions near the C-terminal region and results in GVNPs displaying the inserted sequences on the surface of the nanoparticles. Here, we review the current state of knowledge on GVNP structure and biogenesis as well as available studies on immunogenicity of pathogenic viral, bacterial, and eukaryotic proteins and peptides displayed on the nanoparticles. Recent improvements in genetic tools for bioengineering of GVNPs are discussed, along with future opportunities and challenges for development of vaccines and other applications.

Comparative Molecular Dynamics Simulations of Mitogen-Activated Protein Kinase-Activated Protein Kinase 5

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Inger Lindin

2014-03-01

Full Text Available The mitogen-activated protein kinase-activated protein kinase MK5 is a substrate of the mitogen-activated protein kinases p38, ERK3 and ERK4. Cell culture and animal studies have demonstrated that MK5 is involved in tumour suppression and promotion, embryogenesis, anxiety, cell motility and cell cycle regulation. In the present study, homology models of MK5 were used for molecular dynamics (MD simulations of: (1 MK5 alone; (2 MK5 in complex with an inhibitor; and (3 MK5 in complex with the interaction partner p38α. The calculations showed that the inhibitor occupied the active site and disrupted the intramolecular network of amino acids. However, intramolecular interactions consistent with an inactive protein kinase fold were not formed. MD with p38α showed that not only the p38 docking region, but also amino acids in the activation segment, αH helix, P-loop, regulatory phosphorylation region and the C-terminal of MK5 may be involved in forming a very stable MK5-p38α complex, and that p38α binding decreases the residual fluctuation of the MK5 model. Electrostatic Potential Surface (EPS calculations of MK5 and p38α showed that electrostatic interactions are important for recognition and binding.

INFORMATION DISPLAY: CONSIDERATIONS FOR DESIGNING COMPUTER-BASED DISPLAY SYSTEMS

International Nuclear Information System (INIS)

O'HARA, J.M.; PIRUS, D.; BELTRATCCHI, L.

2004-01-01

This paper discussed the presentation of information in computer-based control rooms. Issues associated with the typical displays currently in use are discussed. It is concluded that these displays should be augmented with new displays designed to better meet the information needs of plant personnel and to minimize the need for interface management tasks (the activities personnel have to do to access and organize the information they need). Several approaches to information design are discussed, specifically addressing: (1) monitoring, detection, and situation assessment; (2) routine task performance; and (3) teamwork, crew coordination, collaborative work

Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.

Science.gov (United States)

Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K

2018-01-01

The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.

A fused selenium-containing protein with both GPx and SOD activities

International Nuclear Information System (INIS)

Yu, Huijun; Ge, Yan; Wang, Ying; Lin, Chi-Tsai; Li, Jing; Liu, Xiaoman; Zang, Tianzhu; Xu, Jiayun; Liu, Junqiu; Luo, Guimin; Shen, Jiacong

2007-01-01

As a safeguard against oxidative stress, the balance between the main antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) was believed to be more important than any single one, for example, dual-functional SOD/CAT enzyme has been proved to have better antioxidant ability than either single enzyme. By combining traditional fusion protein technology with amino acid auxotrophic expression system, we generated a bifunctional enzyme with both GPx and SOD activities. It displayed better antioxidant ability than GPx or SOD. Such dual-functional enzymes could facilitate further studies of the cooperation of GPx and SOD and generation of better therapeutic agents

Coculture of Bifidobacterium longum and Bifidobacterium breve alters their protein expression profiles and enzymatic activities.

Science.gov (United States)

Ruiz, Lorena; Sánchez, Borja; de Los Reyes-Gavilán, Clara G; Gueimonde, Miguel; Margolles, Abelardo

2009-07-31

Some strains of the genus Bifidobacterium are probiotic bacteria commonly added to functional dairy products. The influence of coculturing Bifidobacterium longum NCIMB8809 and Bifidobacterium breve NCIMB8807 on their physiology was studied. 2DE separation of protein extracts, coupled to MS protein analysis allowed the identification of 16 proteins whose expression drastically changed when cells were grown in compartmentalized coculture, compared to monoculture. These included ribosomal proteins and proteins involved in carbohydrate metabolism, gene regulation, cell envelope biogenesis and transport processes. Significant changes in some glycoside-hydrolysing activities (beta-d-xylopyranosidase, alpha-l-arabinofuranosidase and beta-d-glucopyranosidase) were also detected. Furthermore, qRT-PCR experiments using as targets the B. breve genes clgR (transcriptional regulator) clpP1, clpP2 and clpC (chaperone- and protease-encoding genes positively regulated by clgR) supported the proteomic results, the four genes displaying a higher expression level in coculture. This study provides new insights to understand the communication among Bifidobacterium species.

Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology

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Kotaro Sakamoto

2017-09-01

Full Text Available ActRIIB (activin receptor type-2B is an activin receptor subtype constitutively expressed in the whole body, playing a role in cellular proliferation, differentiation, and metabolism. For its various physiological activities, ActRIIB interacts with activin and multiple other ligands including myostatin (MSTN, growth differentiation factor 11 (GDF11, and bone morphogenetic protein 9 (BMP9. Notably, the protein-protein interaction (PPI between ActRIIB and MSTN negatively controls muscular development. Therefore, this PPI has been targeted for effective treatment of muscle degenerative diseases such as muscular dystrophy and sarcopenia. Here, we report the identification of ligand-selective peptidic ActRIIB-antagonists by phage display technology. Our peptides bound to the extracellular domain of ActRIIB, inhibited PPIs between ActRIIB expressed on the cell surface and its ligands, and subsequently suppressed activation of Smad that serves as the downstream signal of the ActRIIB pathway. Interestingly, these peptidic antagonists displayed different ligand selectivities; the AR2mini peptide inhibited multiple ligands (activin A, MSTN, GDF11, and BMP9, AR9 inhibited MSTN and GDF11, while AR8 selectively inhibited MSTN. This is the first report of artificial peptidic ActRIIB-antagonists possessing ligand-selectivity.

Electrochemical Glucose Biosensor Based on Glucose Oxidase Displayed on Yeast Surface.

Science.gov (United States)

Wang, Hongwei; Lang, Qiaolin; Liang, Bo; Liu, Aihua

2015-01-01

The conventional enzyme-based biosensor requires chemical or physical immobilization of purified enzymes on electrode surface, which often results in loss of enzyme activity and/or fractions immobilized over time. It is also costly. A major advantage of yeast surface display is that it enables the direct utilization of whole cell catalysts with eukaryote-produced proteins being displayed on the cell surface, providing an economic alternative to traditional production of purified enzymes. Herein, we describe the details of the display of glucose oxidase (GOx) on yeast cell surface and its application in the development of electrochemical glucose sensor. In order to achieve a direct electrochemistry of GOx, the entire cell catalyst (yeast-GOx) was immobilized together with multiwalled carbon nanotubes on the electrode, which allowed sensitive and selective glucose detection.

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

Science.gov (United States)

Lin, Changsheng; Ear, Jason; Midde, Krishna; Lopez-Sanchez, Inmaculada; Aznar, Nicolas; Garcia-Marcos, Mikel; Kufareva, Irina; Abagyan, Ruben; Ghosh, Pradipta

2014-01-01

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs. PMID:25187647

Development and characterization of membrane surface display system using molecular chaperon, prsA, of Bacillus subtilis

International Nuclear Information System (INIS)

Kim, June-Hyung; Park, In-Suk; Kim, Byung-Gee

2005-01-01

We report a new membrane surface display system based on molecular chaperon, prsA, of Bacillus subtilis. Clostridium thermocellum cellulase, celA, was fused to C-terminal end of PrsA. Cellulase activity of B. subtilis protoplast, which expressed PrsA-CelA was 15 times higher compared to control strain. More than 85% of total cellulase activity was observed in surface displayed format and less than 15% of total cellulase activity was found in supernatant. Flow cytometric analysis of protoplast of PrsA-CelA fusion expressing bacteria provided another proof of uniform expression of fusion protein onto cytoplasmic membrane of B. subtilis. Without lysozyme treatment, only part of cellulase activity (10%) was observed in whole cell fraction

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....

The identification and characterization of nucleic acid chaperone activity of human enterovirus 71 nonstructural protein 3AB.

Science.gov (United States)

Tang, Fenfen; Xia, Hongjie; Wang, Peipei; Yang, Jie; Zhao, Tianyong; Zhang, Qi; Hu, Yuanyang; Zhou, Xi

2014-09-01

Human enterovirus 71 (EV71) belongs to the genus Enterovirus in the family Picornaviridae and has been recognized as one of the most important pathogens that cause emerging infectious disease. Despite of the importance of EV71, the nonstructural protein 3AB from this virus is little understood for its function during EV71 replication. Here we expressed EV71 3AB protein as recombinant protein in a eukaryotic expression system and uncovered that this protein possesses a nucleic acid helix-destabilizing and strand annealing acceleration activity in a dose-dependent manner, indicating that EV71 3AB is a nucleic acid chaperone protein. Moreover, we characterized the RNA chaperone activity of EV71 3AB, and revealed that divalent metal ions, such as Mg(2+) and Zn(2+), were able to inhibit the RNA helix-destabilizing activity of 3AB to different extents. Moreover, we determined that 3B plus the last 7 amino acids at the C-terminal of 3A (termed 3B+7) possess the RNA chaperone activity, and five amino acids, i.e. Lys-80, Phe-82, Phe-85, Tyr-89, and Arg-103, are critical and probably the active sites of 3AB for its RNA chaperone activity. This report reveals that EV71 3AB displays an RNA chaperone activity, adds a new member to the growing list of virus-encoded RNA chaperones, and provides novel knowledge about the virology of EV71. Copyright © 2014 Elsevier Inc. All rights reserved.

Volumetric 3D display with multi-layered active screens for enhanced the depth perception (Conference Presentation)

Science.gov (United States)

Kim, Hak-Rin; Park, Min-Kyu; Choi, Jun-Chan; Park, Ji-Sub; Min, Sung-Wook

2016-09-01

Three-dimensional (3D) display technology has been studied actively because it can offer more realistic images compared to the conventional 2D display. Various psychological factors such as accommodation, binocular parallax, convergence and motion parallax are used to recognize a 3D image. For glass-type 3D displays, they use only the binocular disparity in 3D depth cues. However, this method cause visual fatigue and headaches due to accommodation conflict and distorted depth perception. Thus, the hologram and volumetric display are expected to be an ideal 3D display. Holographic displays can represent realistic images satisfying the entire factors of depth perception. But, it require tremendous amount of data and fast signal processing. The volumetric 3D displays can represent images using voxel which is a physical volume. However, it is required for large data to represent the depth information on voxel. In order to simply encode 3D information, the compact type of depth fused 3D (DFD) display, which can create polarization distributed depth map (PDDM) image having both 2D color image and depth image is introduced. In this paper, a new volumetric 3D display system is shown by using PDDM image controlled by polarization controller. In order to introduce PDDM image, polarization states of the light through spatial light modulator (SLM) was analyzed by Stokes parameter depending on the gray level. Based on the analysis, polarization controller is properly designed to convert PDDM image into sectioned depth images. After synchronizing PDDM images with active screens, we can realize reconstructed 3D image. Acknowledgment This work was supported by `The Cross-Ministry Giga KOREA Project' grant from the Ministry of Science, ICT and Future Planning, Korea

AMP-activated protein kinase regulates lymphocyte responses to metabolic stress but is largely dispensable for immune cell development and function.

Science.gov (United States)

Mayer, Alice; Denanglaire, Sébastien; Viollet, Benoit; Leo, Oberdan; Andris, Fabienne

2008-04-01

AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, represents an energy sensor able to adapt cellular metabolism in response to nutritional environmental variations. TCR stimulation activates AMPK, a regulatory event that is known to stimulate ATP-producing processes, possibly in anticipation of the increased energetic needs associated with cell division and expression of effector function. Taking advantage of the selective expression of the AMPKalpha1 catalytic subunit in lymphoid cells, we have analyzed the in vitro and in vivo capacity of lymphocytes lacking AMPK activity (AMPKalpha1-KO cells) to respond to metabolic stress and to initiate and sustain an immune response. AMPKalpha1-KO cells displayed increasing sensitivity to energetic stress in vitro, and were found unable to maintain adequate ATP levels in response to ATP synthase inhibition. These cells were, however, able to respond to antigen stimulation in vitro, as shown by optimal proliferation and cytokine production. Similarly, AMPKalpha1-KO mice were fully immunocompetent in vivo and displayed normal cell proliferation, humoral, cytotoxic and delayed-type hypersensitivity (DTH) responses following antigen injection. In conclusion, AMPK represents an important enzyme allowing lymphocytes to resist a mild energy crisis in vitro, but is largely dispensable for activation and expression of effector function in response to antigen stimulation.

Fully Screen-Printed, Large-Area, and Flexible Active-Matrix Electrochromic Displays Using Carbon Nanotube Thin-Film Transistors.

Science.gov (United States)

Cao, Xuan; Lau, Christian; Liu, Yihang; Wu, Fanqi; Gui, Hui; Liu, Qingzhou; Ma, Yuqiang; Wan, Haochuan; Amer, Moh R; Zhou, Chongwu

2016-11-22

Semiconducting single-wall carbon nanotubes are ideal semiconductors for printed electronics due to their advantageous electrical and mechanical properties, intrinsic printability in solution, and desirable stability in air. However, fully printed, large-area, high-performance, and flexible carbon nanotube active-matrix backplanes are still difficult to realize for future displays and sensing applications. Here, we report fully screen-printed active-matrix electrochromic displays employing carbon nanotube thin-film transistors. Our fully printed backplane shows high electrical performance with mobility of 3.92 ± 1.08 cm 2 V -1 s -1 , on-off current ratio I on /I off ∼ 10 4 , and good uniformity. The printed backplane was then monolithically integrated with an array of printed electrochromic pixels, resulting in an entirely screen-printed active-matrix electrochromic display (AMECD) with good switching characteristics, facile manufacturing, and long-term stability. Overall, our fully screen-printed AMECD is promising for the mass production of large-area and low-cost flexible displays for applications such as disposable tags, medical electronics, and smart home appliances.

Mitogen-activated protein kinases mediate Mycobacterium ...

Indian Academy of Sciences (India)

2012-01-19

Jan 19, 2012 ... CD44, an adhesion molecule, has been reported to be a binding site for ... receptors in mediating mitogen-activated protein kinase activation. ... surface expression and tumour necrosis factor-alpha levels, ... Abbreviations used: Abs, antibodies; ANOVA, analysis of variance; AP-1, activator protein -1; BCG, ...

Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

Science.gov (United States)

Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J; Mobarec, Juan Carlos; Woodlock, David A; Reynolds, Christopher A; Poyner, David R; Watkins, Harriet A; Ladds, Graham

2016-10-14

The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα s -mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα s and Gα q but also identify a Gα i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα s , Gα i , and Gα q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Glutamate decarboxylase-derived IDDM autoantigens displayed on self-assembled protein nanoparticles

International Nuclear Information System (INIS)

Choi, Hyoung; Ahn, Ji-Young; Sim, Sang Jun; Lee, Jeewon

2005-01-01

The recombinant ferritin heavy chain (FTN-H) formed self-assembled spherical nanoparticles with the size comparable to native one. We tried to express the GAD65 COOH-terminal fragments, i.e., 448-585 (GAD65 448-585 ), 487-585 (GAD65 487-585 ), and 512-585 (GAD65 512-585 ) amino acid fragments, using FTN-H as N-terminus fusion expression partner in Escherichia coli. All of recombinant fusion proteins (FTN-H::GAD65 448-585 , FTN-H::GAD65 487-585 , and FTN-H::GAD65 512-585 ) also formed spherical nanoparticles due probably to the self-assembly function of the fused ferritin heavy chain. The antigenic epitopes within GAD65 448-585 , GAD65 487-585 , and GAD65 512-585 against insulin-dependent diabetes mellitus (IDDM) marker (autoantibodies against GAD65) were localized at the surface of the spherical protein nanoparticles so that anti-GAD65 Ab could recognize them. Protein nanoparticles like FTN-H seem to provide distinct advantages over other inorganic nanoparticles (e.g., Au, Ag, CdSe, etc.) in that through the bacterial synthesis, the active capture probes can be located at the nanoparticle surface with constant orientation/conformation via covalent cross-linking without complex chemistry. Also it is possible for the protein nanoparticles to have uniform particle size, which is rarely achieved in the chemical synthesis of inorganic nanoparticles. Thus, the recombinant ferritin particles can be used as a three-dimensional (spherical) and nanometer-scale probe structure that is a key component in ultra-sensitive protein chip for detecting protein-small molecule interactions and protein-protein interactions

Latest development of display technologies

International Nuclear Information System (INIS)

Gao Hong-Yue; Yao Qiu-Xiang; Liu Pan; Zheng Zhi-Qiang; Liu Ji-Cheng; Zheng Hua-Dong; Zeng Chao; Yu Ying-Jie; Sun Tao; Zeng Zhen-Xiang

2016-01-01

In this review we will focus on recent progress in the field of two-dimensional (2D) and three-dimensional (3D) display technologies. We present the current display materials and their applications, including organic light-emitting diodes (OLEDs), flexible OLEDs quantum dot light emitting diodes (QLEDs), active-matrix organic light emitting diodes (AMOLEDs), electronic paper (E-paper), curved displays, stereoscopic 3D displays, volumetric 3D displays, light field 3D displays, and holographic 3D displays. Conventional 2D display devices, such as liquid crystal devices (LCDs) often result in ambiguity in high-dimensional data images because of lacking true depth information. This review thus provides a detailed description of 3D display technologies. (topical review)

Development of exosome surface display technology in living human cells

International Nuclear Information System (INIS)

Stickney, Zachary; Losacco, Joseph; McDevitt, Sophie; Zhang, Zhiwen; Lu, Biao

2016-01-01

Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

Development of exosome surface display technology in living human cells

Energy Technology Data Exchange (ETDEWEB)

Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu; Zhang, Zhiwen, E-mail: zzhang@scu.edu; Lu, Biao, E-mail: blu2@scu.edu

2016-03-25

Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

Characterization of Anti-Citrinin Specific ScFvs Selected from Non-Immunized Mouse Splenocytes by Eukaryotic Ribosome Display.

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Haiwei Cheng

Full Text Available Single chain variable fragments (scFvs against citrinin (CIT were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA/ CIT-BSA and ovalbumin (OVA/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3 for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109 showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display.

Hybrids of the bHLH and bZIP protein motifs display different DNA-binding activities in vivo vs. in vitro.

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Hiu-Kwan Chow

Full Text Available Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim protein Arnt fused to the leucine zipper (LZ dimerization domain from bZIP (basic region-leucine zipper protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H, transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed, as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (K(d 148.9 nM and 40.2 nM, respectively, but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly alpha-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60-70 aa. Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions.

Inhibition of Plasmepsin V activity demonstrates its essential role in protein export, PfEMP1 display, and survival of malaria parasites

DEFF Research Database (Denmark)

Sleebs, Brad E; Lopaticki, Sash; Marapana, Danushka S

2014-01-01

, Plasmepsin V (PMV). The PMV gene is refractory to deletion, suggesting it is essential, but definitive proof is lacking. Here, we generated a PEXEL-mimetic inhibitor that potently blocks the activity of PMV isolated from P. falciparum and Plasmodium vivax. Assessment of PMV activity in P. falciparum revealed...... surface, and cytoadherence. The inhibitor killed parasites at the trophozoite stage and knockdown of PMV enhanced sensitivity to the inhibitor, while overexpression of PMV increased resistance. This provides the first direct evidence that PMV activity is essential for protein export in Plasmodium spp....... and for parasite survival in human erythrocytes and validates PMV as an antimalarial drug target....

Prazosin Displays Anticancer Activity against Human Prostate Cancers: Targeting DNA, Cell Cycle

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Ssu-Chia Lin

2007-10-01

Full Text Available Quinazoline-based α1,-adrenoceptor antagonists, in particular doxazosin, terazosin, are suggested to display antineoplastic activity against prostate cancers. However, there are few studies elucidating the effect of prazosin. In this study, prazosin displayed antiproliferative activity superior to that of other α1-blockers, including doxazosin, terazosin, tamsulosin, phentolamine. Prazosin induced G2 checkpoint arrest, subsequent apoptosis in prostate cancer PC-3, DU-145, LNCaP cells. In p53-null PC-3 cells, prazosin induced an increase in DNA str, breaks, ATM/ATR checkpoint pathways, leading to the activation of downstream signaling cascades, including Cdc25c phosphorylation at Ser216, nuclear export of Cdc25c, cyclin-dependent kinase (Cdk 1 phosphorylation at Tyr15. The data, together with sustained elevated cyclin A levels (other than cyclin B1 levels, suggested that Cdki activity was inactivated by prazosin. Moreover, prazosin triggered mitochondria-mediated, caspaseexecuted apoptotic pathways in PC-3 cells. The oral administration of prazosin significantly reduced tumor mass in PC-3-derived cancer xenografts in nude mice. In summary, we suggest that prazosin is a potential antitumor agent that induces cell apoptosis through the induction of DNA damage stress, leading to Cdki inactivation, G2 checkpoint arrest. Subsequently, mitochondriamediated caspase cascades are triggered to induce apoptosis in PC-3 cells.

Display and Sexual Activities of the Little Cormorant, Phalacrocorax niger (Ciconiformes: Phalacrocoracidae) in Captivity

International Nuclear Information System (INIS)

Naher, Habibon; Sarker Noor Jahan

2008-01-01

Display and sexual activities of the little cormorant (Phalacrocorax niger) was studied on 19 birds from November 2000 to December 2001. The study was conducted in captivity in the Dhaka Zoo, Mirpur. Displaying behavior commenced in mid February. The male took initiative to select the nest site mostly in trees especially in vertical forked branches. The twigs of peacock flower (Caesalpinia pulcherrima) were used as nesting materials. After the response of the female the bisexual displays started. During this time the male and female always sat together and displayed. Within 1 to 2 days of mutual displays the female ate regurgitated foods from the gular pouch of the male. With this the pair formation occurred permanently. Within 2 to 3 days of pair formation mounting took place. At the end of mounting the male flew away and the female jerked her body and ruffed her feathers. Then the male started to collect nesting materials but both of the male and female placed it together on to the nest, giving more or less a circular cup-like shape. The time required in nest building was 14 to 16 days. (author)

Effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg meat during display storage

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The objective of this study was to evaluate effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg meat during display storage. Two hundred and forty 1-day-old male chicks (5 bird per pen) were randomly subjected to four treatments...

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

2017-01-01

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

Display of a Maize cDNA library on baculovirus infected insect cells

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Jones Ian M

2008-08-01

Full Text Available Abstract Background Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. Results We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 × 105 independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1, was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. Conclusion The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

PTPBR7 binding proteins in myelinating neurons of the mouse brain

NARCIS (Netherlands)

Chesini, I.M.; Debyser, G.; Croes, H.J.E.; Dam, G.B. ten; Devreese, B.; Stoker, A.W.; Hendriks, W.J.A.J.

2011-01-01

Mouse protein tyrosine phosphatase PTPBR7 is a receptor-like, transmembrane protein that is localized on the surface of neuronal cells. Its protein phosphatase activity is reduced upon multimerization, and PTPBR7-deficient mice display motor coordination defects. Extracellular molecules that may

Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

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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.

Structure-Activity Relationship Studies of the Cyclic Depsipeptide Natural Product YM-254890, Targeting the Gq Protein.

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Zhang, Hang; Xiong, Xiao-Feng; Boesgaard, Michael W; Underwood, Christina R; Bräuner-Osborne, Hans; Strømgaard, Kristian

2017-06-07

Extracellular signals perceived by G protein-coupled receptors are transmitted via G proteins, and subsequent intracellular signaling cascades result in a plethora of physiological responses. The natural product cyclic depsipeptides YM-254890 and FR900359 are the only known compounds that specifically inhibit signaling mediated by the G q subfamily. In this study we exploit a newly developed synthetic strategy for this compound class in the design, synthesis, and pharmacological evaluation of eight new analogues of YM-254890. These structure-activity relationship studies led to the discovery of three new analogues, YM-13, YM-14, and YM-18, which displayed potent and selective G q inhibitory activity. This provides pertinent information for the understanding of the G q inhibitory mechanism by this class of compounds and importantly provides a pathway for the development of labeled YM-254890 analogues. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Venom Protein C activators as diagnostic agents for defects of protein C System.

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Ramzan, Faiqah; Asmat, Andleeb

2018-06-18

Background Protein C is a vitamin K dependent plasma zymogen. It prevents clotting by inhibiting clotting by inactivating factor V and factor VIII. Protein C activation pathway involves three steps: (i) Activation of protein C; (ii) Inhibition of coagulation through inactivating factor V and VIII by activated protein C and (iii) Inhibition of activated protein C by plasma protease inhibitors specific for this enzyme. Proteinases converts the zymogen Protein C (PC) of vertebrates into activated PC, which has been detected in several snake venoms. Most PC activators have been purified from venom of snake species belonging to the genera of the Agkistrodon complex. Unlike the physiological thrombin-catalyzed PC activation reaction which requires thrombomodulin as a cofactor, most snake venom activators directly convert the zymogen PC into the catalytically active form which can easily be determined by means of coagulation or chromogenic substrate techniques. Conclusion The fast-acting PC activator Protac® from Agkistrodon contortrix (southern copperhead snake) venom has been found to have broad application in diagnostic practice for the determination of disorders in the PC pathway. Recently, screening assays for the PC pathway have been introduced, based on the observation that the PC pathway is probably the most important physiological barrier against thrombosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

LENUS (Irish Health Repository)

Preston, Roger J S

2012-02-01

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Platelet factor 4 impairs the anticoagulant activity of activated protein C.

LENUS (Irish Health Repository)

Preston, Roger J S

2009-02-27

Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q\\/R679Q and FVa-R306Q\\/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.

Combat vehicle crew helmet-mounted display: next generation high-resolution head-mounted display

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Nelson, Scott A.

1994-06-01

The Combat Vehicle Crew Head-Mounted Display (CVC HMD) program is an ARPA-funded, US Army Natick Research, Development, and Engineering Center monitored effort to develop a high resolution, flat panel HMD for the M1 A2 Abrams main battle tank. CVC HMD is part of the ARPA High Definition Systems (HDS) thrust to develop and integrate small (24 micrometers square pels), high resolution (1280 X 1024 X 6-bit grey scale at 60 frame/sec) active matrix electroluminescent (AMEL) and active matrix liquid crystal displays (AMLCD) for head mounted and projection applications. The Honeywell designed CVC HMD is a next generation head-mounted display system that includes advanced flat panel image sources, advanced digital display driver electronics, high speed (> 1 Gbps) digital interconnect electronics, and light weight, high performance optical and mechanical designs. The resulting dramatic improvements in size, weight, power, and cost have already led to program spin offs for both military and commercial applications.

Outer Membrane Protein 25 of Brucella Activates Mitogen-Activated Protein Kinase Signal Pathway in Human Trophoblast Cells

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Jing Zhang

2017-12-01

Full Text Available Outer membrane protein 25 (OMP25, a virulence factor from Brucella, plays an important role in maintaining the structural stability of Brucella. Mitogen-activated protein kinase (MAPK signal pathway widely exists in eukaryotic cells. In this study, human trophoblast cell line HPT-8 and BALB/c mice were infected with Brucella abortus 2308 strain (S2308 and 2308ΔOmp25 mutant strain. The expression of cytokines and activation of MAPK signal pathway were detected. We found that the expressions of tumor necrosis factor-α, interleukin-1, and interleukin-10 (IL-10 were increased in HPT-8 cells infected with S2308 and 2308ΔOmp25 mutant. S2308 also activated p38 phosphorylation protein, extracellular-regulated protein kinases (ERK, and Jun-N-terminal kinase (JNK from MAPK signal pathway. 2308ΔOmp25 could not activate p38, ERK, and JNK branches. Immunohistochemistry experiments showed that S2308 was able to activate phosphorylation of p38 and ERK in BABL/c mice. However, 2308ΔOmp25 could weakly activate phosphorylation of p38 and ERK. These results suggest that Omp25 played an important role in the process of Brucella activation of the MAPK signal pathway.

TAM receptors, Gas6, and protein S: roles in inflammation and hemostasis.

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van der Meer, Jonathan H M; van der Poll, Tom; van 't Veer, Cornelis

2014-04-17

TAM receptors (Tyro3, Axl, and Mer) belong to a family of receptor tyrosine kinases that have important effects on hemostasis and inflammation. Also, they affect cell proliferation, survival, adhesion, and migration. TAM receptors can be activated by the vitamin K-dependent proteins Gas6 and protein S. Protein S is more commonly known as an important cofactor for protein C as well as a direct inhibitor of multiple coagulation factors. To our knowledge, the functions of Gas6 are limited to TAM receptor activation. When activated, the TAM receptors have effects on primary hemostasis and coagulation and display an anti-inflammatory or a proinflammatory effect, depending on cell type. To comprehend the effects that the TAM receptors and their ligands have on hemostasis and inflammation, we compare studies that report the different phenotypes displayed by mice with deficiencies in the genes of this receptor family and its ligands (protein S(+/-), Gas6(-/-), TAM(-/-), and variations of these). In this manner, we aim to display which features are attributable to the different ligands. Because of the effects TAM receptors have on hemostasis, inflammation, and cancer growth, their modulation could make interesting therapeutic targets in thromboembolic disease, atherosclerosis, sepsis, autoimmune disease, and cancer.

Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

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Tanaka, Tsutomu; Kondo, Akihiko

2015-02-01

In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

Cellular reprogramming through mitogen-activated protein kinases

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Justin eLee

2015-10-01

Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Comparison of NMR and crystal structures highlights conformational isomerism in protein active sites

International Nuclear Information System (INIS)

Serrano, Pedro; Pedrini, Bill; Geralt, Michael; Jaudzems, Kristaps; Mohanty, Biswaranjan; Horst, Reto; Herrmann, Torsten; Elsliger, Marc-André; Wilson, Ian A.; Wüthrich, Kurt

2010-01-01

Tools for systematic comparisons of NMR and crystal structures developed by the JCSG were applied to two proteins with known functions: the T. maritima anti-σ factor antagonist TM1081 and the mouse γ-glutamylamine cyclotransferase A2LD1 (gi:13879369). In an attempt to exploit the complementarity of crystal and NMR data, the combined use of the two structure-determination techniques was explored for the initial steps in the challenge of searching proteins of unknown functions for putative active sites. The JCSG has recently developed a protocol for systematic comparisons of high-quality crystal and NMR structures of proteins. In this paper, the extent to which this approach can provide function-related information on the two functionally annotated proteins TM1081, a Thermotoga maritima anti-σ factor antagonist, and A2LD1 (gi:13879369), a mouse γ-glutamylamine cyclotransferase, is explored. The NMR structures of the two proteins have been determined in solution at 313 and 298 K, respectively, using the current JCSG protocol based on the software package UNIO for extensive automation. The corresponding crystal structures were solved by the JCSG at 100 K and 1.6 Å resolution and at 100 K and 1.9 Å resolution, respectively. The NMR and crystal structures of the two proteins share the same overall molecular architectures. However, the precision of the structure determination along the amino-acid sequence varies over a significantly wider range in the NMR structures than in the crystal structures. Thereby, in each of the two NMR structures about 65% of the residues have displacements below the average and in both proteins the less well ordered residues include large parts of the active sites, in addition to some highly solvent-exposed surface areas. Whereas the latter show increased disorder in the crystal and in solution, the active-site regions display increased displacements only in the NMR structures, where they undergo local conformational exchange on the

[Construction of Lactobacillus rhamnosus GG particles surface display system].

Science.gov (United States)

Su, Runyu; Nie, Boyao; Yuan, Shengling; Tao, Haoxia; Liu, Chunjie; Yang, Bailiang; Wang, Yanchun

2017-01-25

To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.

Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance.

Science.gov (United States)

Chen, Xianzhong

2017-03-04

The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.

Probing the ability of the coat and vertex protein of the membrane-containing bacteriophage PRD1 to display a meningococcal epitope

International Nuclear Information System (INIS)

Huiskonen, Juha T.; Laakkonen, Liisa; Toropainen, Maija; Sarvas, Matti; Bamford, Dennis H.; Bamford, Jaana K.H.

2003-01-01

Bacteriophage PRD1 is an icosahedral dsDNA virus with a diameter of 740 A and an outer protein shell composed of 720 copies of major coat protein P3. Spike complexes at the vertices are composed of a pentameric base (protein P31) and a spike structure (proteins P5 and P2) where the N-terminal region of the trimeric P5 is associated with the base and the C-terminal region of P5 is associated with receptor-binding protein P2. The functionality of proteins P3 and P5 was investigated using insertions and deletions. It was observed that P3 did not tolerate changes whereas P5 tolerated changes much more freely. These properties support the hypothesis that viruses have core structures and functions, which remain stable over time, as well as other elements, responsible for host interactions, which are evolutionally more fluid. The insertional probe used was the apex of exposed loop 4 of group B meningococcal outer membrane protein PorA, a medically important subunit vaccine candidate. It was demonstrated that the epitope could be displayed on the virus surface as part of spike protein P5

Identification and characterisation of the proteins bound by specific phage-displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts.

Science.gov (United States)

de la Cruz, Silvia; Madrid, Raquel; García-García, Aina; Alcocer, Marcos; Martín, Rosario; González, Isabel; García, Teresa

2018-03-01

Almonds and Brazil nuts are widely consumed allergenic nuts whose presence must be declared according to food labelling regulations. Their detection in food products has been recently achieved by ELISA methods with recombinant antibodies (scFv) isolated against complete Brazil nut and almond protein extracts. The screening of phage-scFv libraries against complete protein extracts confers a series of advantages over the use of purified proteins, as recombinant proteins might alter their native folding. However, using this strategy, the nature of the target detected by phage-displayed antibodies remains unknown, and requires further research to identify whether they are nut allergens or other molecules present in the extract, but not related to their allergenic potential. Electrophoretic, chromatographic, immunological and spectrometric techniques revealed that the Brazil nut (BE95) and almond (PD1F6 and PD2C9) specific phage-scFvs detected conformational epitopes of the Brazil nut and almond 11S globulins, recognised by WHO/IUIS as Ber e 2 and Pru du 6 major allergens. Circular dichroism data indicated that severe heat treatment would entail loss of epitope structure, disabling scFv for target detection. The presence of important Brazil nut and almond allergens (Ber e 2 and Pru du 6) in foodstuffs can be determined by using phage-display antibodies BE95, PD1F6 and PD2C9 as affinity probes in ELISA. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

LENUS (Irish Health Repository)

Harmon, Shona

2008-11-07

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

Visual merchandising window display

Directory of Open Access Journals (Sweden)

Opris (Cas. Stanila M.

2013-12-01

Full Text Available Window display plays a major part in the selling strategies; it does not only include the simple display of goods, nowadays it is a form of art, also having the purpose of sustaining the brand image. This article wants to reveal the tools that are essential in creating a fabulous window display. Being a window designer is not an easy job, you have to always think ahead trends, to have a sense of colour, to know how to use light to attract customers in the store after only one glance at the window. The big store window displays are theatre scenes: with expensive backgrounds, special effects and high fashion mannequins. The final role of the displays is to convince customers to enter the store and trigger the purchasing act which is the final goal of the retail activity.

Lambda-Display: A Powerful Tool for Antigen Discovery

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Nicola Gargano

2011-04-01

Full Text Available Since its introduction in 1985, phage display technology has been successfully used in projects aimed at deciphering biological processes and isolating molecules of practical value in several applications. Bacteriophage lambda, representing a classical molecular cloning and expression system has also been exploited for generating large combinatorial libraries of small peptides and protein domains exposed on its capsid. More recently, lambda display has been consistently and successfully employed for domain mapping, antigen discovery and protein interaction studies or, more generally, in functional genomics. We show here the results obtained by the use of large libraries of cDNA and genomic DNA for the molecular dissection of the human B-cell response against complex pathogens, including protozoan parasites, bacteria and viruses. Moreover, by reviewing the experimental work performed in recent investigations we illustrate the potential of lambda display in the diagnostics field and for identifying antigens useful as targets for vaccine development.

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

Science.gov (United States)

Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

2005-01-01

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.

Science.gov (United States)

Karaca, Anara; Malladi, Vijayram Reddy; Zhu, Yan; Tafaj, Olta; Paltrinieri, Elena; Wu, Joy Y; He, Qing; Bastepe, Murat

2018-05-01

GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsα R201H mice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsα R201H mice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsα R201H mice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology. Copyright © 2018 Elsevier Inc. All rights reserved.

A Drosophila protein family implicated in pheromone perception is related to Tay-Sachs GM2-activator protein.

Science.gov (United States)

Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W

2009-01-02

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

A Drosophila Protein Family Implicated in Pheromone Perception Is Related to Tay-Sachs GM2-Activator Protein*

Science.gov (United States)

Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W.

2009-01-01

Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons. PMID:18952610

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

Science.gov (United States)

Prastowo, S.; Widyas, N.

2018-03-01

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

[Peptide phage display in biotechnology and biomedicine].

Science.gov (United States)

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

RNA interference-based (RNAi) suppression of AtMPK6, an Arabidopsis mitogen-activated protein kinase, results in hypersensitivity to ozone and misregulation of AtMPK3

International Nuclear Information System (INIS)

Miles, Godfrey P.; Samuel, Marcus A.; Zhang Yuelin; Ellis, Brian E.

2005-01-01

The recent increase in tropospheric ozone (O 3 ) concentrations promotes additional oxidative stress through the production of reactive oxygen species (ROS) in plant tissues, resulting in the activation of genes whose products enable the stressed cells to retain their integrity and function. This response is made possible by an integration of highly regulated signaling networks that mediate the perception of, and response to, this oxidative assault. In Arabidopsis thaliana, ROS-induced signaling has been shown to flow through a protein phosphorylation cascade involving the mitogen-activated protein kinases (MAPKs) AtMPK3 (MPK3) and AtMPK6 (MPK6). We found that RNAi-mediated silencing of MPK6 renders the plant more sensitive to ozone, as determined by visible leaf damage. The MPK6-RNAi genotype also displayed a more intense and prolonged activation of MPK3 compared to that of WT plants. An MPK3 loss-of-function genotype is similarly very sensitive to ozone, and displays an abnormally prolonged MPK6 activation profile, suggesting reciprocity in regulation between these two MAPKs. - MPK6 is pivotal in the overall response to oxidative stress and regulation of MPK3 in Arabidopsis thaliana

Association of papillomavirus E6 proteins with either MAML1 or E6AP clusters E6 proteins by structure, function, and evolutionary relatedness.

Directory of Open Access Journals (Sweden)

Nicole Brimer

2017-12-01

Full Text Available Papillomavirus E6 proteins bind to LXXLL peptide motifs displayed on targeted cellular proteins. Alpha genus HPV E6 proteins associate with the cellular ubiquitin ligase E6AP (UBE3A, by binding to an LXXLL peptide (ELTLQELLGEE displayed by E6AP, thereby stimulating E6AP ubiquitin ligase activity. Beta, Gamma, and Delta genera E6 proteins bind a similar LXXLL peptide (WMSDLDDLLGS on the cellular transcriptional co-activator MAML1 and thereby repress Notch signaling. We expressed 45 different animal and human E6 proteins from diverse papillomavirus genera to ascertain the overall preference of E6 proteins for E6AP or MAML1. E6 proteins from all HPV genera except Alpha preferentially interacted with MAML1 over E6AP. Among animal papillomaviruses, E6 proteins from certain ungulate (SsPV1 from pigs and cetacean (porpoises and dolphins hosts functionally resembled Alpha genus HPV by binding and targeting the degradation of E6AP. Beta genus HPV E6 proteins functionally clustered with Delta, Pi, Tau, Gamma, Chi, Mu, Lambda, Iota, Dyokappa, Rho, and Dyolambda E6 proteins to bind and repress MAML1. None of the tested E6 proteins physically and functionally interacted with both MAML1 and E6AP, indicating an evolutionary split. Further, interaction of an E6 protein was insufficient to activate degradation of E6AP, indicating that E6 proteins that target E6AP co-evolved to separately acquire both binding and triggering of ubiquitin ligase activation. E6 proteins with similar biological function clustered together in phylogenetic trees and shared structural features. This suggests that the divergence of E6 proteins from either MAML1 or E6AP binding preference is a major event in papillomavirus evolution.

Beyond advertising : large displays for supporting people’s needs and activities in public space

NARCIS (Netherlands)

Kanis, Marije; Groen, Maarten; Meys, Wouter; Slakhorst, Wout; Veenstra, Mettina

2011-01-01

This paper explores how displays can be used to support human needs and activities in public spaces rather than be employed for commercial purposes only. Based on our analysis of screen usage around the world, eight different categories of usage are described and motivated. For the purpose of

Neuronal Functions of Activators of G Protein Signaling

Directory of Open Access Journals (Sweden)

Man K. Tse

2012-05-01

Full Text Available G protein-coupled receptors (GPCRs are one of the most important gateways for signal transduction across the plasma membrane. Over the past decade, several classes of alternative regulators of G protein signaling have been identified and reported to activate the G proteins independent of the GPCRs. One group of such regulators is the activator of G protein signaling (AGS family which comprises of AGS1-10. They have entirely different activation mechanisms for G proteins as compared to the classic model of GPCR-mediated signaling and confer upon cells new avenues of signal transduction. As GPCRs are widely expressed in our nervous system, it is believed that the AGS family plays a major role in modulating the G protein signaling in neurons. In this article, we will review the current knowledge on AGS proteins in relation to their potential roles in neuronal regulations.

Carboxy-terminal truncation activates glp-1 protein to specify vulval fates in Caenorhabditis elegans.

Science.gov (United States)

Mango, S E; Maine, E M; Kimble, J

1991-08-29

The glp-1 and lin-12 genes encode homologous transmembrane proteins that may act as receptors for cell interactions during development. The glp-1 product is required for induction of germ-line proliferation and for embryogenesis. By contrast, lin-12 mediates somatic cell interactions, including those between the precursor cells that form the vulval hypodermis (VPCs). Here we analyse an unusual allele of glp-1, glp-1(q35), which displays a semidominant multivulva phenotype (Muv), as well as the typical recessive, loss-of-function Glp phenotypes (sterility and embryonic lethality). We find that the effects of glp-1(q35) on VPC development mimic those of dominant lin-12 mutations, even in the absence of lin-12 activity. The glp-1(q35) gene bears a nonsense mutation predicted to eliminate the 122 C-terminal amino acids, including a ProGluSerThr (PEST) sequence thought to destabilize proteins. We suggest that the carboxy terminus bears a negative regulatory domain which normally inactivates glp-1 in the VPCs. We propose that inappropriate glp-1(q35) activity can substitute for lin-12 to determine vulval fate, perhaps by driving the VPCs to proliferate.

Petunia nectar proteins have ribonuclease activity.

Science.gov (United States)

Hillwig, Melissa S; Liu, Xiaoteng; Liu, Guangyu; Thornburg, Robert W; Macintosh, Gustavo C

2010-06-01

Plants requiring an insect pollinator often produce nectar as a reward for the pollinator's visitations. This rich secretion needs mechanisms to inhibit microbial growth. In Nicotiana spp. nectar, anti-microbial activity is due to the production of hydrogen peroxide. In a close relative, Petunia hybrida, limited production of hydrogen peroxide was found; yet petunia nectar still has anti-bacterial properties, suggesting that a different mechanism may exist for this inhibition. The nectar proteins of petunia plants were compared with those of ornamental tobacco and significant differences were found in protein profiles and function between these two closely related species. Among those proteins, RNase activities unique to petunia nectar were identified. The genes corresponding to four RNase T2 proteins from Petunia hybrida that show unique expression patterns in different plant tissues were cloned. Two of these enzymes, RNase Phy3 and RNase Phy4 are unique among the T2 family and contain characteristics similar to both S- and S-like RNases. Analysis of amino acid patterns suggest that these proteins are an intermediate between S- and S-like RNases, and support the hypothesis that S-RNases evolved from defence RNases expressed in floral parts. This is the first report of RNase activities in nectar.

Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

International Nuclear Information System (INIS)

Zanzoni, Serena; D’Onofrio, Mariapina; Molinari, Henriette; Assfalg, Michael

2012-01-01

Highlights: ► Bile acid binding proteins from different constructs retain structural integrity. ► NMR 15 N-T 1 relaxation data of BABPs show differences if LVPR extension is present. ► Deviations from a 15 N-T 1 /molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Zanzoni, Serena; D' Onofrio, Mariapina; Molinari, Henriette [Department of Biotechnology, University of Verona, 37134 Verona (Italy); Assfalg, Michael, E-mail: michael.assfalg@univr.it [Department of Biotechnology, University of Verona, 37134 Verona (Italy)

2012-10-26

Highlights: Black-Right-Pointing-Pointer Bile acid binding proteins from different constructs retain structural integrity. Black-Right-Pointing-Pointer NMR {sup 15}N-T{sub 1} relaxation data of BABPs show differences if LVPR extension is present. Black-Right-Pointing-Pointer Deviations from a {sup 15}N-T{sub 1}/molecular-weight calibration curve indicate aggregation. -- Abstract: The use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products.

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts.

Science.gov (United States)

Kassmannhuber, Johannes; Rauscher, Mascha; Schöner, Lea; Witte, Angela; Lubitz, Werner

2017-09-03

In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between -5.6 °C and -6.7 °C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs).

The heat shock protein 90 of Plasmodium falciparum and antimalarial activity of its inhibitor, geldanamycin

Directory of Open Access Journals (Sweden)

Barik Sailen

2003-09-01

Full Text Available Abstract Background The naturally occurring benzoquinone ansamycin compound, geldanamycin (GA, is a specific inhibitor of heat shock protein 90 (Hsp90 and is a potential anticancer agent. Since Plasmodium falciparum has been reported to have an Hsp90 ortholog, we tested the possibility that GA might inhibit it and thereby display antiparasitic activity. Results We provide direct recombinant DNA evidence for the Hsp90 protein of Plasmodium falciparum, the causative agent of fatal malaria. While the mRNA of Hsp90 was mainly expressed in ring and trophozoite stages, the protein was found in all stages, although schizonts contained relatively lower amounts. In vitro the parasitic Hsp90 exhibited an ATP-binding activity that could be specifically inhibited by GA. Plasmodium growth in human erythrocyte culture was strongly inhibited by GA with an IC50 of 20 nM, compared to the IC50 of 15 nM for chloroquine (CQ under identical conditions. When used in combination, the two drugs acted synergistically. GA was equally effective against CQ-sensitive and CQ-resistant strains (3D7 and W2, respectively and on all erythrocytic stages of the parasite. Conclusions Together, these results suggest that an active and essential Hsp90 chaperone cycle exists in Plasmodium and that the ansamycin antibiotics will be an important tool to dissect its role in the parasite. Additionally, the favorable pharmacology of GA, reported in human trials, makes it a promising antimalarial drug.

Immersive virtual walk-through development for tokamak using active head mounted display

International Nuclear Information System (INIS)

Dutta, Pramit

2015-01-01

A fully immersive virtual walk-through of the SST-1 tokamak has been developed. The virtual walkthrough renders the virtual model of SST-1 tokamak through a active stereoscopic head mounted display to visualize the virtual environment. All locations inside and outside of the reactor can be accessed and reviewed. Such a virtual walkthrough provides a 1:1 scale visualization of all components of the tokamak. To achieve such a virtual model, the graphical details of the tokamak CAD model are enhanced. Such enhancements are provided to improve lighting conditions at various locations, texturing of components to have a realistic visual effect and 360° rendering for ease of access. The graphical enhancements also include the redefinition of the facets to optimize the surface triangles to remove lags in display during visual rendering. Two separate algorithms are developed to interact with the virtual model. A fly-by algorithm, developed using C#, uses inputs from a commercial joystick to navigate within the virtual environment. The second algorithm uses the IR and gyroscopic tracking system of the head mounted display to render view as per the current pose of the user within the virtual environment and the direction of view. Such a virtual walk-thorough can be used extensively for design review and integration, review of new components, operator training for remote handling, operations, upgrades of tokamak, etc. (author)

Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C

NARCIS (Netherlands)

Hackeng, T. M.; Hessing, M.; van 't Veer, C.; Meijer-Huizinga, F.; Meijers, J. C.; de Groot, P. G.; van Mourik, J. A.; Bouma, B. N.

1993-01-01

An important feedback mechanism in blood coagulation is supplied by the protein C/protein S anticoagulant pathway. In this study we demonstrate that the binding of human protein S to cultured human umbilical vein endothelial cells (HUVECs) is required for the expression of cofactor activity of

Evolution of phage display technology: from discovery to application.

Science.gov (United States)

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Ahmadzadeh, Vahideh; Akbari, Bahman

2017-03-01

Phage display technology as a selection-based system is an attractive method for evolution of new biological drugs. Unique ability of phage libraries for displaying proteins on bacteriophage surfaces enable them to make a major contribution in diverse fields of researches related to the diagnosis and therapy of diseases. One of the great challenges facing researchers is the modification of phage display technology and the development of new applications. This article reviews the molecular basis of phage display library, and summarizes the novel and specific applications of this technique in the field of biological drugs development including therapeutic antibodies, peptides, vaccines, and catalytic antibodies.

Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

DEFF Research Database (Denmark)

Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

2003-01-01

and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...

Expression, purification and activity determination of the beetle tenebrio molitor antifreeze protein afp84c in escherichia coli

International Nuclear Information System (INIS)

Yang, Q.; Feng, H.

2013-01-01

Summary: A cDNA encoding antifreeze protein (AFP84c) was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 252 bp encodes a protein of 84 amino acid residues and was fused to the expression vectors pMAL-c2X and pMAL-p2X. The expression plasmids pMAL-c2X-afp84c and pMAL-p2X-afp84c were constructed and transformed into Escherischia coli strains TBI, respectively. Strategy of optimization of induction conditions were used for expression of the highly disulfide-bonded beta-helix-contained protein with the activity of antifreeze in pMALTM expression system. The target fusion protein was released from the cytoplasm and periplasm by sonication and cold osmotic shock procedure respectively. Recombinant AFP84c was purified by amylose affinity column. The purified target protein displayed a single band in SDS-PAGE. Expressed AFP84c exhibits to increase low temperature resistance of bacteria. (author)

Display of wasp venom allergens on the cell surface of Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Borodina, Irina; Jensen, B. M.; Søndergaard, Ib

2010-01-01

Background: Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast...... were expressed on the surface as fusions with a-agglutinin complex protein AGA2. The expression was confirmed by fluorescent cytometry (FACS) after staining the cells with antibody against a C-tag attached to the C-terminal end of the allergens. Phospholipase A1 and hyaluronidase retained...... their enzymatic activities. Phospholipase A1 severely inhibited the growth of the yeast cells. Antigen 5 - expressing yeast cells bound IgE antibodies from wasp venom allergic patient sera but not from control sera as demonstrated by FACS. Moreover, antigen 5 - expressing yeast cells were capable of mediating...

Chloroplast-expressed MSI-99 in tobacco improves disease resistance and displays inhibitory effect against rice blast fungus.

Science.gov (United States)

Wang, Yun-Peng; Wei, Zheng-Yi; Zhang, Yu-Ying; Lin, Chun-Jing; Zhong, Xiao-Fang; Wang, Yue-Lin; Ma, Jing-Yong; Ma, Jian; Xing, Shao-Chen

2015-03-02

Rice blast is a major destructive fungal disease that poses a serious threat to rice production and the improvement of blast resistance is critical to rice breeding. The antimicrobial peptide MSI-99 has been suggested as an antimicrobial peptide conferring resistance to bacterial and fungal diseases. Here, a vector harboring the MSI-99 gene was constructed and introduced into the tobacco chloroplast genome via particle bombardment. Transformed plants were obtained and verified to be homoplastomic by PCR and Southern hybridization. In planta assays demonstrated that the transgenic tobacco plants displayed an enhanced resistance to the fungal disease. The evaluation of the antimicrobial activity revealed that the crude protein extracts from the transgenic plants manifested an antimicrobial activity against E. coli, even after incubation at 120 °C for 20 min, indicating significant heat stability of MSI-99. More importantly, the MSI-99-containing protein extracts were firstly proved in vitro and in vivo to display significant suppressive effects on two rice blast isolates. These findings provide a strong basis for the development of new biopesticides to combat rice blast.

Chloroplast-Expressed MSI-99 in Tobacco Improves Disease Resistance and Displays Inhibitory Effect against Rice Blast Fungus

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Yun-Peng Wang

2015-03-01

Full Text Available Rice blast is a major destructive fungal disease that poses a serious threat to rice production and the improvement of blast resistance is critical to rice breeding. The antimicrobial peptide MSI-99 has been suggested as an antimicrobial peptide conferring resistance to bacterial and fungal diseases. Here, a vector harboring the MSI-99 gene was constructed and introduced into the tobacco chloroplast genome via particle bombardment. Transformed plants were obtained and verified to be homoplastomic by PCR and Southern hybridization. In planta assays demonstrated that the transgenic tobacco plants displayed an enhanced resistance to the fungal disease. The evaluation of the antimicrobial activity revealed that the crude protein extracts from the transgenic plants manifested an antimicrobial activity against E. coli, even after incubation at 120 °C for 20 min, indicating significant heat stability of MSI-99. More importantly, the MSI-99-containing protein extracts were firstly proved in vitro and in vivo to display significant suppressive effects on two rice blast isolates. These findings provide a strong basis for the development of new biopesticides to combat rice blast.

Epitope selection from an uncensored peptide library displayed on avian leukosis virus

International Nuclear Information System (INIS)

Khare, Pranay D.; Rosales, Ana G.; Bailey, Kent R.; Russell, Stephen J.; Federspiel, Mark J.

2003-01-01

Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform. In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 x 10 6 different members was generated from a plasmid library of approximately 5 x 10 6 diversity. The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed. No significant sequence censorship was observed in producing the virus display library from the plasmid library. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries

A two-hybrid assay to study protein interactions within the secretory pathway.

Directory of Open Access Journals (Sweden)

Danielle H Dube

Full Text Available Interactions of transcriptional activators are difficult to study using transcription-based two-hybrid assays due to potent activation resulting in false positives. Here we report the development of the Golgi two-hybrid (G2H, a method that interrogates protein interactions within the Golgi, where transcriptional activators can be assayed with negligible background. The G2H relies on cell surface glycosylation to report extracellularly on protein-protein interactions occurring within the secretory pathway. In the G2H, protein pairs are fused to modular domains of the reporter glycosyltransferase, Och1p, and proper cell wall formation due to Och1p activity is observed only when a pair of proteins interacts. Cells containing interacting protein pairs are identified by selectable phenotypes associated with Och1p activity and proper cell wall formation: cells that have interacting proteins grow under selective conditions and display weak wheat germ agglutinin (WGA binding by flow cytometry, whereas cells that lack interacting proteins display stunted growth and strong WGA binding. Using this assay, we detected the interaction between transcription factor MyoD and its binding partner Id2. Interfering mutations along the MyoD:Id2 interaction interface ablated signal in the G2H assay. Furthermore, we used the G2H to detect interactions of the activation domain of Gal4p with a variety of binding partners. Finally, selective conditions were used to enrich for cells encoding interacting partners. The G2H detects protein-protein interactions that cannot be identified via traditional two-hybrid methods and should be broadly useful for probing previously inaccessible subsets of the interactome, including transcriptional activators and proteins that traffic through the secretory pathway.

Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation.

Science.gov (United States)

Scheidegger, F; Ellner, Y; Guye, P; Rhomberg, T A; Weber, H; Augustin, H G; Dehio, C

2009-07-01

The zoonotic pathogen Bartonella henselae (Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.

Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.

Science.gov (United States)

Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel

2016-12-30

GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gα i3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Sequence walkers: a graphical method to display how binding proteins interact with DNA or RNA sequences | Center for Cancer Research

Science.gov (United States)

A graphical method is presented for displaying how binding proteins and other macromolecules interact with individual bases of nucleotide sequences. Characters representing the sequence are either oriented normally and placed above a line indicating favorable contact, or upside-down and placed below the line indicating unfavorable contact. The positive or negative height of each letter shows the contribution of that base to the average sequence conservation of the binding site, as represented by a sequence logo.

Potent antitumor activity of a urokinase-activated engineered anthrax toxin

Science.gov (United States)

Liu, Shihui; Aaronson, Hannah; Mitola, David J.; Leppla, Stephen H.; Bugge, Thomas H.

2003-01-01

The acquisition of cell-surface urokinase plasminogen activator activity is a hallmark of malignancy. We generated an engineered anthrax toxin that is activated by cell-surface urokinase in vivo and displays limited toxicity to normal tissue but broad and potent tumoricidal activity. Native anthrax toxin protective antigen, when administered with a chimeric anthrax toxin lethal factor, Pseudomonas exotoxin fusion protein, was extremely toxic to mice, causing rapid and fatal organ damage. Replacing the furin activation sequence in anthrax toxin protective antigen with an artificial peptide sequence efficiently activated by urokinase greatly attenuated toxicity to mice. In addition, the mutation conferred cell-surface urokinase-dependent toxin activation in vivo, as determined by using a panel of plasminogen, plasminogen activator, plasminogen activator receptor, and plasminogen activator inhibitor-deficient mice. Surprisingly, toxin activation critically depended on both urokinase plasminogen activator receptor and plasminogen in vivo, showing that both proteins are essential cofactors for the generation of cell-surface urokinase. The engineered toxin displayed potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin and could eradicate established solid tumors. This tumoricidal activity depended strictly on tumor cell-surface plasminogen activation. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent.

Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

Science.gov (United States)

Nguyen, Hoang-Minh; Mathiesen, Geir; Stelzer, Elena Maria; Pham, Mai Lan; Kuczkowska, Katarzyna; Mackenzie, Alasdair; Agger, Jane W; Eijsink, Vincent G H; Yamabhai, Montarop; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha

2016-10-04

Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach

Activation of the Unfolded Protein Response Contributes toward the Antitumor Activity of Vorinostat

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Soumen Kahali

2010-01-01

Full Text Available Histone deacetylase (HDAC inhibitors represent an emerging class of anticancer agents progressing through clinical trials. Although their primary target is thought to involve acetylation of core histones, several nonhistone substrates have been identified, including heat shock protein (HSP 90, which may contribute towards their antitumor activity. Glucose-regulated protein 78 (GRP78 is a member of the HSP family of molecular chaperones and plays a central role in regulating the unfolded protein response (UPR. Emerging data suggest that GRP78 is critical in cellular adaptation and survival associated with oncogenesis and may serve as a cancer-specific therapeutic target. On the basis of shared homology with HSP family proteins, we sought to determine whether GRP78 could serve as a molecular target of the HDAC inhibitor vorinostat. Vorinostat treatment led to GRP78 acetylation, dissociation, and subsequent activation of its client protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK. Investigations in a panel of cancer cell lines identified that UPR activation after vorinostat exposure is specific to certain lines. Mass spectrometry performed on immunoprecipitated GRP78 identified lysine-585 as a specific vorinostat-induced acetylation site of GRP78. Downstream activation of the UPR was confirmed, including eukaryotic initiating factor 2α phosphorylation and increase in ATF4 and C/EBP homologous protein expression. To determine the biologic relevance of UPR activation after vorinostat, RNA interference of PERK was performed, demonstrating significantly decreased sensitivity to vorinostat-induced cytotoxicity. Collectively, these findings indicate that GRP78 is a biologic target of vorinostat, and activation of the UPR through PERK phosphorylation contributes toward its antitumor activity.

Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein

NARCIS (Netherlands)

Koppelman, S.J.

1995-01-01

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for

Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

DEFF Research Database (Denmark)

Massip, L; Garand, C; Labbé, A

2010-01-01

show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1......), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease...... activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast...

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries.

Science.gov (United States)

Nuttall, S D; Krishnan, U V; Hattarki, M; De Gori, R; Irving, R A; Hudson, P J

2001-08-01

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

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Tzu-Li Lu

2011-01-01

Full Text Available Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator.

Chimeric Feline Coronaviruses That Encode Type II Spike Protein on Type I Genetic Background Display Accelerated Viral Growth and Altered Receptor Usage▿

Science.gov (United States)

Tekes, Gergely; Hofmann-Lehmann, Regina; Bank-Wolf, Barbara; Maier, Reinhard; Thiel, Heinz-Jürgen; Thiel, Volker

2010-01-01

Persistent infection of domestic cats with feline coronaviruses (FCoVs) can lead to a highly lethal, immunopathological disease termed feline infectious peritonitis (FIP). Interestingly, there are two serotypes, type I and type II FCoVs, that can cause both persistent infection and FIP, even though their main determinant of host cell tropism, the spike (S) protein, is of different phylogeny and displays limited sequence identity. In cell culture, however, there are apparent differences. Type II FCoVs can be propagated to high titers by employing feline aminopeptidase N (fAPN) as a cellular receptor, whereas the propagation of type I FCoVs is usually difficult, and the involvement of fAPN as a receptor is controversial. In this study we have analyzed the phenotypes of recombinant FCoVs that are based on the genetic background of type I FCoV strain Black but encode the type II FCoV strain 79-1146 S protein. Our data demonstrate that recombinant FCoVs expressing a type II FCoV S protein acquire the ability to efficiently use fAPN for host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein. PMID:19906918

Neural Activity Associated with Visual Search for Line Drawings on AAC Displays: An Exploration of the Use of fMRI.

Science.gov (United States)

Wilkinson, Krista M; Dennis, Nancy A; Webb, Christina E; Therrien, Mari; Stradtman, Megan; Farmer, Jacquelyn; Leach, Raevynn; Warrenfeltz, Megan; Zeuner, Courtney

2015-01-01

Visual aided augmentative and alternative communication (AAC) consists of books or technologies that contain visual symbols to supplement spoken language. A common observation concerning some forms of aided AAC is that message preparation can be frustratingly slow. We explored the uses of fMRI to examine the neural correlates of visual search for line drawings on AAC displays in 18 college students under two experimental conditions. Under one condition, the location of the icons remained stable and participants were able to learn the spatial layout of the display. Under the other condition, constant shuffling of the locations of the icons prevented participants from learning the layout, impeding rapid search. Brain activation was contrasted under these conditions. Rapid search in the stable display was associated with greater activation of cortical and subcortical regions associated with memory, motor learning, and dorsal visual pathways compared to the search in the unpredictable display. Rapid search for line drawings on stable AAC displays involves not just the conceptual knowledge of the symbol meaning but also the integration of motor, memory, and visual-spatial knowledge about the display layout. Further research must study individuals who use AAC, as well as the functional effect of interventions that promote knowledge about array layout.

Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity.

Science.gov (United States)

Zamani, Abbas; Benjakul, Soottawat

2016-02-01

Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined. Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH. © 2015 Society of Chemical Industry.

Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus.

Science.gov (United States)

Nhan, Nguyen Thanh; Gonzalez de Valdivia, Ernesto; Gustavsson, Martin; Hai, Truong Nam; Larsson, Gen

2011-04-11

Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes. Both SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis. Apart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein

Evaluation of force-torque displays for use with space station telerobotic activities

Science.gov (United States)

Hendrich, Robert C.; Bierschwale, John M.; Manahan, Meera K.; Stuart, Mark A.; Legendre, A. Jay

1992-01-01

Recent experiments which addressed Space Station remote manipulation tasks found that tactile force feedback (reflecting forces and torques encountered at the end-effector through the manipulator hand controller) does not improve performance significantly. Subjective response from astronaut and non-astronaut test subjects indicated that force information, provided visually, could be useful. No research exists which specifically investigates methods of presenting force-torque information visually. This experiment was designed to evaluate seven different visual force-torque displays which were found in an informal telephone survey. The displays were prototyped in the HyperCard programming environment. In a within-subjects experiment, 14 subjects nullified forces and torques presented statically, using response buttons located at the bottom of the screen. Dependent measures included questionnaire data, errors, and response time. Subjective data generally demonstrate that subjects rated variations of pseudo-perspective displays consistently better than bar graph and digital displays. Subjects commented that the bar graph and digital displays could be used, but were not compatible with using hand controllers. Quantitative data show similar trends to the subjective data, except that the bar graph and digital displays both provided good performance, perhaps do to the mapping of response buttons to display elements. Results indicate that for this set of displays, the pseudo-perspective displays generally represent a more intuitive format for presenting force-torque information.

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

Science.gov (United States)

Suebsuwong, Chalada; Pinkas, Daniel M; Ray, Soumya S; Bufton, Joshua C; Dai, Bing; Bullock, Alex N; Degterev, Alexei; Cuny, Gregory D

2018-02-15

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

A Conserved Epitope Mapped with a Monoclonal Antibody against the VP3 Protein of Goose Parvovirus by Using Peptide Screening and Phage Display Approaches.

Science.gov (United States)

Li, Chenxi; Liu, Hongyu; Li, Jinzhe; Liu, Dafei; Meng, Runze; Zhang, Qingshan; Shaozhou, Wulin; Bai, Xiaofei; Zhang, Tingting; Liu, Ming; Zhang, Yun

2016-01-01

Waterfowl parvovirus (WPV) infection causes high mortality and morbidity in both geese (Anser anser) and Muscovy ducks (Cairina moschata), resulting in significant losses to the waterfowl industries. The VP3 protein of WPV is a major structural protein that induces neutralizing antibodies in the waterfowl. However, B-cell epitopes on the VP3 protein of WPV have not been characterized. To understand the antigenic determinants of the VP3 protein, we used the monoclonal antibody (mAb) 4A6 to screen a set of eight partially expressed overlapping peptides spanning VP3. Using western blotting and an enzyme-linked immunosorbent assay (ELISA), we localized the VP3 epitope between amino acids (aa) 57 and 112. To identify the essential epitope residues, a phage library displaying 12-mer random peptides was screened with mAb 4A6. Phage clone peptides displayed a consensus sequence of YxRFHxH that mimicked the sequence 82Y/FNRFHCH88, which corresponded to amino acid residues 82 to 88 of VP3 protein of WPVs. mAb 4A6 binding to biotinylated fragments corresponding to amino acid residues 82 to 88 of the VP3 protein verified that the 82FxRFHxH88 was the VP3 epitope and that amino acids 82F is necessary to retain maximal binding to mAb 4A6. Parvovirus-positive goose and duck sera reacted with the epitope peptide by dot blotting assay, revealing the importance of these amino acids of the epitope in antibody-epitope binding reactivity. We identified the motif FxRFHxH as a VP3-specific B-cell epitope that is recognized by the neutralizing mAb 4A6. This finding might be valuable in understanding of the antigenic topology of VP3 of WPV.

Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae

Energy Technology Data Exchange (ETDEWEB)

Kojima, Motoki; Akahoshi, Tomohiro; Okamoto, Kenji; Yanase, Hideshi [Tottori Univ. (Japan). Dept. of Chemistry and Biotechnology

2012-11-15

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae. (orig.)

MULTIFUNCTIONAL ADHESIN PROTEINS AND THEIR DISPLAY IN MICROBIAL CELLS

DEFF Research Database (Denmark)

1999-01-01

Recombinant cells expressing a multifunctional adhesin protein derived from a naturally occurring adhesin, containing a binding domain that is capable of binding to an organic receptor and a binding domain that is capable of binding to a compound to which the naturally occurring adhesin protein...... substantially does not bind. The cells or modified adhesin proteins, optionally in immobilized form, are useful for separating organic and inorganic compounds including toxic or precious metals from an environment....

Gamma irradiation effect on soy protein modification, protein - phenolic interaction and antioxidant activity in soybean

International Nuclear Information System (INIS)

Kumari, Sweta; Dahuja, Anil; Vinutha, T.; Singh, Bhupinder

2014-01-01

Soy protein is one of the most important sources of protein to feed the world population in the future. Consumption of soybean quality protein and their texture is dependent on the protein modification. In the present study, four soybean genotypes PL5039 (black), EC 472143 (black), Pusa 9814 (yellow) and SL525 (yellow), differing in their seed coat colour were gamma irradiated at 0.5,1.0, 2.0 and 5.0 kGy and the extent of protein modification and parameters affecting it viz. free phenolics, bound phenolics, lip oxygenase and antioxidant activity were analysed. Modifications of soybean proteins were investigated by chemical analysis and electrophoresis. The irradiation dose of 1.0 kGy showed decreased turbidity, protein oxidation, surface hydrophobicity but increased solubility and sulfhydryl and disulfide contents in all the genotypes. Further, SDS PAGE profile of treated soybean seeds revealed remarkable difference in electrophoretic bands as compared to the untreated seeds. Lipoxygense activity in all the genotypes decreased with increased exposure of gamma irradiation, which produced peroxide products that changes the structural characteristics of soy protein. Free phenolics, bound phenolics and total antioxidant activity measured in terms of FRAP in all the genotypes increased significantly at a dose of 2.0 kGy and it declined at a dose of 5.0 kGy. Antioxidant potential measured in terms of 1,1-diphenyl-2- picrylhydrazyl (DPPH) scavenging activity showed an increasing trend with dose, indicating that radiation processing as a method of food preservation has a positive nutritional implication. Hence, it is suggested that, mild gamma irradiation upto 2.0 kGy may reduce the protein oxidation, enhance the antioxidant activity and improve the soybean protein quality compared to higher dose 5.0 kGy, which reduced the protein quality. (author)

One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA.

Science.gov (United States)

Mochizuki, Yuki; Biyani, Manish; Tsuji-Ueno, Sachika; Suzuki, Miho; Nishigaki, Koichi; Husimi, Yuzuru; Nemoto, Naoto

2011-09-12

A rapid, easy, and robust preparation method for mRNA/cDNA display using a newly designed puromycin-linker DNA is presented. The new linker is structurally simple, easy to synthesize, and cost-effective for use in "in vitro peptide and protein selection". An introduction of RNase T1 nuclease site to the new linker facilitates the easy recovery of mRNA/cDNA displayed protein by an improvement of the efficiency of ligating the linker to mRNAs and efficient release of mRNA/cDNA displayed protein from the solid-phase (magnetic bead). For application demonstration, affinity selections were successfully performed. Furthermore, we introduced a "one-pot" preparation protocol to perform mRNA display easy. Unlike conventional approaches that require tedious and downstream multistep process including purification, this protocol will make the mRNA/cDNA display methods more practical and convenient and also facilitate the development of next-generation, high-throughput mRNA/cDNA display systems amenable to automation.

Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

Science.gov (United States)

Yamaguchi, Hiroshi; Miyazaki, Masaya

2014-02-20

Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

Directory of Open Access Journals (Sweden)

Hiroshi Yamaguchi

2014-02-01

Full Text Available Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

Caged xanthones displaying protein tyrosine phosphatase 1B (PTP1B) inhibition from Cratoxylum cochinchinense.

Science.gov (United States)

Li, Zuo Peng; Lee, Hyeong-Hwan; Uddin, Zia; Song, Yeong Hun; Park, Ki Hun

2018-08-01

Four new caged xanthones (1-4) and two known compounds (5, 6) were isolated from the roots of Cratoxylum cochinchinense, a polyphenol rich plant, collected in China. The structures of the isolated compounds (1-6) were characterized by obtaining their detailed spectroscopic data. In particular, compounds 1 and 6 were fully identified by X-ray crystallographic data. The isolated compounds (1-6) were evaluated against protein tyrosine phosphatase 1B (PTP1B), which plays an important role in diabetes, obesity, and cancer. Among these compounds, 3, 4, and 6 displayed significant inhibition with IC 50 values of 76.3, 43.2, and 6.6 µM, respectively. A detailed kinetic study was conducted by determining K m , V max , and the ratio of K ik and K iv , which revealed that all the compounds behaved as competitive inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

When Heterotrimeric G Proteins Are Not Activated by G Protein-Coupled Receptors: Structural Insights and Evolutionary Conservation.

Science.gov (United States)

DiGiacomo, Vincent; Marivin, Arthur; Garcia-Marcos, Mikel

2018-01-23

Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the "Gα-binding and -activating (GBA) motif". So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subunits in vitro and activates G protein signaling in cells. The importance of this mechanism of signal transduction is highlighted by the fact that its dysregulation underlies human diseases, such as cancer, which has made the proteins attractive new candidates for therapeutic intervention. Here we discuss recent discoveries on the structural basis of GBA-mediated activation of G proteins and its evolutionary conservation and compare them with the better-studied mechanism mediated by GPCRs.

Preferential interactions and the effect of protein PEGylation

DEFF Research Database (Denmark)

Holm, Louise Stenstrup; Thulstrup, Peter Waaben; Kasimova, Marina Robertovna

2015-01-01

enthalpy was decreased to half the value for PEGylated lysozyme. The ratio between calorimetric and van't Hoff enthalpy suggests that our PEGylated lysozyme is a dimer. CONCLUSION/SIGNIFICANCE: The PEGylated model protein displayed similar stability responses to the addition of preferentially active...

A New Approach for Designing a Potentially Vaccine Candidate against Urinary Tract Infection by Using Protein Display on Lactobacillus

Directory of Open Access Journals (Sweden)

Gholamreza Goudarzi

2015-10-01

Full Text Available Background: The prevalence of Urinary Tract Infection (UTI is really high in the world. Escherichia coli is a major agent of UTI. One of the strategies for decreasing UTI infections is vaccine development. As the attachment is a really important stage in colonization and infection, at- tachment inhibition has an applied strategy.  FimH protein is a major factor during bacterial colonization in urinary tract and could be used as a vaccine. Thus, it was considered in this research as a candidate anti- gen.Methods: The sequences of fimH and acmA genes were used for de- signing a synthetic gene. It was cloned to pET23a expression vector and transformed  to E. coli (DE3 Origami.  To confirm the expression  of recombinant  protein,  SDS-PAGE  and western  blotting  methods  were used.  Subsequently,  recombinant  protein  was  purified.  On  the  other hand, Lactobacillus reuteri was cultured and mixed with FimH / AcmA recombinant  protein. The rate of protein localization  on lactobacillus surface was assessed using ELISA method.Results: It was showed that the recombinant protein was expressed inE. coli (DE3 Origami and purified by affinity chromatography. More- over, this protein could be localized on lactobacillus surface by 5 days. Conclusion:  In current study,  a fusion recombinant  protein was pre- pared and displayed on L. reuteri surface. This strain could be used for animal  experiment  as  a  competitor  against  Uropathogenic   E.  coli (UPEC. Using manipulated probiotics strains instead of antibiotic ther- apy could decrease the antibiotic consumption  and reduce multi-drug resistant strains.

A Novel Yeast Surface Display Method for Large-Scale Screen Inhibitors of Sortase A.

Science.gov (United States)

Wu, Lin; Li, Huijun; Tang, Tianle

2017-01-24

Fluorescence resonance energy transfer substrates of sortase A are too expensive to be used to roughly screen high-throughput sortase A inhibitors. This makes therapeutic strategies difficult to realize in a clinical therapeutic use. Instead, we design here an LPETG-EGFP (leucine, proline, glutamic, threonine and glycine-enhanced green fluorescence) protein displayed on a yeast surface as a substrate by adaptively reducing the cost. We do this by optimizing the induction conditions of sortase A expression in Escherichia coli DE3(BL21) and catalyzing LPETG proteins, which are displayed on surface of Pichia pastoris . Different expression conditions of sortase A include: induction temperature (22 °C, 28 °C, 37 °C and 40 °C), induction time (4 h, 5 h, 6 h and 7 h) and induction concentration of isopropyl β-d-thiogalactoside IPTG (0.25 mmol/L, 0.5 mmol/L, 1 mmol/L, and 2 mmol/L). The fluorescence change of the LPETG-EGFP protein on the surface of P. pastoris over time was detected by flow cytometry and fluorescence spectrophotometry, and then the sensitivities of the two methods were compared. Using berberine chloride as an inhibitor, the activity of sortase A was investigated with the substrates of LPETG-EGFP protein, and compared to Dabcyl-QALPETGEE-Edans. A high yield of sortase A was achieved by inducing 1.0 mmol/L IPTG at 28 °C for 6 h. The intensity of green fluorescence of substrates displayed on the yeast surface was increased over time, while the stability was decreased slightly. Both fluorescence spectrophotometery and flow cytometry were fit for detection because of their high sensitivity. We utilized two different substrates of sortase A to investigate sortase A activity, which resulted in the increase of fluorescence intensity with respect to the increased time of growth. However, the method with Dabcyl-QALPETGEE-Edans as its substrate was more robust. Thus, the method described in this paper is a simple and cheap method which is very suitable for

A Novel Yeast Surface Display Method for Large-Scale Screen Inhibitors of Sortase A

Directory of Open Access Journals (Sweden)

Lin Wu

2017-01-01

Full Text Available Fluorescence resonance energy transfer substrates of sortase A are too expensive to be used to roughly screen high-throughput sortase A inhibitors. This makes therapeutic strategies difficult to realize in a clinical therapeutic use. Instead, we design here an LPETG-EGFP (leucine, proline, glutamic, threonine and glycine-enhanced green fluorescence protein displayed on a yeast surface as a substrate by adaptively reducing the cost. We do this by optimizing the induction conditions of sortase A expression in Escherichia coli DE3(BL21 and catalyzing LPETG proteins, which are displayed on surface of Pichia pastoris. Different expression conditions of sortase A include: induction temperature (22 °C, 28 °C, 37 °C and 40 °C, induction time (4 h, 5 h, 6 h and 7 h and induction concentration of isopropyl β-d-thiogalactoside IPTG (0.25 mmol/L, 0.5 mmol/L, 1 mmol/L, and 2 mmol/L. The fluorescence change of the LPETG-EGFP protein on the surface of P. pastoris over time was detected by flow cytometry and fluorescence spectrophotometry, and then the sensitivities of the two methods were compared. Using berberine chloride as an inhibitor, the activity of sortase A was investigated with the substrates of LPETG-EGFP protein, and compared to Dabcyl-QALPETGEE-Edans. A high yield of sortase A was achieved by inducing 1.0 mmol/L IPTG at 28 °C for 6 h. The intensity of green fluorescence of substrates displayed on the yeast surface was increased over time, while the stability was decreased slightly. Both fluorescence spectrophotometery and flow cytometry were fit for detection because of their high sensitivity. We utilized two different substrates of sortase A to investigate sortase A activity, which resulted in the increase of fluorescence intensity with respect to the increased time of growth. However, the method with Dabcyl-QALPETGEE-Edans as its substrate was more robust. Thus, the method described in this paper is a simple and cheap method which is very

Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis.

Science.gov (United States)

Holst, B; Hastrup, H; Raffetseder, U; Martini, L; Schwartz, T W

2001-06-08

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.

Nitric oxide stress and activation of AMP-activated protein kinase impair β-cell sarcoendoplasmic reticulum calcium ATPase 2b activity and protein stability.

Science.gov (United States)

Tong, X; Kono, T; Evans-Molina, C

2015-06-18

The sarcoendoplasmic reticulum Ca(2+) ATPase 2b (SERCA2b) pump maintains a steep Ca(2+) concentration gradient between the cytosol and ER lumen in the pancreatic β-cell, and the integrity of this gradient has a central role in regulated insulin production and secretion, maintenance of ER function and β-cell survival. We have previously demonstrated loss of β-cell SERCA2b expression under diabetic conditions. To define the mechanisms underlying this, INS-1 cells and rat islets were treated with the proinflammatory cytokine interleukin-1β (IL-1β) combined with or without cycloheximide or actinomycin D. IL-1β treatment led to increased inducible nitric oxide synthase (iNOS) gene and protein expression, which occurred concurrently with the activation of AMP-activated protein kinase (AMPK). IL-1β led to decreased SERCA2b mRNA and protein expression, whereas time-course experiments revealed a reduction in protein half-life with no change in mRNA stability. Moreover, SERCA2b protein but not mRNA levels were rescued by treatment with the NOS inhibitor l-NMMA (NG-monomethyl L-arginine), whereas the NO donor SNAP (S-nitroso-N-acetyl-D,L-penicillamine) and the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) recapitulated the effects of IL-1β on SERCA2b protein stability. Similarly, IL-1β-induced reductions in SERCA2b expression were rescued by pharmacological inhibition of AMPK with compound C or by transduction of a dominant-negative form of AMPK, whereas β-cell death was prevented in parallel. Finally, to determine a functional relationship between NO and AMPK signaling and SERCA2b activity, fura-2/AM (fura-2-acetoxymethylester) Ca(2+) imaging experiments were performed in INS-1 cells. Consistent with observed changes in SERCA2b expression, IL-1β, SNAP and AICAR increased cytosolic Ca(2+) and decreased ER Ca(2+) levels, suggesting congruent modulation of SERCA activity under these conditions. In aggregate, these results show that SERCA2b

Functional and biochemical analysis of Chlamydia trachomatis MurC, an enzyme displaying UDP-N-acetylmuramate:amino acid ligase activity.

Science.gov (United States)

Hesse, Lars; Bostock, Julieanne; Dementin, Sebastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Chopra, Ian

2003-11-01

Chlamydiae are unusual obligate intracellular bacteria that cause serious infections in humans. Chlamydiae contain genes that appear to encode products with peptidoglycan biosynthetic activity. The organisms are also susceptible to antibiotics that inhibit peptidoglycan synthesis. However, chlamydiae do not synthesize detectable peptidoglycan. The paradox created by these observations is known as the chlamydial anomaly. The MurC enzyme of chlamydiae, which is synthesized as a bifunctional MurC-Ddl product, is expected to possess UDP-N-acetylmuramate (UDP-MurNAc):L-alanine ligase activity. In this paper we demonstrate that the MurC domain of the Chlamydia trachomatis bifunctional protein is functionally expressed in Escherichia coli, since it complements a conditional lethal E. coli mutant possessing a temperature-sensitive lesion in MurC. The recombinant MurC domain was overexpressed in and purified from E. coli. It displayed in vitro ATP-dependent UDP-MurNAc:L-alanine ligase activity, with a pH optimum of 8.0 and dependence upon magnesium ions (optimum concentration, 20 mM). Its substrate specificity was studied with three amino acids (L-alanine, L-serine, and glycine); comparable Vmax/Km values were obtained. Our results are consistent with the synthesis of a muramic acid-containing polymer in chlamydiae with UDP-MurNAc-pentapeptide as a precursor molecule. However, due to the lack of specificity of MurC activity in vitro, it is not obvious which amino acid is present in the first position of the pentapeptide.

Energy transfer at the active sites of heme proteins

International Nuclear Information System (INIS)

Dlott, D.D.; Hill, J.R.

1995-01-01

Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes

New ultraportable display technology and applications

Science.gov (United States)

Alvelda, Phillip; Lewis, Nancy D.

1998-08-01

MicroDisplay devices are based on a combination of technologies rooted in the extreme integration capability of conventionally fabricated CMOS active-matrix liquid crystal display substrates. Customized diffraction grating and optical distortion correction technology for lens-system compensation allow the elimination of many lenses and systems-level components. The MicroDisplay Corporation's miniature integrated information display technology is rapidly leading to many new defense and commercial applications. There are no moving parts in MicroDisplay substrates, and the fabrication of the color generating gratings, already part of the CMOS circuit fabrication process, is effectively cost and manufacturing process-free. The entire suite of the MicroDisplay Corporation's technologies was devised to create a line of application- specific integrated circuit single-chip display systems with integrated computing, memory, and communication circuitry. Next-generation portable communication, computer, and consumer electronic devices such as truly portable monitor and TV projectors, eyeglass and head mounted displays, pagers and Personal Communication Services hand-sets, and wristwatch-mounted video phones are among the may target commercial markets for MicroDisplay technology. Defense applications range from Maintenance and Repair support, to night-vision systems, to portable projectors for mobile command and control centers.

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.

Activity-based in vitro selection of T4 DNA ligase

International Nuclear Information System (INIS)

Takahashi, Fumio; Funabashi, Hisakage; Mie, Masayasu; Endo, Yaeta; Sawasaki, Tatsuya; Aizawa, Masuo; Kobatake, Eiry

2005-01-01

Recent in vitro methodologies for selection and directed evolution of proteins have concentrated not only on proteins with affinity such as single-chain antibody but also on enzymes. We developed a display technology for selection of T4 DNA ligase on ribosome because an in vitro selection method for DNA ligase had never been developed. The 3' end of mRNA encoding the gene of active or inactive T4 DNA ligase-spacer peptide fusion protein was hybridized to dsDNA fragments with cohesive ends, the substrate of T4 DNA ligase. After in vitro translation of the mRNA-dsDNA complex in a rabbit reticulocyte system, a mRNA-dsDNA-ribosome-ligase complex was produced. T4 DNA ligase enzyme displayed on a ribosome, through addition of a spacer peptide, is able to react with dsDNA in the complex. The complex expressing active ligase was biotinylated by ligation with another biotinylated dsDNA probe and selected with streptavidin-coated magnetic beads. We effectively selected active T4 DNA ligase from a small amount of protein. The gene of the active T4 DNA ligase was enriched 40 times from a mixture of active and inactive genes using this selection strategy. This ribosomal display strategy may have high potential to be useful for selection of other enzymes associated with DNA

Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer

Energy Technology Data Exchange (ETDEWEB)

Purcell, James W; Davis, Jefferson; Reddy, Mamatha; Martin, Shamra; Samayoa, Kimberly; Vo, Hung; Thomsen, Karen; Bean, Peter; Kuo, Wen Lin; Ziyad, Safiyyah; Billig, Jessica; Feiler, Heidi S; Gray, Joe W; Wood, Kenneth W; Cases, Sylvaine

2009-06-10

Ispinesib (SB-715992) is a potent inhibitor of kinesin spindle protein (KSP), a kinesin motor protein essential for the formation of a bipolar mitotic spindle and cell cycle progression through mitosis. Clinical studies of ispinesib have demonstrated a 9% response rate in patients with locally advanced or metastatic breast cancer, and a favorable safety profile without significant neurotoxicities, gastrointestinal toxicities or hair loss. To better understand the potential of ispinesib in the treatment of breast cancer we explored the activity of ispinesib alone and in combination several therapies approved for the treatment of breast cancer. We measured the ispinesib sensitivity and pharmacodynamic response of breast cancer cell lines representative of various subtypes in vitro and as xenografts in vivo, and tested the ability of ispinesib to enhance the anti-tumor activity of approved therapies. In vitro, ispinesib displayed broad anti-proliferative activity against a panel of 53 breast cell-lines. In vivo, ispinesib produced regressions in each of five breast cancer models, and tumor free survivors in three of these models. The effects of ispinesib treatment on pharmacodynamic markers of mitosis and apoptosis were examined in vitro and in vivo, revealing a greater increase in both mitotic and apoptotic markers in the MDA-MB-468 model than in the less sensitive BT-474 model. In vivo, ispinesib enhanced the anti-tumor activity of trastuzumab, lapatinib, doxorubicin, and capecitabine, and exhibited activity comparable to paclitaxel and ixabepilone. These findings support further clinical exploration of KSP inhibitors for the treatment of breast cancer.

Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

Science.gov (United States)

Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi

2003-01-01

Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

Plastic substrates for active matrix liquid crystal display incapable of withstanding processing temperature of over 200 C and method of fabrication

Energy Technology Data Exchange (ETDEWEB)

Carey, P.G.; Smith, P.M.; Havens, J.H.; Jones, P.

1999-01-05

Bright-polarizer-free, active-matrix liquid crystal displays (AMLCDs) are formed on plastic substrates. The primary components of the display are a pixel circuit fabricated on one plastic substrate, an intervening liquid-crystal material, and a counter electrode on a second plastic substrate. The-pixel circuit contains one or more thin-film transistors (TFTs) and either a transparent or reflective pixel electrode manufactured at sufficiently low temperatures to avoid damage to the plastic substrate. Fabrication of the TFTs can be carried out at temperatures less than 100 C. The liquid crystal material is a commercially made nematic curvilinear aligned phase (NCAP) film. The counter electrode is comprised of a plastic substrate coated with a transparent conductor, such as indium-doped tin oxide (ITO). By coupling the active matrix with NCAP, a high-information content can be provided in a bright, fully plastic package. Applications include any low cost portable electronics containing flat displays where ruggedization of the display is desired. 12 figs.

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

Energy Technology Data Exchange (ETDEWEB)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

2012-03-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

International Nuclear Information System (INIS)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

2012-01-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H 2 O 2 ) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H 2 O 2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

International Nuclear Information System (INIS)

Galvão, C.W.; Souza, E.M.; Etto, R.M.; Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G.; Schumacher, J.; Buck, M.; Steffens, M.B.R.

2012-01-01

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs ) can interact with the H. seropedicae RecA protein (RecA Hs ) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs . RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

Directory of Open Access Journals (Sweden)

C.W. Galvão

2012-12-01

Full Text Available DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs can interact with the H. seropedicaeRecA protein (RecA Hs and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA, inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae.

Science.gov (United States)

Galvão, C W; Souza, E M; Etto, R M; Pedrosa, F O; Chubatsu, L S; Yates, M G; Schumacher, J; Buck, M; Steffens, M B R

2012-12-01

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX Hs) can interact with the H. seropedicaeRecA protein (RecA Hs) and that RecA Hs possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX Hs inhibited 90% of the RecA Hs DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA Hs. RecA Hs ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX Hs was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX Hs protein negatively modulates the RecA Hs activities by protein-protein interactions and also by DNA-protein interactions.

Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans

OpenAIRE

Shibasaki, Seiji; Karasaki, Miki; Tafuku, Senji; Aoki, Wataru; Sewaki, Tomomitsu; Ueda, Mitsuyoshi

2014-01-01

Abstract Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later chall...

A New Approach for Designing A Potentially Vaccine Candidate against Urinary Tract Infection by Using Protein Display on Lacto-bacillus Surface

Directory of Open Access Journals (Sweden)

Jalil Fallah Mehrabadi

2013-07-01

Full Text Available Background: The prevalence of Urinary Tract Infection (UTI is really high in the world. Escherichia coli is a major agent of UTI. One of the strategies for decreasing UTI infections is vaccine development. As the attachment is a really important stage in colonization and infection, at­tachment inhibition has an applied strategy. FimH protein is a major factor during bacterial colonization in urinary tract and could be used as a vaccine. Thus, it was considered in this research as a candidate antigen. Methods: The sequences of fimH and acmA genes were used for designing a synthetic gene. It was cloned to pET23a expression vector and transformed to E. coli (DE3 Origami. To confirm the expression of recombinant protein, SDS-PAGE and western blotting methods were used. Subsequently, recombinant protein was purified. On the other hand, Lactobacillus reuteri was cultured and mixed with FimH / AcmA recombinant protein. The rate of protein localization on lactobacillus surface was assessed using ELISA method. Results: It was showed that the recombinant protein was expressed in E. coli (DE3 Origami and purified by affinity chromatography. Moreover, this protein could be localized on lactobacillus surface by 5 days. Conclusion: In current study, a fusion recombinant protein was pre­pared and displayed on L. reuteri surface. This strain could be used for animal experiment as a competitor against Uropathogenic E. coli (UPEC. Using manipulated probiotics strains instead of antibiotic ther­apy could decrease the antibiotic consumption and reduce multi-drug resistant strains.

An anti-tumor protein produced by Trichinella spiralis and identified by screening a T7 phage display library, induces apoptosis in human hepatoma H7402 cells

Science.gov (United States)

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic m...

Survey of standards for electronic image displays

Science.gov (United States)

Rowe, William A.

1996-02-01

Electronic visual displays have been evolving from the 1960's basis of cathode ray tube (CRT) technology. Now, many other technologies are also available, including both flat panels and projection displays. Standards for these displays are being developed at both the national level and the international levels. Standards activity within the United States is in its infancy and is fragmented according to the inclination of each of the standards developing organizations. The latest round of flat panel display technology was primarily developed in Japan. Initially standards arose from component vendor-to-OEM customer relationships. As a result, Japanese standards for components are the best developed. The Electronics Industries Association of Japan (EIAJ) is providing their standards to the International Electrotechnical Commission (IEC) for adoption. On the international level, professional societies such as the human factors society (hfs) and the International Organization for Standardization (ISO) have completed major standards, hfs developed the first ergonomic standard hfs-100 and the ISO has developed some sections of a broader ergonomic standard ISO 9241. This paper addresses the organization of standards activity. Active organizations and their areas of focus are identified. The major standards that have been completed or are in development are described. Finally, suggestions for improving the this standards activity are proposed.

Soya bean Gα proteins with distinct biochemical properties exhibit differential ability to complement Saccharomyces cerevisiae gpa1 mutant.

Science.gov (United States)

Roy Choudhury, Swarup; Wang, Yuqi; Pandey, Sona

2014-07-01

Signalling pathways mediated by heterotrimeric G-proteins are common to all eukaryotes. Plants have a limited number of each of the G-protein subunits, with the most elaborate G-protein network discovered so far in soya bean (Glycine max, also known as soybean) which has four Gα, four Gβ and ten Gγ proteins. Biochemical characterization of Gα proteins from plants suggests significant variation in their properties compared with the well-characterized non-plant proteins. Furthermore, the four soya bean Gα (GmGα) proteins exhibit distinct biochemical activities among themselves, but the extent to which such biochemical differences contribute to their in vivo function is also not known. We used the yeast gpa1 mutant which displays constitutive signalling and growth arrest in the pheromone-response pathway as an in vivo model to evaluate the effect of distinct biochemical activities of GmGα proteins. We showed that specific GmGα proteins can be activated during pheromone-dependent receptor-mediated signalling in yeast and they display different strengths towards complementation of yeast gpa1 phenotypes. We also identified amino acids that are responsible for differential complementation abilities of specific Gα proteins. These data establish that specific plant Gα proteins are functional in the receptor-mediated pheromone-response pathway in yeast and that the subtle biochemical differences in their activity are physiologically relevant.

Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.

OpenAIRE

Martin, M E; Piette, J; Yaniv, M; Tang, W J; Folk, W R

1988-01-01

The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription fac...

Protein stability and enzyme activity at extreme biological temperatures

International Nuclear Information System (INIS)

Feller, Georges

2010-01-01

Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 0 C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

Adolescent sympathetic activity and salivary C-reactive protein: The effects of parental behavior.

Science.gov (United States)

Nelson, Benjamin W; Byrne, Michelle L; Simmons, Julian G; Whittle, Sarah; Schwartz, Orli S; Reynolds, Eric C; O'Brien-Simpson, Neil M; Sheeber, Lisa; Allen, Nicholas B

2017-10-01

This study utilized a novel multisystem approach to investigate the effect of observed parental behavior on the relationship between biological mechanisms associated with disease processes (i.e., autonomic physiology and immune response) among their adolescent children. Thirty-three adolescents (23 males), aged 11-13, and their parents participated in a laboratory session in which adolescents provided baseline measures of autonomic (sympathetic) activity, and adolescents and 1 parent participated in a laboratory based dyadic conflict resolution interaction task. This included 3 male parent/male adolescent dyads, 20 female parent/male adolescent dyads, 3 male parent/female adolescent dyads, and 7 female parent/female adolescent dyads. Approximately 3 years later, adolescents provided a salivary measure of C-Reactive Protein (sCRP) to index inflammation. Analyses revealed a positive association between sympathetic activity and sCRP, as well as a moderating role of positive parental behavior in this relationship, such that the association between sympathetic activity and sCRP was greater among adolescents whose parents displayed shorter duration of positive affect. Overall findings indicate parental behavior may influence the association between adolescent sympathetic activity and inflammatory processes. These findings have important implications for understanding the impact of psychosocial factors on biological mechanisms of disease. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

KAUST Repository

Haidar, Malak

2017-09-08

One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKAis an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.

Pro-region engineering for improved yeast display and secretion of brain derived neurotrophic factor.

Science.gov (United States)

Burns, Michael L; Malott, Thomas M; Metcalf, Kevin J; Puguh, Arthya; Chan, Jonah R; Shusta, Eric V

2016-03-01

Brain derived neurotrophic factor (BDNF) is a promising therapeutic candidate for a variety of neurological diseases. However, it is difficult to produce as a recombinant protein. In its native mammalian context, BDNF is first produced as a pro-protein with subsequent proteolytic removal of the pro-region to yield mature BDNF protein. Therefore, in an attempt to improve yeast as a host for heterologous BDNF production, the BDNF pro-region was first evaluated for its effects on BDNF surface display and secretion. Addition of the wild-type pro-region to yeast BDNF production constructs improved BDNF folding both as a surface-displayed and secreted protein in terms of binding its natural receptors TrkB and p75, but titers remained low. Looking to further enhance the chaperone-like functions provided by the pro-region, two rounds of directed evolution were performed, yielding mutated pro-regions that further improved the display and secretion properties of BDNF. Subsequent optimization of the protease recognition site was used to control whether the produced protein was in pro- or mature BDNF forms. Taken together, we have demonstrated an effective strategy for improving BDNF compatibility with yeast protein engineering and secretion platforms. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mucosal vaccination by adenoviruses displaying reovirus sigma 1

Energy Technology Data Exchange (ETDEWEB)

Weaver, Eric A. [Department of Internal Medicine, Division of Infectious Diseases, Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902 (United States); Camacho, Zenaido T. [Department of Cell Biology, Department of Natural Sciences, Western New Mexico University, Silver City, NM 88062 (United States); Hillestad, Matthew L. [Nephrology Training Program, Mayo Clinic, Rochester, MN 55902 (United States); Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J. [Virology and Gene Therapy Graduate Program, Mayo Clinic, Rochester, MN 55902 (United States); Mercier, George T. [Department of Physics, University of Houston, Houston, TX 77004 (United States); Barry, Michael A., E-mail: mab@mayo.edu [Department of Internal Medicine, Division of Infectious Diseases, Translational Immunovirology and Biodefense Program, Mayo Clinic, Rochester, MN 55902 (United States); Department of Immunology and Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55902 (United States)

2015-08-15

We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5.

Mucosal vaccination by adenoviruses displaying reovirus sigma 1

International Nuclear Information System (INIS)

Weaver, Eric A.; Camacho, Zenaido T.; Hillestad, Matthew L.; Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J.; Mercier, George T.; Barry, Michael A.

2015-01-01

We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5

The potent, indirect adenosine monophosphate-activated protein kinase activator R419 attenuates mitogen-activated protein kinase signaling, inhibits nociceptor excitability, and reduces pain hypersensitivity in mice

Directory of Open Access Journals (Sweden)

Galo L. Mejia

2016-07-01

Full Text Available Abstract. There is a great need for new therapeutics for the treatment of pain. A possible avenue to development of such therapeutics is to interfere with signaling pathways engaged in peripheral nociceptors that cause these neurons to become hyperexcitable. There is strong evidence that mitogen-activated protein kinases and phosphoinositide 3-kinase (PI3K/mechanistic target of rapamycin signaling pathways are key modulators of nociceptor excitability in vitro and in vivo. Activation of adenosine monophosphate-activated protein kinase (AMPK can inhibit signaling in both of these pathways, and AMPK activators have been shown to inhibit nociceptor excitability and pain hypersensitivity in rodents. R419 is one of, if not the most potent AMPK activator described to date. We tested whether R419 activates AMPK in dorsal root ganglion (DRG neurons and if this leads to decreased pain hypersensitivity in mice. We find that R419 activates AMPK in DRG neurons resulting in decreased mitogen-activated protein kinase signaling, decreased nascent protein synthesis, and enhanced P body formation. R419 attenuates nerve growth factor (NGF-induced changes in excitability in DRG neurons and blocks NGF-induced mechanical pain amplification in vivo. Moreover, locally applied R419 attenuates pain hypersensitivity in a model of postsurgical pain and blocks the development of hyperalgesic priming in response to both NGF and incision. We conclude that R419 is a promising lead candidate compound for the development of potent and specific AMPK activation to inhibit pain hypersensitivity as a result of injury.

Protein C activity and antigen levels in childhood

NARCIS (Netherlands)

van Teunenbroek, A.; Peters, M.; Sturk, A.; Borm, J. J.; Breederveld, C.

1990-01-01

Hereditary protein C deficiency is an important risk factor for thrombosis. To enable its diagnosis shortly after birth, we determined reference values of protein C antigen and activity levels for the first 3 months of life. To establish an age-related range of protein C levels we also determined

3D display system using monocular multiview displays

Science.gov (United States)

Sakamoto, Kunio; Saruta, Kazuki; Takeda, Kazutoki

2002-05-01

A 3D head mounted display (HMD) system is useful for constructing a virtual space. The authors have researched the virtual-reality systems connected with computer networks for real-time remote control and developed a low-priced real-time 3D display for building these systems. We developed a 3D HMD system using monocular multi-view displays. The 3D displaying technique of this monocular multi-view display is based on the concept of the super multi-view proposed by Kajiki at TAO (Telecommunications Advancement Organization of Japan) in 1996. Our 3D HMD has two monocular multi-view displays (used as a visual display unit) in order to display a picture to the left eye and the right eye. The left and right images are a pair of stereoscopic images for the left and right eyes, then stereoscopic 3D images are observed.

Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

Science.gov (United States)

Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

2017-03-01

Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and

Protein-directed in situ synthesis of platinum nanoparticles with superior peroxidase-like activity, and their use for photometric determination of hydrogen peroxide

International Nuclear Information System (INIS)

Chen, Lijian; Wang, Nan; Wang, Xindong; Ai, Shiyun

2013-01-01

Platinum nanoparticles (Pt-NPs) with sizes in the range from 10 to 30 nm were synthesized using protein-directed one-pot reduction. The model globular protein bovine serum albumin (BSA) was exploited as the template, and the resulting BSA/Pt-NPs were studied by transmission electron microscopy, energy dispersive X-ray spectroscopy, and resonance Rayleigh scattering spectroscopy. The modified nanoparticles display a peroxidase-like activity that was exploited in a rapid method for the colorimetric determination of hydrogen peroxide which can be detected in the 50 μM to 3 mM concentration range. The limit of detection is 7.9 μM, and the lowest concentration that can be visually detected is 200 μM. (author)

The RecX protein interacts with the RecA protein and modulates its activity in Herbaspirillum seropedicae

Energy Technology Data Exchange (ETDEWEB)

Galvão, C.W. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Souza, E.M. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Etto, R.M. [Departamento de Biologia Estrutural, Molecular e Genética, Universidade Estadual de Ponta Grossa, Ponta Grossa, PR (Brazil); Pedrosa, F.O.; Chubatsu, L.S.; Yates, M.G. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil); Schumacher, J.; Buck, M. [Department of Life Sciences, Imperial College London, London (United Kingdom); Steffens, M.B.R. [Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR (Brazil)

2012-10-15

DNA repair is crucial to the survival of all organisms. The bacterial RecA protein is a central component in the SOS response and in recombinational and SOS DNA repairs. The RecX protein has been characterized as a negative modulator of RecA activity in many bacteria. The recA and recX genes of Herbaspirillum seropedicae constitute a single operon, and evidence suggests that RecX participates in SOS repair. In the present study, we show that the H. seropedicae RecX protein (RecX{sub Hs}) can interact with the H. seropedicae RecA protein (RecA{sub Hs}) and that RecA{sub Hs} possesses ATP binding, ATP hydrolyzing and DNA strand exchange activities. RecX{sub Hs} inhibited 90% of the RecA{sub Hs} DNA strand exchange activity even when present in a 50-fold lower molar concentration than RecA{sub Hs}. RecA{sub Hs} ATP binding was not affected by the addition of RecX, but the ATPase activity was reduced. When RecX{sub Hs} was present before the formation of RecA filaments (RecA-ssDNA), inhibition of ATPase activity was substantially reduced and excess ssDNA also partially suppressed this inhibition. The results suggest that the RecX{sub Hs} protein negatively modulates the RecA{sub Hs} activities by protein-protein interactions and also by DNA-protein interactions.

Circular displays: control/display arrangements and stereotype strength with eight different display locations.

Science.gov (United States)

Chan, Alan H S; Hoffmann, Errol R

2015-01-01

Two experiments are reported that were designed to investigate control/display arrangements having high stereotype strengths when using circular displays. Eight display locations relative to the operator and control were tested with rotational and translational controls situated on different planes according to the Frame of Reference Transformation Tool (FORT) model of Wickens et al. (2010). (Left. No, Right! Development of the Frame of Reference Transformation Tool (FORT), Proceedings of the Human Factors and Ergonomics Society 54th Annual Meeting, 54: 1022-1026). In many cases, there was little effect of display locations, indicating the importance of the Worringham and Beringer (1998. Directional stimulus-response compatibility: a test of three alternative principles. Ergonomics, 41(6), 864-880) Visual Field principle and an extension of this principle for rotary controls (Hoffmann and Chan (2013). The Worringham and Beringer 'visual field' principle for rotary controls. Ergonomics, 56(10), 1620-1624). The initial indicator position (12, 3, 6 and 9 o'clock) had a major effect on control/display stereotype strength for many of the six controls tested. Best display/control arrangements are listed for each of the different control types (rotational and translational) and for the planes on which they are mounted. Data have application where a circular display is used due to limited display panel space and applies to space-craft, robotics operators, hospital equipment and home appliances. Practitioner Summary: Circular displays are often used when there is limited space available on a control panel. Display/control arrangements having high stereotype strength are listed for four initial indicator positions. These arrangements are best for design purposes.

Dyes for displays

Science.gov (United States)

Claussen, U.

1984-01-01

The improvement of contrast and visibility of LCD by two different means was undertaken. The two methods are: (1) development of fluorescent dyes to increase the visibility of fluorescent activated displays (FLAD); and (2) development of dichroic dyes to increase the contrast of displays. This work was done in close cooperation with the electronic industry, where the newly synthesized dyes were tested. The targets for the chemical synthesis were selected with the help of computer model calculations. A marketable range of dyes was developed. Since the interest of the electronic industries concerning FLAD was low, the investigations were stopped. Dichroic dyes, especially black mixtures with good light fastness, order parameter, and solubility in nematic phases were developed. The application of these dyes is restricted to indoor use because of an increase of viscosity below -10 C. Applications on a technical scale, e.g., for the automotive industry, will be possible if the displays work at temperatures down to -40 C. This problem requires a complex optimization of the dye/nematic phase system.

Advanced and tendencies in the development of display technologies

Science.gov (United States)

Kompanets, I. N.

2006-06-01

Advances and key display applications are discussed. Computer, compact mobile, TV and collective large screen displays are mentioned. Flat panel displays step on CRT devices to leave them behind in 2007. Materials, active matricies and applications of bright radiative field emission and organic LED displays are developing successively and pressing other technologies to be used in photo-cameras, cellular phones, auto-cars and avionics. Progress in flexible screens can substantially extend the display design and application soon. 3D display systems are under intensive development, and laser is an important unit in some vaiants of holographic and volumetric 3D displays. Value forecast of different display markets is presented.

The analysis of false prolongation of the activated partial thromboplastin time (activator: silica): Interference of C-reactive protein.

Science.gov (United States)

Liu, Jie; Li, Fanfan; Shu, Kuangyi; Chen, Tao; Wang, Xiaoou; Xie, Yaoqi; Li, Shanshan; Zhang, Zhaohua; Jin, Susu; Jiang, Minghua

2018-05-13

To investigate the effect of C-reactive protein on the activated partial thromboplastin time (APTT) (different activators) in different detecting systems. The C-reactive protein and coagulation test of 112 patients with the infectious disease were determined by automation protein analyzer IMMAG 800 and automation coagulation analyzer STA-R Evolution, respectively. The pooled plasma APTT with different concentrations of C-reactive protein was measured by different detecting system: STA-R Evolution (activator: silica, kaolin), Sysmex CS-2000i (activator: ellagic acid), and ACL TOP 700 (activator: colloidal silica). In addition, the self-made platelet lysate (phospholipid) was added to correct the APTT prolonged by C-reactive protein (150 mg/L) on STA-R Evolution (activator: silica) system. The good correlation between C-reactive protein and APTT was found on the STA-R Evolution (activator: silica) system. The APTT on the STA-R Evolution (activator: silica) system was prolonged by 24.6 second, along with increasing C-reactive protein concentration. And the APTT of plasma containing 150 mg/L C-reactive protein was shortened by 3.4-6.9 second when the plasma was mixed with self-made platelet lysate. However, the APTT was prolonged unobviously on other detecting systems including STA-R Evolution (activator: kaolin), Sysmex CS-2000i, and ACL TOP 700. C-reactive protein interferes with the detection of APTT, especially in STA-R Evolution (activator: silica) system. The increasing in C-reactive protein results in a false prolongation of the APTT (activator: silica), and it is most likely that C-reactive protein interferes the coagulable factor binding of phospholipid. © 2018 Wiley Periodicals, Inc.

DUMA - a program to display distributions in hexagonal geometry

International Nuclear Information System (INIS)

Tran Quoc Dung; Makai, M.

1987-09-01

DUMA program displays hexagonal structures applied in WWER-440 reactors or one or two distributions in them. It helps users to display either integer, literal or real arrays in an arbitrary hexagonal structure. Possible applications: displaying reactor core layout, power distribution or activity measurements. (author)

The functional significance of the autolysis loop in protein C and activated protein C.

Science.gov (United States)

Yang, Likui; Manithody, Chandrashekhara; Rezaie, Alireza R

2005-07-01

The autolysis loop of activated protein C (APC) is five residues longer than the autolysis loop of other vitamin K-dependent coagulation proteases. To investigate the role of this loop in the zymogenic and anticoagulant properties of the molecule, a protein C mutant was constructed in which the autolysis loop of the protein was replaced with the corresponding loop of factor X. The protein C mutant was activated by thrombin with approximately 5-fold higher rate in the presence of Ca2+. Both kinetics and direct binding studies revealed that the Ca2+ affinity of the mutant has been impaired approximately 3-fold. The result of a factor Va degradation assay revealed that the anticoagulant function of the mutant has been improved 4-5-fold in the absence but not in the presence of protein S. The improvement was due to a better recognition of both the P1-Arg506 and P1-Arg306 cleavage sites by the mutant protease. However, the plasma half-life of the mutant was markedly shortened due to faster inactivation by plasma serpins. These results suggest that the autolysis loop of protein C is critical for the Ca(2+)-dependence of activation by thrombin. Moreover, a longer autolysis loop in APC is not optimal for interaction with factor Va in the absence of protein S, but it contributes to the lack of serpin reactivity and longer half-life of the protease in plasma.

mRNA Display Selection of a High-Affinity, Modification-Specific Phospho-IκBα-Binding Fibronectin

Science.gov (United States)

Olson, C. Anders; Liao, Hsiang-I; Sun, Ren; Roberts, Richard W.

2009-01-01

The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we have evolved novel, modification-specific ligands that target phosphorylated IκBα. To do this, we employed mRNA display-based in vitro selection using a 30-trillion-member protein library based on the fibronectin type III domain. The selection yielded one fibronectin molecule, 10C17C25, that binds a phospho-IκBα peptide with Kd = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated IκBα from mammalian cell extract and stabilizes phospho-IκBα in vivo. We also incorporated 10C17C25 into a FRET indicator that detects IκB kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors. PMID:18590330

mRNA display selection of a high-affinity, modification-specific phospho-IkappaBalpha-binding fibronectin.

Science.gov (United States)

Olson, C Anders; Liao, Hsiang-I; Sun, Ren; Roberts, Richard W

2008-08-15

The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we have evolved novel, modification-specific ligands that target phosphorylated IkappaBalpha. To do this, we employed mRNA display-based in vitro selection using a 30-trillion-member protein library based on the fibronectin type III domain. The selection yielded one fibronectin molecule, 10C17C25, that binds a phospho-IkappaBalpha peptide with K d = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated IkappaBalpha from mammalian cell extract and stabilizes phospho-IkappaBalpha in vivo. We also incorporated 10C17C25 into a FRET indicator that detects IkappaB kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors.

Mitogen-activated protein kinase signaling in plants

DEFF Research Database (Denmark)

Rodriguez, Maria Cristina Suarez; Petersen, Morten; Mundy, John

2010-01-01

crossinhibition, feedback control, and scaffolding. Plant MAPK cascades regulate numerous processes, including stress and hormonal responses, innate immunity, and developmental programs. Genetic analyses have uncovered several predominant MAPK components shared by several of these processes including...... of substrate proteins, whose altered activities mediate a wide array of responses, including changes in gene expression. Cascades may share kinase components, but their signaling specificity is maintained by spaciotemporal constraints and dynamic protein-protein interactions and by mechanisms that include...

Plastic substrates for active matrix liquid crystal display incapable of withstanding processing temperature of over 200.degree. C and method of fabrication

Energy Technology Data Exchange (ETDEWEB)

Carey, Paul G. (Mountain View, CA); Smith, Patrick M. (San Ramon, CA); Havens, John (San Diego, CA); Jones, Phil (Marlborough, GB)

1999-01-01

Bright-polarizer-free, active-matrix liquid crystal displays (AMLCDs) are formed on plastic substrates. The primary components of the display are a pixel circuit fabricated on one plastic substrate, an intervening liquid-crystal material, and a counter electrode on a second plastic substrate. The-pixel circuit contains one or more thin-film transistors (TFTs) and either a transparent or reflective pixel electrode manufactured at sufficiently low temperatures to avoid damage to the plastic substrate. Fabrication of the TFTs can be carried out at temperatures less than 100.degree. C. The liquid crystal material is a commercially made nematic curvilinear aligned phase (NCAP) film. The counter electrode is comprised of a plastic substrate coated with a transparent conductor, such as indium-doped tin oxide (ITO). By coupling the active matrix with NCAP, a high-information content can be provided in a bright, fully plastic package. Applications include any low cost portable electronics containing flat displays where ruggedization of the display is desired.

Development of an activity-based probe for acyl-protein thioesterases

Science.gov (United States)

Garland, Megan; Schulze, Christopher J.; Foe, Ian T.; van der Linden, Wouter A.; Child, Matthew A.

2018-01-01

Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation–PATs, APTs and PPTs–will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite Toxoplasma gondii. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity in situ in mammalian cells, to detect an APT in T. gondii (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe. PMID:29364904

The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

Science.gov (United States)

Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

2018-04-20

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

Science.gov (United States)

Chiu, Shao-Chih; Chen, Jo-Mei Maureen; Wei, Tong-You Wade; Cheng, Tai-Shan; Wang, Ya-Hui Candice; Ku, Chia-Feng; Lian, Chiao-Hsuan; Liu, Chun-Chih Jared; Kuo, Yi-Chun; Yu, Chang-Tze Ricky

2014-09-01

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis. Copyright © 2014 the American Physiological Society.

Chimeric microbial rhodopsins for optical activation of Gs-proteins

Science.gov (United States)

Yoshida, Kazuho; Yamashita, Takahiro; Sasaki, Kengo; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

2017-01-01

We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins. PMID:29362703

An Advanced Diagnostic Display for Core Protection Calculator System

Energy Technology Data Exchange (ETDEWEB)

Kim, Ji-Hyeon; Jeong, See-Chae; Sohn, Se-Do [Korea Power Engineering Company, Daejeon (Korea, Republic of)

2008-10-15

The main purpose of a Nuclear Power Plant Instrumentation and Control (I and C) Display System is to provide operator's interface for I and C systems. The CPCS display(Shin-Kori 1 and 2) provides operators with 1) plant monitoring values of field input and algorithm variables that reflect the reactor core conditions, 2) operation values that operators can change and 3) CPCS status. It will be an optimal case if operators can understand the plant (including CPCS itself) condition intuitively with the displayed values but it is not easy in CPCS. For example, if the CPCS Channel Trouble light is lit, operators need some amount of time to investigate what caused the trouble light because there are more than hundred causes that can generate the channel trouble. If a Display supports diagnostic information that shows what cause the displayed alarms, it will greatly help operators in easy understanding the CPCS status. To provide these diagnostic information, this paper suggests an active self-explanatory display mechanism. This self-explanatory diagnostic display mechanism utilizes an ontology in XML that describes parent child, sibling relationships of display variables, through which in-depth, in-breadth diagnostic tracking is possible. This paper consists of two parts. First, the key features of CPCS Flat Panel Display System (FPDS) are described. Second, the features of active self explanatory diagnostic display are discussed.

An Advanced Diagnostic Display for Core Protection Calculator System

International Nuclear Information System (INIS)

Kim, Ji-Hyeon; Jeong, See-Chae; Sohn, Se-Do

2008-01-01

The main purpose of a Nuclear Power Plant Instrumentation and Control (I and C) Display System is to provide operator's interface for I and C systems. The CPCS display(Shin-Kori 1 and 2) provides operators with 1) plant monitoring values of field input and algorithm variables that reflect the reactor core conditions, 2) operation values that operators can change and 3) CPCS status. It will be an optimal case if operators can understand the plant (including CPCS itself) condition intuitively with the displayed values but it is not easy in CPCS. For example, if the CPCS Channel Trouble light is lit, operators need some amount of time to investigate what caused the trouble light because there are more than hundred causes that can generate the channel trouble. If a Display supports diagnostic information that shows what cause the displayed alarms, it will greatly help operators in easy understanding the CPCS status. To provide these diagnostic information, this paper suggests an active self-explanatory display mechanism. This self-explanatory diagnostic display mechanism utilizes an ontology in XML that describes parent child, sibling relationships of display variables, through which in-depth, in-breadth diagnostic tracking is possible. This paper consists of two parts. First, the key features of CPCS Flat Panel Display System (FPDS) are described. Second, the features of active self explanatory diagnostic display are discussed

The effect of display movement angle, indicator type and display location on control/display stereotype strength.

Science.gov (United States)

Hoffmann, Errol R; Chan, Alan H S

2017-08-01

Much research on stereotype strength relating display and control movements for displays moving in the vertical or horizontal directions has been reported. Here we report effects of display movement angle, where the display moves at angles (relative to the vertical) of between 0° and 180°. The experiment used six different controls, four display locations relative to the operator and three types of indicator. Indicator types were included because of the strong effects of the 'scale-side principle' that are variable with display angle. A directional indicator had higher stereotype strength than a neutral indicator, and showed an apparent reversal in control/display stereotype direction beyond an angle of 90°. However, with a neutral indicator this control reversal was not present. Practitioner Summary: The effects of display moving at angles other than the four cardinal directions, types of control, location of display and types of indicator are investigated. Indicator types (directional and neutral) have an effect on stereotype strength and may cause an apparent control reversal with change of display movement angle.

Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

DEFF Research Database (Denmark)

Sayed, M; Kim, S O; Salh, B S

2000-01-01

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

Histone acetyltransferase inhibitors antagonize AMP-activated protein kinase in postmortem glycolysis

Directory of Open Access Journals (Sweden)

Qiong Li

2017-06-01

Full Text Available Objective The purpose of this study was to investigate the influence of AMP-activated protein kinase (AMPK activation on protein acetylation and glycolysis in postmortem muscle to better understand the mechanism by which AMPK regulates postmortem glycolysis and meat quality. Methods A total of 32 mice were randomly assigned to four groups and intraperitoneally injected with 5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside (AICAR, a specific activator of AMPK, AICAR and histone acetyltransferase inhibitor II, or AICAR, Trichostatin A (TSA, an inhibitor of histone deacetylase I and II and Nicotinamide (NAM, an inhibitor of the Sirt family deacetylases. After mice were euthanized, the Longissimus dorsi muscle was collected at 0 h, 45 min, and 24 h postmortem. AMPK activity, protein acetylation and glycolysis in postmortem muscle were measured. Results Activation of AMPK by AICAR significantly increased glycolysis in postmortem muscle. At the same time, it increased the total acetylated proteins in muscle 45 min postmortem. Inhibition of protein acetylation by histone acetyltransferase inhibitors reduced AMPK activation induced increase in the total acetylated proteins and glycolytic rate in muscle early postmortem, while histone deacetylase inhibitors further promoted protein acetylation and glycolysis. Several bands of proteins were detected to be differentially acetylated in muscle with different glycolytic rates. Conclusion Protein acetylation plays an important regulatory role in postmortem glycolysis. As AMPK mediates the effects of pre-slaughter stress on postmortem glycolysis, protein acetylation is likely a mechanism by which antemortem stress influenced postmortem metabolism and meat quality though the exact mechanism is to be elucidated.

Activated Protein C Drives the Hyperfibrinolysis of Acute Traumatic Coagulopathy.

Science.gov (United States)

Davenport, Ross A; Guerreiro, Maria; Frith, Daniel; Rourke, Claire; Platton, Sean; Cohen, Mitchell; Pearse, Rupert; Thiemermann, Chris; Brohi, Karim

2017-01-01

Major trauma is a leading cause of morbidity and mortality worldwide with hemorrhage accounting for 40% of deaths. Acute traumatic coagulopathy exacerbates bleeding, but controversy remains over the degree to which inhibition of procoagulant pathways (anticoagulation), fibrinogen loss, and fibrinolysis drive the pathologic process. Through a combination of experimental study in a murine model of trauma hemorrhage and human observation, the authors' objective was to determine the predominant pathophysiology of acute traumatic coagulopathy. First, a prospective cohort study of 300 trauma patients admitted to a single level 1 trauma center with blood samples collected on arrival was performed. Second, a murine model of acute traumatic coagulopathy with suppressed protein C activation via genetic mutation of thrombomodulin was used. In both studies, analysis for coagulation screen, activated protein C levels, and rotational thromboelastometry (ROTEM) was performed. In patients with acute traumatic coagulopathy, the authors have demonstrated elevated activated protein C levels with profound fibrinolytic activity and early depletion of fibrinogen. Procoagulant pathways were only minimally inhibited with preservation of capacity to generate thrombin. Compared to factors V and VIII, proteases that do not undergo activated protein C-mediated cleavage were reduced but maintained within normal levels. In transgenic mice with reduced capacity to activate protein C, both fibrinolysis and fibrinogen depletion were significantly attenuated. Other recognized drivers of coagulopathy were associated with less significant perturbations of coagulation. Activated protein C-associated fibrinolysis and fibrinogenolysis, rather than inhibition of procoagulant pathways, predominate in acute traumatic coagulopathy. In combination, these findings suggest a central role for the protein C pathway in acute traumatic coagulopathy and provide new translational opportunities for management of

Amended Final Report - Antibodies to Radionuclides. Engineering by Surface Display for Immunosensors

Energy Technology Data Exchange (ETDEWEB)

Blake, Diane A. [Tulane Univ., New Orleans, LA (United States)

2013-06-14

The relatively new techniques of antibody display, which permit molecular engineering of antibody structure and function, have the potential to revolutionize the way scientists generate binding proteins for specific applications. However, the skills required to efficiently use antibody display techniques have proven difficult for other laboratories to acquire without hands-on training and exchange of laboratory personnel. This research project is designed bring important expertise in antibody display to the State of Louisiana while pursuing a project with direct relevance to the DOE’s EM program.

Recent progress in Bacillus subtilis spore-surface display: concept, progress, and future.

Science.gov (United States)

Wang, He; Wang, Yunxiang; Yang, Ruijin

2017-02-01

With the increased knowledge on spore structure and advances in biotechnology engineering, the newly developed spore-surface display system confers several inherent advantages over other microbial cell-surface display systems including enhanced stability and high safety. Bacillus subtilis is the most commonly used Bacillus species for spore-surface display. The expression of heterologous antigen or protein on the surface of B. subtilis spores has now been practiced for over a decade with noteworthy success. As an update and supplement to other previous reviews, we comprehensively summarize recent studies in the B. subtilis spore-surface display technique. We focus on its benefits as well as the critical factors affecting its display efficiency and offer suggestions for the future success of this field.

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

DEFF Research Database (Denmark)

John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

2010-01-01

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

Site-specific incorporation of redox active amino acids into proteins

Science.gov (United States)

Alfonta, Lital [San Diego, CA; Schultz, Peter G [La Jolla, CA; Zhang, Zhiwen [San Diego, CA

2009-02-24

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Site-specific incorporation of redox active amino acids into proteins

Energy Technology Data Exchange (ETDEWEB)

Alfonta, Lital; Schultz, Peter G.; Zhang, Zhiwen

2017-10-10

Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate redox active amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with redox active amino acids using these orthogonal pairs.

Peptide inhibitors of botulinum neurotoxin by mRNA display

International Nuclear Information System (INIS)

Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H.

2005-01-01

Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs

The contact activation proteins: a structure/function overview

NARCIS (Netherlands)

Meijers, J. C.; McMullen, B. A.; Bouma, B. N.

1992-01-01

In recent years, extensive knowledge has been obtained on the structure/function relationships of blood coagulation proteins. In this overview, we present recent developments on the structure/function relationships of the contact activation proteins: factor XII, high molecular weight kininogen,

Active learning in optics and photonics: Liquid Crystal Display in the do-it-yourself

Science.gov (United States)

Vauderwange, Oliver; Haiss, Ulrich; Wozniak, Peter; Israel, Kai; Curticapean, Dan

2015-10-01

Monitors are in the center of media productions and hold an important function as the main visual interface. Tablets and smartphones are becoming more and more important work tools in the media industry. As an extension to our lecture contents an intensive discussion of different display technologies and its applications is taking place now. The established LCD (Liquid Crystal Display) technology and the promising OLED (Organic Light Emitting Diode) technology are in the focus. The classic LCD is currently the most important display technology. The paper will present how the students should develop sense for display technologies besides the theoretical scientific basics. The workshop focuses increasingly on the technical aspects of the display technology and has the goal of deepening the students understanding of the functionality by building simple Liquid Crystal Displays by themselves. The authors will present their experience in the field of display technologies. A mixture of theoretical and practical lectures has the goal of a deeper understanding in the field of digital color representation and display technologies. The design and development of a suitable learning environment with the required infrastructure is crucial. The main focus of this paper is on the hands-on optics workshop "Liquid Crystal Display in the do-it-yourself".

Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

International Nuclear Information System (INIS)

Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

2007-01-01

Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe

Vivo-morpholinos induced transient knockdown of physical activity related proteins.

Directory of Open Access Journals (Sweden)

David P Ferguson

Full Text Available Physical activity is associated with disease prevention and overall wellbeing. Additionally there has been evidence that physical activity level is a result of genetic influence. However, there has not been a reliable method to silence candidate genes in vivo to determine causal mechanisms of physical activity regulation. Vivo-morpholinos are a potential method to transiently silence specific genes. Thus, the aim of this study was to validate the use of Vivo-morpholinos in a mouse model for voluntary physical activity with several sub-objectives. We observed that Vivo-morpholinos achieved between 60-97% knockdown of Drd1-, Vmat2-, and Glut4-protein in skeletal muscle, the delivery moiety of Vivo-morpholinos (scramble did not influence physical activity and that a cocktail of multiple Vivo-morpholinos can be given in a single treatment to achieve protein knockdown of two different targeted proteins in skeletal muscle simultaneously. Knocking down Drd1, Vmat2, or Glut4 protein in skeletal muscle did not affect physical activity. Vivo-morpholinos injected intravenously alone did not significantly knockdown Vmat2-protein expression in the brain (p = 0.28. However, the use of a bradykinin analog to increase blood-brain-barrier permeability in conjunction with the Vivo-morpholinos significantly (p = 0.0001 decreased Vmat2-protein in the brain with a corresponding later over-expression of Vmat2 coincident with a significant (p = 0.0016 increase in physical activity. We conclude that Vivo-morpholinos can be a valuable tool in determining causal gene-phenotype relationships in whole animal models.

Association of the macrophage activating factor (MAF) precursor activity with polymorphism in vitamin D-binding protein.

Science.gov (United States)

Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Kubo, Shinichi; Hori, Hitoshi

2004-01-01

Serum vitamin D-binding protein (Gc protein or DBP) is a highly expressed polymorphic protein, which is a precursor of the inflammation-primed macrophage activating factor, GcMAF, by a cascade of carbohydrate processing reactions. In order to elucidate the relationship between Gc polymorphism and GcMAF precursor activity, we estimated the phagocytic ability of three homotypes of Gc protein, Gc1F-1F, Gc1S-1S and Gc2-2, through processing of their carbohydrate moiety. We performed Gc typing of human serum samples by isoelectric focusing (IEF). Gc protein from human serum was purified by affinity chromatography with 25-hydroxyvitamin D3-sepharose. A phagocytosis assay of Gc proteins, modified using beta-glycosidase and sialidase, was carried out. The Gc1F-1F phenotype was revealed to possess Galbeta1-4GalNAc linkage by the analysis of GcMAF precursor activity using beta1-4 linkage-specific galactosidase from jack bean. The GcMAF precursor activity of the Gc1F-1F phenotype was highest among three Gc homotypes. The Gc polymorphism and carbohydrate diversity of Gc protein are significant for its pleiotropic effects.

Animal and Plant Proteins as Precursors of Peptides with ACE Inhibitory Activity – An in silico Strategy of Protein Evaluation

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Anna Iwaniak

2009-01-01

Full Text Available This paper presents a modern in silico approach useful in the evaluation of proteins as a source of ACE inhibitors. All protein sequences analyzed were derived from the BIOPEP database. To determine the protein value, the following criteria of evaluation were applied: the profile of potential biological (ACE inhibitory activity of a protein, the frequency of the occurrence of fragments with ACE inhibitory activity (A and the potential biological activity of a protein (B. The results, based on a statistical analysis, indicate that milk proteins can be a better source of ACE inhibitors than wheat gliadins. Moreover, all analyzed gliadins possessed more potent ACE inhibitors than chicken meat proteins. No significant differences were observed when comparing A values between soy globulins and β-lactoglobulins. Although criteria such as the profile of potential biological activity of protein, as well as parameters A and B, can be suitable tools in protein evaluation, the proteolytic digestion of protein needs to be considered. Moreover, computerised methods of classifying proteins according to different algorithms are often subjective due to discretion in interpretation of the results.

PENURUNAN KADAR PROTEIN LIMBAH CAIR TAHU DENGAN PEMANFAATAN KARBON BAGASSE TERAKTIVASI (Protein Reduction of Tofu Wastewater Using Activated Carbon Bagasse

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Candra Purnawan

2014-10-01

Full Text Available ABSTRAK Penurunan kadar protein limbah tahu telah dilakukan dengan pemanfaatan karbon Bagasse teraktivasi. Tujuan dari penelitian ini adalah untuk mengetahui kondisi optimum dari karbon teraktivasi NaOH dan H2SO4 dalam menurunkan kadar protein limbah cair tahu dan mengetahui jenis isoterm adsorpsi dari karbon aktif yang digunakan untuk menyerap protein limbah cair tahu. Hasil penelitian menunjukkan konsentrasi NaOH yang optimum untuk aktivasi karbon aktif 15%, massa optimum karbon bagasse teraktivasi NaOH adalah 2 g dan penurunan kadar proteinnya 71,95%, sedangkan massa optimum karbon bagasse teraktivasi H2SO4 adalah 1 g dengan penurunan kadar protein sebesar 38,19%. Waktu kontak optimum karbon bagasse teraktivasi  NaOH dan H2SO4 adalah 12 jam. Adsorpsi protein oleh karbon bagasse teraktivasi NaOH mengikuti isoterm adsorpsi Langmuir dan Freundlich sedangkan karbon bagasse teraktivasi H2SO4 dominan mengikuti isoterm Freundlich.   ABSTRACT The protein reduction of tofu wastewater using activated carbon from bagasse  had been conducted. The purposes of this research were to analysis optimum condition of activated carbon bagsse using NaOH and H2SO4 for reduction protein in tofu wastewater, and analysis adsorption isotherm of activated carbon with protein. The result showed that optimum mass of carbon bagasse activated NaOH was  2 g with 71.95% protein reduction, while carbon bagasse activated H2SO4 has 1 g with 38.19% protein reduction. The optimum contact time between protein and activated carbon (with NaOH and H2SO4 was happened in 12 hours. Adsorption protein with carbon bagasse activated NaOH had followed Langmuir and Freundlich adsorption isotherm, while adsorption with carbon bagasse activated H2SO4 dominantlyhad followed Freundlich adsorption isotherm

Unsolicited displays of insights

DEFF Research Database (Denmark)

Brouwer, Catherine E.

2015-01-01

This study is based on videorecorded interactional data from a specific type of institutional setting which consists of a variety of 'language stimulation activities' for bilingual children in Danish preschools. Bilingual children, with a variety of linguistic backgrounds, take part in these acti......This study is based on videorecorded interactional data from a specific type of institutional setting which consists of a variety of 'language stimulation activities' for bilingual children in Danish preschools. Bilingual children, with a variety of linguistic backgrounds, take part...... in these activities in small groups together with a specialized preschool teacher. One pervasive feature of this kind of data is the ongoing orientation to, and guidance from the adult towards the children on what the main business of their interaction is - what they relevantly are doing. In this light, the paper......: Unsolicited displays may lead to side sequences, they may lead to a shift in the main business of the talk, or they may be explicitly or implicitly ignored. The paper discusses whether and how these unsolicited displays of understanding then can be thought of as leading to opportunities for (language...

Enhanced Adsorption and Recovery of Uranyl Ions by NikR Mutant-Displaying Yeast

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Kouichi Kuroda

2014-04-01

Full Text Available Uranium is one of the most important metal resources, and the technology for the recovery of uranyl ions (UO22+ from aqueous solutions is required to ensure a semi-permanent supply of uranium. The NikR protein is a Ni2+-dependent transcriptional repressor of the nickel-ion uptake system in Escherichia coli, but its mutant protein (NikRm is able to selectively bind uranyl ions in the interface of the two monomers. In this study, NikRm protein with ability to adsorb uranyl ions was displayed on the cell surface of Saccharomyces cerevisiae. To perform the binding of metal ions in the interface of the two monomers, two metal-binding domains (MBDs of NikRm were tandemly fused via linker peptides and displayed on the yeast cell surface by fusion with the cell wall-anchoring domain of yeast α-agglutinin. The NikRm-MBD-displaying yeast cells with particular linker lengths showed the enhanced adsorption of uranyl ions in comparison to the control strain. By treating cells with citrate buffer (pH 4.3, the uranyl ions adsorbed on the cell surface were recovered. Our results indicate that the adsorption system by yeast cells displaying tandemly fused MBDs of NikRm is effective for simple and concentrated recovery of uranyl ions, as well as adsorption of uranyl ions.

In vitro evolution and affinity-maturation with Coliphage qβ display.

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Claudia Skamel

Full Text Available The Escherichia coli bacteriophage, Qβ (Coliphage Qβ, offers a favorable alternative to M13 for in vitro evolution of displayed peptides and proteins due to high mutagenesis rates in Qβ RNA replication that better simulate the affinity maturation processes of the immune response. We describe a benchtop in vitro evolution system using Qβ display of the VP1 G-H loop peptide of foot-and-mouth disease virus (FMDV. DNA encoding the G-H loop was fused to the A1 minor coat protein of Qβ resulting in a replication-competent hybrid phage that efficiently displayed the FMDV peptide. The surface-localized FMDV VP1 G-H loop cross-reacted with the anti-FMDV monoclonal antibody (mAb SD6 and was found to decorate the corners of the Qβ icosahedral shell by electron microscopy. Evolution of Qβ-displayed peptides, starting from fully degenerate coding sequences corresponding to the immunodominant region of VP1, allowed rapid in vitro affinity maturation to SD6 mAb. Qβ selected under evolutionary pressure revealed a non-canonical, but essential epitope for mAb SD6 recognition consisting of an Arg-Gly tandem pair. Finally, the selected hybrid phages induced polyclonal antibodies in guinea pigs with good affinity to both FMDV and hybrid Qβ-G-H loop, validating the requirement of the tandem pair epitope. Qβ-display emerges as a novel framework for rapid in vitro evolution with affinity-maturation to molecular targets.

Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.

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Pascual, Elena; Mata, Carlos P; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L; Castón, José R

2015-03-01

Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign

Correlation between protein kinase C alpha activity and membrane phase behavior.

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Micol, V; Sánchez-Piñera, P; Villalaín, J; de Godos, A; Gómez-Fernández, J C

1999-02-01

Lipid activation of protein kinase C alpha (PKC alpha) was studied by using a model mixture containing 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1, 2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), and 1, 2-dimyristoyl-sn-glycerol (1,2-DMG). This lipid mixture was physically characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and 31P-nuclear magnetic resonance (31P-NMR). Based on these techniques, a phase diagram was constructed by keeping a constant DMPC/DMPS molar ratio of 4:1 and changing the concentration of 1,2-DMG. This phase diagram displayed three regions and two compounds: compound 1 (C1), with 45 mol% 1,2-DMG, and compound 2 (C2), with 60 mol% 1,2-DMG. When the phase diagram was elaborated in the presence of Ca2+ and Mg2+, at concentrations similar to those used in the PKC alpha activity assay, the boundaries between the regions changed slightly and C1 had 35 mol% 1,2-DMG. The activity of PKC alpha was studied at several temperatures and at different concentrations of 1,2-DMG, with a maximum of activity reached at 30 mol% 1,2-DMG and lower values at higher concentrations. In the presence of Ca2+ and Mg2+, maximum PKC alpha activity occurred at concentrations of 1,2-DMG that were close to the boundary in the phase diagram between region 1, where compound C1 and the pure phospholipid coexisted in the gel phase, and region 2, where compounds C1 and C2 coexisted. These results suggest that the membrane structure corresponding to a mixture of 1,2-DMG/phospholipid complex and free phospholipid is better able to support the activity of PKC alpha than the 1,2-DMG/phospholipid complex alone.

Image quality affected by diffraction of aperture structure arrangement in transparent active-matrix organic light-emitting diode displays.

Science.gov (United States)

Tsai, Yu-Hsiang; Huang, Mao-Hsiu; Jeng, Wei-de; Huang, Ting-Wei; Lo, Kuo-Lung; Ou-Yang, Mang

2015-10-01

Transparent display is one of the main technologies in next-generation displays, especially for augmented reality applications. An aperture structure is attached on each display pixel to partition them into transparent and black regions. However, diffraction blurs caused by the aperture structure typically degrade the transparent image when the light from a background object passes through finite aperture window. In this paper, the diffraction effect of an active-matrix organic light-emitting diode display (AMOLED) is studied. Several aperture structures have been proposed and implemented. Based on theoretical analysis and simulation, the appropriate aperture structure will effectively reduce the blur. The analysis data are also consistent with the experimental results. Compared with the various transparent aperture structure on AMOLED, diffraction width (zero energy position of diffraction pattern) of the optimize aperture structure can be reduced 63% and 31% in the x and y directions in CASE 3. Associated with a lenticular lens on the aperture structure, the improvement could reach to 77% and 54% of diffraction width in the x and y directions. Modulation transfer function and practical images are provided to evaluate the improvement of image blurs.

Effector and naturally occurring regulatory T cells display no abnormalities in activation induced cell death in NOD mice.

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Ayelet Kaminitz

Full Text Available BACKGROUND: Disturbed peripheral negative regulation might contribute to evolution of autoimmune insulitis in type 1 diabetes. This study evaluates the sensitivity of naïve/effector (Teff and regulatory T cells (Treg to activation-induced cell death mediated by Fas cross-linking in NOD and wild-type mice. PRINCIPAL FINDINGS: Both effector (CD25(-, FoxP3(- and suppressor (CD25(+, FoxP3(+ CD4(+ T cells are negatively regulated by Fas cross-linking in mixed splenocyte populations of NOD, wild type mice and FoxP3-GFP trangeneess. Proliferation rates and sensitivity to Fas cross-linking are dissociated in Treg cells: fast cycling induced by IL-2 and CD3/CD28 stimulation improve Treg resistance to Fas-ligand (FasL in both strains. The effector and suppressor CD4(+ subsets display balanced sensitivity to negative regulation under baseline conditions, IL-2 and CD3/CD28 stimulation, indicating that stimulation does not perturb immune homeostasis in NOD mice. Effective autocrine apoptosis of diabetogenic cells was evident from delayed onset and reduced incidence of adoptive disease transfer into NOD.SCID by CD4(+CD25(- T cells decorated with FasL protein. Treg resistant to Fas-mediated apoptosis retain suppressive activity in vitro. The only detectable differential response was reduced Teff proliferation and upregulation of CD25 following CD3-activation in NOD mice. CONCLUSION: These data document negative regulation of effector and suppressor cells by Fas cross-linking and dissociation between sensitivity to apoptosis and proliferation in stimulated Treg. There is no evidence that perturbed AICD in NOD mice initiates or promotes autoimmune insulitis.

Hypocholesterolaemic and antioxidant activities of chickpea (Cicer arietinum L.) protein hydrolysates.

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Yust, María del Mar; Millán-Linares, María del Carmen; Alcaide-Hidalgo, Juan María; Millán, Francisco; Pedroche, Justo

2012-07-01

Some dietary proteins possess biological properties which make them potential ingredients of functional or health-promoting foods. Many of these properties are attributed to bioactive peptides that can be released by controlled hydrolysis using exogenous proteases. The aim of this work was to test the improvement of hypocholesterolaemic and antioxidant activities of chickpea protein isolate by means of hydrolysis with alcalase and flavourzyme. All hydrolysates tested exhibited better hypocholesterolaemic activity when compared with chickpea protein isolate. The highest cholesterol micellar solubility inhibition (50%) was found after 60 min of treatment with alcalase followed by 30 min of hydrolysis with flavourzyme. To test antioxidant activity of chickpea proteins three methods were used: β-carotene bleaching method, reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging effect since antioxidant activity of protein hydrolysates may not be attributed to a single mechanism. Chickpea hydrolysates showed better antioxidant activity in all assays, especially reducing power and DPPH scavenging effect than chickpea protein isolate. The results of this study showed the good potential of chickpea protein hydrolysates as bioactive ingredients. The highest bioactive properties could be obtained by selecting the type of proteases and the hydrolysis time. Copyright © 2012 Society of Chemical Industry.

GridOrbit public display

DEFF Research Database (Denmark)

Ramos, Juan David Hincapie; Tabard, Aurélien; Bardram, Jakob

2010-01-01

We introduce GridOrbit, a public awareness display that visualizes the activity of a community grid used in a biology laboratory. This community grid executes bioin-formatics algorithms and relies on users to donate CPU cycles to the grid. The goal of GridOrbit is to create a shared awareness about...

Probing ADAMTS13 substrate specificity using phage display.

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Karl C Desch

Full Text Available Von Willebrand factor (VWF is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2' and P11', for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13-VWF exosite interactions outside of VWF73.

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

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Thakkar, Vidhi D; Cox, Robert M; Sawatsky, Bevan; da Fontoura Budaszewski, Renata; Sourimant, Julien; Wabbel, Katrin; Makhsous, Negar; Greninger, Alexander L; von Messling, Veronika; Plemper, Richard K

2018-04-15

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design. IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral

Binding of phage displayed Bacillus subtilis lipase A to a phosphonate suicide inhibitor

NARCIS (Netherlands)

Dröge, M.J; Ruggeberg, C.J.; van der Sloot, Almer Martinus; Schimmel, J.; Dijkstra, Durk; Verhaert, R.M D; Reetz, M.T.; Quax, Wim; Droge, MJ; Dijkstra, DS

2003-01-01

Phage display can be used as a protein engineering tool to select proteins with desirable binding properties from a library of randomly constructed mutants. Here, we describe the development of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has marked properties

Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

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Mikael S Lindström

Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

Energy Technology Data Exchange (ETDEWEB)

Smith, Jordan N.; Tyrrell, Kimberly J.; Hansen, Joshua R.; Thomas, Dennis G.; Murphree, Taylor A.; Shukla, Anil K.; Luders, Teresa; Madden, James M.; Li, Yunying; Wright, Aaron T.; Piehowski, Paul D.

2017-12-06

Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from rats over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of protein

Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

Science.gov (United States)

Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

2017-08-15

The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Voice-activated intelligent radiologic image display

International Nuclear Information System (INIS)

Fisher, P.

1989-01-01

The authors present a computer-based expert computer system called Mammo-Icon, which automatically assists the radiologist's case analysis by reviewing the trigger phrase output of a commercially available voice transcription system in he domain of mammography. A commercially available PC-based voice dictation system is coupled to an expert system implemented on a microcomputer. Software employs the LISP and C computer languages. Mammo-Icon responds to the trigger phrase output of a voice dictation system with a textual discussion of the potential significance of the findings that have been described and a display of reference images that may help the radiologist to confirm a suspected diagnosis or consider additional diagnoses. This results in automatic availability of potentially useful computer-based expert advice, making such systems much more likely to be used in routine clinical practice

Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.

Science.gov (United States)

Mohamad, Saharuddin Bin; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-01-01

Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF. Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity. Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

Nanobody-Displaying Flagellar Nanotubes.

Science.gov (United States)

Klein, Ágnes; Kovács, Mátyás; Muskotál, Adél; Jankovics, Hajnalka; Tóth, Balázs; Pósfai, Mihály; Vonderviszt, Ferenc

2018-02-26

In this work we addressed the problem how to fabricate self-assembling tubular nanostructures displaying target recognition functionalities. Bacterial flagellar filaments, composed of thousands of flagellin subunits, were used as scaffolds to display single-domain antibodies (nanobodies) on their surface. As a representative example, an anti-GFP nanobody was successfully inserted into the middle part of flagellin replacing the hypervariable surface-exposed D3 domain. A novel procedure was developed to select appropriate linkers required for functional internal insertion. Linkers of various lengths and conformational properties were chosen from a linker database and they were randomly attached to both ends of an anti-GFP nanobody to facilitate insertion. Functional fusion constructs capable of forming filaments on the surface of flagellin-deficient host cells were selected by magnetic microparticles covered by target GFP molecules and appropriate linkers were identified. TEM studies revealed that short filaments of 2-900 nm were formed on the cell surface. ITC and fluorescent measurements demonstrated that the fusion protein exhibited high binding affinity towards GFP. Our approach allows the development of functionalized flagellar nanotubes against a variety of important target molecules offering potential applications in biosensorics and bio-nanotechnology.

Intracellular protein delivery activity of peptides derived from insulin-like growth factor binding proteins 3 and 5

International Nuclear Information System (INIS)

Goda, Natsuko; Tenno, Takeshi; Inomata, Kosuke; Shirakawa, Masahiro; Tanaka, Toshiki; Hiroaki, Hidekazu

2008-01-01

Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20-150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs

Dimerization Efficiency of Canine Distemper Virus Matrix Protein Regulates Membrane-Budding Activity.

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Bringolf, Fanny; Herren, Michael; Wyss, Marianne; Vidondo, Beatriz; Langedijk, Johannes P; Zurbriggen, Andreas; Plattet, Philippe

2017-08-15

Paramyxoviruses rely on the matrix (M) protein to orchestrate viral assembly and budding at the plasma membrane. Although the mechanistic details remain largely unknown, structural data suggested that M dimers and/or higher-order oligomers may facilitate membrane budding. To gain functional insights, we employed a structure-guided mutagenesis approach to investigate the role of canine distemper virus (CDV) M protein self-assembly in membrane-budding activity. Three six-alanine-block (6A-block) mutants with mutations located at strategic oligomeric positions were initially designed. While the first one includes residues potentially residing at the protomer-protomer interface, the other two display amino acids located within two distal surface-exposed α-helices proposed to be involved in dimer-dimer contacts. We further focused on the core of the dimeric interface by mutating asparagine 138 (N138) to several nonconservative amino acids. Cellular localization combined with dimerization and coimmunopurification assays, performed under various denaturing conditions, revealed that all 6A-block mutants were impaired in self-assembly and cell periphery accumulation. These phenotypes correlated with deficiencies in relocating CDV nucleocapsid proteins to the cell periphery and in virus-like particle (VLP) production. Conversely, all M-N138 mutants remained capable of self-assembly, though to various extents, which correlated with proper accumulation and redistribution of nucleocapsid proteins at the plasma membrane. However, membrane deformation and VLP assays indicated that the M-N138 variants exhibiting the most reduced dimerization propensity were also defective in triggering membrane remodeling and budding, despite proper plasma membrane accumulation. Overall, our data provide mechanistic evidence that the efficiency of CDV M dimerization/oligomerization governs both cell periphery localization and membrane-budding activity. IMPORTANCE Despite the availability of

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

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Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

Touch sensitive electrorheological fluid based tactile display

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Liu, Yanju; Davidson, Rob; Taylor, Paul

2005-12-01

A tactile display is programmable device whose controlled surface is intended to be investigated by human touch. It has a great number of potential applications in the field of virtual reality and elsewhere. In this research, a 5 × 5 tactile display array including electrorheological (ER) fluid has been developed and investigated. Force responses of the tactile display array have been measured while a probe was moved across the upper surface. The purpose of this was to simulate the action of touch performed by human finger. Experimental results show that the sensed surface information could be controlled effectively by adjusting the voltage activation pattern imposed on the tactels. The performance of the tactile display is durable and repeatable. The touch sensitivity of this ER fluid based tactile display array has also been investigated in this research. The results show that it is possible to sense the touching force normal to the display's surface by monitoring the change of current passing through the ER fluid. These encouraging results are helpful for constructing a new type of tactile display based on ER fluid which can act as both sensor and actuator at the same time.

Specific chlamydial inclusion membrane proteins associate with active Src family kinases in microdomains that interact with the host microtubule network.

Science.gov (United States)

Mital, Jeffrey; Miller, Natalie J; Fischer, Elizabeth R; Hackstadt, Ted

2010-09-01

Chlamydiae are Gram-negative obligate intracellular bacteria that cause diseases with significant medical and economic impact. Chlamydia trachomatis replicates within a vacuole termed an inclusion, which is extensively modified by the insertion of a number of bacterial effector proteins known as inclusion membrane proteins (Incs). Once modified, the inclusion is trafficked in a dynein-dependent manner to the microtubule-organizing centre (MTOC), where it associates with host centrosomes. Here we describe a novel structure on the inclusion membrane comprised of both host and bacterial proteins. Members of the Src family of kinases are recruited to the chlamydial inclusion in an active form. These kinases display a distinct, localized punctate microdomain-like staining pattern on the inclusion membrane that colocalizes with four chlamydial inclusion membrane proteins (Incs) and is enriched in cholesterol. Biochemical studies show that at least two of these Incs stably interact with one another. Furthermore, host centrosomes associate with these microdomain proteins in C. trachomatis-infected cells and in uninfected cells exogenously expressing one of the chlamydial effectors. Together, the data suggest that a specific structure on the C. trachomatis inclusion membrane may be responsible for the known interactions of chlamydiae with the microtubule network and resultant effects on centrosome stability.

Orally active-targeted drug delivery systems for proteins and peptides.

Science.gov (United States)

Li, Xiuying; Yu, Miaorong; Fan, Weiwei; Gan, Yong; Hovgaard, Lars; Yang, Mingshi

2014-09-01

In the past decade, extensive efforts have been devoted to designing 'active targeted' drug delivery systems (ATDDS) to improve oral absorption of proteins and peptides. Such ATDDS enhance cellular internalization and permeability of proteins and peptides via molecular recognition processes such as ligand-receptor or antigen-antibody interaction, and thus enhance drug absorption. This review focuses on recent advances with orally ATDDS, including ligand-protein conjugates, recombinant ligand-protein fusion proteins and ligand-modified carriers. In addition to traditional intestinal active transport systems of substrates and their corresponding receptors, transporters and carriers, new targets such as intercellular adhesion molecule-1 and β-integrin are also discussed. ATDDS can improve oral absorption of proteins and peptides. However, currently, no clinical studies on ATDDS for proteins and peptides are underway, perhaps due to the complexity and limited knowledge of transport mechanisms. Therefore, more research is warranted to optimize ATDDS efficiency.

Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells.

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Caligiuri, Alessandra; Bertolani, Cristiana; Guerra, Cristina Tosti; Aleffi, Sara; Galastri, Sara; Trappoliere, Marco; Vizzutti, Francesco; Gelmini, Stefania; Laffi, Giacomo; Pinzani, Massimo; Marra, Fabio

2008-02-01

Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.

Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

Science.gov (United States)

Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

2014-01-17

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

NIST display colorimeter calibration facility

Science.gov (United States)

Brown, Steven W.; Ohno, Yoshihiro

2003-07-01

A facility has been developed at the National Institute of Standards and Technology (NIST) to provide calibration services for color-measuring instruments to address the need for improving and certifying the measurement uncertainties of this type of instrument. While NIST has active programs in photometry, flat panel display metrology, and color and appearance measurements, these are the first services offered by NIST tailored to color-measuring instruments for displays. An overview of the facility, the calibration approach, and associated uncertainties are presented. Details of a new tunable colorimetric source and the development of new transfer standard instruments are discussed.

Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores

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De Felice Maurilio

2010-01-01

Full Text Available Abstract Background The bacterial endospore (spore has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the Bacillus subtilis spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen Helicobater acinonychis. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen H. pylori. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections. Results We expressed UreA from H. acinonychis on the B. subtilis spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 × 103 recombinant molecules per spore, whereas when fused to CotC, although most efficiently expressed (7-15 × 103 recombinant molecules per spore and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed. Conclusion UreA was efficiently expressed on the spore coat of B. subtilis when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.

Expression and display of UreA of Helicobacter acinonychis on the surface of Bacillus subtilis spores.

Science.gov (United States)

Hinc, Krzysztof; Isticato, Rachele; Dembek, Marcin; Karczewska, Joanna; Iwanicki, Adam; Peszyńska-Sularz, Grazyna; De Felice, Maurilio; Obuchowski, Michał; Ricca, Ezio

2010-01-18

The bacterial endospore (spore) has recently been proposed as a new surface display system. Antigens and enzymes have been successfully exposed on the surface layers of the Bacillus subtilis spore, but only in a few cases the efficiency of expression and the effective surface display and have been determined. We used this heterologous expression system to produce the A subunit of the urease of the animal pathogen Helicobater acinonychis. Ureases are multi-subunit enzymes with a central role in the virulence of various bacterial pathogens and necessary for colonization of the gastric mucosa by the human pathogen H. pylori. The urease subunit UreA has been recognized as a major antigen, able to induce high levels of protection against challenge infections. We expressed UreA from H. acinonychis on the B. subtilis spore coat by using three different spore coat proteins as carriers and compared the efficiency of surface expression and surface display obtained with the three carriers. A combination of western-, dot-blot and immunofluorescence microscopy allowed us to conclude that, when fused to CotB, UreA is displayed on the spore surface (ca. 1 x 10(3) recombinant molecules per spore), whereas when fused to CotC, although most efficiently expressed (7-15 x 10(3) recombinant molecules per spore) and located in the coat layer, it is not displayed on the surface. Experiments with CotG gave results similar to those with CotC, but the CotG-UreA recombinant protein appeared to be partially processed. UreA was efficiently expressed on the spore coat of B. subtilis when fused to CotB, CotC or CotG. Of these three coat proteins CotC allows the highest efficiency of expression, whereas CotB is the most appropriate for the display of heterologous proteins on the spore surface.

The Cytotoxicity of Elderberry Ribosome-Inactivating Proteins Is Not Solely Determined by Their Protein Translation Inhibition Activity.

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Chenjing Shang

Full Text Available Although the protein translation inhibition activity of ribosome inactivating proteins (RIPs is well documented, little is known about the contribution of the lectin chain to the biological activity of these proteins. In this study, we compared the in vitro and intracellular activity of several S. nigra (elderberry RIPs and non-RIP lectins. Our data demonstrate that RIPs from elderberry are much more toxic to HeLa cells than to primary fibroblasts. Differences in the cytotoxicity between the elderberry proteins correlated with differences in glycan specificity of their lectin domain, cellular uptake efficiency and intracellular destination. Despite the fact that the bulk of the RIPs accumulated in the lysosomes and partly in the Golgi apparatus, we could demonstrate effective inhibition of protein synthesis in cellula. As we also observed cytotoxicity for non-RIP lectins, it is clear that the lectin chain triggers additional pathways heralding cell death. Our data suggest that one of these pathways involves the induction of autophagy.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

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Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient production of D-tagatose using a food-grade surface display system.

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Liu, Yi; Li, Sha; Xu, Hong; Wu, Lingtian; Xu, Zheng; Liu, Jing; Feng, Xiaohai

2014-07-16

D-tagatose, a functional sweetener, is commonly transformed from D-galactose by L-arabinose isomerase (L-AI). In this study, a novel type of biocatalyst, L-AI from Lactobacillus fermentum CGMCC2921 displayed on the spore surface of Bacillus subtilis 168, was developed for producing D-tagatose. The anchored L-AI, exhibiting the relatively high bioactivity, suggested that the surface display system using CotX as the anchoring protein was successfully constructed. The stability of the anchored L-AI was significantly improved. Specifically, the consolidation of thermal stability representing 87% of relative activity was retained even at 80 °C for 30 min, which remarkably favored the production of D-tagatose. Under the optimal conditions, the robust spores can convert 75% D-galactose (100 g/L) into D-tagatose after 24 h, and the conversion rate remained at 56% at the third cycle. Therefore, this biocatalysis system, which could express the target enzyme on the food-grade vector, was an alternative method for the value-added production of D-tagatose.

Pharmacokinetics of activated protein C in guinea pigs

International Nuclear Information System (INIS)

Berger, H. Jr.; Kirstein, C.G.; Orthner, C.L.

1991-01-01

Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of 125 I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of 125 I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. (Abstract Truncated)

Transthyretin knockout mice display decreased susceptibility to AMPA-induced neurodegeneration

DEFF Research Database (Denmark)

Nunes, Ana Filipa; Montero, Maria; Franquinho, Filipa

2009-01-01

Transthyretin (TTR) has been regarded as a neuroprotective protein given that TTR knockout (KO) mice display increased susceptibility for amyloid beta deposition and memory deficits during aging. In parallel, TTR KO mice have increased levels of neuropeptide Y (NPY), which promotes neuroprotectio...

Hepatic protein synthetic activity in vivo after ethanol administration

International Nuclear Information System (INIS)

Donohue, T.M. Jr.; Sorrell, M.F.; Tuma, D.J.

1987-01-01

Hepatic protein synthetic activity in vivo was measured by the incorporation of [ 3 H]puromycin into elongating nascent polypeptides of rat liver to form peptidyl-[ 3 H]puromycin. Our initial experiments showed that saturating doses of [ 3 H]puromycin were achieved at 3-6 mumol/100 g body weight, and that maximum labeling of nascent polypeptides was obtained 30 min after injection of the labeled precursor. Labeled puromycin was found to be suitable for measuring changes in the status of protein synthesis, since the formation of the peptidyl-[ 3 H]puromycin was decreased in fasted animals and was increased in rats pretreated with L-tryptophan. [ 3 H]Puromycin incorporation into polypeptides was then measured after acute ethanol administration as well as after prolonged consumption of ethanol which was administered as part of a liquid diet for 31 days. Acute alcohol treatment caused no significant change in [ 3 H]puromycin incorporation into liver polypeptides. In rats exposed to chronic ethanol feeding, peptidyl-[3H]puromycin formation, when expressed per mg of protein, was slightly lower compared to pair-fed controls, but was unchanged compared to chow-fed animals. When the data were expressed per mg of DNA or per 100 g body wt, no differences in protein synthetic activity were observed among the three groups. These findings indicate that neither acute nor chronic alcohol administration significantly affects protein synthetic activity in rat liver. They further suggest that accumulation of protein in the liver, usually seen after prolonged ethanol consumption, is apparently not reflected by an alteration of hepatic protein synthesis

Characterization of two bacterial hydroxynitrile lyases with high similarity to cupin superfamily proteins

NARCIS (Netherlands)

Hussain, Z.; Wiedner, R.; Steiner, K.; Hajek, T.; Avi, M.; Hecher, B.; Sessitsch, A.; Schwab, H.

2012-01-01

Hydroxynitrile lyases (HNLs) catalyze the cleavage of cyanohydrins. In the reverse reaction, they catalyze the formation of carbon-carbon bonds by enantioselective condensation of hydrocyanic acid with carbonyls. In this study, we describe two proteins from endophytic bacteria that display activity

Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

NARCIS (Netherlands)

Koppelman, S.J.

1995-01-01

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic

[G-protein potentiates the activation of TNF-alpha on calcium-activated potassium channel in ECV304].

Science.gov (United States)

Lin, L; Zheng, Y; Qu, J; Bao, G

2000-06-01

Observe the effect of tumor necrosis factor-alpha (TNF-alpha) on calcium-activated potassium channel in ECV304 and the possible involvement of G-protein mediation in the action of TNF-alpha. Using the cell-attached configuration of patch clamp technique. (1) the activity of high-conductance calcium-activated potassium channel (BKca) was recorded. Its conductance is (202.54 +/- 16.62) pS; (2) the activity of BKca was potentiated by 200 U/ml TNF-alpha; (3) G-protein would intensify this TNF-alpha activation. TNF-alpha acted on vascular endothelial cell ECV304 could rapidly activate the activity of BKca. Opening of BKca resulted in membrane hyper-polarization which could increase electro-chemical gradient for the resting Ca2+ influx and open leakage calcium channel, thus resting cytoplasmic free Ca2+ concentration could be elevated. G-protein may exert an important regulation in this process.

Unfolded protein response and activated degradative pathways regulation in GNE myopathy.

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Honghao Li

Full Text Available Although intracellular beta amyloid (Aβ accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP deposition including unfolded protein response (UPR, ubiquitin proteasome system (UPS activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94, glucose-regulated protein 78 (GRP78, calreticulin and calnexin and valosin containing protein (VCP were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.

The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span.

Science.gov (United States)

Vasicova, Pavla; Lejskova, Renata; Malcova, Ivana; Hasek, Jiri

2015-11-01

Stationary-growth-phase Saccharomyces cerevisiae yeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. In S. cerevisiae stationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Our in vivo analyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficient cox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

International Nuclear Information System (INIS)

Nakatsu, Yusuke; Kotake, Yaichiro; Hino, Atsuko; Ohta, Shigeru

2008-01-01

AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release

Heterologous expression of enterocin A, a bacteriocin from Enterococcus faecium, fused to a cellulose-binding domain in Escherichia coli results in a functional protein with inhibitory activity against Listeria.

Science.gov (United States)

Klocke, Michael; Mundt, Kerstin; Idler, Frank; Jung, Sabrina; Backhausen, Jan E

2005-06-01

The genes for the bacteriocins enterocin A and B were isolated from Enterococcus faecium ATB 197a. Using the pET37b(+) vector, the enterocin genes were fused to an Escherichia coli specific export signal sequence, a cellulose-binding domain (CBD(cenA)) and a S-tag under the control of a T7lac promotor. The constructs were subsequently cloned into E. coli host cells. The expression of the recombinant enterocins had different effects on both the host cells and other Gram-positive bacteria. The expression of entA in Esc. coli led to the synthesis and secretion of functional active enterocin A fusion proteins, which were active against some Gram-positive indicator bacteria, but did not influence the viability of the host cells. In contrast, the expression of enterocin B fusion proteins led to a reduced viability of the host cells, indicating a misfolding of the protein or interference with the cellular metabolism of Esc. coli. Indicator strains of Gram-positive bacteria were not inhibited by purified enterocin B fusion proteins. However, recombinant enterocin B displayed inhibitory activity after the proteolytic cleavage of the fused peptides.

Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

Science.gov (United States)

Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

2014-01-01

Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

A histone-like protein of mycobacteria possesses ferritin superfamily protein-like activity and protects against DNA damage by Fenton reaction.

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Masaki Takatsuka

Full Text Available Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+ into Fe(3+ and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1, a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c, Mycobacterium tuberculosis (Rv2986c, and Mycobacterium leprae (ML1683; ML-LBP were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

High Resolution Displays Using NCAP Liquid Crystals

Science.gov (United States)

Macknick, A. Brian; Jones, Phil; White, Larry

1989-07-01

Nematic curvilinear aligned phase (NCAP) liquid crystals have been found useful for high information content video displays. NCAP materials are liquid crystals which have been encapsulated in a polymer matrix and which have a light transmission which is variable with applied electric fields. Because NCAP materials do not require polarizers, their on-state transmission is substantially better than twisted nematic cells. All dimensional tolerances are locked in during the encapsulation process and hence there are no critical sealing or spacing issues. By controlling the polymer/liquid crystal morphology, switching speeds of NCAP materials have been significantly improved over twisted nematic systems. Recent work has combined active matrix addressing with NCAP materials. Active matrices, such as thin film transistors, have given displays of high resolution. The paper will discuss the advantages of NCAP materials specifically designed for operation at video rates on transistor arrays; applications for both backlit and projection displays will be discussed.

Anticariogenic and Hemolytic Activity of Selected Seed Protein Extracts In vitro conditions.

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Kalpesh B Ishnava

2014-10-01

Full Text Available This study aimed to assess the anticariogenic and hemolytic activity of crude plant seed protein extracts against tooth decaying bacteria.The proteins from seeds of 12 different plants were extracted and used for antimicrobial assay against six different organisms. The extraction was carried out in 10mM of sodium phosphate buffer (pH 7.0. Protein concentrations were determined as described by Bradford method. Anticariogenic activity was studied by agar well diffusion method and Minimum Inhibitory Concentration (MIC was evaluated by the two-fold serial broth dilution method. Hemolytic activity, treatment of proteinase K and Kinetic study in Mimusops elengi crude seed protein extract.The anticariogenic assay demonstrated the activity of Mimusops elengi against Staphylococcus aureus and Streptococcus pyogenes. A minor activity of Glycine wightii against Streptococcus mutans was also found. The protein content of Mimusops elengi seed protein extract was 5.84mg/ml. The MIC values for Staphylococcus aureus and Streptococcus pyogenes against Mimusops elengi seed protein extract were 364.36μg/ml and 182.19μg/ml, respectively. Kinetic study further elucidated the mode of inhibition in the presence of the Mimusops elengi plant seed protein with respect to time. The concentration of crude extract which gave 50% hemolysis compared to Triton X-100 treatment (HC50 value was 1.58 mg/ml; which is more than five times larger than that of the MIC. Treatment with proteinase K of the Mimusops elengi seed protein resulted in absence of the inhibition zone; which clearly indicates that the activity was only due to protein.Our results showed the prominence of Mimusops elengi plant seed protein extract as an effective herbal medication against tooth decaying bacteria.

Nucleolar re-activation is delayed in mouse embryos cloned from two different cell lines

DEFF Research Database (Denmark)

Svarcova, Olga; Dinnyes, A.; Polgar, Z.

2009-01-01

displayed early NPBs transformation. In conclusion, despite normal onset of EGA in cloned embryos, activation of functional nucleoli was one cell cycle delayed in NT embryos. NT-MEF embryos displayed normal targeting but delayed activation of nucleolar proteins. Contrary, in NT-HM1 embryos, both......Aim of this study was to evaluate and compare embryonic genome activation (EGA) in mouse embryos of different origin using nucleolus as a marker. Early and late 2-cell and late 4-cell stage embryos, prepared by in vitro fertilization (IVF), parthenogenetic activation (PG), and nuclear transfer...... ofmouse embryonic fibroblast (MEF) and mouse HM1 emryonic stem cells (HM1), were processed for autoradiography following 3H-uridine incubation (transcriptional activity), transmission electron microscopy (ultrastructure) and immunofluorescence (nucleolar proteins; upstream binding factor, UBF...

Oral Immunization Against Candidiasis Using Lactobacillus casei Displaying Enolase 1 from Candida albicans.

Science.gov (United States)

Shibasaki, Seiji; Karasaki, Miki; Tafuku, Senji; Aoki, Wataru; Sewaki, Tomomitsu; Ueda, Mitsuyoshi

2014-01-01

Candidiasis is a common fungal infection that is prevalent in immunocompromised individuals. In this study, an oral vaccine against Candida albicans was developed by using the molecular display approach. Enolase 1 protein (Eno1p) of C. albicans was expressed on the Lactobacillus casei cell surface by using poly-gamma-glutamic acid synthetase complex A from Bacillus subtilis as an anchoring protein. The Eno1p-displaying L. casei cells were used to immunize mice, which were later challenged with a lethal dose of C. albicans. The data indicated that the vaccine elicited a strong IgG response and increased the survival rate of the vaccinated mice. Furthermore, L. casei acted as a potent adjuvant and induced high antibody titers that were comparable to those induced by strong adjuvants such as the cholera toxin. Overall, the molecular display method can be used to rapidly develop vaccines that can be conveniently administered and require minimal processing.

Quinoline-3-carboxamide Derivatives as Potential Cholesteryl Ester Transfer Protein Inhibitors

Directory of Open Access Journals (Sweden)

Jing-Kang Shen

2012-05-01

Full Text Available A series of novel quinoline-3-carboxamide derivatives 10–17 and 23–27 were designed and synthesized as cholesteryl ester transfer protein (CETP inhibitors. All of them exhibited activity against CETP. Particularly, compounds 24 and 26 displayed the best activity against CETP with the same inhibitory rate of 80.1%.

A novel protease activity assay using a protease-responsive chaperone protein

International Nuclear Information System (INIS)

Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

2009-01-01

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

A novel protease activity assay using a protease-responsive chaperone protein

Energy Technology Data Exchange (ETDEWEB)

Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

2009-06-05

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

TOPICAL REVIEW: Protein stability and enzyme activity at extreme biological temperatures

Science.gov (United States)

Feller, Georges

2010-08-01

Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 °C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins.

In vitro Fab display: a cell-free system for IgG discovery

Science.gov (United States)

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

Science.gov (United States)

Babon, Aurélie; Wurceldorf, Thibault; Almunia, Christine; Pichard, Sylvain; Chenal, Alexandre; Buhot, Cécile; Beaumelle, Bruno; Gillet, Daniel

2016-06-15

We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Modulation of catalytic activity in multi-domain protein tyrosine phosphatases.

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Lalima L Madan

Full Text Available Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1 domains, while the membrane-distal (D2 domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A. While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

Autostereoscopic display technology for mobile 3DTV applications

Science.gov (United States)

Harrold, Jonathan; Woodgate, Graham J.

2007-02-01

Mobile TV is now a commercial reality, and an opportunity exists for the first mass market 3DTV products based on cell phone platforms with switchable 2D/3D autostereoscopic displays. Compared to conventional cell phones, TV phones need to operate for extended periods of time with the display running at full brightness, so the efficiency of the 3D optical system is key. The desire for increased viewing freedom to provide greater viewing comfort can be met by increasing the number of views presented. A four view lenticular display will have a brightness five times greater than the equivalent parallax barrier display. Therefore, lenticular displays are very strong candidates for cell phone 3DTV. Selection of Polarisation Activated Microlens TM architectures for LCD, OLED and reflective display applications is described. The technology delivers significant advantages especially for high pixel density panels and optimises device ruggedness while maintaining display brightness. A significant manufacturing breakthrough is described, enabling switchable microlenses to be fabricated using a simple coating process, which is also readily scalable to large TV panels. The 3D image performance of candidate 3DTV panels will also be compared using autostereoscopic display optical output simulations.

NK-like homeodomain proteins activate NOTCH3-signaling in leukemic T-cells

International Nuclear Information System (INIS)

Nagel, Stefan; Scherr, Michaela; MacLeod, Roderick AF; Venturini, Letizia; Przybylski, Grzegorz K; Grabarczyk, Piotr; Meyer, Corinna; Kaufmann, Maren; Battmer, Karin; Schmidt, Christian A; Drexler, Hans G

2009-01-01

Homeodomain proteins control fundamental cellular processes in development and in cancer if deregulated. Three members of the NK-like subfamily of homeobox genes (NKLs), TLX1, TLX3 and NKX2-5, are implicated in T-cell acute lymphoblastic leukemia (T-ALL). They are activated by particular chromosomal aberrations. However, their precise function in leukemogenesis is still unclear. Here we screened further NKLs in 24 T-ALL cell lines and identified the common expression of MSX2. The subsequent aim of this study was to analyze the role of MSX2 in T-cell differentiation which may be disturbed by oncogenic NKLs. Specific gene activity was examined by quantitative real-time PCR, and globally by expression profiling. Proteins were analyzed by western blot, immuno-cytology and immuno-precipitation. For overexpression studies cell lines were transduced by lentiviruses. Quantification of MSX2 mRNA in primary hematopoietic cells demonstrated higher levels in CD34+ stem cells as compared to peripheral blood cells and mature CD3+ T-cells. Furthermore, analysis of MSX2 expression levels in T-cell lines after treatment with core thymic factors confirmed their involvement in regulation. These results indicated that MSX2 represents an hematopoietic NKL family member which is downregulated during T-cell development and may functionally substituted by oncogenic NKLs. For functional analysis JURKAT cells were lentivirally transduced, overexpressing either MSX2 or oncogenic TLX1 and NKX2-5, respectively. These cells displayed transcriptional activation of NOTCH3-signaling, including NOTCH3 and HEY1 as analyzed by gene expression profiling and quantitative RT-PCR, and consistently attenuated sensitivity to gamma-secretase inhibitor as analyzed by MTT-assays. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with NOTCH-pathway repressors, SPEN/MINT/SHARP and TLE1/GRG1, representing a potential mechanism for (de)regulation. Finally, elevated expression of NOTCH3

Effect of display location on control-display stereotype strength for translational and rotational controls with linear displays.

Science.gov (United States)

Chan, Alan H S; Hoffmann, Errol R

2015-01-01

Experiments were designed to investigate the effects of control type and display location, relative to the operator, on the strength of control/display stereotypes. The Worringham and Beringer Visual Field principle and an extension of this principle for rotary controls (Hoffmann E.R., and Chan A.H.S. 2013). "The Worringham and Beringer 'Visual Field' Principle for Rotary Controls. Ergonomics." 56 (10): 1620-1624) indicated that, for a number of different control types (rotary and lever) on different planes, there should be no significant effect of the display location relative to the seated operator. Past data were surveyed and stereotype strengths listed. Experiments filled gaps where data are not available. Six different control types and seven display locations were used, as in the Frame of Reference Transformation Tool (FORT) model of Wickens et al. (Wickens, C.D., Keller, J.W., and Small, R.L. (2010). "Left. No, Right! Development of the Frame of Reference Transformation Tool (FORT)." Proceedings of the Human Factors and Ergonomics Society 54th Annual Meeting September 2010, 54: 1022-1026). Control/display arrangements with high stereotype strengths were evaluated yielding data for designers of complex control/display arrangements where the control and display are in different planes and for where the operator is moving. It was found possible to predict display/control arrangements with high stereotype strength, based on past data. Practitioner Summary: Controls and displays in complex arrangements need to have high compatibility. These experiments provide arrangements for six different controls (rotary and translational) and seven different display locations relative to the operator.

Heat Shock Proteins and Mitogen-activated Protein Kinases in Steatotic Livers Undergoing Ischemia-Reperfusion: Some Answers

Science.gov (United States)

Massip-Salcedo, Marta; Casillas-Ramirez, Araní; Franco-Gou, Rosah; Bartrons, Ramón; Ben Mosbah, Ismail; Serafin, Anna; Roselló-Catafau, Joan; Peralta, Carmen

2006-01-01

Ischemic preconditioning protects steatotic livers against ischemia-reperfusion (I/R) injury, but just how this is achieved is poorly understood. Here, I/R or preconditioning plus I/R was induced in steatotic and nonsteatotic livers followed by investigating the effect of pharmacological treatments that modulate heat shock proteins (HSPs) and mitogen-activated protein kinases (MAPKs). MAPKs, HSPs, protein kinase C, and transaminase levels were measured after reperfusion. We report that preconditioning increased HSP72 and heme-oxygenase-1 (HO-1) at 6 and 24 hours of reperfusion, respectively. Unlike nonsteatotic livers, steatotic livers benefited from HSP72 activators (geranylgeranylacetone) throughout reperfusion. This protection seemed attributable to HO-1 induction. In steatotic livers, preconditioning and geranylgeranylacetone treatment (which are responsible for HO-1 induction) increased protein kinase C activity. HO-1 activators (cobalt(III) protoporphyrin IX) protected both liver types. Preconditioning reduced p38 MAPK and c-Jun N-terminal kinase (JNK), resulting in HSP72 induction though HO-1 remained unmodified. Like HSP72, both p38 and JNK appeared not to be crucial in preconditioning, and inhibitors of p38 (SB203580) and JNK (SP600125) were less effective against hepatic injury than HO-1 activators. These results provide new data regarding the mechanisms of preconditioning and may pave the way to the development of new pharmacological strategies in liver surgery. PMID:16651615

FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans

Directory of Open Access Journals (Sweden)

Ineke Dhondt

2016-09-01

Full Text Available Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditis elegans and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory.

Proteins labelling with 125I and experimental determination of their specific activity

International Nuclear Information System (INIS)

Caro, R.A.; Ciscato, V.A.; Giacomini, S.M.V. de; Quiroga, S.; Radicella, R.

1975-11-01

A standardization of the labelling technique of proteins with 125 I and the control of the obtained products, principally their specific activities was performed, in order to utilize them correctly in radioimmunoassays. The quantities of chloramine-T and sodium metabisulphite were lowered, with regard to the original method, to 3.6 and 9.6 μg respectively. Under these conditions, optimal yields and radioiodinated proteins with good immunological activities were obtained. It was found that the specific activity calculated, as usual, from the yield obtained by electrophoresis, is higher than the real value. For these reasons the yields and the corresponding specific activities were determined from ascending chromatographies performed with 70 per cent methanol as solvent, during two hours in darkness. The radioimmunoassay displacement curves obtained with proteins labelled which the proposed method and the specific activities of which were calculated from their radiochromatographic patterns, were reproducible and gave a percentage of bound radioiodinated protein in the absence of cold protein of 50 +- 4. (author) [es

Potential anti-cholinesterase and β-site amyloid precursor protein cleaving enzyme 1 inhibitory activities of cornuside and gallotannins from Cornus officinalis fruits.

Science.gov (United States)

Bhakta, Himanshu Kumar; Park, Chan Hum; Yokozawa, Takako; Tanaka, Takashi; Jung, Hyun Ah; Choi, Jae Sue

2017-07-01

Cholinesterase (ChE) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors are promising agents for the treatment of Alzheimer's disease (AD). In the present study, we examined the inhibitory activity of seven compounds isolated from the fruits of Cornus officinalis, cornuside, polymeric proanthocyanidins, 1,2,3-tri-O-galloyl-β-D-glucose, 1,2,3,6-tetra-O-galloyl-β-D-glucose, tellimagrandin I, tellimagrandin II, and isoterchebin, against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE1. All of the compounds displayed concentration-dependent in vitro inhibitory activity toward the ChEs and BACE1. Among them, tellimagrandin II exhibited the best inhibitory activity toward ChEs, whereas the best BACE1 inhibitor was 1,2,3,6-tetra-O-galloyl-β-D-glucose. Isoterchebin and polymeric proanthocyanidins were also significant ChE inhibitors. The kinetic and docking studies demonstrated that all compounds interacted with both the catalytic active sites and the peripheral anionic sites of the ChEs and BACE1. Tellimagrandin II, isoterchebin, and the polymeric proanthocyanidins exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. In conclusion, we identified significant ChE and BACE1 inhibitors from Corni Fructus that could have value as new multi-targeted compounds for anti-AD agents.

Displays in scintigraphy

International Nuclear Information System (INIS)

Todd-Pokropek, A.E.; Pizer, S.M.

1977-01-01

Displays have several functions: to transmit images, to permit interaction, to quantitate features and to provide records. The main characteristics of displays used for image transmission are their resolution, dynamic range, signal-to-noise ratio and uniformity. Considerations of visual acuity suggest that the display element size should be much less than the data element size, and in current practice at least 256X256 for a gamma camera image. The dynamic range for image transmission should be such that at least 64 levels of grey (or equivalent) are displayed. Scanner displays are also considered, and in particular, the requirements of a whole-body camera are examined. A number of display systems and devices are presented including a 'new' heated object colour display system. Interaction with displays is considered, including background subtraction, contrast enhancement, position indication and region-of-interest generation. Such systems lead to methods of quantitation, which imply knowledge of the expected distributions. Methods for intercomparing displays are considered. Polaroid displays, which have for so long dominated the field, are in the process of being replaced by stored image displays, now that large cheap memories exist which give an equivalent image quality. The impact of this in nuclear medicine is yet to be seen, but a major effect will be to enable true quantitation. (author)

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

Science.gov (United States)

Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

2018-06-01

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

Directory of Open Access Journals (Sweden)

Johanna M Mattsson

Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

Comparison between medium-chain acyl-CoA dehydrogenase mutant proteins overexpressed in bacterial and mammalian cells

DEFF Research Database (Denmark)

Jensen, T G; Bross, P; Andresen, B S

1995-01-01

." Upon expression in E. coli, these mutant proteins produce activity levels in the range of the wild-type enzyme only if the chaperonins GroESL are co-overproduced. When overexpressed in COS cells, the pure folding mutants display enzyme activities comparable to the wild-type enzyme. The results suggest...

Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.

Science.gov (United States)

Shrestha, Madan K; Peri, Irena; Smirnoff, Patricia; Birk, Yehudith; Golan-Goldhirsh, Avi

2002-09-25

The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.

Protein Hydrolysis from Catfish Prepared by Papain Enzyme and Antioxidant Activity of Hydrolyzate

Directory of Open Access Journals (Sweden)

Ace Baehaki

2015-12-01

Full Text Available The objective of this research was to make a protein hydrolysates from catfish (Pangasius pangasius enzymatically using papain enzyme and analyzed the antioxidant activity of protein hydrolysates produced. The research used the method completely randomized design with two replications the treatment were the difference concentration of the papain enzyme (0%, 1%, 2%, 3%, 4%, 5%, and 6%. The parameters of research were antioxidative activity using DPPH (2,2-difenil-1–pikrilhidrazil, protein content, and molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. The results showed that catfish protein hydrolysates prepared by papain enzyme has antioxidative activity. The highest degree of hydrolysis was 71.98% at enzyme concentration of 6%. Based on the DPPH scavenging method catfish protein hydrolysates has the antioxidative activity with the value 37.85-67.62%. The protein content of catfish protein hydrolysates were 20.86-54.47 mg/ml. The molecular weight of catfish protein hydrolyzates were 11.90-65.20 kDa.

Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro.

Science.gov (United States)

Chellaiah, Meenakshi A; Schaller, Michael D

2009-08-01

PTP-PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP-PEST on dephosphorylation of c-Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over-expression of PTP-PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP-PEST over-expression as compared with control osteoclasts. Cells expressing constitutively active-Src (527YDeltaF) simulate the effects mediated by PTP-PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src-mediated phosphorylation of cortactin and WASP as well as the formation of WASP.cortactin.Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP-PEST/Src, in addition to its effect on the inhibition of the post-translational modification of small GTP-binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates.

Hewlett-Packard's Approaches to Full Color Reflective Displays

Science.gov (United States)

Gibson, Gary

2012-02-01

Reflective displays are desirable in applications requiring low power or daylight readability. However, commercial reflective displays are currently either monochrome or capable of only dim color gamuts. Low cost, high-quality color technology would be rapidly adopted in existing reflective display markets and would enable new solutions in areas such as retail pricing and outdoor digital signage. Technical breakthroughs are required to enable bright color gamuts at reasonable cost. Pixel architectures that rely on pure reflection from a single layer of side-by-side primary-color sub-pixels use only a fraction of the display area to reflect incident light of a given color and are, therefore, unacceptably dark. Reflective devices employing stacked color primaries offer the possibility of a somewhat brighter color gamut but can be more complex to manufacture. In this talk, we describe HP's successes in addressing these fundamental challenges and creating both high performance stacked-primary reflective color displays as well as inexpensive single layer prototypes that provide good color. Our stacked displays utilize a combination of careful light management techniques, proprietary high-contrast electro-optic shutters, and highly transparent active-matrix TFT arrays based on transparent metal oxides. They also offer the possibility of relatively low cost manufacturing through roll-to-roll processing on plastic webs. To create even lower cost color displays with acceptable brightness, we have developed means for utilizing photoluminescence to make more efficient use of ambient light in a single layer device. Existing reflective displays create a desired color by reflecting a portion of the incident spectrum while absorbing undesired wavelengths. We have developed methods for converting the otherwise-wasted absorbed light to desired wavelengths via tailored photoluminescent composites. Here we describe a single active layer prototype display that utilizes these materials

Tumour exosomes display different differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness

DEFF Research Database (Denmark)

Whitehead, Bradley Joseph; Wu, Linping; Hvam, Michael Lykke

2015-01-01

(QNM AFM) to determine size and nanomechanical properties. Effect of HCV-29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC) monolayer integrity was determined by transendothelial electrical resistance (TEER) measurements and transport was determined by flow cytometry. Complement......). Malignant cell-derived exosomes activated complement through calcium-sensitive pathways in a concentration-dependent manner. Conclusions : Malignant (metastatic and non-metastatic) cell line exosomes display a markedly reduced stiffness and adhesion but an increased complement activation compared to non...

Probing intracellular motor protein activity using an inducible cargo trafficking assay

NARCIS (Netherlands)

L.C. Kapitein (Lukas); M.A. Schlager (Max); W.A. van der Zwan (Wouter); P. Wulf (Phebe); N. Keijzer (Nanda); C.C. Hoogenraad (Casper)

2010-01-01

textabstractAlthough purified cytoskeletal motor proteins have been studied extensively with the use of in vitro approaches, a generic approach to selectively probe actin and microtubule-based motor protein activity inside living cells is lacking. To examine specific motor activity inside living

Identification and characterization of Ixodes scapularis antigens that elicit tick immunity using yeast surface display.

Directory of Open Access Journals (Sweden)

Tim J Schuijt

2011-01-01

Full Text Available Repeated exposure of rabbits and other animals to ticks results in acquired resistance or immunity to subsequent tick bites and is partially elicited by antibodies directed against tick antigens. In this study we demonstrate the utility of a yeast surface display approach to identify tick salivary antigens that react with tick-immune serum. We constructed an Ixodes scapularis nymphal salivary gland yeast surface display library and screened the library with nymph-immune rabbit sera and identified five salivary antigens. Four of these proteins, designated P8, P19, P23 and P32, had a predicted signal sequence. We generated recombinant (r P8, P19 and P23 in a Drosophila expression system for functional and immunization studies. rP8 showed anti-complement activity and rP23 demonstrated anti-coagulant activity. Ixodes scapularis feeding was significantly impaired when nymphs were fed on rabbits immunized with a cocktail of rP8, rP19 and rP23, a hall mark of tick-immunity. These studies also suggest that these antigens may serve as potential vaccine candidates to thwart tick feeding.

Probing ADAMTS13 Substrate Specificity using Phage Display

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Desch, Karl C.; Kretz, Colin; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

2015-01-01

Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73. PMID:25849793

Protein implicated in nonsyndromic mental retardation regulates protein kinase A (PKA) activity

KAUST Repository

Altawashi, Azza; Jung, Sung Yun; Liu, Dou; Su, Bing; Qin, Jun

2012-01-01

capacitytoformdendritesandsynapsesinculture. Atthebiochemical level,CC2D1Atransduces signals to the cyclic adenosine 3?,5?-monophosphate (cAMP)-protein kinase A (PKA) pathway during neuronal cell differentiation. PKA activity is compromised, and the translocation of its catalytic subunit

Reassessing the Potential Activities of Plant CGI-58 Protein

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Khatib, Abdallah; Arhab, Yani; Bentebibel, Assia; Abousalham, Abdelkarim; Noiriel, Alexandre

2016-01-01

Comparative Gene Identification-58 (CGI-58) is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL), the initial enzyme responsible for the triacylglycerol (TAG) catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed. PMID:26745266

Reassessing the Potential Activities of Plant CGI-58 Protein.

Directory of Open Access Journals (Sweden)

Abdallah Khatib

Full Text Available Comparative Gene Identification-58 (CGI-58 is a widespread protein found in animals and plants. This protein has been shown to participate in lipolysis in mice and humans by activating Adipose triglyceride lipase (ATGL, the initial enzyme responsible for the triacylglycerol (TAG catabolism cascade. Human mutation of CGI-58 is the cause of Chanarin-Dorfman syndrome, an orphan disease characterized by a systemic accumulation of TAG which engenders tissue disorders. The CGI-58 protein has also been shown to participate in neutral lipid metabolism in plants and, in this case, a mutation again provokes TAG accumulation. Although its roles as an ATGL coactivator and in lipid metabolism are quite clear, the catalytic activity of CGI-58 is still in question. The acyltransferase activities of CGI-58 have been speculated about, reported or even dismissed and experimental evidence that CGI-58 expressed in E. coli possesses an unambiguous catalytic activity is still lacking. To address this problem, we developed a new set of plasmids and site-directed mutants to elucidate the in vivo effects of CGI-58 expression on lipid metabolism in E. coli. By analyzing the lipid composition in selected E. coli strains expressing CGI-58 proteins, and by reinvestigating enzymatic tests with adequate controls, we show here that recombinant plant CGI-58 has none of the proposed activities previously described. Recombinant plant and mouse CGI-58 both lack acyltransferase activity towards either lysophosphatidylglycerol or lysophosphatidic acid to form phosphatidylglycerol or phosphatidic acid and recombinant plant CGI-58 does not catalyze TAG or phospholipid hydrolysis. However, expression of recombinant plant CGI-58, but not mouse CGI-58, led to a decrease in phosphatidylglycerol in all strains of E. coli tested, and a mutation of the putative catalytic residues restored a wild-type phenotype. The potential activities of plant CGI-58 are subsequently discussed.

Regulatory crosstalk by protein kinases on CFTR trafficking and activity

Science.gov (United States)

Farinha, Carlos Miguel; Swiatecka-Urban, Agnieszka; Brautigan, David; Jordan, Peter

2016-01-01

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a member of the ATP binding cassette (ABC) transporter superfamily that functions as a cAMP-activated chloride ion channel in fluid-transporting epithelia. There is abundant evidence that CFTR activity (i.e. channel opening and closing) is regulated by protein kinases and phosphatases via phosphorylation and dephosphorylation. Here, we review recent evidence for the role of protein kinases in regulation of CFTR delivery to and retention in the plasma membrane. We review this information in a broader context of regulation of other transporters by protein kinases because the overall functional output of transporters involves the integrated control of both their number at the plasma membrane and their specific activity. While many details of the regulation of intracellular distribution of CFTR and other transporters remain to be elucidated, we hope that this review will motivate research providing new insights into how protein kinases control membrane transport to impact health and disease.

Advances in phage display technology for drug discovery.

Science.gov (United States)

Omidfar, Kobra; Daneshpour, Maryam

2015-06-01

Over the past decade, several library-based methods have been developed to discover ligands with strong binding affinities for their targets. These methods mimic the natural evolution for screening and identifying ligand-target interactions with specific functional properties. Phage display technology is a well-established method that has been applied to many technological challenges including novel drug discovery. This review describes the recent advances in the use of phage display technology for discovering novel bioactive compounds. Furthermore, it discusses the application of this technology to produce proteins and peptides as well as minimize the use of antibodies, such as antigen-binding fragment, single-chain fragment variable or single-domain antibody fragments like VHHs. Advances in screening, manufacturing and humanization technologies demonstrate that phage display derived products can play a significant role in the diagnosis and treatment of disease. The effects of this technology are inevitable in the development pipeline for bringing therapeutics into the market, and this number is expected to rise significantly in the future as new advances continue to take place in display methods. Furthermore, a widespread application of this methodology is predicted in different medical technological areas, including biosensing, monitoring, molecular imaging, gene therapy, vaccine development and nanotechnology.

Wolfram Syndrome protein, Miner1, regulates sulphydryl redox status, the unfolded protein response, and Ca2+ homeostasis.

Science.gov (United States)

Wiley, Sandra E; Andreyev, Alexander Y; Divakaruni, Ajit S; Karisch, Robert; Perkins, Guy; Wall, Estelle A; van der Geer, Peter; Chen, Yi-Fan; Tsai, Ting-Fen; Simon, Melvin I; Neel, Benjamin G; Dixon, Jack E; Murphy, Anne N

2013-06-01

Miner1 is a redox-active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1(-/-) mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca(2+) stores, a dramatic increase in mitochondrial Ca(2+) load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD(+)/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti-oxidant N-acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Selective radiolabeling of cell surface proteins to a high specific activity

International Nuclear Information System (INIS)

Thompson, J.A.; Lau, A.L.; Cunningham, D.D.

1987-01-01

A procedure was developed for selective radiolabeling of membrane proteins on cells to higher specific activities than possible with available techniques. Cell surface amino groups were derivatized with 125 I-(hydroxyphenyl)propionyl groups via 125 I-sulfosuccinimidyl (hydroxyphenyl)propionate ( 125 II-sulfo-SHPP). This reagent preferentially labeled membrane proteins exposed at the cell surface of erythrocytes as assessed by the degree of radiolabel incorporation into erythrocyte ghost proteins and hemoglobin. Comparison with the lactoperoxidase-[ 125 I]iodide labeling technique revealed that 125 I-sulfo-SHPP labeled cell surface proteins to a much higher specific activity and hemoglobin to a much lower specific activity. Additionally, this reagent was used for selective radiolabeling of membrane proteins on the cytoplasmic face of the plasma membrane by blocking exofacial amino groups with uniodinated sulfo-SHPP, lysing the cells, and then incubating them with 125 I-sulfo-SHPP. Exclusive labeling of either side of the plasma membrane was demonstrated by the labeling of some marker proteins with well-defined spacial orientations on erythroctyes. Transmembrane proteins such as the epidermal growth factor receptor on cultured cells could also be labeled differentially from either side of the plasma membrane

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.

Monocular display unit for 3D display with correct depth perception

Science.gov (United States)

Sakamoto, Kunio; Hosomi, Takashi

2009-11-01

A study of virtual-reality system has been popular and its technology has been applied to medical engineering, educational engineering, a CAD/CAM system and so on. The 3D imaging display system has two types in the presentation method; one is a 3-D display system using a special glasses and the other is the monitor system requiring no special glasses. A liquid crystal display (LCD) recently comes into common use. It is possible for this display unit to provide the same size of displaying area as the image screen on the panel. A display system requiring no special glasses is useful for a 3D TV monitor, but this system has demerit such that the size of a monitor restricts the visual field for displaying images. Thus the conventional display can show only one screen, but it is impossible to enlarge the size of a screen, for example twice. To enlarge the display area, the authors have developed an enlarging method of display area using a mirror. Our extension method enables the observers to show the virtual image plane and to enlarge a screen area twice. In the developed display unit, we made use of an image separating technique using polarized glasses, a parallax barrier or a lenticular lens screen for 3D imaging. The mirror can generate the virtual image plane and it enlarges a screen area twice. Meanwhile the 3D display system using special glasses can also display virtual images over a wide area. In this paper, we present a monocular 3D vision system with accommodation mechanism, which is useful function for perceiving depth.

Activity of cAMP-dependent protein kinases and cAMP-binding proteins of rat kidney cytosol during dehydration

International Nuclear Information System (INIS)

Zelenina, M.N.; Solenov, E.I.; Ivanova, L.N.

1985-01-01

The activity of cAMP-dependent protein kinases, the binding of cAMP, and the spectrum of cAMP-binding proteins in the cytosol of the renal papilla was studied in intact rats and in rats after 24 h on a water-deprived diet. It was found that the activation of protein kinases by 10 -6 M cAMP is significantly higher in the experimental animals than in the intact animals. In chromatography on DEAE-cellulose, the positions of the peaks of specific reception of cAMP corresponded to the peaks of the regulatory subunits of cAMP-dependent protein kinases of types I and II. In this case, in intact animals more than 80% of the binding activity was detected in peaks II, whereas in rats subjected to water deprivation, more than 60% of the binding was observed in peak I. The general regulatory activity of the cytosol was unchanged in the experimental animals in comparison with intact animals. It is suggested that during dehydration there is an induction of the synthesis of the regulatory subunit of type I cAMP-dependent protein kinase in the renal papilla

Activated protein C attenuates acute ischaemia reperfusion injury in skeletal muscle.

LENUS (Irish Health Repository)

Dillon, J P

2012-02-03

Activated protein C (APC) is an endogenous anti-coagulant with anti-inflammatory properties. The purpose of the present study was to evaluate the effects of activated protein C in the setting of skeletal muscle ischaemia reperfusion injury (IRI). IRI was induced in rats by applying rubber bands above the levels of the greater trochanters bilaterally for a period of 2h followed by 12h reperfusion. Treatment groups received either equal volumes of normal saline or activated protein C prior to tourniquet release. Following 12h reperfusion, muscle function was assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Activated protein C significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet to dry ratio and electrical properties of skeletal muscle. Further in vitro work was carried out on neutrophils isolated from healthy volunteers to determine the direct effect of APC on neutrophil function. The effects of APC on TNF-alpha stimulated neutrophils were examined by measuring CD18 expression as well as reactive oxygen species generation. The in vitro work demonstrated a reduction in CD18 expression and reactive oxygen species generation. We conclude that activated protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury and that this is in part mediated by a direct inhibitory effect on neutrophil activation.

An oral vaccine against candidiasis generated by a yeast molecular display system.

Science.gov (United States)

Shibasaki, Seiji; Aoki, Wataru; Nomura, Takashi; Miyoshi, Ayuko; Tafuku, Senji; Sewaki, Tomomitsu; Ueda, Mitsuyoshi

2013-12-01

Enolase 1 (Eno1p) of Candida albicans is an immunodominant antigen. However, conventional technologies for preparing an injectable vaccine require purification of the antigenic protein and preparation of an adjuvant. To develop a novel type of oral vaccine against candidiasis, we generated Saccharomyces cerevisiae cells that display the Eno1p antigen on their surfaces. Oral delivery of the engineered S. cerevisiae cells prolonged survival rate of mice that were subsequently challenged with C. albicans. Given that a vaccine produced using molecular display technology avoids the need for protein purification, this oral vaccine offers a promising alternative to the use of conventional and injectable vaccines for preventing a range of infectious diseases. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

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

Secreted Immunomodulatory Proteins of Staphylococcus aureus Activate Platelets and Induce Platelet Aggregation.

Science.gov (United States)

Binsker, Ulrike; Palankar, Raghavendra; Wesche, Jan; Kohler, Thomas P; Prucha, Josephine; Burchhardt, Gerhard; Rohde, Manfred; Schmidt, Frank; Bröker, Barbara M; Mamat, Uwe; Pané-Farré, Jan; Graf, Anica; Ebner, Patrick; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-01

Staphylococcus aureus can cause bloodstream infections associated with infective endocarditis (IE) and disseminated intravascular coagulopathy (DIC). Both complications involve platelets. In view of an increasing number of antibiotic-resistant strains, new approaches to control systemic S. aureus infection are gaining importance. Using a repertoire of 52 recombinant S. aureus proteins in flow cytometry-based platelet activation and aggregation assays, we identified, in addition to the extracellular adherence protein Eap, three secreted staphylococcal proteins as novel platelet activating proteins. Eap and the chemotaxis inhibitory protein of S. aureus (CHIPS), the formyl peptide receptor-like 1 inhibitory protein (FLIPr) and the major autolysin Atl induced P-selectin expression in washed platelets and platelet-rich plasma. Similarly, AtlA, CHIPS and Eap induced platelet aggregation in whole blood. Fluorescence microscopy illustrated that P-selectin expression is associated with calcium mobilization and re-organization of the platelet actin cytoskeleton. Characterization of the functionally active domains of the major autolysin AtlA and Eap indicates that the amidase domain of Atl and the tandem repeats 3 and 4 of Eap are crucial for platelet activation. These results provide new insights in S. aureus protein interactions with platelets and identify secreted proteins as potential treatment targets in case of antibiotic-resistant S. aureus infection. Schattauer GmbH Stuttgart.

Degradation of brown adipocyte purine nucleotides regulates uncoupling protein 1 activity

Directory of Open Access Journals (Sweden)

Tobias Fromme

2018-02-01

Full Text Available Objective: Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation. Methods: With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist. Results: Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered uncoupling protein 1 mediated uncoupled respiration. Conclusion: Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to uncoupling protein 1 activation. Keywords: Purine nucleotides, Uncoupling protein 1, Brown adipose tissue, Non-shivering thermogenesis, HILIC-MS/MS, Guanosine monophosphate reductase

Antioxidant Activity of Coconut (Cocos nucifera L.) Protein Fractions.

Science.gov (United States)

Li, Yan; Zheng, Yajun; Zhang, Yufeng; Xu, Jianguo; Gao, Gang

2018-03-20

Coconut cake is an abundant and good potential edible protein source. However, until now it has not been extensively used in the food industry. To promote its usage, the characterization, nutrition value and antioxidant activity of coconut cake protein fractions (albumin, globulin, prolamine, glutelin-1 and glutelin-2) were studied. Results revealed that all the albumin, globulin, glutelin-1 and glutelin-2 fractions showed a high nutrition value. The prolamine, glutelin-1 and glutelin-2 all exhibited good radical scavenging activity and reducing power, and the globulin and prolamine showed high ion chelating ability (89.14-80.38%). Moreover, all the fractions except glutelin-2 could effectively protect DNA against oxidative damage. Several peptides containing five to eight amino acids with antioxidant activity were also identified by LC-MS/MS from the globulin and glutelin-2 fractions. The results demonstrated that the coconut cake protein fractions have potential usages in functional foods.

Syncytin-1, an endogenous retroviral protein, triggers the activation of CRP via TLR3 signal cascade in glial cells.

Science.gov (United States)

Wang, Xiuling; Liu, Zhongchun; Wang, Peigang; Li, Shan; Zeng, Jie; Tu, Xiaoning; Yan, Qiujin; Xiao, Zheman; Pan, Mengxian; Zhu, Fan

2018-01-01

Schizophrenia is a devastating psychiatric disorder that impacts on social functioning and quality of life, and there is accumulating evidence that inflammation is a potential pathogenic mechanism of schizophrenia. However, the mechanism of inflammation possibly occurred in schizophrenia has not been well understood. The endogenous retroviral protein syncytin-1 and inflammatory marker CRP are both abnormally expressed in schizophrenia patients. CRP is one of the markers of bacterial infection generally. Less clear is whether virus or viral protein can trigger the activation of CRP. Here, we detected a robust increase of the levels of syncytin-1 and CRP in schizophrenia patients, and displayed a positive correlation and marked consistency between expressions of syncytin-1 and CRP in schizophrenia patients. Furthermore, overexpression of syncytin-1 significantly elevated the levels of CRP, TLR3, and IL-6 in both human microglia and astrocytes. TLR3 deficiency impaired the expressions of CRP and IL-6 induced by syncytin-1. Importantly, we observed a cellular co-localization and a direct interaction between syncytin-1 and TLR3. Additionally, knockdown of IL-6 inhibited the syncytin-1-induced CRP expression. Thus, the totality of these results showed that viral protein syncytin-1 could trigger the activation of CRP, which might explain the elevated CRP in sterile inflammation and exhibit a novel mechanism for regulation of inflammation by syncytin-1 in schizophrenia. Copyright © 2017 Elsevier Inc. All rights reserved.

L-Alanylglutamine inhibits signaling proteins that activate protein degradation, but does not affect proteins that activate protein synthesis after an acute resistance exercise.

Science.gov (United States)

Wang, Wanyi; Choi, Ran Hee; Solares, Geoffrey J; Tseng, Hung-Min; Ding, Zhenping; Kim, Kyoungrae; Ivy, John L

2015-07-01

Sustamine™ (SUS) is a dipeptide composed of alanine and glutamine (AlaGln). Glutamine has been suggested to increase muscle protein accretion; however, the underlying molecular mechanisms of glutamine on muscle protein metabolism following resistance exercise have not been fully addressed. In the present study, 2-month-old rats climbed a ladder 10 times with a weight equal to 75 % of their body mass attached at the tail. Rats were then orally administered one of four solutions: placebo (PLA-glycine = 0.52 g/kg), whey protein (WP = 0.4 g/kg), low dose of SUS (LSUS = 0.1 g/kg), or high dose of SUS (HSUS = 0.5 g/kg). An additional group of sedentary (SED) rats was intubated with glycine (0.52 g/kg) at the same time as the ladder-climbing rats. Blood samples were collected immediately after exercise and at either 20 or 40 min after recovery. The flexor hallucis longus (FHL), a muscle used for climbing, was excised at 20 or 40 min post exercise and analyzed for proteins regulating protein synthesis and degradation. All supplements elevated the phosphorylation of FOXO3A above SED at 20 min post exercise, but only the SUS supplements significantly reduced the phosphorylation of AMPK and NF-kB p65. SUS supplements had no effect on mTOR signaling, but WP supplementation yielded a greater phosphorylation of mTOR, p70S6k, and rpS6 compared with PLA at 20 min post exercise. However, by 40 min post exercise, phosphorylation of mTOR and rpS6 in PLA had risen to levels not different than WP. These results suggest that SUS blocks the activation of intracellular signals for MPB, whereas WP accelerates mRNA translation.

Liposomal packaging generates Wnt protein with in vivo biological activity.

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Nathan T Morrell

2008-08-01

Full Text Available Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.

Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF1

OpenAIRE

Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

2008-01-01

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of pu...

HTLV-2B Tax oncoprotein is modified by ubiquitination and sumoylation and displays intracellular localization similar to its homologue HTLV-1 Tax

International Nuclear Information System (INIS)

Turci, Marco; Lodewick, Julie; Righi, Paola; Polania, Angela; Romanelli, Maria Grazia; Bex, Francoise; Bertazzoni, Umberto

2009-01-01

HTLV-1 is more pathogenic than HTLV-2B. The difference is generally attributed to the properties of their individual transactivating Tax proteins. By using internal Flag-6His tagged Tax-1 and Tax-2B, which display transcriptional activities comparable to the untagged proteins and can be recognized by a single anti-Flag antibody, we demonstrate that Tax-2B is modified by ubiquitination and sumoylation. In addition, Tax2B is distributed in punctuate nuclear structures that include the RelA subunit of NF-κB, as has been previously demonstrated for Tax-1

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

KAUST Repository

Diaz Galicia, Miriam Escarlet

2018-05-01

Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

Activation of protein kinase C inhibits synthesis and release of decidual prolactin

International Nuclear Information System (INIS)

Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

1986-01-01

Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

Phage display of the serpin alpha-1 proteinase inhibitor randomized at consecutive residues in the reactive centre loop and biopanned with or without thrombin.

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Benjamin M Scott

Full Text Available In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2-P1 yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352-356 (P7-P3 was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7-P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1 as a serpin amenable to phage display and suggest the utility of this approach for the selection