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Sample records for formed synthetic peptide

  1. Synthetic Channel-forming Peptides and Ion Selectivity

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    @@ Introduction Peptides made up of alternating L- and D- amino acids can form β-helices as in gramicidin A or cyclic peptides that aggregate to form tubes[1]. In both cases the structures are hollow with all the side chains projecting outwards. Kennedy et al. [2] postulated that peptides having the (LLLD)n configuration could form helices with every fourth side chain projecting inward.It is a fact that synthetic N-formyl-( LeuSerLeuGly)6-OH, when added to a lipid bilayer, dimerizes, to form ion channels having conductances greater than that of gramicidin.

  2. Synthetic β-sheet forming peptide amphiphiles for treatment of fungal keratitis.

    Science.gov (United States)

    Wu, Hong; Ong, Zhan Yuin; Liu, Shaoqiong; Li, Yan; Wiradharma, Nikken; Yang, Yi Yan; Ying, Jackie Y

    2015-03-01

    Fungal keratitis is a leading cause of ocular morbidity. It is frequently misdiagnosed as bacterial keratitis, causing a delay in proper treatment. Furthermore, due to the lack of safe and effective anti-fungal agents for clinical use, treatment of fugal keratitis remains a challenge. In recent years, antimicrobial peptides (AMPs) have received considerable attention as potent and broad-spectrum antimicrobial agents with the potential to overcome antibiotics resistance. We previously reported the design of short synthetic β-sheet forming peptides (IKIK)2-NH2 and (IRIK)2-NH2 with excellent antimicrobial activities and selectivities against various clinically relevant microorganisms, including Gram-positive Staphylococcus epidermidis and Staphylococcus aureus, Gram-negative Escherichia coli and Pseudomonas aeruginosa, and yeast Candida albicans (C. albicans). In this study, we evaluated the application of the two most promising synthetic β-sheet forming peptide candidates for in vivo fungal keratitis treatment in comparison with the commercially available amphotericin B. It was found that topical solutions of the designed peptides are safe, and as effective as the clinically used amphotericin B. Compared to the costly and unstable amphotericin B, (IKIK)2-NH2 and (IRIK)2-NH2 are water-soluble, less expensive and stable. Thus, the synthetic β-sheet forming peptides are presented as promising candidates for the treatment of fungal keratitis.

  3. Synthetic molecular evolution of pore-forming peptides by iterative combinatorial library screening.

    Science.gov (United States)

    Krauson, Aram J; He, Jing; Wimley, Andrew W; Hoffmann, Andrew R; Wimley, William C

    2013-04-19

    We previously reported the de novo design of a combinatorial peptide library that was subjected to high-throughput screening to identify membrane-permeabilizing antimicrobial peptides that have β-sheet-like secondary structure. Those peptides do not form discrete pores in membranes but instead partition into membrane interfaces and cause transient permeabilization by membrane disruption, but only when present at high concentration. In this work, we used a consensus sequence from that initial screen as a template to design an iterative, second generation library. In the 24-26-residue, 16,200-member second generation library we varied six residues. Two diad repeat motifs of alternating polar and nonpolar amino acids were preserved to maintain a propensity for non-helical secondary structure. We used a new high-throughput assay to identify members that self-assemble into equilibrium pores in synthetic lipid bilayers. This screen was done at a very stringent peptide to lipid ratio of 1:1000 where most known membrane-permeabilizing peptides, including the template peptide, are not active. In a screen of 10,000 library members we identified 16 (~0.2%) that are equilibrium pore-formers at this high stringency. These rare and highly active peptides, which share a common sequence motif, are as potent as the most active pore-forming peptides known. Furthermore, they are not α-helical, which makes them unusual, as most of the highly potent pore-forming peptides are amphipathic α-helices. Here we demonstrate that this synthetic molecular evolution-based approach, taken together with the new high-throughput tools we have developed, enables the identification, refinement, and optimization of unique membrane active peptides.

  4. Effect of Diaminopropionic acid (Dap) on the Biophysical Properties of a Modified Synthetic Channel-Forming Peptide

    OpenAIRE

    Bukovnik, Urska; Sala-Rabanal, Monica; Francis, Simonne; Frazier, Shawnalea J.; Schultz, Bruce D.; Nichols, Colin G.; John M Tomich

    2013-01-01

    Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, ...

  5. Synthetic peptides for antibody production

    NARCIS (Netherlands)

    Zegers, N.D.

    1995-01-01

    Synthetic peptides are useful tools for the generation of antibodies. The use of antibodies as specific reagents in inununochemical assays is widely applied. In this chapter, the application of synthetic peptides for the generation of antibodies is described. The different steps that lead to the uni

  6. Channel protein engineering: Synthetic 22-mer peptide from the primary structure of the voltage-sensitive sodium channel forms ionic channels in lipid bilayers

    OpenAIRE

    1988-01-01

    A synthetic 22-mer peptide that mimics the sequence of a putative pore segment of the voltage-dependent sodium channel forms transmembrane ionic channels in lipid bilayers. Several features of the authentic sodium channel are exhibited by the synthetic peptide: (i) The single channel conductance of the most frequent event is 20 pS in 0.5 M NaCl. (ii) The single channel open and closed lifetimes are in the ms time range. (iii) The synthetic channel discriminates cations over anions but is nons...

  7. Effect of stereochemistry, chain length and sequence pattern on antimicrobial properties of short synthetic β-sheet forming peptide amphiphiles.

    Science.gov (United States)

    Ong, Zhan Yuin; Cheng, Junchi; Huang, Yuan; Xu, Kaijin; Ji, Zhongkang; Fan, Weimin; Yang, Yi Yan

    2014-01-01

    In the face of mounting global antibiotics resistance, the identification and development of membrane-active antimicrobial peptides (AMPs) as an alternative class of antimicrobial agent have gained significant attention. The physical perturbation and disruption of microbial membranes by the AMPs have been proposed to be an effective means to overcome conventional mechanisms of drug resistance. Recently, we have reported the design of a series of short synthetic β-sheet folding peptide amphiphiles comprised of recurring (X1Y1X2Y2)n-NH2 sequences where X: hydrophobic amino acids, Y: cationic amino acids and n: number of repeat units. In efforts to investigate the effects of key parameters including stereochemistry, chain length and sequence pattern on antimicrobial effects, systematic d-amino acid substitutions of the lead peptides (IRIK)2-NH2 (IK8-all L) and (IRVK)3-NH2 (IK12-all L) were performed. It was found that the corresponding D-enantiomers exhibited stronger antimicrobial activities with minimal or no change in hemolytic activities, hence translating very high selectivity indices of 407.0 and >9.8 for IK8-all D and IK12-all D respectively. IK8-all D was also demonstrated to be stable to degradation by broad spectrum proteases trypsin and proteinase K. The membrane disrupting bactericidal properties of IK8-all D effectively prevented drug resistance development and inhibited the growth of various clinically isolated MRSA, VRE, Acinetobacter baumanni, Pseudomonas aeruginosa, Cryptococcus. neoformans and Mycobacterium tuberculosis. Significant reduction in intracellular bacteria counts was also observed following treatment with IK8-all D in the Staphylococcus. aureus infected mouse macrophage cell line RAW264.7 (P < 0.01). These results suggest that the d-amino acids substituted β-sheet forming peptide IK8-all D with its enhanced antimicrobial activities and improved protease stability, is a promising therapeutic candidate with potential to combat

  8. Detection of alpha-helical coiled-coil dimer formation by spin-labeled synthetic peptides: a model parallel coiled-coil peptide and the antiparallel coiled coil formed by a replica of the ProP C-terminus.

    Science.gov (United States)

    Hillar, Alexander; Tripet, Brian; Zoetewey, David; Wood, Janet M; Hodges, Robert S; Boggs, Joan M

    2003-12-30

    Electron paramagnetic resonance spectroscopy was used to determine relative peptide orientation within homodimeric, alpha-helical coiled-coil structures. Introduction of cysteine (Cys) residues into peptides/proteins for spin labeling allows detection of their oligomerization from exchange broadening or dipolar interactions between residues within 25 A of each other. Two synthetic peptides containing Cys substitutions were used: a 35-residue model peptide and the 30-residue ProP peptide. The model peptide is known to form a stable, parallel homodimeric coiled coil, which is partially destabilized by Cys substitutions at heptad a and d positions (peptides C30a and C33d). The ProP peptide, a 30-residue synthetic peptide, corresponds to residues 468-497 of osmoregulatory transporter ProP from Escherichia coli. It forms a relatively unstable, homodimeric coiled coil that is predicted to be antiparallel in orientation. Cys was introduced in heptad g positions of the ProP peptide, near the N-terminus (K473C, creating peptide C473g) or closer to the center of the sequence (E480C, creating peptide C480g). In contrast to the destabilizing effect of Cys substitution at the core heptad a or d positions of model peptides C30a and C33d, circular dichroism spectroscopy showed that Cys substitutions at the heptad g positions of the ProP peptide had little or no effect on coiled-coil stability. Thermal denaturation analysis showed that spin labeling increased the stability of the coiled coil for all peptides. Strong exchange broadening was detected for both C30a and C33d, in agreement with a parallel structure. EPR spectra of C480g had a large hyperfine splitting of about 90 G, indicative of strong dipole-dipole interactions and a distance between spin-labeled residues of less than 9 A. Spin-spin interactions were much weaker for C473g. These results supported the hypothesis that the ProP peptide primarily formed an antiparallel coiled coil, since formation of a parallel dimer

  9. Characterization of Synthetic Peptides by Mass Spectrometry

    DEFF Research Database (Denmark)

    Prabhala, Bala K; Mirza, Osman; Højrup, Peter;

    2015-01-01

    Mass spectrometry (MS) is well suited for analysis of the identity and purity of synthetic peptides. The sequence of a synthetic peptide is most often known, so the analysis is mainly used to confirm the identity and purity of the peptide. Here, simple procedures are described for MALDI-TOF-MS an......Mass spectrometry (MS) is well suited for analysis of the identity and purity of synthetic peptides. The sequence of a synthetic peptide is most often known, so the analysis is mainly used to confirm the identity and purity of the peptide. Here, simple procedures are described for MALDI...

  10. Secondary structure of fluorescence labelled synthetic peptides

    CERN Document Server

    Martin, A S

    2000-01-01

    A series of eight synthetic oligopeptides has been prepared and their secondary structures investigated using various techniques. The project represents a continuation of an investigation into thermally induced changes in secondary structure. Following the previously reported results, the change in structure was initially thought to represent a change from an alpha-helix at low temperature to 3 sub 1 sub 0 -helix at high temperature. However, the results reported herein suggest the peptides retain an alpha-helical configuration at all temperatures studied, but that this helix can adopt at least two related forms. The difference in the structures relates to the nature of the H-bonds which may or may not involve an additional interaction from water molecules or side-chains. The peptides were encouraged to adopt a helical configuration by the inclusion of alpha- aminoisobutyric acid (Aib) residues. Also, modified forms of glutamic acid were included in the sequences. These had pendant donor (4-methoxy naphthalen...

  11. Synthetic Peptides as Receptors in Affinity Sensors: A Feasibility Study

    NARCIS (Netherlands)

    Heuvel, van den Dave J.; Kooyman, Rob P.H.; Drijfhout, Jan Wouter; Welling, Gjalt W.

    1993-01-01

    A relatively simple method for immobilizing synthetic peptides as a receptor onto a gold surface using the self-assembling monolayer (SAM) technique has been investigated. A synthetic peptide with an amino acid sequence similar to the 9-21 gD sequence of herpes simplex virus type 1 was modified with

  12. Cellular recognition of synthetic peptide amphiphiles in supported bioartificial membranes

    Science.gov (United States)

    Pakalns, Teika

    The goal of this study was to demonstrate that lipidated cell adhesion peptides could form well-ordered biomimetic surfaces that were capable of influencing cellular behavior in a controlled and specific manner. The first step taken was to covalently link synthetic dialkyl tails to the amino-termini of the collagen-derived peptide IV-H1 (amino acid sequence GVKGDKGNPGWPGAP) and the well-known tripeptide Arg-Gly-Asp (RGD) to produce amino-coupled peptide amphiphiles. Other spatial orientations of RGD were also generated by coupling tails to the carboxyl-terminus to give carboxyl-coupled RGD amphiphiles and to both the amino- and carboxyl-termini to give looped RGD amphiphiles. The next step taken was to let the peptide amphiphile self-assemble along with methyl ester-capped dialkyl tails into mixed films. It was found that all the peptide amphiphiles formed stable monolayers at the air-water interface in a Langmuir trough. IV-H1 amphiphiles and carboxyl-coupled and looped RGD amphiphiles deposited well as Langmuir-Blodgett mixed films on solid surfaces at all peptide concentrations, but aminocoupled RGD amphiphiles did not deposit well at high RGD concentrations. FT-IR studies of films containing RGD amphiphiles showed that amino-coupled RGD head groups formed the strongest lateral hydrogen bonds. The final step was to study cellular response to mixed films containing IV-H1 or RGD amphiphiles. The spreading of melanoma cells was influenced by both the molar concentration and spatial orientation of the amphiphilic peptides. Cells spread on IV-H1 and looped RGD films in a concentration-dependent manner, but spread indiscriminately on carboxyl-coupled RGD films and did not spread at all on well-deposited amino-coupled RGD films. The specificity of the cellular response to looped RGD amphiphiles was investigated. Control films of looped Arg-Gly-Glu (RGE) amphiphiles inhibited the adhesion and spreading of melanoma and endothelial cells, and antibody inhibition of the

  13. Synthetic peptides for the immunodiagnosis of hepatitis A virus infection.

    Science.gov (United States)

    Gauna, A; Losada, S; Lorenzo, M; Bermúdez, H; Toledo, M; Pérez, H; Chacón, E; Noya, O

    2015-12-01

    VP1, VP2 and VP3 molecules of hepatitis A virus are exposed capsid proteins that have shown to be antigenic and are used for diagnosis in recombinant-antigen commercial kits. In this study, we developed a sequence analysis in order to predict diagnostic peptide epitopes, followed by their spot synthesis on functionalized cellulose paper (Pepscan). This paper with synthetic peptides was tested against a sera pool of hepatitis A patients. Two peptide sequences, that have shown an antigenic recognition, were selected for greater scale synthesis on resin. A dimeric form of one of these peptides (IMT-1996), located in the C-Terminus region of protein VP1, was antigenic with a recognition frequency of 87-100% of anti-IgG antibodies and 100% of anti-IgM antibodies employing the immunological assays MABA and ELISA. We propose peptide IMT-1996, with less than twenty residues, as a cheaper alternative for prevalence studies and diagnosis of hepatitis A infection.

  14. Construction of synthetic dermis and skin based on a self-assembled peptide hydrogel scaffold.

    Science.gov (United States)

    Kao, Bunsho; Kadomatsu, Koichi; Hosaka, Yoshiaki

    2009-09-01

    Using biocompatible peptide hydrogel as a scaffold, we prepared three-dimensional synthetic skin that does not contain animal-derived materials or pathogens. The present study investigated preparation methods, proliferation, and functional expression of fibroblasts in the synthetic dermis and differentiation of keratinocytes in the epidermis. Synthetic dermis was prepared by mixing fibroblasts with peptide hydrogel, and synthetic skin was prepared by forming an epidermal layer using keratinocytes on the synthetic dermis. A fibroblast-rich foamy layer consisting of homogeneous peptide hydrogel subsequently formed in the synthetic dermis, with fibroblasts aggregating in clusters within the septum. The epidermis consisted of three to five keratinocyte layers. Immunohistochemical staining showed human type I collagen, indicating functional expression around fibroblasts in the synthetic dermis, keratinocyte differentiation in the epidermis, and expression of basement membrane proteins. The number of fibroblasts tended to increase until the second week and was maintained until the fourth week, but rapidly decreased in the fifth week. In the synthetic dermis medium, the human type I collagen concentration increased after the second week to the fifth week. These findings suggest that peptide hydrogel acts as a synthetic skin scaffold that offers a platform for the proliferation and functional expression of fibroblasts and keratinocytes.

  15. Synthetic peptide vaccines: palmitoylation of peptide antigens by an thioester bond increases immunogenicity

    NARCIS (Netherlands)

    Beekman, N.J.C.M.; Schaaper, W.M.M.; Tesser, G.I.; Dalsgaard, K.; Langeveld, J.P.M.; Boshuizen, R.S.; Meloen, R.H.

    1997-01-01

    Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts

  16. Biomathematical description of synthetic peptide libraries.

    Directory of Open Access Journals (Sweden)

    Timo Sieber

    Full Text Available Libraries of randomised peptides displayed on phages or viral particles are essential tools in a wide spectrum of applications. However, there is only limited understanding of a library's fundamental dynamics and the influences of encoding schemes and sizes on their quality. Numeric properties of libraries, such as the expected number of different peptides and the library's coverage, have long been in use as measures of a library's quality. Here, we present a graphical framework of these measures together with a library's relative efficiency to help to describe libraries in enough detail for researchers to plan new experiments in a more informed manner. In particular, these values allow us to answer-in a probabilistic fashion-the question of whether a specific library does indeed contain one of the "best" possible peptides. The framework is implemented in a web-interface based on two packages, discreteRV and peptider, to the statistical software environment R. We further provide a user-friendly web-interface called PeLiCa (Peptide Library Calculator, http://www.pelica.org, allowing scientists to plan and analyse their peptide libraries.

  17. Organometallic-Peptide Bioconjugates: Synthetic Strategies and Medicinal Applications.

    Science.gov (United States)

    Albada, Bauke; Metzler-Nolte, Nils

    2016-10-12

    Peptides are important biological molecular entities in biomedical research. They can be prepared in a large variety of shapes, with a host of chemical functions, and tailored for specific applications. Organometallic medicinal chemistry is a relatively young field that explores biomedical and bioanalytical applications of organometallic complexes, that is, metal compounds with at least one direct, covalent metal-carbon bond. The conjugation of peptides to such medicinally active organometallic moieties started only about 20 years ago, and it has been very beneficial for the development of bioorganometallic chemistry in general. Similarly, the biomedical properties of peptides have been altered by their conjugation to organometallic (OM) moieties. In this review, synthetic methods by which OM moieties can be conjugated to peptides via a carbon-metal bond are described, and selected medicinal applications of such conjugates are discussed. Inorganic coordination complexes between metal ions and peptides are excluded from this review. Also, the labeling of peptides with radiometals and applications of radiolabeled peptides will not be treated herein. First, modifications of the peptide backbone (either N- or C-terminally, or both) with organometallic moieties will be described, including the insertion of OM moieties as part of the peptide backbone. Then side-chain modifications will be reported, among them the most recent strategies for chemoselective arene metalation on peptides. Finally, approaches by which multiple metalation can be achieved are explored. In each section, selected examples of biological applications are highlighted.

  18. Protein- and peptide-modified synthetic polymeric biomaterials.

    Science.gov (United States)

    Krishna, Ohm D; Kiick, Kristi L

    2010-01-01

    This review presents an overview on bio-hybrid approaches of integrating the structural and functional features of proteins and peptides with synthetic polymers and the resulting unique properties in such hybrids, with a focus on bioresponsive/bioactive systems with biomaterials applications. The review is divided in two broad sections. First, we describe several examples of bio-hybrids produced by combining versatile synthetic polymers with proteins/enzymes and drugs that have resulted in (1) hybrid materials based on responsive polymers, (2) responsive hydrogels based on enzyme-catalyzed reactions, protein-protein interactions and protein-drug sensing, and (3) dynamic hydrogels based on conformational changes of a protein. Next, we present hybrids produced by combining synthetic polymers with peptides, classified based on the properties of the peptide domain: (1) peptides with different conformations, such as alpha-helical, coiled-coil, and beta-sheet; (2) peptides derived from structural protein domains such as silk, elastin, titin, and collagen; and (3) peptides with other biofunctional properties such as cell-binding domains and enzyme-recognized degradation domains. (c) 2010 Wiley Periodicals, Inc.

  19. Antimicrobial peptides: natural templates for synthetic membrane-active compounds.

    Science.gov (United States)

    Giuliani, A; Pirri, G; Bozzi, A; Di Giulio, A; Aschi, M; Rinaldi, A C

    2008-08-01

    The innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (AMPs) to resist microbial invasion. Crafted by evolution into an extremely diversified array of sequences and folds, AMPs do share a common amphiphilic 3-D arrangement. This feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. This minireview reports on current understanding of the modes of interaction of AMPs with biological and model membranes, especially focusing on recent insights into the folding and oligomerization requirements of peptides to bind and insert into lipid membranes and exert their antibiotic effects. Given the potential of AMPs to be developed into a new class of anti-infective agents, emphasis is placed on how the information on peptide-membrane interactions could direct the design and selection of improved biomimetic synthetic peptides with antibiotic properties.

  20. Immunomodulatory effects of endogenous and synthetic peptides activating opioid receptors.

    Science.gov (United States)

    Pomorska, Dorota K; Gach, Katarzyna; Janecka, Anna

    2014-01-01

    The main role of endogenous opioid peptides is the modulation of pain. Opioid peptides exert their analgesic activity by binding to the opioid receptors distributed widely in the central nervous system (CNS). However, opioid receptors are also found on tissues and organs outside the CNS, including the cells of the immune system, indicating that opioids are capable of exerting additional effects in periphery. Morphine, which is a gold standard in the treatment of chronic pain, is well-known for its immunosuppressive effects. Much less is known about the immunomodulatory effects exerted by endogenous (enkephalins, endorphins, dynorphins and endomorphins) and synthetic peptides activating opioid receptors. In this review we tried to summarize opioid peptide-mediated modulation of immune cell functions which can be stimulatory as well as inhibitory.

  1. Hybrid Block Copolymers Constituted by Peptides and Synthetic Polymers: An Overview of Synthetic Approaches, Supramolecular Behavior and Potential Applications

    Directory of Open Access Journals (Sweden)

    Jordi Puiggalí

    2013-02-01

    Full Text Available Hybrid block copolymers based on peptides and synthetic polymers, displaying different types of topologies, offer new possibilities to integrate the properties and functions of biomacromolecules and synthetic polymers in a single hybrid material. This review provides a current status report of the field concerning peptide-synthetic polymer hybrids. The first section is focused on the different synthetic approaches that have been used within the last three years for the preparation of peptide-polymer hybrids having different topologies. In the last two sections, the attractive properties, displayed in solution or in the solid state, together with the potential applications of this type of macromolecules or supramolecular systems are highlighted.

  2. Synthetic cationic peptide IDR-1018 modulates human macrophage differentiation.

    Directory of Open Access Journals (Sweden)

    Olga M Pena

    Full Text Available Macrophages play a critical role in the innate immune response. To respond in a rapid and efficient manner to challenges in the micro-environment, macrophages are able to differentiate towards classically (M1 or alternatively (M2 activated phenotypes. Synthetic, innate defense regulators (IDR peptides, designed based on natural host defence peptides, have enhanced immunomodulatory activities and reduced toxicity leading to protection in infection and inflammation models that is dependent on innate immune cells like monocytes/macrophages. Here we tested the effect of IDR-1018 on macrophage differentiation, a process essential to macrophage function and the immune response. Using transcriptional, protein and systems biology analysis, we observed that differentiation in the presence of IDR-1018 induced a unique signature of immune responses including the production of specific pro and anti-inflammatory mediators, expression of wound healing associated genes, and increased phagocytosis of apoptotic cells. Transcription factor IRF4 appeared to play an important role in promoting this IDR-1018-induced phenotype. The data suggests that IDR-1018 drives macrophage differentiation towards an intermediate M1-M2 state, enhancing anti-inflammatory functions while maintaining certain pro-inflammatory activities important to the resolution of infection. Synthetic peptides like IDR-1018, which act by modulating the immune system, could represent a powerful new class of therapeutics capable of treating the rising number of multidrug resistant infections as well as disorders associated with dysregulated immune responses.

  3. A synthetic peptide derivative that is a cholecystokinin receptor antagonist.

    Science.gov (United States)

    Lignon, M F; Galas, M C; Rodriguez, M; Laur, J; Aumelas, A; Martinez, J

    1987-05-25

    So far, there are no known peptidic effective receptor antagonists of both peripheral and central effects of cholecystokinin (CCK). Here, we describe a synthetic peptide derivative of CCK, t-butyloxycarbonyl-Tyr(SO3-)-Met-Gly-D-Trp-Nle-Asp 2-phenylethyl ester 1 (where Nle is norleucine), which is a potent CCK receptor antagonist. In rat and guinea pig dispersed pancreatic acini, this peptide derivative did not alter amylase secretion, but was able to antagonize the stimulation caused by cholecystokinin-related agonists. It caused a parallel rightward shift in the dose-response curve for the stimulation of amylase secretion with half-maximal inhibition of CCK-8-stimulated amylase release at a concentration of about 0.1 microM. Compound 1 was able to inhibit the binding of labeled CCK-9 (the C-terminal nonapeptide of CCK) to rat and guinea pig pancreatic acini (IC50 = 5 X 10(-8) M) as well as to guinea pig cerebral cortical membranes (IC50 = 5 X 10(-7) M). These results indicate that Compound 1 is a potent competitive CCK receptor antagonist.

  4. Boolean logic functions of a synthetic peptide network.

    Science.gov (United States)

    Ashkenasy, Gonen; Ghadiri, M Reza

    2004-09-15

    Living cells can process rapidly and simultaneously multiple extracellular input signals through the complex networks of evolutionary selected biomolecular interactions and chemical transformations. Recent approaches to molecular computation have increasingly sought to mimic or exploit various aspects of biology. A number of studies have adapted nucleic acids and proteins to the design of molecular logic gates and computational systems, while other works have affected computation in living cells via biochemical pathway engineering. Here we report that de novo designed synthetic peptide networks can also mimic some of the basic logic functions of the more complex biological networks. We show that segments of a small network whose graph structure is composed of five nodes and 15 directed edges can express OR, NOR, and NOTIF logic.

  5. Helical peptide-polyamine and -polyether conjugates as synthetic ionophores.

    Science.gov (United States)

    Benincasa, Monica; Francescon, Marco; Fregonese, Massimo; Gennaro, Renato; Pengo, Paolo; Rossi, Paola; Scrimin, Paolo; Tecilla, Paolo

    2015-12-01

    Two new synthetic ionophores in which the hydrophobic portion is represented by a short helical Aib-peptide (Aib=α-amino-isobutyric acid) and the hydrophilic one is a poly-amino (1a) or a polyether (1b) chain have been prepared. The two conjugates show a high ionophoric activity in phospholipid membranes being able to efficiently dissipate a pH gradient and, in the case of 1b, to transport Na(+) across the membrane. Bioactivity evaluation of the two conjugates shows that 1a has a moderate antimicrobial activity against a broad spectrum of microorganisms and it is able to permeabilize the inner and the outer membrane of Escherichia coli cells.

  6. Synthesis of biologically important neutral amylo-β peptide by using improved Fmoc solid-phase peptide synthetic strategy.

    Science.gov (United States)

    Selvam, R; Sudha, E; Rajkumar, P R; Subashchandran, K P

    2015-04-01

    The 10 amino acid sequence of the biologically important neutral amylo-β peptide has equally hydrophilic and hydrophobic properties, which reduces the coupling efficiency during its synthesis and reduces the final yield of the peptide, and is therefore classified as a "difficult peptide sequence." The method presented here minimizes the synthetic problems by the introduction of improved Fmoc chemistry and effective hydroxybenzotriazole (HoBt), diisopropylcarbodiimide (DIC)-coupling and activation strategies. In addition, we developed a PS-TPGD resin as a solid support for the synthesis of specific neutral peptides, which is still a challenge to peptide chemistry. The most essential biologically active neutral amylo-β peptide (KVKRIILARS) was successfully synthesized, and some synthetic modification was performed using the Fmoc solid-phase peptide synthesis (SPPS) method for purity and yield improvement. Graphical abstractᅟ.

  7. Monoclonal antibodies against a synthetic peptide from human immunodeficiency virus type 1 Nef protein

    DEFF Research Database (Denmark)

    Steinaa, L; Wulff, A M; Saermark, T

    1994-01-01

    Monoclonal antibodies against a synthetic peptide (aa 138-152) from HIV-1 Nef protein were produced and characterized. Three hybridoma lines producing monoclonal antibodies (MAbs) against the synthetic peptide were generated by fusion between P3-X63 Ag8.653 myeloma cells and BALB/c splenocytes fr...

  8. Pilot study on peptide purity—synthetic human C-peptide

    Science.gov (United States)

    Josephs, R. D.; Li, M.; Song, D.; Daireaux, A.; Choteau, T.; Stoppacher, N.; Westwood, S.; Wielgosz, R.; Xiao, P.; Liu, Y.; Gao, X.; Zhang, C.; Zhang, T.; Mi, W.; Quan, C.; Huang, T.; Li, H.; Melanson, J. E.; Ün, I.; Gören, A. C.; Quaglia, M.; Warren, J.

    2017-01-01

    Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a pilot study, CCQM-P55.2, was coordinated by the Bureau International des Poids et Mesures (BIPM) and the Chinese National Institute of Metrology (NIM). Four Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of human C-peptide (hCP) present as the main component in the comparison sample for CCQM-P55.2. The comparison samples were prepared from synthetic human hCP purchased from a commercial supplier and used as provided without further treatment or purification. hCP was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of short (up to 5 kDa), non-cross-linked synthetic peptides/proteins. It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used a quantitative nuclear magnetic resonance spectroscopy (qNMR) corrected for peptide impurities. Other participants provided results obtained by peptide impurity corrected amino acid analysis (PICAA) or elemental analysis (PICCHN). It was decided to assign reference values based on the KCRVs of CCQM-K115 for both the hCP mass fraction and the mass fraction of the peptide related impurities as indispensable contributor regardless of the use of PICAA, mass balance or any other approach to determine the hCP purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the hCP mass fraction. In particular it allows participants to demonstrate the efficacy of their implementation of peptide related impurity identification and quantification. The assessment of the mass fraction of peptide impurities is based on the assumption that only the most exhaustive and

  9. Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

    DEFF Research Database (Denmark)

    Rønn, L C; Doherty, P; Holm, A;

    2000-01-01

    The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently...... identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons...... from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1...

  10. Synthetic approaches to peptides containing the L-Gln-L-Val-D(S)-Dmt motif.

    Science.gov (United States)

    Suaifan, Ghadeer A R Y; Arafat, Tawfiq; Threadgill, Michael D

    2007-05-15

    The pseudoprolines S-Dmo (5,5-dimethyl-4-oxaproline) and R-Dmt (5,5-dimethyl-4-thiaproline) have been used to study the effects of forcing a fully cis conformation in peptides. Synthesis of peptides containing these (which have the same configuration as L-Pro) is straightforward. However, synthesis of peptides containing S-Dmt is difficult, owing to the rapid cyclisation of L-Aaa-S-Dmt amides and esters to form the corresponding diketopiperazines (DKP); thus the intermediacy of L-Aaa-S-Dmt amides and esters must be avoided in the synthetic sequence. Peptides containing the L-Gln-L-Val-D(S)-Dmt motif are particularly difficult, owing to the insolubility of coupling partners containing Gln. Introduction of Gln as N-Boc-pyroglutamate overcame the latter difficulty and the dipeptide active ester BocPygValOC(6)F(5) coupled in good yield with S-DmtOH. BocPygVal-S- DmtNH(CH(2))(2)C(6)H(4)NO(2) was converted quantitatively to BocGlnVal-S-DmtNH(CH(2))(2)C(6)H(4)NO(2) with ammonia, demonstrating the utility of this approach. Two peptide derivatives (CbzSerLysLeuGlnVal-S-DmtNH(CH(2))(2)C(6)H(4)NO(2) and CbzSerSerLysLeuGlnVal-S- DmtNH(CH(2))(2)C(6)H(4)NO(2)) were assembled, using these new methods of coupling a dipeptide acid active ester with S-DmtOH and introduction of Gln as Pyg, followed by conventional peptide couplings. The presence of the Val caused these peptides to be cleaved very slowly by prostate-specific antigen (PSA) at Leu Gln, rather than the expected Gln Val.

  11. Dual host-defence functions of SPLUNC2/PSP and synthetic peptides derived from the protein.

    Science.gov (United States)

    Gorr, Sven-Ulrik; Abdolhosseini, Mahsa; Shelar, Anuradha; Sotsky, Julie

    2011-08-01

    PSP (parotid secretory protein)/SPLUNC2 (short palate, lung and nasal epithelium clone 2) is expressed in human salivary glands and saliva. The protein exists as an N-glycosylated and non-glycosylated form and both appear to induce agglutination of bacteria, a major antibacterial function for salivary proteins. Both forms of PSP/SPLUNC2 bind LPS (lipopolysaccharide), suggesting that the protein may also play an anti-inflammatory role. Based on the predicted structure of PSP/SPLUNC2 and the location of known antibacterial and anti-inflammatory peptides in BPI (bactericidal/permeability-increasing protein) and LBP (LPS-binding protein), we designed GL13NH2 and GL13K, synthetic peptides that capture these proposed functions of PSP/SPLUNC2. GL13NH3 agglutinates bacteria, leading to increased clearance by macrophages and reduced spread of infection in a plant model. GL13K kills bacteria with a minimal inhibitory concentration of 5-10 μg/ml, kills bacteria in biofilm and retains activity in 150 mM NaCl and 50% saliva. Both peptides block endotoxin action, but only GL13K appears to bind endotoxin. The peptides do not cause haemolysis, haemagglutination in serum, inhibit mammalian cell proliferation or induce an inflammatory response in macrophages. These results suggest that the GL13NH2 and the modified peptide GL13K capture the biological activity of PSP/SPLUNC2 and can serve as lead compounds for the development of novel antimicrobial and anti-inflammatory peptides.

  12. Fibrils from designed non-amyloid-related synthetic peptides induce AA-amyloidosis during inflammation in an animal model.

    Directory of Open Access Journals (Sweden)

    Per Westermark

    Full Text Available BACKGROUND: Mouse AA-amyloidosis is a transmissible disease by a prion-like mechanism where amyloid fibrils act by seeding. Synthetic peptides with no amyloid relationship can assemble into amyloid-like fibrils and these may have seeding capacity for amyloid proteins. PRINCIPAL FINDINGS: Several synthetic peptides, designed for nanotechnology, have been examined for their ability to produce fibrils with Congo red affinity and concomitant green birefringence, affinity for thioflavin S and to accelerate AA-amyloidosis in mice. It is shown that some amphiphilic fibril-forming peptides not only produced Congo red birefringence and showed affinity for thioflavin S, but they also shortened the lag phase for systemic AA-amyloidosis in mice when they were given intravenously at the time of inflammatory induction with silver nitride. Peptides, not forming amyloid-like fibrils, did not have such properties. CONCLUSIONS: These observations should caution researchers and those who work with synthetic peptides and their derivatives to be aware of the potential health concerns.

  13. Key comparison study on peptide purity—synthetic human C-peptide

    Science.gov (United States)

    Josephs, R. D.; Li, M.; Song, D.; Westwood, S.; Stoppacher, N.; Daireaux, A.; Choteau, T.; Wielgosz, R.; Xiao, P.; Liu, Y.; Gao, X.; Zhang, C.; Zhang, T.; Mi, W.; Quan, C.; Huang, T.; Li, H.; Flatschart, R.; Borges Oliveira, R.; Melanson, J. E.; Ohlendorf, R.; Henrion, A.; Kinumi, T.; Wong, L.; Liu, Q.; Oztug Senal, M.; Vatansever, B.; Ün, I.; Gören, A. C.; Akgöz, M.; Quaglia, M.; Warren, J.

    2017-01-01

    Under the auspices of the Protein Analysis Working Group (PAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) a key comparison, CCQM-K115, was coordinated by the Bureau International des Poids et Mesures (BIPM) and the Chinese National Institute of Metrology (NIM). Eight Metrology Institutes or Designated Institutes and the BIPM participated. Participants were required to assign the mass fraction of human C-peptide (hCP) present as the main component in the comparison sample for CCQM-K115. The comparison samples were prepared from synthetic human hCP purchased from a commercial supplier and used as provided without further treatment or purification. hCP was selected to be representative of the performance of a laboratory's measurement capability for the purity assignment of short (up to 5 kDa), non-cross-linked synthetic peptides/proteins. It was anticipated to provide an analytical measurement challenge representative for the value-assignment of compounds of broadly similar structural characteristics. The majority of participants used a peptide impurity corrected amino acid analysis (PICAA) approach as the amount of material that has been provided to each participant (25 mg) is insufficient to perform a full mass balance based characterization of the material by a participating laboratory. The coordinators, both the BIPM and the NIM, were the laboratories to use the mass balance approach as they had more material available. It was decided to propose KCRVs for both the hCP mass fraction and the mass fraction of the peptide related impurities as indispensable contributor regardless of the use of PICAA, mass balance or any other approach to determine the hCP purity. This allowed participants to demonstrate the efficacy of their implementation of the approaches used to determine the hCP mass fraction. In particular it allows participants to demonstrate the efficacy of their implementation of peptide related impurity identification and quantification

  14. The Contribution of DOPA to Substrate-Peptide Adhesion and Internal Cohesion of Mussel-Inspired Synthetic Peptide Films.

    Science.gov (United States)

    Anderson, Travers H; Yu, Jing; Estrada, Abril; Hammer, Malte U; Waite, J Herbert; Israelachvili, Jacob N

    2010-12-08

    Mussels use a variety of 3, 4-dihydroxyphenyl-l-alanine (DOPA) rich proteins specifically tailored to adhering to wet surfaces. Synthetic polypeptide analogues of adhesive mussel foot proteins (specifically mfp-3) are used to study the role of DOPA in adhesion. The mussel-inspired peptide is a random copolymer of DOPA and N(5) -(2-hydroxyethyl)-l-glutamine synthesized with DOPA concentrations of 0-27 mol% and molecular weights of 5.9-7.1 kDa. Thin films (3-5 nm thick) of the mussel-inspired peptide are used in the surface forces apparatus (SFA) to measure the force-distance profiles and adhesion and cohesion energies of the films in an acetate buffer. The adhesion energies of the mussel-inspired peptide films to mica and TiO(2) surfaces increase with DOPA concentration. The adhesion energy to mica is 0.09 μJ m(-2) mol(DOPA) (-1) and does not depend on contact time or load. The adhesion energy to TiO(2) is 0.29 μJ m(-2) mol(DOPA) (-1) for short contact times and increases to 0.51 μJ m(-2) mol(DOPA) (-1) for contact times >60 min in a way suggestive of a phase transition within the film. Oxidation of DOPA to the quinone form, either by addition of periodate or by increasing the pH, increases the thickness and reduces the cohesion of the films. Adding thiol containing polymers between the oxidized films recovers some of the cohesion strength. Comparison of the mussel-inspired peptide films to previous studies on mfp-3 thin films show that the strong adhesion and cohesion in mfp-3 films can be attributed to DOPA groups favorably oriented within or at the interface of these films.

  15. Use of Synthetic Peptides to Study Structure-Function Relationships of Matrix Metalloproteinases and Their Substrates.

    Science.gov (United States)

    Netzel-Arnett, Sarah Joann

    The matrix metalloproteinases (MMPs) are a family of zinc proteinases that is collectively capable of degrading the major components of the extracellular matrix. A variety of synthetic peptides has been prepared which are models for the human MMP and their substrates to study structure -function relationships in this enzyme-substrate system. To elucidate the sequence specificity of the MMP, the k _{cat}/K_ M values for the hydrolysis of over 50 synthetic octapeptides has been investigated. Similarities, as well as distinct differences have been found between the individual MMP with the largest differences occurring at subsites P_1, P_1^' and P_3 ^'. Based on these data, quenched -fluorescence substrates with optimized sequences have been developed for five human MMP. The key features of these heptapeptides are a tryptophan on the P_ n^' side and a dinitrophenol quenching group on the amino terminus. To assess the role of the triple helical conformation in the collagenase-collagen system, a series of triple helical peptides has been prepared and shown to compete with collagen in collagenase assays. This provides evidence for the existence of a triple helical recognition site distinct from the active site. All of the MMP are secreted as zymogens and it has been postulated that the portion of the propeptide surrounding a critical cysteine is responsible for maintaining latency. Conformational energy calculations and mutagenesis studies have suggested that this region adopts a specific conformation that stabilizes the latent form. Peptide models of this region of the propeptide have been prepared and shown to inhibit the MMP. CD and NMR studies, however, have failed to provide evidence for the predicted peptide conformation. Thus, the observed inhibition may reflect their propensity to adopt the propeptide conformation upon binding to the enzyme.

  16. Synthetic lung surfactants containing SP-B and SP-C peptides plus novel phospholipase-resistant lipids or glycerophospholipids

    Science.gov (United States)

    Notter, Robert H.; Gupta, Rohun; Schwan, Adrian L.; Wang, Zhengdong; Shkoor, Mohanad Gh

    2016-01-01

    instillation in future clinical applications. Discussion Our findings support the potential of dual-peptide synthetic lipid/peptide surfactants containing S-MB DATK + SP-Css ion-lock 1 for treating diseases of surfactant deficiency or dysfunction. Moreover, phospholipase-resistant dual-peptide surfactants containing DEPN-8/PG-1 may have particular applications in treating direct forms of ARDS where endogenous phospholipases are present in the lungs.

  17. Building parity between brand and generic peptide products: Regulatory and scientific considerations for quality of synthetic peptides.

    Science.gov (United States)

    Wu, Larisa C; Chen, Fu; Lee, Sau L; Raw, Andre; Yu, Lawrence X

    2017-02-25

    Peptides are a fast growing segment in the pharmaceutical industry. Consequently, the industry and regulatory agencies are increasing their focus on the regulatory path and quality considerations for peptide development and manufacturing. Although most peptides are synthetic, manufactured by solid phase synthesis, nevertheless they are complex molecules with challenging quality and regulatory aspects. This paper provides a structured overview of relevant quality issues for chemically synthesized peptides used as active pharmaceutical ingredients (API) in drug products. It addresses the unique characteristics of peptides pertaining to structural and physicochemical characterization, manufacturing and in process controls, impurities and aggregates arising from manufacturing and storage, along with their potential impact on safety (including immunogenicity) and efficacy of the peptide drug products.

  18. Tritium labelling of PACAP-38 using a synthetic diiodinated precursor peptide

    DEFF Research Database (Denmark)

    Pedersen, Martin Holst Friborg; Baun, Michael

    2012-01-01

    In the interest of developing efficient methods for tritium labelling peptides, we here demonstrate the successful labelling of PACAP-38 (pituitary adenylate cyclase-activating polypeptide), a 38-mer peptide, using a synthetic diiodinated PACAP-38 precursor. In this example, we employ standard...... hydrogenation chemistry with the use of a heterogeneous palladium catalyst and carrier-free tritium gas on a tritium manifold system....

  19. Experimental inhibition of peptide fibrillogenesis by synthetic peptides, carbohydrates and drugs.

    Science.gov (United States)

    Srinivasan, Alagiri

    2012-01-01

    Peptide fibrillogenesis generally begins by the transformation of normally soluble proteins into elongated aggregates which are called as amyloid. These fibrils mainly consist of ß-sheets. They share certain common characteristics such as a cross-ß x-ray diffraction pattern, association with other common proteins and typical staining by the dye Congo Red. The individual form of the deposit consists of a disease-specific peptide/protein. The disease-specific protein serves as the basis for the classification of the amyloids. The association of fibril-forming peptides/proteins with diseases makes them primary disease-targets. Understanding the molecular interactions involved in the fibril formation becomes the foremost requirement to characterize the target. Interference with these interactions of ß-sheets in vitro prevents and sometimes reverses the fibril assembly. A small molecule capable of interfering with the formation of fibril could have therapeutic applications in these diseases. This anti-aggregation approach appears to be a viable treatment option. A search for such a molecule is pursued actively world over. All types of compounds and approaches to slow down or prevent the aggregation process have been described in the literature. These efforts are reviewed in this chapter.

  20. High Specific Selectivity and Membrane-Active Mechanism of Synthetic Cationic Hybrid Antimicrobial Peptides Based on the Peptide FV7.

    Science.gov (United States)

    Tan, Tingting; Wu, Di; Li, Weizhong; Zheng, Xin; Li, Weifen; Shan, Anshan

    2017-02-06

    Hybrid peptides integrating different functional domains of peptides have many advantages, such as remarkable antimicrobial activity, lower hemolysis and ideal cell selectivity, compared with natural antimicrobial peptides. FV7 (FRIRVRV-NH₂), a consensus amphiphilic sequence was identified as being analogous to host defense peptides. In this study, we designed a series of hybrid peptides FV7-LL-37 (17-29) (FV-LL), FV7-magainin 2 (9-21) (FV-MA) and FV7-cecropin A (1-8) (FV-CE) by combining the FV7 sequence with the small functional sequences LL-37 (17-29) (LL), magainin 2 (9-21) (MA) and cecropin A (1-8) (CE) which all come from well-described natural peptides. The results demonstrated that the synthetic hybrid peptides, in particular FV-LL, had potent antibacterial activities over a wide range of Gram-negative and Gram-positive bacteria with lower hemolytic activity than other peptides. Furthermore, fluorescent spectroscopy indicated that the hybrid peptide FV-LL exhibited marked membrane destruction by inducing outer and inner bacterial membrane permeabilization, while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that FV-LL damaged membrane integrity by disrupting the bacterial membrane. Inhibiting biofilm formation assays also showed that FV-LL had similar anti-biofilm activity compared with the functional peptide sequence FV7. Synthetic cationic hybrid peptides based on FV7 could provide new models for combining different functional domains and demonstrate effective avenues to screen for novel antimicrobial agents.

  1. High Specific Selectivity and Membrane-Active Mechanism of Synthetic Cationic Hybrid Antimicrobial Peptides Based on the Peptide FV7

    Science.gov (United States)

    Tan, Tingting; Wu, Di; Li, Weizhong; Zheng, Xin; Li, Weifen; Shan, Anshan

    2017-01-01

    Hybrid peptides integrating different functional domains of peptides have many advantages, such as remarkable antimicrobial activity, lower hemolysis and ideal cell selectivity, compared with natural antimicrobial peptides. FV7 (FRIRVRV-NH2), a consensus amphiphilic sequence was identified as being analogous to host defense peptides. In this study, we designed a series of hybrid peptides FV7-LL-37 (17–29) (FV-LL), FV7-magainin 2 (9–21) (FV-MA) and FV7-cecropin A (1–8) (FV-CE) by combining the FV7 sequence with the small functional sequences LL-37 (17–29) (LL), magainin 2 (9–21) (MA) and cecropin A (1–8) (CE) which all come from well-described natural peptides. The results demonstrated that the synthetic hybrid peptides, in particular FV-LL, had potent antibacterial activities over a wide range of Gram-negative and Gram-positive bacteria with lower hemolytic activity than other peptides. Furthermore, fluorescent spectroscopy indicated that the hybrid peptide FV-LL exhibited marked membrane destruction by inducing outer and inner bacterial membrane permeabilization, while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that FV-LL damaged membrane integrity by disrupting the bacterial membrane. Inhibiting biofilm formation assays also showed that FV-LL had similar anti-biofilm activity compared with the functional peptide sequence FV7. Synthetic cationic hybrid peptides based on FV7 could provide new models for combining different functional domains and demonstrate effective avenues to screen for novel antimicrobial agents. PMID:28178190

  2. Biomimeticity in tissue engineering scaffolds through synthetic peptide modifications-altering chemistry for enhanced biological response.

    Science.gov (United States)

    Sreejalekshmi, Kumaran G; Nair, Prabha D

    2011-02-01

    Biomimetic and bioactive biomaterials are desirable as tissue engineering scaffolds by virtue of their capability to mimic natural environments of the extracellular matrix. Biomimeticity has been achieved by the incorporation of synthetic short peptide sequences into suitable materials either by surface modification or by bulk incorporation. Research in this area has identified several novel synthetic peptide segments, some of them with cell-specific interactions, which may serve as potential candidates for use in explicit tissue applications. This review focuses on the developments and prospective directions of incorporating short synthetic peptide sequences onto scaffolds for tissue engineering, with emphasis on the chemistry of peptide immobilization and subsequent cell responses toward modified scaffolds. The article provides a decision-tree-type flow chart indicating the most probable cellular events on a given peptide-modified scaffold along with the consolidated list of synthetic peptide sequences, supports as well as cell types used in various tissue engineering studies, and aims to serve as a quick reference guide to peptide chemists and material scientists interested in the field.

  3. Encoding physico-chemical cues in synthetic hydrogels by triple helix assembly of collagen mimetic peptides

    Science.gov (United States)

    Stahl, Patrick

    The ECM is a complex natural system evolved to promote proliferation and differentiation of cells during tissue development. In order to create synthetic biomaterials for studying cell-scaffold interactions and ultimately for engineering tissues, scientists strive to recapitulate many characteristics of ECM by developing hydrogels that contain mechanical cues and biochemical signals such as adhesion moieties and cell growth factors. While synthetic hydrogels bypass limitations of naturally-derived materials (e.g. transfer of pathogens), nature provides inspiration to enhance the functionality of synthetic hydrogels through biomimetic approaches. The collagen triple helix is the basis for the supramolecular structure of collagen in the ECM, and its adaptation in collagen mimetic peptides (CMPs) has provided hybridization mechanisms that can be employed in the formation and functionalization of synthetic hydrogels. The aim of this dissertation is to develop novel poly(ethylene glycol) (PEG)-based hydrogels that employ CMP triple helix assembly as a non-covalent yet target-specific tool to encode physical and chemical cues into the hydrogel with spatial control. We demonstrate that multi-arm PEG functionalized with CMPs form hydrogels supported by physical crosslinks mediated by CMP triple helix. Particle tracking microrheology shows that these physical crosslinks are sensitive to temperature as well as addition of exogenous CMPs that can disrupt crosslinks by competing for triple helix formation. This physical crosslink disruption enables the modulation of bulk hydrogel elasticity and the introduction of local stiffness gradients in PEG-CMP hydrogels. We also present photopolymerized PEG diacrylate (PEGDA) hydrogels displaying CMPs that can be further conjugated to CMPs with bioactive moieties via triple helix hybridization. Encoding these hydrogels with cell-adhesive CMPs induces cell spreading and proliferation. We further demonstrate generation of gradients and

  4. Immune response to synthetic peptides of dengue prM protein.

    Science.gov (United States)

    Vázquez, Susana; Guzmán, María Guadalupe; Guillen, Gerardo; Chinea, Glay; Pérez, Ana Beatriz; Pupo, Maritza; Rodriguez, Rosmary; Reyes, Osvaldo; Garay, Hilda Elisa; Delgado, Iselys; García, Gissel; Alvarez, Mayling

    2002-03-15

    The immunological activities of five synthetic peptides of the prM protein of dengue-2 (DEN-2) virus containing B cell epitopes were evaluated in BALB/c mice. Two peptides elicited neutralizing antibodies against all four DEN serotypes. Virus-specific proliferative responses were demonstrated in mice immunized with four of the five peptides, demonstrating the presence of T cell epitopes. Mice immunized with three of the five peptides conjugated with bovine albumin showed statistically significant levels (Pdevelopment of anti-flaviviral vaccines.

  5. Systemic Antibacterial Activity of Novel Synthetic Cyclic Peptides

    Science.gov (United States)

    Dartois, Véronique; Sanchez-Quesada, Jorge; Cabezas, Edelmira; Chi, Ellen; Dubbelde, Chad; Dunn, Carrie; Granja, Juan; Gritzen, Colleen; Weinberger, Dana; Ghadiri, M. Reza; Parr, Thomas R.

    2005-01-01

    Cyclic peptides with an even number of alternating d,l-α-amino acid residues are known to self-assemble into organic nanotubes. Such peptides previously have been shown to be stable upon protease treatment, membrane active, and bactericidal and to exert antimicrobial activity against Staphylococcus aureus and other gram-positive bacteria. The present report describes the in vitro and in vivo pharmacology of selected members of this cyclic peptide family. The intravenous (i.v.) efficacy of six compounds with MICs of less than 12 μg/ml was tested in peritonitis and neutropenic-mouse thigh infection models. Four of the six peptides were efficacious in vivo, with 50% effective doses in the peritonitis model ranging between 4.0 and 6.7 mg/kg against methicillin-sensitive S. aureus (MSSA). In the thigh infection model, the four peptides reduced the bacterial load 2.1 to 3.0 log units following administration of an 8-mg/kg i.v. dose. Activity against methicillin-resistant S. aureus was similar to MSSA. The murine pharmacokinetic profile of each compound was determined following i.v. bolus injection. Interestingly, those compounds with poor efficacy in vivo displayed a significantly lower maximum concentration of the drug in serum and a higher volume of distribution at steady state than compounds with good therapeutic properties. S. aureus was unable to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations, in agreement with the proposed interaction with multiple components of the bacterial membrane canopy. Although additional structure-activity relationship studies are required to improve the therapeutic window of this class of antimicrobial peptides, our results suggest that these amphipathic cyclic d,l-α-peptides have potential for systemic administration and treatment of otherwise antibiotic-resistant infections. PMID:16048940

  6. Impairment of IFN-gamma response to synthetic peptides of Mycobacterium tuberculosis in a 7-day whole blood assay.

    Directory of Open Access Journals (Sweden)

    Hannah Priyadarshini Gideon

    Full Text Available Studies on Mycobacterium tuberculosis (MTB antigens are of interest in order to improve vaccine efficacy and to define biomarkers for diagnosis and treatment monitoring. The methodologies used for these investigations differ greatly between laboratories and discordant results are common. The IFN-gamma response to two well characterized MTB antigens ESAT-6 and CFP-10, in the form of recombinant proteins and synthetic peptides, was evaluated in HIV-1 uninfected persons in both long-term (7 day and 24 hour, commercially available QuantiFERON TB Gold in Tube (QFT-GIT, whole blood assays. Our findings showed differences in the IFN-gamma response between 24 hour and 7 day cultures, with recombinant proteins inducing a significantly higher response than the peptide pools in 7 day whole blood assays. The activity of peptides and recombinant proteins did not differ in 24 hour whole blood or peripheral blood mononuclear cell (PBMC based assays, nor in the ELISpot assay. Further analysis by SELDI-TOF mass spectrometry showed that the peptides are degraded over the course of 7 days of incubation in whole blood whilst the recombinant proteins remain intact. This study therefore demonstrates that screening antigenic candidates as synthetic peptides in long-term whole blood assays may underestimate immunogenicity.

  7. Design and Engineering Strategies for Synthetic Antimicrobial Peptides

    Science.gov (United States)

    Tossi, Alessandro

    Thousands of antimicrobial peptides (AMPs) of prokaryotic, fungal, plant, or animal origin have been identified, and their potential as lead compounds for the design of novel therapeutic agents in the treatment of infection, for stimulating the immune system, or in countering septic shock has been widely recognized. Added to this is their possible use in prophylaxis of infectious diseases for animal or plant protection, for disinfection of surgical instruments or industrial surfaces, and for food preservation among other commercially important applications. Since the early eighties, AMPs have been subject to a vast number of studies aimed at understanding what determines their potency and spectrum of activities against bacterial or fungal pathogens, and at maximizing these while limiting cytotoxic activities toward host cells. Much research has also been directed toward understanding specific mechanisms of action underlying the antimicrobial activity and selectivity, to be able to redesign the peptides for optimal performance. A central theme in the mode of action of many AMPs is their dynamic interaction with biological membranes, which involves various properties of these peptides such as, among others, surface hydrophobicity and polarity, charge, structure, and induced conformational variations. These features are often intimately interconnected so that engineering peptides to independently adjust any one property in particular is not an easy task. However, solid-phase peptide synthesis allows the use of a large repertoire of nonproteinogenic amino acids that can be used in the rational design of peptides to finely tune structural and physicochemical properties and precisely probe structure-function relationships.

  8. Characterization of antibodies to synthetic nerve growth factor (NGF) and proNGF peptides.

    Science.gov (United States)

    Ebendal, T; Persson, H; Larhammar, D; Lundströmer, K; Olson, L

    1989-03-01

    Sequence data for the mature nerve growth factor (NGF) protein and its precursor are available from molecular cloning of the NGF gene in several species, including mice, humans, rats, and chickens. Hydrophilicity analysis of the predicted rat and chicken prepro-NGF was carried out to locate putative antigenic determinants. Eight peptides were selected and synthesized based on hydrophilicity profiles. Two peptides represent sequences in the rat (and mouse) pro-NGF, one peptide (our peptide P3) represents a highly conserved region of the mature NGF protein (identical in humans, mice, rats, and chickens), two peptides are specific for the mature chicken NGF, and the remaining three peptides are specific for the mature rat NGF (each with only one amino acid substitution compared with corresponding segments of the mouse NGF). For immunization, the peptides were conjugated to keyhold limpet hemocyanin and used to produce antisera in rabbits. After bleeding, peptide-specific antibodies were purified on affinity columns prepared by coupling each of the synthetic peptides. The different peptide antisera and affinity-purified antibodies then were characterized by enzyme-linked immunoassay (ELISA) and immunohistochemistry of the male mouse submandibular gland, a rich exocrine source of NGF. ELISA analysis showed that all peptide antisera bound two to four orders of magnitude better than normal rabbit serum to a coat of their proper peptide. The higher binding was retained by the purified peptide antibodies compared with normal rabbit immunoglobulin. Specific tests, in which one peptide antiserum was checked against different peptide coats in the ELISA, also showed two to four orders of magnitude higher binding of antibodies to the proper synthetic peptide. The peptide antibodies also were tested for their ability to bind to native mouse beta NGF coated to the immunoplates. Only antibodies raised to the conserved P3 peptide recognized native NGF to an extent similar to that

  9. A new synthetic peptide having two target of antibacterial action in E. coli ML35

    Directory of Open Access Journals (Sweden)

    Hernando Curtidor

    2016-12-01

    Full Text Available The increased resistance of microorganisms to the different antimicrobials available to today has highlighted the need to find new therapeutic agents, including natural and/or synthetic antimicrobial peptides. This study has evaluated the antimicrobial activity of synthetic peptide 35409 (RYRRKKKMKKALQYIKLLKE against S. aureus ATCC 29213, P. aeruginosa ATCC 15442 and E. coli ML 35 (ATCC 43827. The results have shown that peptide 35409 inhibited the growth of these three bacterial strains, having 16 fold greater activity against E. coli and P. aeruginosa, but requiring less concentration regarding E. coli (22 µM. When analyzing this activity against E. coli compared to time taken, it was found that this peptide inhibited bacterial growth during the first 60 min and reduced CFU/mL 1 log after 120 min had elapsed. This antimicrobial peptide permeabilized the E. coli membrane by interaction with membrane phospholipids, mainly phosphatidylethanolamine, inhibited cell division and induced filamentation, suggesting two different targets of action within a bacterial cell. Cytotoxicity studies revealed that peptide 35409 had low hemolytic activity and was not cytotoxic for two human cell lines. We would thus propose, in the light of these findings, that the peptide 35409 sequence should provide a promising template for designing broad-spectrum antimicrobial peptides.

  10. Fiber formation of a synthetic spider peptide derived from Nephila clavata.

    Science.gov (United States)

    Hidaka, Yuji; Kontani, Ko-Ichi; Taniguchi, Rina; Saiki, Masatoshi; Yokoi, Sayoko; Yukuhiro, Kenji; Yamaguchi, Hiroshi; Miyazawa, Mitsuhiro

    2011-01-01

    Dragline silk is a high-performance biopolymer with exceptional mechanical properties. Artificial spider dragline silk is currently prepared by a recombinant technique or chemical synthesis. However, the recombinant process is costly and large-sized synthetic peptides are needed for fiber formation. In addition, the silk fibers that are produced are much weaker than a fiber derived from a native spider. In this study, a small peptide was chemically synthesized and examined for its ability to participate in fiber formation. A short synthetic peptide derived from Nephila clavata was prepared by a solid-phase peptide method, based on a prediction using the hydrophobic parameter of each individual amino acid residue. After purification of the spider peptide, fiber formation was examined under several conditions. Fiber formation proceeded in the acidic pH range, and larger fibers were produced when organic solvents such as trifluoroethanol and acetonitrile were used at an acidic pH. Circular dichroism measurements of the spider peptide indicate that the peptide has a beta-sheet structure and that the formation of a beta-sheet structure is required for the spider peptide to undergo fiber formation.

  11. Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity

    DEFF Research Database (Denmark)

    Beekman, N.J.C.M.; Schaaper, W.M.M.; Tesser, G.I.

    1997-01-01

    or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin...

  12. Synthetic methodology for the preparation of nucleic acid containing peptides

    NARCIS (Netherlands)

    Heden-van Noort, Gerbrand Jan van der

    2012-01-01

    Dit proefschrift beschrijft de ontwikkeling van nieuwe methoden voor de synthese van hybride biomoleculen die samengesteld zijn uit een peptide- en een nucleïnezuurfragment. Zulke hybride moleculen komen in de natuur voor en hebben belangrijke functies. In dit proefschrift wordt aandacht besteed aan

  13. Synthetic lung surfactants containing SP-B and SP-C peptides plus novel phospholipase-resistant lipids or glycerophospholipids

    Directory of Open Access Journals (Sweden)

    Robert H. Notter

    2016-10-01

    9:1 DEPN-8:PG-1 or 5:3:2 DPPC:POPC:POPG had the greatest in vivo activity in improving arterial oxygenation and dynamic lung compliance in ventilated animals with ARDS. Saline dispersions of these dual-peptide synthetic surfactants were also found to have shear viscosities comparable to or below those of current animal-derived surfactant drugs, supporting their potential ease of deliverability by instillation in future clinical applications. Discussion Our findings support the potential of dual-peptide synthetic lipid/peptide surfactants containing S-MB DATK + SP-Css ion-lock 1 for treating diseases of surfactant deficiency or dysfunction. Moreover, phospholipase-resistant dual-peptide surfactants containing DEPN-8/PG-1 may have particular applications in treating direct forms of ARDS where endogenous phospholipases are present in the lungs.

  14. Identification of human nonpancreatic-type ribonuclease by antibodies obtained against a synthetic peptide.

    Science.gov (United States)

    Bravo, M I; Cuchillo, C M; Nogués, M V

    1995-09-01

    An antibody that recognizes human nonpancreatic-type ribonuclease was obtained by immunizing a rabbit with a 14-residue synthetic peptide corresponding to the N-terminal sequence of eosinophil-derived neurotoxin which is identical to human liver ribonuclease. This amino acid sequence is unique to this protein. The anti N-peptide antibody was purified by protein A-Sepharose and by using ELISA and SDS-PAGE immunoblot techniques, the antibody reactivity against EDN and partially purified nonpancreatic-type ribonucleases from human plasma and urine was observed. Cross-reactivity with bovine pancreatic ribonuclease A and other proteins was not detected. In addition, the activity of the nonpancreatic-type ribonuclease was not affected by the antibody. The immune response was elicited without the need for a carrier protein showing that the N-terminal sequence of nonpancreatic ribonuclease contains a specific epitope. This antibody can be used for the immunological identification of both the native and denatured forms of this type of enzyme.

  15. Using synthetic peptides to benchmark peptide identification software and search parameters for MS/MS data analysis

    Directory of Open Access Journals (Sweden)

    Andreas Quandt

    2014-12-01

    Full Text Available Tandem mass spectrometry and sequence database searching are widely used in proteomics to identify peptides in complex mixtures. Here we present a benchmark study in which a pool of 20,103 synthetic peptides was measured and the resulting data set was analyzed using around 1800 different software and parameter set combinations. The results indicate a strong relationship between the performance of an analysis workflow and the applied parameter settings. We present and discuss strategies to optimize parameter settings in order to significantly increase the number of correctly assigned fragment ion spectra and to make the analysis method robust.

  16. A molecular dynamics and circular dichroism study of a novel synthetic antimicrobial peptide

    Science.gov (United States)

    Rodina, N. P.; Yudenko, A. N.; Terterov, I. N.; Eliseev, I. E.

    2013-08-01

    Antimicrobial peptides are a class of small, usually positively charged amphiphilic peptides that are used by the innate immune system to combat bacterial infection in multicellular eukaryotes. Antimicrobial peptides are known for their broad-spectrum antimicrobial activity and thus can be used as a basis for a development of new antibiotics against multidrug-resistant bacteria. The most challengeous task on the way to a therapeutic use of antimicrobial peptides is a rational design of new peptides with enhanced activity and reduced toxicity. Here we report a molecular dynamics and circular dichroism study of a novel synthetic antimicrobial peptide D51. This peptide was earlier designed by Loose et al. using a linguistic model of natural antimicrobial peptides. Molecular dynamics simulation of the peptide folding in explicit solvent shows fast formation of two antiparallel beta strands connected by a beta-turn that is confirmed by circular dichroism measurements. Obtained from simulation amphipatic conformation of the peptide is analysed and possible mechanism of it's interaction with bacterial membranes together with ways to enhance it's antibacterial activity are suggested.

  17. Creating Protein Affinity Reagents by Combining Peptide Ligands on Synthetic DNA Scaffolds

    Science.gov (United States)

    Williams, Berea A. R.; Diehnelt, Chris W.; Belcher, Paul; Greving, Matthew; Woodbury, Neal W.; Johnston, Stephen A.; Chaput, John C.

    2009-01-01

    A full understanding of the proteome will require ligands to all of the proteins encoded by genomes. While antibodies represent the principle affinity reagents used to bind proteins, their limitations have created a need for new ligands to large numbers of proteins. Here we propose a general concept to obtain protein affinity reagents that avoids animal immunization and iterative selection steps. Central to this process is the idea that small peptide libraries contain sequences that will bind to independent regions on a protein surface, and that these ligands can be combined on synthetic scaffolds to create high affinity bivalent reagents. To demonstrate the feasibility of this approach, an array of 4,000 unique 12-mer peptides was screened to identify sequences that bind to non-overlapping sites on the yeast regulatory protein Gal80. Individual peptide ligands were screened at different distances using a novel DNA linking strategy to identify the optimal peptide pair and peptide pair separation distance required to transform two weaker ligands into a single high affinity protein capture reagent. A synthetic antibody or synbody was created with 5 nM affinity to Gal80 that functions in conventional ELISA and pull-down assays. We validated our synthetic antibody approach by creating a second synbody to human transferrin. In both cases, we observed an increase in binding affinity of ∼1000-fold (ΔΔG = ∼4.1 kcal/mol) between the individual peptides and final bivalent synbody construct. PMID:19894711

  18. Effect of synthetic antimicrobial peptides on Naegleria fowleri trophozoites.

    Science.gov (United States)

    Tiewcharoen, Supathra; Phurttikul, Watchara; Rabablert, Jundee; Auewarakul, Prasert; Roytrakul, Sittiruk; Chetanachan, Pruksawan; Atithep, Thassanant; Junnu, Virach

    2014-05-01

    We evaluated the effect of tritrpticin, lactoferrin, killer decapeptide and scrambled peptide in vitro against Naegleria fowleri trophozoites compared with amphotericin B. Tritrpticin (100 microg/ml) caused apoptosis of N. fowleri trophozoites (2x10(5) cells/ml), while lactoferrin, killer decapeptide and scrambled peptide did not. On Gormori trichrome staining, tritrpticin affected the elasticity of the surface membrane and reduced the size of the nuclei of N. fowleri trophozoites. The ultrastructure surface membrane and food cup formation of the trophozoites were 100% inhibited. These results are consistent with inhibition of the nfa1, Mp2CL5 of the treated trophozoite, which plays a role in food cup formation. Tritrpticin 100 microg/ml was not toxic against SK-N-MC cells. Our findings suggest tritrpticin has activity against the surface membrane and nfa1 and Mp2CL5 of N. fowleri trophozoites and could be developed as a potential therapeutic agent.

  19. Antimicrobial polymers as synthetic mimics of host-defense peptides.

    Science.gov (United States)

    Kuroda, Kenichi; Caputo, Gregory A

    2013-01-01

    Antibiotic-resistant bacteria 'superbugs' are an emerging threat to public health due to the decrease in effective antibiotics as well as the slowed pace of development of new antibiotics to replace those that become ineffective. The need for new antimicrobial agents is a well-documented issue relating to world health. Tremendous efforts have been given to developing compounds that not only show high efficacy, but also those that are less susceptible to resistance development in the bacteria. However, the development of newer, stronger antibiotics which can overcome these acquired resistances is still a scientific challenge because a new mode of antimicrobial action is likely required. To that end, amphiphilic, cationic polymers have emerged as a promising candidate for further development as an antimicrobial agent with decreased potential for resistance development. These polymers are designed to mimic naturally occurring host-defense antimicrobial peptides which act on bacterial cell walls or membranes. Antimicrobial-peptide mimetic polymers display antibacterial activity against a broad spectrum of bacteria including drug-resistant strains and are less susceptible to resistance development in bacteria. These polymers also showed selective activity to bacteria over mammalian cells. Antimicrobial polymers provide a new molecular framework for chemical modification and adaptation to tune their biological functions. The peptide-mimetic design of antimicrobial polymers will be versatile, generating a new generation of antibiotics toward implementation of polymers in biomedical applications. Copyright © 2012 Wiley Periodicals, Inc.

  20. Synthetic peptide inhibitors of DNA replication in Staphylococcus aureus

    DEFF Research Database (Denmark)

    Løbner-Olesen, Anders; Kjelstrup, Susanne

    of clinically important pathogens and is essential for bacterial proliferation. The bacterial replication apparatus fulfill the requirements for a good drug target. The replisome of S. aureus consists of 5 different subunits (2, PolC2, 4, δ and δ`) who’s organization depends on multiple protein......-protein interactions. Centrally in the replisome is the -clamp where to multiple proteins binds through a conserved motif. We have identified the protein-protein interactions in the replisome of S. aureus by use of a bacterial two-hybrid system. A reverse bacterial two-hybrid system (R-BTH) based on Pyr......N (), DnaB and DnaX (). Three peptides identified as inhibitors of DnaN have been purified. Two of these peptides inhibited growth as well as DNA replication in S. aureus. The minimal inhibitory concentration (MIC) of the peptides was approximately 50 g/ml. Overexpression of DnaN reduced the inhibitory...

  1. Assessment of protective immune responses against hydatid disease in sheep by immunization with synthetic peptide antigens.

    Science.gov (United States)

    Woollard, D J; Heath, D D; Lightowlers, M W

    2000-08-01

    Four synthetic peptides which comprise the immunodominant linear epitopes of the EG95 recombinant protein, were investigated for their ability to induce host-protective immunity against Echinococcus granulosus in sheep. Sheep were immunized with either free peptide or peptide conjugated to diphtheria toxoid and challenge infected with E. granulosus eggs. All of the peptides elicited specific antibody, but these did not kill the parasite in in vitro culture assays, nor did the peptides induce protection against challenge infection. In contrast, anti-EG95 antibodies affinity purified against each of the 4 peptides were lethal to the parasite in in vitro culture. These affinity-purified antibodies were shown to contain specific antibody to both peptide and EG95. In in vitro inhibition assays, the peptides did not diminish anti-EG95 antibody binding to EG95 or parasite lysis in oncosphere killing assays. These results suggest that the fine specificities of antibodies raised against the recombinant protein are different to those raised against the peptide immunogens and that the majority of the antibody induced by vaccination with EG95 is raised against conformational determinants.

  2. Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide

    NARCIS (Netherlands)

    Villen, J.; Borras, E.; Schaaper, W.M.M.; Meloen, R.H.; Davila, M.; Domingo, E.; Giralt, E.; Andreu, D.

    2002-01-01

    Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on

  3. Synthetic peptide with inhibin-like activity preferentially inhibits follitropin secretion in comparison with lutropin-releasing hormone antagonists

    Energy Technology Data Exchange (ETDEWEB)

    Sairam, M.R.; Ramasharma, K.; Li, C.H.

    1987-04-01

    Biological activity of a synthetic peptide with inhibin-like activity under in vitro and in vivo conditions was compared with three highly potent synthetic lutropin-releasing hormone antagonists. Unlike the synthetic lutropin-releasing hormone antagonists, which effectively inhibited both lutropin and follitropin secretion from the pituitary, the inhibin-like peptide showed a preferential effect by inhibiting follitropin release both in vitro and in vivo. Thus, small peptides such as inhibin-like peptide with a sequence unrelated to lutropin-releasing hormone may provide a basis for design of selective inhibitors of gonadotropin release. FSH and LH were measured by radioimmunoassay.

  4. Micropatterning proteins and synthetic peptides on solid supports: a novel application for microelectronics fabrication technology.

    Science.gov (United States)

    Britland, S; Perez-Arnaud, E; Clark, P; McGinn, B; Connolly, P; Moores, G

    1992-01-01

    In this paper, we describe a method for immobilizing proteins and synthesizing peptides in micrometer-dimension patterns on solid supports. Microelectronics fabrication technology was adapted and used to lithographically direct the location of immobilization of proteins on appropriately derivatized surfaces. As examples, we micropatterned the protein bovine serum albumin (BSA) and the enzyme horseradish peroxidase (HRP). The catalytic activity of HRP was shown to be retained after being cross-linked to the support. When coupled with solid-phase peptide synthesis, the technique allowed synthetic peptides to be constructed in patterns again having micrometer dimensions. Synthetic polypeptides, polylysine, were constructed in patterns with dimensions that approached the practical limit of resolution for optical lithography at 1-2 microns. The patterns of immobilized molecules and synthetic peptides were visualized using histochemical methods together with light and fluorescence microscopy. The protein and peptide patterning technique described here is an advance in the field of bioelectronics. In particular, it should now be possible to devise novel methods for interfacing with biological systems and constructing new devices for incorporation into miniaturized biosensors.

  5. Human T-cell recognition of synthetic peptides representing conserved and variant sequences from the merozoite surface protein 2 of Plasmodium falciparum

    DEFF Research Database (Denmark)

    Theander, T G; Hviid, L; Dodoo, D

    1997-01-01

    Merozoite surface protein 2 (MSP2) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the peripheral blood mononuclear cell (PBMC) response to synthetic peptides corresponding to conserved and variant regions of the FCQ-27 allelic form of MSP2 in Ghanaian individuals...

  6. Synthetic peptides and ribosomal proteins as substrate for 60S ribosomal protein kinase from yeast cells

    DEFF Research Database (Denmark)

    Grankowski, N; Gasior, E; Issinger, O G

    1993-01-01

    Kinetic studies on the 60S protein kinase were conducted with synthetic peptides and ribosomal proteins as substrate. Peptide RRREEESDDD proved to be the best synthetic substrate for this enzyme. The peptide has a sequence of amino acids which most closely resembles the structure of potential...... phosphorylation sites in natural substrates, i.e., acidic ribosomal proteins. The superiority of certain kinetic parameters for 60S kinase obtained with the native whole 80S ribosomes over those of the isolated fraction of acidic ribosomal proteins indicates that the affinity of 60S kinase to the specific protein...... substrate not only depends on the structure of the polypeptide chain around the target amino acid but also on its native structure within the 80S ribosome....

  7. Application of synthetic peptides in development of a serologic method for laboratory diagnosis of schistosomiasis mansoni

    Directory of Open Access Journals (Sweden)

    Edward José de Oliveira

    2006-10-01

    Full Text Available The immunoreactivity of seven peptides synthesized from Schistosoma mansoni proteins, was evaluated by dot-blot and ELISA assays using two different sensitization methodologies. The best results were obtained on wells of the Costar 3590 microplates coated with peptides P1, P2, P3, P6, and P7 using conventional methodology. The signals increased considerably (p < 0.0003 on wells sensitized with P1 to P6 using alternative methodology. In contrast, the well coated with peptide P7 presented lower signal when compared with conventional methodology (p = 0.0019. These results, establish the basis for the application of synthetic peptides for laboratory diagnosis of schistosomiasis mansoni.

  8. Effect of Synthetic Truncated Apolipoprotein C-I Peptide on Plasma Lipoprotein Cholesterol in Nonhuman Primates

    Directory of Open Access Journals (Sweden)

    Rampratap S. Kushwaha

    2004-01-01

    Full Text Available The present studies were conducted to determine whether a synthetic truncated apoC-I peptide that inhibits CETP activity in baboons would raise plasma HDL cholesterol levels in nonhuman primates with low HDL levels. We used 2 cynomolgus monkeys and 3 baboons fed a cholesterol- and fat-enriched diet. In cynomolgus monkeys, we injected synthetic truncated apoC-I inhibitor peptide at a dose of 20 mg/kg and, in baboons, at doses of 10, 15, and 20 mg/kg at weekly intervals. Blood samples were collected 3 times a week and VLDL + LDL and HDL cholesterol concentrations were measured. In cynomolgus monkeys, administration of the inhibitor peptide caused a rapid decrease in VLDL + LDL cholesterol concentrations (30%–60% and an increase in HDL cholesterol concentrations (10%–20%. VLDL + LDL cholesterol concentrations returned to baseline levels in approximately 15 days. In baboons, administration of the synthetic inhibitor peptide caused a decrease in VLDL + LDL cholesterol (20%–60% and an increase in HDL cholesterol (10%–20%. VLDL + LDL cholesterol returned to baseline levels by day 21, whereas HDL cholesterol concentrations remained elevated for up to 26 days. ApoA-I concentrations increased, whereas apoE and triglyceride concentrations decreased. Subcutaneous and intravenous administrations of the inhibitor peptide had similar effects on LDL and HDL cholesterol concentrations. There was no change in body weight, food consumption, or plasma IgG levels of any baboon during the study. These studies suggest that the truncated apoC-I peptide can be used to raise HDL in humans.

  9. Immunodiagnosis of human neurocysticercosis using a synthetic peptide selected by phage-display.

    Science.gov (United States)

    Hell, R C R; Amim, P; de Andrade, H M; de Avila, R A M; Felicori, L; Oliveira, A G; Oliveira, C A; Nascimento, E; Tavares, C A P; Granier, C; Chávez-Olórtegui, C

    2009-04-01

    The usefulness of a synthetic peptide in the serodiagnosis of Taenia solium human neurocysticercosis (NC) has been evaluated. Phage-displayed peptides were screened with human antibodies to scolex protein antigen from cysticercus cellulosae (SPACc). One clone was found to interact specifically with anti-SPACc IgGs. The corresponding synthetic peptide was found to be recognized in ELISA by NC patient's sera. The study was carried out with sera from 28 confirmed NC patients, 13 control sera and 73 sera from patients suffering from other infectious diseases. A 93% sensibility and a 94.3% specificity was achieved. Figures of 89% and 31.4% of sensibility and specificity were obtained in a SPACc-based ELISA. Immunoblotting of SPACc with anti-peptide antibodies revealed a single band of approximately 45 kDa in 1D and four 45 kDa isoforms in 2D-gel electrophoresis. A strong and specific immunostaining in the fibers beneath the suckers, at the base of the rostellum, and in the tissue surrounding the scolex of cysticerci was observed by immunomicroscopy. Our results show that a peptide-based immunodiagnostic of neurocisticercosis can be envisioned.

  10. Synthetic mimic of antimicrobial peptide with nonmembrane-disrupting antibacterial properties.

    Science.gov (United States)

    Gabriel, Gregory J; Madkour, Ahmad E; Dabkowski, Jeffrey M; Nelson, Christopher F; Nüsslein, Klaus; Tew, Gregory N

    2008-11-01

    Polyguanidinium oxanorbornene ( PGON) was synthesized from norbornene monomers via ring-opening metathesis polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly encourage the examination of other novel guanidino containing macromolecules as powerful and selective antimicrobial agents.

  11. Synthetic Toll like receptor-4 (TLR-4 agonist peptides as a novel class of adjuvants.

    Directory of Open Access Journals (Sweden)

    Arulkumaran Shanmugam

    Full Text Available BACKGROUND: Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS, a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants. METHODOLOGY/PRINCIPAL FINDINGS: We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-κB nuclear translocation analyses in HEK-BLUE™-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate the identification of synthetic peptides that mimic LPS by interacting with TLR-4. This LPS mimotope-TLR-4 interaction will allow for the development and use of these peptides as a new class of adjuvants, namely TLR-4 agonists.

  12. Selection of Small Synthetic Antimicrobial Peptides Inhibiting Xanthomonas citri subsp. citri Causing Citrus Canker

    Science.gov (United States)

    Choi, Jeahyuk; Park, Euiho; Lee, Se-Weon; Hyun, Jae-Wook; Baek, Kwang-Hyun

    2017-01-01

    Citrus canker disease decreases the fruit quality and yield significantly, furthermore, emerging of streptomycin-resistant pathogens threatens the citrus industry seriously because of a lack of proper control agents. Small synthetic antimicrobial peptides (AMPs) could be a promising alternative. Fourteen hexapeptides were selected by using positional scanning of synthetic peptide combinatorial libraries. Each hexapeptide showed different antimicrobial spectrum against Bacillus, Pseudomonas, Xanthomonas, and Candida species. Intriguingly, BHC10 showed bactericidal activity exclusively on Xanthomonas citri subsp. citri (Xcc), while BHC7 was none-active exclusively against two Pseudomonas spp. at concentration of 100 μg/ml suggesting potential selectivity constrained in hexapeptide frame. Three hexapeptides, BHC02, 06 and 11, showed bactericidal activities against various Xcc strains at concentration of 10 μg/ml. When they were co-infiltrated with pathogens into citrus leaves the disease progress was suppressed significantly. Further study would be needed to confirm the actual disease control capacity of the selected hexapeptides. PMID:28167892

  13. Application of synthetic peptides for detection of anti-citrullinated peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Holm, Bettina Eide; Slot, Ole

    2016-01-01

    Anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis (RA) and represent an important tool for the serological diagnosis of RA. In this study, we describe ACPA reactivity to overlapping citrullinated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-derived peptides...... and that the presence of Epstein-Barr virus may play a role in the induction of these autoantibodies....

  14. Cationic Synthetic Peptides: Assessment of Their Antimicrobial Potency in Liquid Preserved Boar Semen

    OpenAIRE

    Stephanie Speck; Alexandre Courtiol; Christof Junkes; Margitta Dathe; Karin Müller; Martin Schulze

    2014-01-01

    Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional ...

  15. IMMUNOLOGICAL CHARACTERISTIC OF SYNTHETIC PEPTIDES SIMILAR TO ACTUAL HIV ANTIGEN DETERMINANTS

    Directory of Open Access Journals (Sweden)

    S. V. Korobova

    2016-01-01

    Full Text Available The development of HIV vaccine remains an important goal in prophylaxis and therapy of HIV/ AIDS epidemics. There are various approaches for development of а candidate vaccine based on induction of neutralizing antibodies and cell-mediated immunity. Synthetic peptides are considered promising vaccine antigens since they are capable of activating both humoral and cellular immune response. HIV-1 envelope gp120 is the target for neutralizing antiviral antibodies. The V3 region of the HIV-1 gp120 is highly immunogenic and important for the virus-coreceptor interaction. In a RV144 vaccine trial, the levels of vaccine-induced IgG antibodies recognizing V1V2 regions from multiple HIV-1 subtypes show inverse correlations with a risk for HIV-1 infection. Meanwhile, HIV is characterized by high diversity. The consensus and mosaic immunogens are complete but artificial proteins, which are computationally designed to elicit immune responses with improved cross-reactive broadness. We have been studied immunogenic properties of synthetic peptides derived from V1, V2, V3 loop regions of the consensus M HIV1 (CON-S sequence group of the gp 120 envelope protein and V3 loop derived from a Russian RUA022a2 isolate. These peptides specifically reacted to HIV-positive sera in ELISA, thus indicating their similarity to appropriate HIV proteins. The peptides proved to be weakly immunogenic. Therefore, Freund complete adjuvant was used to enhance peptide immunogenicity. To assess the immunogenicity, the mice were immunized with a peptide mixture. Antibodies have been developed to every peptide from the mixture, being, predominantly, of IgG isotype. The antibody titers depended on the length of peptide sequences. However, the sera from immunized mice did not have a HIV neutralizing activity. The serum neutralization was assessed by pseudovirus-based assay, using a molecular clone of virus isolates CAP 45.2.00.G3 and QH.209.14.M.EnvA2. The virus neutralization is a

  16. Interaction of antibodies to synthetic peptides of proNGF with in vitro synthesized NGF precursors.

    Science.gov (United States)

    Dicou, E

    1989-09-25

    Sera raised against three synthetic peptides that reproduce sequences of the pro-nerve growth factor (proNGF) protein were tested in immunoprecipitation experiments using in vitro translation products of SP6-directed NGF mRNA in a rabbit reticulocyte lysate. The interaction of these antibodies with bacterially synthesized chimeric preproNGF was also examined. Digestion of the translation products by the gamma-subunit generated the 22 and 18 kDa intermediates. A predominant 13 kDa intermediate was obtained after digestion of translation products in wheat germ extract. This is shown to be the N-terminal peptide by immunoprecipitation with an anti-peptide serum. These antibodies may be used to detect NGF precursor cleavage products in vivo.

  17. Surface chemical immobilization of bioactive peptides on synthetic polymers for cardiac tissue engineering.

    Science.gov (United States)

    Rosellini, Elisabetta; Cristallini, Caterina; Guerra, Giulio D; Barbani, Niccoletta

    2015-01-01

    The aim of this work was the development of new synthetic polymeric systems, functionalized by surface chemical modification with bioactive peptides, for myocardial tissue engineering. Polycaprolactone and a poly(ester-ether-ester) block copolymer synthesized in our lab, polycaprolactone-poly(ethylene oxide)-polycaprolactone (PCL-PEO-PCL), were used as the substrates to be modified. Two pentapeptides, H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) from fibronectin and H-Tyr-Ile-Gly-Ser-Arg-OH (YIGSR) from laminin, were used for the functionalization. Polymeric membranes were obtained by casting from solutions and then functionalized by means of alkaline hydrolysis and subsequent coupling of the bioactive molecules through 1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride/N-hydroxysuccinimide chemistry. The hydrolysis conditions, in terms of hydrolysis time, temperature, and sodium hydroxide concentration, were optimized for the two materials. The occurrence of the coupling reaction was demonstrated by infrared spectroscopy, as the presence on the functionalized materials of the absorption peaks typical of the two peptides. The peptide surface density was determined by chromatographic analysis and the distribution was studied by infrared chemical imaging. The results showed a nearly homogeneous peptide distribution, with a density above the minimum value necessary to promote cell adhesion. Preliminary in vitro cell culture studies demonstrated that the introduction of the bioactive molecules had a positive effect on improving C2C12 myoblasts growth on the synthetic materials.

  18. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Directory of Open Access Journals (Sweden)

    Nazila Amini

    2014-06-01

    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  19. Synthetic mimics of antimicrobial peptides--a versatile ring-opening metathesis polymerization based platform for the synthesis of selective antibacterial and cell-penetrating polymers.

    Science.gov (United States)

    Lienkamp, Karen; Tew, Gregory N

    2009-11-09

    Natural macromolecules exhibit an extensive arsenal of properties, many of which have proven difficult to recapitulate in simpler synthetic systems. Over the last couple of years, foldamers have emerged as one important step toward increased functionality in synthetic systems. While the great majority of work in this area has focused on folded structures, hence the name, more recent progress has centered on polymers that mimic protein function. These efforts have resulted in the design of relatively simple macromolecules; one example are the synthetic mimics of antimicrobial peptides (SMAMPs) that capture the central physicochemical features of their natural archetypes irrespective of the specific folded form. Here we present our recent efforts to create polymers which display biological activity similar to natural proteins, including antimicrobial and cell-penetrating peptides.

  20. Selection of glutamate-rich protein long synthetic peptides for vaccine development: antigenicity and relationship with clinical protection and immunogenicity

    DEFF Research Database (Denmark)

    Theisen, M; Dodoo, D; Toure-Balde, A;

    2001-01-01

    Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat reg...... antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination against P. falciparum malaria....

  1. Comparison of adjuvant formulations for cytotoxic T cell induction using synthetic peptides.

    Science.gov (United States)

    Hioe, C E; Qiu, H; Chend, P D; Bian, Z; Li, M L; Li, J; Singh, M; Kuebler, P; McGee, P; O'Hagan, D; Zamb, T; Koff, W; Allsopp, C; Wang, C Y; Nixon, D F

    1996-04-01

    We have investigated the capacity of synthetic peptides delivered in different adjuvant formulations to induce cytotoxic T lymphocyte (CTL) responses to a class I H-2Kd-restricted Plasmodium berghei circumsporozoite epitope, CS 252-260. Using three immunogen formulations: soybean emulsion; Montanide ISA720; and lipopeptide (P3-CS), we first evaluated the effects of immunization routes on CTL induction. No CTL response was induced in mice immunized s.c. or i.p. with CS peptide formulated in soybean emulsion. In contrast, immunization with lipopeptide P3-CS either s.c. or i.p. effectively primed for CTL. Interestingly, CS peptide emulsified in Montanide ISA720 induced a CTL response only when delivered s.c. and not i.p., indicating the critical influence of immunization routes on CTL induction. We then compared the effectiveness of eight adjuvant formulations to induce CTL response following a single s.c. immunization. Notably, lipopeptide P3-CS and CS peptide admixed with P3 or POE lipid molecules stimulated a vigorous CTL response. However, only mice immunized with P3-CS and CS peptide admixed with P3 molecule generated long-lived CTL which persisted in vivo for 5 months. Thus, based on a simultaneous comparison of the different adjuvant formulations, we demonstrated that the conjugated and unconjugated P3 lipopeptides were the most effective immunogens for eliciting primary and memory CTL in mice.

  2. Oral and parenteral immunization with synthetic retro-inverso peptides induce antibodies that cross-react with native peptides and parent antigens.

    Science.gov (United States)

    Fischer, Peter; Comis, Alfio; Tyler, Margaret; Howden, Merlin

    2007-06-01

    The objective of this study was to determine whether certain retro-inverso peptides have the potential to act as synthetic vaccines in mice, when immunized by injection or orally. Immunization of mice parenterally with conjugates of three such retro-inverso peptides and orally with the unconjugated peptides elicited generally high titres of anti-peptide antibodies. Antibodies against the same three peptides cross-reacted by binding strongly in ELISA to the native peptides and vice versa, regardless of the mode of immunization. Antibodies against a retro-inverso diphtheria peptide also reacted strongly with diphtheria toxin. Seven of 8 mice, immunized by injection of the conjugate of a retro-inverso derivative of robustoxin [a lethal spider (Atrax robustus) venom toxin] were protected from challenge involving injection with twice the minimum lethal dose of A. robustus venom containing the toxin.

  3. Transgenic Brassica juncea plants expressing MsrA1, a synthetic cationic antimicrobial peptide, exhibit resistance to fungal phytopathogens.

    Science.gov (United States)

    Rustagi, Anjana; Kumar, Deepak; Shekhar, Shashi; Yusuf, Mohd Aslam; Misra, Santosh; Sarin, Neera Bhalla

    2014-06-01

    Cationic antimicrobial peptides (CAPs) have shown potential against broad spectrum of phytopathogens. Synthetic versions with desirable properties have been modeled on these natural peptides. MsrA1 is a synthetic chimera of cecropin A and melittin CAPs with antimicrobial properties. We generated transgenic Brassica juncea plants expressing the msrA1 gene aimed at conferring fungal resistance. Five independent transgenic lines were evaluated for resistance to Alternaria brassicae and Sclerotinia sclerotiorum, two of the most devastating pathogens of B. juncea crops. In vitro assays showed inhibition by MsrA1 of Alternaria hyphae growth by 44-62 %. As assessed by the number and size of lesions and time taken for complete leaf necrosis, the Alternaria infection was delayed and restricted in the transgenic plants with the protection varying from 69 to 85 % in different transgenic lines. In case of S. sclerotiorum infection, the lesions were more severe and spread profusely in untransformed control compared with transgenic plants. The sclerotia formed in the stem of untransformed control plants were significantly more in number and larger in size than those present in the transgenic plants where disease protection of 56-71.5 % was obtained. We discuss the potential of engineering broad spectrum biotic stress tolerance by transgenic expression of CAPs in crop plants.

  4. Aspartate-bond isomerization affects the major conformations of synthetic peptides.

    Science.gov (United States)

    Szendrei, G I; Fabian, H; Mantsch, H H; Lovas, S; Nyéki, O; Schön, I; Otvos, L

    1994-12-15

    The aspartic acid bond changes to an beta-aspartate bond frequently as a side-reaction during peptide synthesis and often as a post-translational modification of proteins. The formation of beta-asparate bonds is reported to play a major role not only in protein metabolism, activation and deactivation, but also in pathological processes such as deposition of the neuritic plaques of Alzheimer's disease. Recently, we reported how conformational changes following the aspartic-acid-bond isomerization may help the selective aggregation and retention of the amyloid beta peptide in affected brains (Fabian et al., 1994). In the current study we used circular dichroism, Fourier-transform infrared spectroscopy, and molecular modeling to characterize the general effect of the beta-aspartate-bond formation on the conformation of five sets of synthetic model peptides. Each of the non-modified, parent peptides has one of the major secondary structures as the dominant spectroscopically determined conformation: a type I beta turn, a type II beta turn, short segments of alpha or 3(10) helices, or extended beta strands. We found that both types of turn structures are stabilized by the aspartic acid-bond isomerization. The isomerization at a terminal position did not affect the helix propensity, but placing it in mid-chain broke both the helix and the beta-pleated sheet with the formation of reverse turns. The alteration of the geometry of the lowest energy reverse turn was also supported by molecular dynamics calculations. The tendency of the aspartic acid-bond isomerization to stabilize turns is very similar to the effect of incorporating sugars into synthetic peptides and suggests a common feature of these post-translational modifications in defining the secondary structure of protein fragments.

  5. Inhalation therapy with the synthetic TIP-like peptide AP318 attenuates pulmonary inflammation in a porcine sepsis model

    OpenAIRE

    Hartmann, Erik K; Ziebart, Alexander; Thomas, Rainer; Liu, Tanghua; Schad, Arno; Tews, Martha; Moosmann, Bernd; Kamuf, Jens; Duenges, Bastian; Thal, Serge C.; David, Matthias

    2015-01-01

    Background The lectin-like domain of TNF-α can be mimicked by synthetic TIP peptides and represents an innovative pharmacologic option to treat edematous respiratory failure. TIP inhalation was shown to reduce pulmonary edema and improve gas exchange. In addition to its edema resolution effect, TIP peptides may exert some anti-inflammatory properties. The present study therefore investigates the influence of the inhaled TIP peptide AP318 on intrapulmonary inflammatory response in a porcine mo...

  6. DNA-like double helix formed by peptide nucleic acid

    DEFF Research Database (Denmark)

    Wittung, P; Nielsen, Peter E.; Buchardt, O;

    1994-01-01

    Although the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic...... acid (PNA) is a DNA analogue with a backbone consisting of N-(2-aminoethyl)glycine units (Fig. 1) which has been shown to mimic DNA in forming Watson-Crick complementary duplexes with normal DNA. Using circular dichroism spectroscopy we show here that two complementary PNA strands can hybridize to one...

  7. Synthetic Smac Peptide Enhances Chemo-sensitivity of Bladder Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Jing WANG; Fuqing ZENG; Liang WANG; Zhaohui ZHU; Guosong JIANG

    2008-01-01

    The effects of synthetic Smac peptide (SmacN7) on chemotherapeutic sensitivity of bladder cancer cells were investigated. SmacN7 penetratin peptide was synthesized and delivered into T24 cells. MTT assay was used to evaluate the viability of T24 cells induced by low-dosage of MMC. Flow cytometry was used to analyze the proportions of apoptosis. Western blot was used to detect the expression of XIAP and Caspase-3. The activity of Caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined. The results showed that SmacN7 penetratin peptide could successfully interact with endogenous XIAP, increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time-dependent manner. An obvious down-regulation of XIAP expression and up-regulation of Caspase-3 was identified by Western blot. The activity of Caspase-3 in experimental group was significantly increased as compared with that in the control group. As compared with MMC group, the viability of T24 cells in SmacN7 penetratin peptide + MMC group was markedly decreased to 2.22 and 3.61 folds at 24h and 48h respectively. It was concluded that SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. These findings indicate that SmacN7 penetratin peptide may be a very promising ageut for bladder cancer treatment when used in combination with chemotherapy.

  8. Phase-I study of synthetic muc1 peptides in breast-cancer.

    Science.gov (United States)

    Xing, P; Michael, M; Apostolopoulos, V; Prenzoska, J; Marshall, C; Bishop, J; McKenzie, I

    1995-06-01

    Exposed peptides in the repeat (VNTR) protein core of human mucin 1 (MUC1) could be a target for immunotherapy, as it is highly immunogenic in mice and a human cytotoxic T lymphocytes to MUC1 recognise the peptide. On this basis 13 patients were immunised with a MUC1 peptide - a 20 amino acids dimer conjugated with diphtheria toroid as carrier. In patients with established breast cancer increasing doses (0.15 mg, 0.25 mg, 0.5 mg, 1.0 mg) were used at 2 week intervals (3 injections). No toxicity was found, other than for DTH reaction to the diphtheria carrier; weak antibody and T cell proliferative responses were seen and weak DTH reaction in proportion of patients. The MUC1 peptide appears to be safe but in the form used was not highly immunogenic.

  9. Boronic acid functionalized peptidyl synthetic lectins: Combinatorial library design, peptide sequencing, and selective glycoprotein recognition

    Science.gov (United States)

    Bicker, Kevin L.; Sun, Jing; Lavigne, John J.; Thompson, Paul R.

    2011-01-01

    Aberrant glycosylation of cell membrane and secreted glycoproteins is a hallmark of various disease states, including cancer. The natural lectins currently used in the recognition of these glycoproteins are costly, difficult to produce, and unstable towards rigorous use. Herein we describe the design and synthesis of several boronic acid functionalized peptide-based synthetic lectin (SL) libraries, as well as the optimized methodology for obtaining peptide sequences of these SLs. SL libraries were subsequently used to identify SLs with as high as 5-fold selectivity for various glycoproteins. SLs will inevitably find a role in cancer diagnositics, given that they do not suffer from the drawbacks of natural lectins and that the combinatorial nature of these libraries allows for the identification of an SL for nearly any glycosylated biomolecule. PMID:21405093

  10. Synthetic peptides derived from ribosomal proteins of Leishmania spp. in mucocutaneous leishmaniasis: Diagnostic usefulness.

    Science.gov (United States)

    Florez, Magda Melissa; de Oliveira, Camila Indiani; Puerta, Concepción; Guzmán, Fanny; Ayala, Martha; Montoya, Gladis; Delgado, Gabriela

    2017-07-28

    The serological diagnostic methods currently available for mucocutaneous leishmaniasis (MCL) lack specificity when complete parasites are used; however, such specificity increases when protein fractions are used. Ribosomal proteins have been reported to induce antibodies in animal and humans infected with the parasite, making them a worth candidate to assess its diagnosis potential. This study was thus aimed at evaluating synthetic peptides derived from Leishmania braziliensis ribosomal proteins S25 and S5 as antigen candidates for diagnosing MCL by ELISA; 4 of these 21 peptides (P4, P6, P19 and P21) had the greatest sensitivity (21.7%, 13.04%, 20% and 20%, respectively) as well as having 95%, 100%, 100% and 82.5% specificity, respectively. The study revealed the limited usefulness of the peptides being studied as a diagnostic tool in the conditions used here, because its low sensitivity, but it is worth highlighting that the use of peptides as antigen in the serodiagnosis of MCL may overcome the cross reaction presented with other antigens, thus avoiding false positives. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. NMR structure of mussel mytilin, and antiviral-antibacterial activities of derived synthetic peptides

    OpenAIRE

    Roch, Philippe; Yang, Y.; Toubiana, Mylene; Aumelas, A

    2008-01-01

    Mytilin is a 34-residue antibacterial peptide from the mussel Mytilus galloprovincialis, which in addition possesses in vitro antiviral activity. The three-dimensional solution structure of the synthetic mytilin was established by using 1H NMR and consists of the common cysteine-stabilized alpha beta motif close to the one observed in the mussel defensin MGD-1. Mytilin is characterized by 8 cysteines engaged in four disulfide bonds (2-27, 6-29, 10-31, and 15-34) only involving the beta-strand...

  12. Anticancer activity of a synthetic peptide derived from harmoniasin, an antibacterial peptide from the ladybug Harmonia axyridis.

    Science.gov (United States)

    Kim, In-Woo; Lee, Joon Ha; Kwon, Young-Nam; Yun, Eun-Young; Nam, Sung-Hee; Ahn, Mi-Young; Kang, Dong-Chul; Hwang, Jae Sam

    2013-08-01

    Harmoniasin is a defensin-like antimicrobial peptide identified from the ladybug Harmonia axyridis. Among the synthetic homodimer peptide analogues derived from harmoniasin, HaA4 has been found to have antibacterial activity without hemolytic activity. In this study, we investigated whether HaA4 has anticancer activity against human leukemia cell lines such as U937 and Jurkat cells. HaA4 manifested cytotoxicity and decreased the cell viability of U937 and Jurkat cells in MTS assay and LDH release assay. We found that HaA4 induced apoptotic and necrotic cell death of the leukemia cells using flow cytometric analysis, acridine orange/ethidium bromide staining and nucleosomal fragmentation of genomic DNA. Activation of caspase-7 and -9 and fragmentation of poly (ADP-ribose) polymerase was detected in the HaA4-treated leukemia cells, suggesting induction of a caspase-dependent apoptosis pathway by HaA4. Caspase-dependent apoptosis was further confirmed by reversal of the HaA4-induced viability reduction by treatment of Z-VAD-FMK, a pan-caspase inhibitor. In conclusion, HaA4 caused necrosis and caspase-dependent apoptosis in both U937 and Jurkat leukemia cells, which suggests potential utility of HaA4 as a cancer therapeutic agent.

  13. Synthetic Random Copolymers as a Molecular Platform To Mimic Host-Defense Antimicrobial Peptides.

    Science.gov (United States)

    Takahashi, Haruko; Caputo, Gregory A; Vemparala, Satyavani; Kuroda, Kenichi

    2017-05-17

    Synthetic polymers have been used as a molecular platform to develop host-defense antimicrobial peptide (AMP) mimetics which are effective in killing drug-resistant bacteria. In this topical review, we will discuss the AMP-mimetic design and chemical optimization strategies as well as the biological and biophysical implications of AMP mimicry by synthetic polymers. Traditionally, synthetic polymers have been used as a chemical means to replicate the chemical functionalities and physicochemical properties of AMPs (e.g., cationic charge, hydrophobicity) to recapitulate their mode of action. However, we propose a new perception that AMP-mimetic polymers are an inherently bioactive platform as whole molecules, which mimic more than the side chain functionalities of AMPs. The tunable nature and chemical simplicity of synthetic random polymers facilitate the development of potent, cost-effective, broad-spectrum antimicrobials. The polymer-based approach offers the potential for many antimicrobial applications to be used directly in solution or attached to surfaces to fight against drug-resistant bacteria.

  14. Chitosan-Poly (I:C-PADRE Based Nanoparticles as Delivery Vehicles for Synthetic Peptide Vaccines

    Directory of Open Access Journals (Sweden)

    Jorge F. Correia-Pinto

    2015-09-01

    Full Text Available The safety and precision of peptide antigens has prompted the search for adjuvants capable of increasing the immune response against these intrinsically poorly immunogenic antigens. The integration of both immunostimulants and peptide antigens within nanometric delivery systems for their co-delivery to immune cells is a promising vaccination strategy. With this in mind, the potential synergistic effect of the immunostimulant poly (I:C (pIC and a T-Helper peptide (PADRE, integrated into a chitosan (CS based nanostructure, was explored. The value of this nanostructured combination of materials was assessed for a peptide antigen (1338aa derived from the HPV-16 L2 protein. These nanoparticles, produced by ionic gelation technique, exhibited a nanometric size (<300 nm, a high positive surface charge (>40 mV and high pIC association efficiency (>96%. They also showed capacity for the association of both the 1338aa and PADRE peptides. The influence of the presence of pIC and PADRE in the nanocomposition, as well as that of the peptide presentation form (encapsulated versus surface adsorbed on the antibody induction was evaluated in a preliminary in vivo study. The data obtained highlights the possibility to engineer nanoparticles through the rational combination of a number of adjuvant molecules together with the antigen.

  15. Chitosan-Poly (I:C)-PADRE Based Nanoparticles as Delivery Vehicles for Synthetic Peptide Vaccines.

    Science.gov (United States)

    Correia-Pinto, Jorge F; Csaba, Noemi; Schiller, John T; Alonso, Maria J

    2015-01-01

    The safety and precision of peptide antigens has prompted the search for adjuvants capable of increasing the immune response against these intrinsically poorly immunogenic antigens. The integration of both immunostimulants and peptide antigens within nanometric delivery systems for their co-delivery to immune cells is a promising vaccination strategy. With this in mind, the potential synergistic effect of the immunostimulant poly (I:C) (pIC) and a T-Helper peptide (PADRE), integrated into a chitosan (CS) based nanostructure, was explored. The value of this nanostructured combination of materials was assessed for a peptide antigen (1338aa) derived from the HPV-16 L2 protein. These nanoparticles, produced by ionic gelation technique, exhibited a nanometric size (40 mV) and high pIC association efficiency (>96%). They also showed capacity for the association of both the 1338aa and PADRE peptides. The influence of the presence of pIC and PADRE in the nanocomposition, as well as that of the peptide presentation form (encapsulated versus surface adsorbed) on the antibody induction was evaluated in a preliminary in vivo study. The data obtained highlights the possibility to engineer nanoparticles through the rational combination of a number of adjuvant molecules together with the antigen.

  16. Human T-cell recognition of synthetic peptides representing conserved and variant sequences from the merozoite surface protein 2 of Plasmodium falciparum.

    Science.gov (United States)

    Theander, T G; Hviid, L; Dodoo, D; Afari, E A; Jensen, J B; Rzepczyk, C M

    1997-06-01

    Merozoite surface protein 2 (MSP2) is a malaria vaccine candidate currently undergoing clinical trials. We analyzed the peripheral blood mononuclear cell (PBMC) response to synthetic peptides corresponding to conserved and variant regions of the FCQ-27 allelic form of MSP2 in Ghanaian individuals from an area of hyperendemic malaria transmission and in Danes without exposure to malaria. PBMC from 20-39% of Ghanaians responded to each of the peptides by proliferation and 29-36% had PBMC which produced interferon-gamma (IFN-gamma) in response to peptide stimulation. In Danes, there was no proliferation to two of the peptides and only PBMC from 5% of the individuals proliferated to the other three peptides. IFN-gamma production was not detected to any peptide. In both Danes and Ghanaians in only a few instances was IL-4 detected in the PBMC cultures. Overall PBMC from 79% of the Ghanaians responded by proliferation and/or cytokine secretion to at least one of three peptides tested, whereas responses were only observed in 14% of Danes (P = 0.002). These data suggest that the Ghanaians had expanded peripheral blood T-cell populations recognizing the peptides as a result of natural infection. The findings are encouraging for the development of a vaccine based on these T-epitope containing regions of MSP2, as the peptides were broadly recognized suggesting that they can bind to diverse HLA alleles and also because they include conserved MSP2 sequences. Immunisation with a vaccine construct incorporating the sequences present in these peptides could thus be expected to be immunogenic in a high percentage of individuals and lead to the establishment of memory T-cells, which can be boosted through natural infection.

  17. Detection of Cancer Cell Death Mediated by a Synthetic Granzyme B-like Peptide Fluorescent Conjugate and the same Peptide Binding in Bacteria.

    Science.gov (United States)

    Lo, Wai Chun Jennifer; Luther, Donald Gene

    2014-03-01

    Granzyme-mediated apoptosis, supported by pore-forming perforin, plays an important role in CD8+ T lymphocytes (CTL)-dependent cellular immunity protection against both cancer and viral infection. Quantitative and qualitative problems with CTL are potential contributing factors to disease progression. The feasibility of developing CTL-independent cellular immunity is desired but must first overcome the barrier of CTL-independent target cell recognition. Granzyme B with its strong pro-apoptotic activity in many different target cells is investigated for use in the CTL-independent cellular immunity approach, and granzyme B or its bioactive peptides without the enzymatic activity are more desirable for use. Native granzyme B with enzymatic activity is usually investigated in cancer cells for its mediation of apoptosis by detection of DNA fragmentation. Detection of cell death mediated by such peptides in cancer cells is needed to demonstrate the potential therapeutic purposes. We show with never-before-seen microscopic images using fluorescence microscopy that a synthetic granzyme B-like peptide fluorescent conjugate (GP1R) can: 1) mediate cell death of different cancer cells via membrane extrusion, 2) bind to constitutively expressed binding targets in different cancer cells and bacteria, and 3) promote bacterial phagocytosis. The putative binding targets may serve as a universal pathologic biomarker detectable by GP1R. Our data taken together demonstrate the potential applications of GP1R for use in CTL-independent target cell recognition and target cell death induction. It may lead to development of rapid targeted detection and new treatment of cancer, viral and bacterial infections. The new treatment may show mutual benefits for two or more diseases.

  18. Viral replication modulated by synthetic peptide derived from hepatitis B virus X protein

    Institute of Scientific and Technical Information of China (English)

    Chang-Zheng Song; Qing-Wei Wang; Chang-Cheng Song; Zeng-Liang Bai

    2004-01-01

    AIM: A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx.METHODS: We synthesized the fully conserved peptide (CP)with nine residues, FVLGGCRHK. HBV-producing 2.2.15 cells were treated with or without 3.5 μM CP for 36 hours.Quantitative detection of viral DNA was performed by realtime PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence.Flow cytometry was performed to detect cell cycle and apoptosis.RESULTS: Both extracellular and intracellular copies of HBV DNA per ml were significantly increased atter incubation with 3.5 μM of CP. HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control cells. CP infiuenced negatively the extracellular viral gene products, and 3.5 μM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5 μM CP could efficiently increase the level of intracellular HBeAg.Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2.15 cells with or without CP.CONCLUSION: Together with the resulte generated from the synthetic peptide, we address that the conserved region,a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.

  19. Spermicidal efficacy of VRP, a synthetic cationic antimicrobial peptide, inducing apoptosis and membrane disruption.

    Science.gov (United States)

    Ghosh, Prasanta; Bhoumik, Arpita; Saha, Sudipta; Mukherjee, Sandipan; Azmi, Sarfuddin; Ghosh, Jimut K; Dungdung, Sandhya R

    2017-04-14

    Presently available contraceptives are mostly hormonal or detergent in nature with numerous side effects like irritation, lesion, inflammation in vagina, alteration of body homeostasis, etc. Antimicrobial peptides with spermicidal activity but without adverse effects may be suitable alternatives. In the present study, spermicidal activity of a cationic antimicrobial peptide VRP on human spermatozoa has been elucidated. Progressive forward motility of human spermatozoa was instantly stopped after 100 μM VRP treatment and at 350 μM, all kinds of sperm motility ceased within 20 s as assessed by the Sander-Cramer assay. The spermicidal effect was confirmed by eosin-nigrosin assay and HOS test. VRP treatment (100 μM) in human spermatozoa induced both the intrinsic and extrinsic pathways of apoptosis. TUNEL assay showed VRP treatment significantly disrupted the DNA integrity and changed the mitochondrial membrane permeability as evident from MPTP assay. AFM and SEM results depicted ultra structural changes including disruption of the acrosomal cap and plasma membrane of the head and midpiece region after treatment with 350 μM VRP. MTT assay showed after treatments with 100 and 350 μM of VRP for 24 hr, a substantial amount of Lactobacillus acidophilus (about 90% and 75%, respectively) remained viable. Hence, VRP being a small synthetic peptide with antimicrobial and spermicidal activity but tolerable to normal vaginal microflora, may be a suitable target for elucidating its contraceptive potentiality. © 2017 Wiley Periodicals, Inc.

  20. Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean.

    Directory of Open Access Journals (Sweden)

    Bing Wang

    Full Text Available Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL. A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed.

  1. Biomimetic modification of synthetic hydrogels by incorporation of adhesive peptides and calcium phosphate nanoparticles: in vitro evaluation of cell behavior

    Directory of Open Access Journals (Sweden)

    M Bongio

    2011-12-01

    Full Text Available The ultimate goal of this work was to develop a biocompatible and biomimetic in situ crosslinkable hydrogel scaffold with an instructive capacity for bone regenerative treatment. To this end, synthetic hydrogels were functionalized with two key components of the extracellular matrix of native bone tissue, i.e. the three-amino acid peptide sequence RGD (which is the principal integrin-binding domain responsible for cell adhesion and survival of anchorage-dependent cells and calcium phosphate (CaP nanoparticles in the form of hydroxyapatite (which are similar to the inorganic phase of bone tissue. Rat bone marrow osteoblast-like cells (OBLCs were encapsulated in four different biomaterials (plain oligo(poly(ethylene glycol fumarate (OPF, RGD-modified OPF, OPF enriched with CaP nanoparticles and RGD-modified OPF enriched with CaP nanoparticles and cell survival, cell spreading, proliferation and mineralized matrix formation were determined via cell viability assay, histology and biochemical analysis for alkaline phosphatase activity and calcium. This study showed that RGD peptide sequences promoted cell spreading in OPF hydrogels and hence play a crucial role in cell survival during the early stage of culture, whereas CaP nanoparticles significantly enhanced cell-mediated hydrogel mineralization. Although cell spreading and proliferation activity were inhibited, the combined effect of RGD peptide sequences and CaP nanoparticles within OPF hydrogel systems elicited a better biological response than that of the individual components. Specifically, both a sustained cell viability and mineralized matrix production mediated by encapsulated OBLCs were observed within these novel biomimetic composite systems.

  2. Antagonism of human formyl peptide receptor 1 with natural compounds and their synthetic derivatives.

    Science.gov (United States)

    Schepetkin, Igor A; Khlebnikov, Andrei I; Kirpotina, Liliya N; Quinn, Mark T

    2016-08-01

    Formyl peptide receptor 1 (FPR1) regulates a wide variety of neutrophil functional responses and plays an important role in inflammation and the pathogenesis of various diseases. To date, a variety of natural and synthetic molecules have been identified as FPR1 ligands. Here, we review current knowledge on natural products and natural product-inspired small molecules reported to antagonize and/or inhibit the FPR1-mediated responses. Based on this literature, additional screening of selected commercially available natural compounds for their ability to inhibit fMLF-induced Ca(2+) mobilization in human neutrophils and FPR1 transfected HL-60 cells, and pharmacophore modeling, natural products with potential as FPR1 antagonists are considered and discussed in this review. The identification and characterization of natural products that antagonize FPR1 activity may have potential for the development of novel therapeutics to limit or alter the outcome of inflammatory processes. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Synthetic protein scaffolds based on peptide motifs and cognate adaptor domains for improving metabolic productivity

    Directory of Open Access Journals (Sweden)

    Anselm H.C. Horn

    2015-11-01

    Full Text Available The efficiency of many cellular processes relies on the defined interaction among different proteins within the same metabolic or signaling pathway. Consequently, a spatial colocalization of functionally interacting proteins has frequently emerged during evolution. This concept has been adapted within the synthetic biology community for the purpose of creating artificial scaffolds. A recent advancement of this concept is the use of peptide motifs and their cognate adaptor domains. SH2, SH3, GBD, and PDZ domains have been used most often in research studies to date. The approach has been successfully applied to the synthesis of a variety of target molecules including catechin, D-glucaric acid, H2, hydrochinone, resveratrol, butyrate, gamma-aminobutyric acid, and mevalonate. Increased production levels of up to 77-fold have been observed compared to non-scaffolded systems. A recent extension of this concept is the creation of a covalent linkage between peptide motifs and adaptor domains, which leads to a more stable association of the scaffolded systems and thus bears the potential to further enhance metabolic productivity.

  4. Evaluation of Suppression of Synthetic Paraffinic Kerosene (SPK) Fuel Fires with Aqueous Film Forming Foam (AFFF)

    Science.gov (United States)

    2009-12-01

    FIRES WITH AQUEOUS FILM FORMING FOAM ( AFFF ...performance of Aqueous Film-Forming Foam ( AFFF ) on synthetic fuel fires to aid Air Force firefighters’ response to an incident. Results show that AFFF can...hydrocarbons. Aqueous Film Forming Foam ( AFFF ) is used by Air Force fire departments to extinguish fuel spill fires involving jet fuel (JP-8), diesel,

  5. [Immunization with synthetic peptide anti-tick Rhipicephalus (Boophilus) microplus SBm7462 via mucosal routes].

    Science.gov (United States)

    Carvalho, Gabriel D; Peconick, Ana P; Medeiros, Carla L; Vargas-V, Marlene I; Patarroyo, Joaquín H

    2008-09-01

    The mucosal immunization consists on antigen administration in these surfaces it is a not invasive method, inductor of local and systemic immune response. This work evaluated the immune response of the synthetic vaccine anti-tick Rhipicephalus (Boophilus) microplus SBm7462®, via oral and nasal routes. Were used 60 BALB/c mice, divided in three groups of 20 animals each (I: oral immunization; II: nasal immunization; III: animals not immunized). Nine and 15 days after the first and the second immunization was collected, separately for each group, T cells from the immunized animals, cultivating them per 10 days. After the incubation, were determined the percentage of cellular viability and the specific nature of this T cells, which had held as memory T cell. The averages of T cell SBm7462-reagents had been submitted to the analysis of variance and comparison for Tukey test, 5% of probability. Group II presented higher cellular proliferation "in vitro". For ELISA test, were positive only two animals in group I and eight in group II. The oral and nasal routs alternatively viable for immunization with the synthetic peptide SBm7462®, however require greater number of doses to induce responses with high levels of immunoglobulins.

  6. Generation of recombinant antibodies to rat GABAA receptor subunits by affinity selection on synthetic peptides.

    Directory of Open Access Journals (Sweden)

    Sujatha P Koduvayur

    Full Text Available The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

  7. Functional and structural characterization of a synthetic peptide representing the N-terminal domain of prokaryotic pyruvate dehydrogenase

    NARCIS (Netherlands)

    Hengeveld, A.F.; Mierlo, van C.P.M.; Hooven, van den H.W.; Visser, A.J.W.G.; Kok, de A.

    2002-01-01

    A synthetic peptide (Nterm-E1p) is used to characterize the structure and function of the N-terminal region (amino acid residues 4-45) of the pyruvate dehydrogenase component (E1p) from the pyruvate dehydrogenase multienzyme complex (PDHC) from Azotobacter vinelandii. Activity and binding studies es

  8. Enhanced Antimicrobial Activity of AamAP1-Lysine, a Novel Synthetic Peptide Analog Derived from the Scorpion Venom Peptide AamAP1

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    Ammar Almaaytah

    2014-04-01

    Full Text Available There is great interest in the development of antimicrobial peptides as a potentially novel class of antimicrobial agents. Several structural determinants are responsible for the antimicrobial and cytolytic activity of antimicrobial peptides. In our study, a new synthetic peptide analog, AamAP1-Lysine from the naturally occurring scorpion venom antimicrobial peptide AamAP1, was designed by modifying the parent peptide in order to increase the positive charge and optimize other physico-chemical parameters involved in antimicrobial activity. AamAP1-Lysine displayed potent antibacterial activity against Gram-positive and Gram-negative bacteria. The minimum inhibitory concentration was in the range of 5 to 15 µM with a 10 fold increase in potency over the parent peptide. The hemolytic and antiproliferative activity of AamAP1-Lysine against eukaryotic mammalian cells was minimal at the concentration range needed to inhibit bacterial growth. The antibacterial mechanism analysis indicated that AamAP1-Lysine is probably inducing bacterial cell death through membrane damage and permeabilization determined by the release of β-galactosidase enzyme from peptide treated E. coli cells. DNA binding studies revealed that AamAP1-Lysine caused complete retardation of DNA migration and could display intracellular activities in addition to the membrane permeabilization mode of action reported earlier. In conclusion, AamAP1-Lysine could prove to be a potential candidate for antimicrobial drug development in future studies.

  9. Immunization of rabbits with synthetic peptides derived from a highly conserved β-sheet epitope region underneath the receptor binding site of influenza A virus

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    Ideno S

    2013-11-01

    forming the β-sheet structure. Both peptides were then coupled to keyhole limpet hemocyanin, and the peptides, alone or in combination, were used to immunize rabbits. The resulting antibody responses were examined by enzyme-linked immunosorbent assay. The upper peptide, but not the lower peptide, elicited antibodies that were reactive to HA. Interestingly, the use of both peptides together could elicit antibodies with a higher reactivity to HA than either peptide alone. The antibodies were found to react to HA at the N-terminus of the upper peptide, which is exposed at the surface of trimeric HA on influenza virions. Discussion: The higher production of HA-reactive antibodies following immunization with both peptides suggests that the upper peptide forms the effective epitope structure in the binding state, and the lower peptide enhances the production of HA antibodies. This study could be the first step towards the development of pandemic viral vaccines that can be produced within short time periods. Keywords: influenza A, hemagglutinin, epitope, synthetic peptide, rabbit

  10. Immunodominant epitopes mapped by synthetic peptides on the capsid protein of avian hepatitis E virus are non-protective.

    Science.gov (United States)

    Guo, Hailong; Zhou, E M; Sun, Z F; Meng, X J

    2008-03-01

    Avian hepatitis E virus (avian HEV) was recently discovered in chickens with hepatitis-splenomegaly syndrome in the United States. The open reading frame 2 (ORF2) protein of avian HEV has been shown to cross-react with human and swine HEV ORF2 proteins, and immunodominant antigenic epitopes on avian HEV ORF2 protein were identified in the predicted antigenic domains by synthetic peptides. However, whether these epitopes are protective against avian HEV infection has not been investigated. In this study, groups of chickens were immunized with keyhole limpet hemocyanin (KLH)-conjugated peptides and recombinant avian HEV ORF2 antigen followed by challenge with avian HEV virus to assess the protective capacity of these peptides containing the epitopes. While avian HEV ORF2 protein showed complete protection against infection, viremia and fecal virus shedding were found in all peptide-immunized chickens. Using purified IgY from normal, anti-peptide, and anti-avian HEV ORF2 chicken sera, an in-vitro neutralization and in-vivo monitoring assay was performed to further evaluate the neutralizing ability of anti-peptide IgY. Results showed that none of the anti-peptide IgY can neutralize avian HEV in vitro, as viremia, fecal virus shedding, and seroconversion appeared similarly in chickens inoculated with avian HEV mixed with anti-peptide IgY and chickens inoculated with avian HEV mixed with normal IgY. As expected, chickens inoculated with the avian HEV and anti-avian HEV ORF2 IgY mixture did not show detectable avian HEV infection. Taken together, the results of this study demonstrated that immunodominant epitopes on avian HEV ORF2 protein identified by synthetic peptides are non-protective, suggesting protective neutralizing epitope on avian HEV ORF2 may not be linear as is human HEV.

  11. Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction

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    Takahiro Ochiya

    2015-01-01

    Full Text Available The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy.

  12. Selective detection of carbohydrates and their peptide conjugates by ESI-MS using synthetic quaternary ammonium salt derivatives of phenylboronic acids.

    Science.gov (United States)

    Kijewska, Monika; Kuc, Adam; Kluczyk, Alicja; Waliczek, Mateusz; Man-Kupisinska, Aleksandra; Lukasiewicz, Jolanta; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2014-06-01

    We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation.

  13. Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae.

    Science.gov (United States)

    Rebollar, Aarón; Marcos, Jose F; López-García, Belén

    2014-11-01

    The rice blast disease caused by Magnaporthe oryzae is one of the most devastating diseases of cultivated rice. One of the most important stages in the infective cycle of M. oryzae is the formation of the dome-shaped structure called appressorium. The purpose of the present study was to identify novel peptides to control the rice blast disease by blocking the appressorium formation through screening of a synthetic peptide combinatorial library. As result of the screening, a set of 29 putative bioactive peptides were identified, synthesized and assayed in comparison with the previously identified peptide PAF104. The peptides MgAPI24, MgAPI40 and MgAPI47 showed improved inhibitory activity on the M. oryzae appressorium formation. Our data show that these peptides have a differential effect on two developmental structures: appressoria and appressorium-like structures. Antimicrobial assays against M. oryzae and other non-target microorganisms showed a weak or no toxicity of these peptides, demonstrating their specific activity blocking the appressorium formation. Therefore, the outcome of this research would be useful in the development of novel target-oriented peptides to use in plant protection.

  14. Synthetic osteogenic extracellular matrix formed by coated silicon dioxide nanosprings

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    Hass Jamie L

    2012-01-01

    Full Text Available Abstract Background The design of biomimetic materials that parallel the morphology and biology of extracellular matrixes is key to the ability to grow functional tissues in vitro and to enhance the integration of biomaterial implants into existing tissues in vivo. Special attention has been put into mimicking the nanostructures of the extracellular matrix of bone, as there is a need to find biomaterials that can enhance the bonding between orthopedic devices and this tissue. Methods We have tested the ability of normal human osteoblasts to propagate and differentiate on silicon dioxide nanosprings, which can be easily grown on practically any surface. In addition, we tested different metals and metal alloys as coats for the nanosprings in tissue culture experiments with bone cells. Results Normal human osteoblasts grown on coated nanosprings exhibited an enhanced rate of propagation, differentiation into bone forming cells and mineralization. While osteoblasts did not attach effectively to bare nanowires grown on glass, these cells propagated successfully on nanosprings coated with titanium oxide and gold. We observed a 270 fold increase in the division rate of osteoblasts when grow on titanium/gold coated nanosprings. This effect was shown to be dependent on the nanosprings, as the coating by themselves did not alter the growth rate of osteoblast. We also observed that titanium/zinc/gold coated nanosprings increased the levels of osteoblast production of alkaline phosphatase seven folds. This result indicates that osteoblasts grown on this metal alloy coated nanosprings are differentiating to mature bone making cells. Consistent with this hypothesis, we showed that osteoblasts grown on the same metal alloy coated nanosprings have an enhanced ability to deposit calcium salt. Conclusion We have established that metal/metal alloy coated silicon dioxide nanosprings can be used as a biomimetic material paralleling the morphology and biology of

  15. System and method for forming synthetic protein crystals to determine the conformational structure by crystallography

    Science.gov (United States)

    Craig, George D.; Glass, Robert; Rupp, Bernhard

    1997-01-01

    A method for forming synthetic crystals of proteins in a carrier fluid by use of the dipole moments of protein macromolecules that self-align in the Helmholtz layer adjacent to an electrode. The voltage gradients of such layers easily exceed 10.sup.6 V/m. The synthetic protein crystals are subjected to x-ray crystallography to determine the conformational structure of the protein involved.

  16. Molecular cloning, expression and in vitro analysis of soluble cationic synthetic antimicrobial peptide from salt-inducible Escherichia coli GJ1158

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    Jawahar Babu Peravali

    2013-01-01

    Full Text Available Antimicrobial peptides are the upcoming therapeutic molecules as alternative drugs to the existing antibiotics owing to their potent action against pathogenic microorganisms. In this study, to obtain an antimicrobial peptide with a broad range of activity, the synthetic cationic antimicrobial peptide was designed by using in silico tools viz., antimicrobial peptide database, protparam, hierarchical neural network. Later, the peptide was translated back into a core nucleotide sequence and the gene for the peptide was constructed by overlapping PCR. The amplified gene was cloned into pRSET–A vector and transformed into salt inducible expression host E. coli GJ1158. The expression results show high yields of soluble recombinant fusion peptide (0.52 g/L from salt-inducible E. coli. The recombinant peptide was purified by the IMAC purification system and cleaved by enterokinase. The digested product was further purified and 0.12 g/L of biologically active recombinant cationic antimicrobial peptide was obtained. In vitro analysis of the purified peptide demonstrated high antimicrobial activity against both Gram positive and Gram negative bacteria devoid of hemolytic activity. Therefore, this synthetic cationic antimicrobial peptide could serves as an promising agent over chemical antibiotics. In this study, a synthetic cationic antimicrobial peptide was designed, cloned and expressed from salt-inducible E. coli GJ1158 using cost effective media in the large scale production of antimicrobial peptide and its biological activity was analysed against different Gram positive and negative organisms.

  17. Osteoinductive Effects of Free and Immobilized Bone Forming Peptide-1 on Human Adipose-Derived Stem Cells.

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    Wenyue Li

    Full Text Available Most synthetic polymeric materials currently used for bone tissue engineering lack specific signals through which cells can identify and interact with the surface, resulting in incompatibility and compromised osteogenic activity. Soluble inductive factors also have issues including a short half-live in vivo. Bone forming peptide-1 is a truncated peptide from the immature form of bone morphogenetic protein-7 (BMP-7 that displays higher osteogenic activity than full-length, mature BMP-7. In this study, we used a mussel-inspired immobilization strategy mediated by polymerization of dopamine to introduce recently discovered stimulators of bone forming peptide-1 (BFP-1 onto the surface of poly-lactic-co-glycolic acid (PLGA substrate to form a biomaterial that overcomes these challenges. Human adipose-derived stem cells (hASCs, being abundant and easy accessible, were used to test the osteogenic activity of BFP-1 and the novel biomaterial. Under osteoinductive conditions, cells treated with both BFP-1 alone and BFP-1-coated biomaterials displayed elevated expression of the osteogenic markers alkaline phosphatase (ALP, osteocalcin (OC, and RUNX2. Furthermore, hASCs associated with poly-dopamine-assisted BFP-1-immobilized PLGA (pDA-BFP-1-PLGA scaffolds promoted in vivo bone formation in nude mice. Our novel materials may hold great promise for future bone tissue engineering applications.

  18. Protein C-terminal labeling and biotinylation using synthetic peptide and split-intein.

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    Gerrit Volkmann

    Full Text Available BACKGROUND: Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling. METHODOLOGY: A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein. CONCLUSIONS: We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.

  19. Antibacterial Activity of Synthetic Peptides Derived from Lactoferricin against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212

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    María A. León-Calvijo

    2015-01-01

    Full Text Available Peptides derived from human and bovine lactoferricin were designed, synthesized, purified, and characterized using RP-HPLC and MALDI-TOF-MS. Specific changes in the sequences were designed as (i the incorporation of unnatural amino acids in the sequence, the (ii reduction or (iii elongation of the peptide chain length, and (iv synthesis of molecules with different number of branches containing the same sequence. For each peptide, the antibacterial activity against Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 was evaluated. Our results showed that Peptides I.2 (RWQWRWQWR and I.4 ((RRWQWR4K2Ahx2C2 exhibit bigger or similar activity against E. coli (MIC 4–33 μM and E. faecalis (MIC 10–33 μM when they were compared with lactoferricin protein (LF and some of its derivate peptides as II.1 (FKCRRWQWRMKKLGA and IV.1 (FKCRRWQWRMKKLGAPSITCVRRAE. It should be pointed out that Peptides I.2 and I.4, containing the RWQWR motif, are short and easy to synthesize; our results demonstrate that it is possible to design and obtain synthetic peptides that exhibit enhanced antibacterial activity using a methodology that is fast and low-cost and that allows obtaining products with a high degree of purity and high yield.

  20. Serologic reactivity of a synthetic peptide from human immunodeficiency virus type 1 gp41 with sera from a Mexican population.

    Science.gov (United States)

    Gevorkian, G; Soler, C; Viveros, M; Padilla, A; Govezensky, T; Larralde, C

    1996-01-01

    The reactivities of 1,172 serum samples obtained from asymptomatic human immunodeficiency virus type 1 (HIV-1)-positive and HIV-1-negative individuals residing in Mexico to a synthetic disulfide-looped peptide from the HIV-1 gp41 (amino acids 602 to 616 [IWGCSGKLICTTAVP] were examined by an enzyme-linked immunoadsorbent assay (ELISA) procedure. Antibodies to the synthetic peptide were detected in 261 of 268 serum samples from HIV-positive individuals (sensitivity, 97.4%). The peptide also reacted with 12 of 904 serum samples from control HIV-negative individuals (specificity, 98.7%). Western blots (immunoblots) of four of the seven serum samples that produced false-negative results in the ELISA showed that three of them reacted weakly with gp41 and strongly with gp120, p55, and/or p24. Potential diagnostic difficulties raised by the reported C1q binding capacity of this peptide were also evaluated: few and weak false-positive results were found among sera from patients with rheumatoid arthritis (1 of 31) and neurocysticercosis (2 of 111). In fact, strong reactivity with the peptide spotted an undetected HIV infection underlying clinical neurocysticercosis. PMID:8914754

  1. Cationic synthetic peptides: assessment of their antimicrobial potency in liquid preserved boar semen.

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    Stephanie Speck

    Full Text Available Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW and a helical magainin II amide analog (MK5E was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs and Staphylococcus aureus ATCC 29213 (MK5E. We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.

  2. Cationic synthetic peptides: assessment of their antimicrobial potency in liquid preserved boar semen.

    Science.gov (United States)

    Speck, Stephanie; Courtiol, Alexandre; Junkes, Christof; Dathe, Margitta; Müller, Karin; Schulze, Martin

    2014-01-01

    Various semen extender formulas are in use to maintain sperm longevity and quality whilst acting against bacterial contamination in liquid sperm preservation. Aminoglycosides are commonly supplemented to aid in the control of bacteria. As bacterial resistance is increasing worldwide, antimicrobial peptides (AMPs) received lively interest as alternatives to overcome multi-drug resistant bacteria. We investigated, whether synthetic cationic AMPs might be a suitable alternative for conventional antibiotics in liquid boar sperm preservation. The antibacterial activity of two cyclic AMPs (c-WWW, c-WFW) and a helical magainin II amide analog (MK5E) was studied in vitro against two Gram-positive and eleven Gram-negative bacteria. Isolates included ATCC reference strains, multi-resistant E. coli and bacteria cultured from boar semen. Using broth microdilution, minimum inhibitory concentrations were determined for all AMPs. All AMPs revealed activity towards the majority of bacteria but not against Proteus spp. (all AMPs) and Staphylococcus aureus ATCC 29213 (MK5E). We could also demonstrate that c-WWW and c-WFW were effective against bacterial growth in liquid preserved boar semen in situ, especially when combined with a small amount of gentamicin. Our results suggest that albeit not offering a complete alternative to traditional antibiotics, the use of AMPs offers a promising solution to decrease the use of conventional antibiotics and thereby limit the selection of multi-resistant strains.

  3. Constitutive expression of transgenes encoding derivatives of the synthetic antimicrobial peptide BP100: impact on rice host plant fitness

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    Nadal Anna

    2012-09-01

    Full Text Available Abstract Background The Biopeptide BP100 is a synthetic and strongly cationic α-helical undecapeptide with high, specific antibacterial activity against economically important plant-pathogenic bacteria, and very low toxicity. It was selected from a library of synthetic peptides, along with other peptides with activities against relevant bacterial and fungal species. Expression of the BP100 series of peptides in plants is of major interest to establish disease-resistant plants and facilitate molecular farming. Specific challenges were the small length, peptide degradation by plant proteases and toxicity to the host plant. Here we approached the expression of the BP100 peptide series in plants using BP100 as a proof-of-concept. Results Our design considered up to three tandemly arranged BP100 units and peptide accumulation in the endoplasmic reticulum (ER, analyzing five BP100 derivatives. The ER retention sequence did not reduce the antimicrobial activity of chemically synthesized BP100 derivatives, making this strategy possible. Transformation with sequences encoding BP100 derivatives (bp100der was over ten-fold less efficient than that of the hygromycin phosphotransferase (hptII transgene. The BP100 direct tandems did not show higher antimicrobial activity than BP100, and genetically modified (GM plants constitutively expressing them were not viable. In contrast, inverted repeats of BP100, whether or not elongated with a portion of a natural antimicrobial peptide (AMP, had higher antimicrobial activity, and fertile GM rice lines constitutively expressing bp100der were produced. These GM lines had increased resistance to the pathogens Dickeya chrysanthemi and Fusarium verticillioides, and tolerance to oxidative stress, with agronomic performance comparable to untransformed lines. Conclusions Constitutive expression of transgenes encoding short cationic α-helical synthetic peptides can have a strong negative impact on rice fitness. However, GM

  4. Cooperative effects in differentiation and proliferation between PDGF-BB and matrix derived synthetic peptides in human osteoblasts

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    Vordemvenne Thomas

    2011-11-01

    Full Text Available Abstract Background Enhancing osteogenic capabilities of bone matrix for the treatment of fractures and segmental defects using growth factors is an active area of research. Recently, synthetic peptides like AC- 100, TP508 or p-15 corresponding to biologically active sequences of matrix proteins have been proven to stimulate bone formation. The platelet-derived growth factor (PDGF BB has been identified as an important paracrine factor in early bone healing. We hypothesized that the combined use of PDGF-BB with synthetic peptides could result in an increase in proliferation and calcification of osteoblast-like cells. Methods Osteoblast-like cell cultures were treated with PDGF and synthetic peptides, singly and as combinations, and compared to non-treated control cell cultures. The cultures were evaluated at days 2, 5, and 10 in terms of cell proliferation, calcification and gene expression of alkaline phosphate, collagen I and osteocalcin. Results Experimental findings revealed that the addition of PDGF, p-15 and TP508 and combinations of PDGF/AC-100, PDGF/p-15 and PDGF/TP508 resulted in an increase in proliferating osteoblasts, especially in the first 5 days of cultivation. Proliferation did not significantly differ between single factors and factor combinations (p > 0.05. The onset of calcification in osteoblasts occurred earlier and was more distinct compared to the corresponding control or PDGF stimulation alone. Significant difference was found for the combined use of PDGF/p-15 and PDGF/AC-100 (p Conclusions Our findings indicate that PDGF exhibits cooperative effects with synthetic peptides in differentiation and proliferation. These cooperative effects cause a significant early calcification of osteoblast-like cells (p

  5. AMBER: An X-band FMCW digital beam forming synthetic aperture radar for a tactical UAV

    NARCIS (Netherlands)

    Graaf, M.W. van der; Otten, M.P.G.; Huizing, A.G.; Tan, R.G.; Caro Cuenca, M.; Ruizenaar, M.G.A.

    2013-01-01

    An X-band Digital Array Synthetic Aperture Radar for a Short Range Tactical UAV is presented. This system is demonstrated on a manned helicopter and motor glider. The Frequency Modulated Continuous Wave radar principle in combination with digital beam forming over 24 receive channels is used to meet

  6. AMBER: An X-band FMCW digital beam forming synthetic aperture radar for a tactical UAV

    NARCIS (Netherlands)

    Graaf, M.W. van der; Otten, M.P.G.; Huizing, A.G.; Tan, R.G.; Caro Cuenca, M.; Ruizenaar, M.G.A.

    2013-01-01

    An X-band Digital Array Synthetic Aperture Radar for a Short Range Tactical UAV is presented. This system is demonstrated on a manned helicopter and motor glider. The Frequency Modulated Continuous Wave radar principle in combination with digital beam forming over 24 receive channels is used to meet

  7. NMR structure of mussel mytilin, and antiviral-antibacterial activities of derived synthetic peptides.

    Science.gov (United States)

    Roch, Philippe; Yang, Yinshan; Toubiana, Mylène; Aumelas, André

    2008-01-01

    Mytilin is a 34-residue antibacterial peptide from the mussel Mytilus galloprovincialis, which in addition possesses in vitro antiviral activity. The three-dimensional solution structure of the synthetic mytilin was established by using 1H NMR and consists of the common cysteine-stabilized alphabeta motif close to the one observed in the mussel defensin MGD-1. Mytilin is characterized by 8 cysteines engaged in four disulfide bonds (2-27, 6-29, 10-31, and 15-34) only involving the beta-strand II. Hydrophilic and hydrophobic areas of mytilin account for 63% and 37%, respectively, a ratio very close to that of MGD-1 (64% and 36%). One linear and three cyclic fragments were designed from the interstrand loop sequence known to retain the biological activities in MGD-1. Only the fragment of 10 amino acids (C10C) constrained by two disulfide bonds in a stable beta-hairpin structure was able to inhibit the mortality of Palaemon serratus shrimp injected with white spot syndrome virus (WSSV). Fifty percent inhibition was obtained by in vitro pre-incubation of WSSV with 45 microM of C10C compared with 7 microM for mytilin. Interaction between the fragment and the virus occurred very rapidly as 40% survival was recorded after only 1 min of pre-incubation. In addition, C10C was capable of inhibiting in vitro growth of Vibrio splendidus LGP32 (MIC 125 microM), Vibrio anguillarum (MIC 2mM), Micrococcus lysodeikticus and Escherichia coli (MIC 1mM). Destroying the cysteine-stabilized alphabeta structure or shortening the C10C fragment to the C6C fragment with only one disulfide bond resulted in loss of both antiviral and antibacterial activities. Increasing the positive net charge did not enforce the antibacterial activity and completely suppressed the antiviral one. The C10C-designed peptide from mytilin appeared comparable in composition and structure with protegrin, tachyplesin and polyphemusin.

  8. Specific binding of DNA to aggregated forms of Alzheimer's disease amyloid peptides.

    Science.gov (United States)

    Camero, Sergio; Ayuso, Jose M; Barrantes, Alejandro; Benítez, María J; Jiménez, Juan S

    2013-04-01

    Anomalous protein aggregation is closely associated to age-related mental illness. Extraneuronal plaques, mainly composed of aggregated amyloid peptides, are considered as hallmarks of Alzheimer's disease. According to the amyloid cascade hypothesis, this disease starts as a consequence of an abnormal processing of the amyloid precursor protein resulting in an excess of amyloid peptides. Nuclear localization of amyloid peptide aggregates together with amyloid-DNA interaction, have been repeatedly reported. In this paper we have used surface plasmon resonance and electron microscopy to study the structure and behavior of different peptides and proteins, including β-lactoglobulin, bovine serum albumin, myoglobin, histone, casein and the amyloid-β peptides related to Alzheimer's disease Aβ25-35 and Aβ1-40. The main purpose of this study is to investigate whether proneness to DNA interaction is a general property displayed by aggregated forms of proteins, or it is an interaction specifically related to the aggregated forms of those particular proteins and peptides related to neurodegenerative diseases. Our results reveal that those aggregates formed by amyloid peptides show a particular proneness to interact with DNA. They are the only aggregated structures capable of binding DNA, and show more affinity for DNA than for other polyanions like heparin and polyglutamic acid, therefore strengthening the hypothesis that amyloid peptides may, by means of interaction with nuclear DNA, contribute to the onset of Alzheimer's disease.

  9. Synthetic Long Peptide Derived from Mycobacterium tuberculosis Latency Antigen Rv1733c Protects against Tuberculosis.

    Science.gov (United States)

    Coppola, Mariateresa; van den Eeden, Susan J F; Wilson, Louis; Franken, Kees L M C; Ottenhoff, Tom H M; Geluk, Annemieke

    2015-09-01

    Responsible for 9 million new cases of active disease and nearly 2 million deaths each year, tuberculosis (TB) remains a global health threat of overwhelming dimensions. Mycobacterium bovis BCG, the only licensed vaccine available, fails to confer lifelong protection and to prevent reactivation of latent infection. Although 15 new vaccine candidates are now in clinical trials, an effective vaccine against TB remains elusive, and new strategies for vaccination are vital. BCG vaccination fails to induce immunity against Mycobacterium tuberculosis latency antigens. Synthetic long peptides (SLPs) combined with adjuvants have been studied mostly for therapeutic cancer vaccines, yet not for TB, and proved to induce efficient antitumor immunity. This study investigated an SLP derived from Rv1733c, a major M. tuberculosis latency antigen which is highly expressed by "dormant" M. tuberculosis and well recognized by T cells from latently M. tuberculosis-infected individuals. In order to assess its in vivo immunogenicity and protective capacity, Rv1733c SLP in CpG was administered to HLA-DR3 transgenic mice. Immunization with Rv1733c SLP elicited gamma interferon-positive/tumor necrosis factor-positive (IFN-γ(+)/TNF(+)) and IFN-γ(+) CD4(+) T cells and Rv1733c-specific antibodies and led to a significant reduction in the bacterial load in the lungs of M. tuberculosis-challenged mice. This was observed both in a pre- and in a post-M. tuberculosis challenge setting. Moreover, Rv1733c SLP immunization significantly boosted the protective efficacy of BCG, demonstrating the potential of M. tuberculosis latency antigens to improve BCG efficacy. These data suggest a promising role for M. tuberculosis latency antigen Rv1733c-derived SLPs as a novel TB vaccine approach, both in a prophylactic and in a postinfection setting.

  10. Spectral and biological evaluation of a synthetic antimicrobial peptide derived from 1-aminocyclohexane carboxylic acid.

    Science.gov (United States)

    Abercrombie, J J; Leung, Kai P; Chai, Hanbo; Hicks, Rickey P

    2015-03-15

    Ac-GF(A6c)G(A6c)K(A6c)G(A6c)F(A6c)G(A6c)GK(A6c)KKKK-amide (A6c=1-aminocyclohexane carboxylic acid) is a synthetic antimicrobial peptide (AMP) that exhibits in vitro inhibitory activity against drug resistant strains of Staphylococcus aureus, Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, and Enterococcus faecium at concentrations ranging from 10.9 to 43μM. Spectroscopic investigations were conducted to determine how this AMP interacts with simple membrane model systems in order to provide insight into possible mechanisms of action. CD and 2D-(1)H NMR experiments indicated this AMP on binding to SDS and DPC micelles adopts conformations with varying percentages of helical and random coil conformers. CD investigations in the presence of three phospholipid SUVs consisting of POPC, 4:1 POPC/POPG, and 60% POPE/21%POPG/19%POPC revealed: (1) The interactions occurring with POPC SUVs have minimal effect on the conformational diversity of the AMP yielding conformations similar to those observed in buffer. (2) The interactions with 4:1 POPC/POPG, and 60% POPE/21%POPG/19%POPC SUVs exhibited a greater influence on the percentage of different conformers contributing to the CD spectra. (3) The presence of a high of percentage of helical conformers was not observed in the presence of SUVs as was the case with micelles. This data indicates that the diversity of surface bound conformations adopted by this AMP are very different from the diversity of conformations adopted by this AMP on insertion into the lipid bilayer. CD spectra of this AMP in the presence of SUVs consisting of LPS isolated from P. aeruginosa, K. pneumoniae and Escherichia coli exhibited characteristics associated with various helical conformations.

  11. Cytological profile of antibacterial FtsZ inhibitors and synthetic peptide MciZ

    Directory of Open Access Journals (Sweden)

    Lidia Araujo-Bazan

    2016-10-01

    Full Text Available Cell division protein FtsZ is the organizer of the cytokinetic ring in almost all bacteria and a target for the discovery of new antibacterial agents that are needed to counter widespread antibiotic resistance. Bacterial cytological profiling, using quantitative microscopy, is a powerful approach for identifying the mechanism of action of antibacterial molecules affecting different cellular pathways. We have determined the cytological profile on Bacillus subtilis cells of a selection of small molecule inhibitors targeting FtsZ on different binding sites. FtsZ inhibitors lead to long undivided cells, impair the normal assembly of FtsZ into the midcell Z-rings, induce aberrant ring distributions, punctate FtsZ foci, membrane spots and also modify nucleoid length. Quantitative analysis of cell and nucleoid length combined, or the Z-ring distribution, allows categorizing FtsZ inhibitors and to distinguish them from antibiotics with other mechanisms of action, which should be useful for identifying new antibacterial FtsZ inhibitors. Biochemical assays of FtsZ polymerization and GTPase activity combined explain the cellular effects of the FtsZ polymer stabilizing agent PC190723 and its fragments. MciZ is a 40-aminoacid endogenous inhibitor of cell division normally expressed during sporulation in B. subtilis. Using FtsZ cytological profiling we have determined that exogenous synthetic MciZ is an effective inhibitor of B. subtilis cell division, Z-ring formation and localization. This finding supports our cell-based approach to screen for FtsZ inhibitors and opens new possibilities for peptide inhibitors of bacterial cell division.

  12. Insights from Synthetic Star-forming Regions: I. Reliable Mock Observations from SPH Simulations

    CERN Document Server

    Koepferl, Christine M; Dale, James E; Biscani, Francesco

    2016-01-01

    Through synthetic observations of a hydrodynamical simulation of an evolving star-forming region, we assess how the choice of observational techniques affects the measurements of properties which trace star formation. Testing and calibrating observational measurements requires synthetic observations which are as realistic as possible. In this part of the paper series (Paper I), we explore different techniques for how to map the distributions of densities and temperatures from the particle-based simulations onto a Voronoi mesh suitable for radiative transfer and consequently explore their accuracy. We further test different ways to set up the radiative transfer in order to produce realistic synthetic observations. We give a detailed description of all methods and ultimately recommend techniques. We have found that the flux around 20 microns is strongly overestimated when blindly coupling the dust radiative transfer temperature with the hydrodynamical gas temperature. We find that when instead assuming a consta...

  13. Glucagon-like peptide-1 synthetic analogs: new therapeutic agents for use in the treatment of diabetes mellitus.

    Science.gov (United States)

    Holz, George G; Chepurny, Oleg G

    2003-11-01

    Glucagon-like peptide-1-(7-36)-amide (GLP-1) is a potent blood glucose-lowering hormone now under investigation for use as a therapeutic agent in the treatment of type 2 (adult onset) diabetes mellitus. GLP-1 binds with high affinity to G protein-coupled receptors (GPCRs) located on pancreatic beta-cells, and it exerts insulinotropic actions that include the stimulation of insulin gene transcription, insulin biosynthesis, and insulin secretion. The beneficial therapeutic action of GLP-1 also includes its ability to act as a growth factor, stimulating formation of new pancreatic islets (neogenesis) while slowing beta-cell death (apoptosis). GLP-1 belongs to a large family of structurally-related hormones and neuropeptides that include glucagon, secretin, GIP, PACAP, and VIP. Biosynthesis of GLP-1 occurs in the enteroendocrine L-cells of the distal intestine, and the release of GLP-1 into the systemic circulation accompanies ingestion of a meal. Although GLP-1 is inactivated rapidly by dipeptidyl peptidase IV (DDP-IV), synthetic analogs of GLP-1 exist, and efforts have been directed at engineering these peptides so that they are resistant to enzymatic hydrolysis. Additional modifications of GLP-1 incorporate fatty acylation and drug affinity complex (DAC) technology to improve serum albumin binding, thereby slowing renal clearance of the peptides. NN2211, LY315902, LY307161, and CJC-1131 are GLP-1 synthetic analogs that reproduce many of the biological actions of GLP-1, but with a prolonged duration of action. AC2993 (Exendin-4) is a naturally occurring peptide isolated from the lizard Heloderma, and it acts as a high affinity agonist at the GLP-1 receptor. This review summarizes structural features and signal transduction properties of GLP-1 and its cognate beta-cell GPCR. The usefulness of synthetic GLP-1 analogs as blood glucose-lowering agents is discussed, and the applicability of GLP-1 as a therapeutic agent for treatment of type 2 diabetes is highlighted.

  14. Quality evaluation of synthetic quorum sensing peptides used in R & D

    Institute of Scientific and Technical Information of China (English)

    Frederick Verbeke; Evelien Wynendaele; Sarah Braet; Matthias DHondt; Bart De Spiegeleer

    2015-01-01

    Peptides are becoming an important class of molecules in the pharmaceutical field. Closely related peptide-impurities in peptides are inherent to the synthesis approach and have demonstrated to potentially mask biomedical experimental results. Quorum sensing peptides are attracting high interest in R&D and therefore a representative set of quorum sensing peptides, with a requested purity of at least 95.0%, was evaluated for their purity and nature of related impurities. In-house quality control (QC) revealed a large discrepancy between the purity levels as stated on the supplier’s certificate of analysis and our QC results. By using our QC analysis flowchart, we demonstrated that only 44.0% of the peptides met the required purity. The main compound of one sample was even found to have a different structure compared to the desired peptide. We also found that the majority of the related impurities were lacking amino acid(s) in the desired peptide sequence. Relying on the certificates of analysis as provided by the supplier might have serious consequences for peptide research, and peptide-researchers should implement and maintain a thorough in-house QC.

  15. Quality evaluation of synthetic quorum sensing peptides used in R&D

    Directory of Open Access Journals (Sweden)

    Frederick Verbeke

    2015-06-01

    Full Text Available Peptides are becoming an important class of molecules in the pharmaceutical field. Closely related peptide-impurities in peptides are inherent to the synthesis approach and have demonstrated to potentially mask biomedical experimental results. Quorum sensing peptides are attracting high interest in R&D and therefore a representative set of quorum sensing peptides, with a requested purity of at least 95.0%, was evaluated for their purity and nature of related impurities. In-house quality control (QC revealed a large discrepancy between the purity levels as stated on the supplier׳s certificate of analysis and our QC results. By using our QC analysis flowchart, we demonstrated that only 44.0% of the peptides met the required purity. The main compound of one sample was even found to have a different structure compared to the desired peptide. We also found that the majority of the related impurities were lacking amino acid(s in the desired peptide sequence. Relying on the certificates of analysis as provided by the supplier might have serious consequences for peptide research, and peptide-researchers should implement and maintain a thorough in-house QC.

  16. A Novel Monoclonal Antibody Against a Synthetic Peptide from β-Actin can React with its Corresponding Protein.

    Science.gov (United States)

    Amini, Nazila; Bayat, Ali-Ahmad; Zarei, Omid; Hadavi, Reza; Mahmoudian, Jafar; Mahmoudi, Ahmad R; Darzi, Maryam; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

    2015-01-01

    Actin is one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells with important roles in many cell functions. Antibodies against β-actin and other housekeeping gene-encoded proteins are used as internal loading controls in Western blot analyses. The aim of this study was to produce a monoclonal antibody (mAb) against a synthetic peptide derived from N-terminal region of β-actin and to study its reactivity with different organisms. A synthetic peptide, derived from β-actin, was designed and used to produce a mAb by hybridoma technology. The produced antibody (clone 4E5- A10) was purified by an affinity chromatography column followed by characterization of purified mAb using SDS-PAGE, ELISA and Western blot. Our results showed that 4E5-A10 was an IgM and had desired purity and excellent reactivity with the immunizing peptide with an affinity constant of 2.7x10(8) M(-1)>. It could detect a band of about 45 kDa, corresponding to β-actin, in Western blot. Furthermore, it could react in a more sensitive manner and with a wider range of organisms than a known commercial anti β-actin antibody. Our data suggest that 4E5-A10 can act as a sensitive probe for detection of β-actin as an internal loading control, for a wide range of organisms, in Western blot analyses.

  17. Functionalized D-form self-assembling peptide hydrogels for bone regeneration

    Directory of Open Access Journals (Sweden)

    He B

    2016-04-01

    Full Text Available Bin He,1 Yunsheng Ou,1 Ao Zhou,1 Shuo Chen,1 Weikang Zhao,1 Jinqiu Zhao,2 Hong Li,3 Yong Zhu,1 Zenghui Zhao,1 Dianming Jiang1 1Department of Orthopedics, 2Department of Infectious Diseases, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China; 3School of Physical Science and Technology, Sichuan University, Chengdu, People’s Republic of China Abstract: Bone defects are very common in orthopedics, and there is great need to develop suitable bone grafts for transplantation in vivo. However, current bone grafts still encounter some limitations, including limited availability, immune rejection, poor osteoinduction and osteoconduction, poor biocompatibility and degradation properties, etc. Self-assembling peptide nanofiber scaffolds have emerged as an important substrate for cell culture and bone regeneration. We report on the structural features (eg, Congo red staining, circular dichroism spectroscopy, transmission electron microscopy, and rheometry assays and osteogenic ability of D-RADA16-RGD peptide hydrogels (with or without basic fibroblast growth factor due to the better stability of peptide bonds formed by these peptides compared with those formed by L-form peptides, and use them to fill the femoral condyle defect of Sprague Dawley rat model. The bone morphology change, two-dimensional reconstructions using microcomputed tomography, quantification of the microcomputed tomography analyses as well as histological analyses have demonstrated that RGD-modified D-form peptide scaffolds are able to enhance extensive bone regeneration. Keywords: bone defect, functionalized D-form self-assembling peptide, D-RADA16-RGD, peptide hydrogel, bone regeneration

  18. Synthetic

    Directory of Open Access Journals (Sweden)

    Anna Maria Manferdini

    2010-06-01

    Full Text Available Traditionally materials have been associated with a series of physical properties that can be used as inputs to production and manufacturing. Recently we witnessed an interest in materials considered not only as ‘true matter’, but also as new breeds where geometry, texture, tooling and finish are able to provoke new sensations when they are applied to a substance. These artificial materials can be described as synthetic because they are the outcome of various qualities that are not necessarily true to the original matter, but they are the combination of two or more parts, whether by design or by natural processes. The aim of this paper is to investigate the potential of architectural surfaces to produce effects through the invention of new breeds of artificial matter, using micro-scale details derived from Nature as an inspiration.

  19. Searching for Synthetic Antimicrobial Peptides: An Experiment for Organic Chemistry Students

    Science.gov (United States)

    Vasquez, Thomas E., Jr.; Saldan~a, Cristina; Muzikar, Katy A.; Mashek, Debra; Liu, Jane M.

    2016-01-01

    This laboratory experiment provides undergraduate students enrolled in organic chemistry the opportunity to design and synthesize their own peptide, which is then tested for antimicrobial activity. After reading a primary scientific paper on antimicrobial peptides, students design and synthesize their own hexapeptide that they hypothesize will…

  20. Searching for Synthetic Antimicrobial Peptides: An Experiment for Organic Chemistry Students

    Science.gov (United States)

    Vasquez, Thomas E., Jr.; Saldan~a, Cristina; Muzikar, Katy A.; Mashek, Debra; Liu, Jane M.

    2016-01-01

    This laboratory experiment provides undergraduate students enrolled in organic chemistry the opportunity to design and synthesize their own peptide, which is then tested for antimicrobial activity. After reading a primary scientific paper on antimicrobial peptides, students design and synthesize their own hexapeptide that they hypothesize will…

  1. Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides

    Directory of Open Access Journals (Sweden)

    Protti Maria

    2005-12-01

    Full Text Available Abstract Background MHC class I-peptide tetramers are currently utilised to characterize CD8+ T cell responses at single cell level. The generation and use of MHC class II tetramers to study antigen-specific CD4+ T cells appears less straightforward. Most MHC class II tetramers are produced with a homogeneously built-in peptide, reducing greatly their flexibility of use. We attempted the generation of "empty" functional HLA-DR*1101 tetramers, receptive for loading with synthetic peptides by incubation. No such reagent is in fact available for this HLA-DR allele, one of the most frequent in the Caucasian population. Results We compared soluble MHC class II-immunoglobulin fusion proteins (HLA-DR*1101-Ig with soluble MHC class II protein fused with an optimised Bir site for enzymatic biotynilation (HLA-DR*1101-Bir, both produced in insect cells. The molecules were multimerised by binding fluorochrome-protein A or fluorochrome-streptavidin, respectively. We find that HLA-DR*1101-Bir molecules are superior to the HLA-DR*1101-Ig ones both in biochemical and functional terms. HLA-DR*1101-Bir molecules can be pulsed with at least three different promiscuous peptide epitopes, derived from Tetanus Toxoid, influenza HA and the tumour associated antigen MAGE-3 respectively, to stain specific CD4+ T cells. Both staining temperature and activation state of CD4+ T cells are critical for the binding of peptide-pulsed HLA-DR*1101-Bir to the cognate TCR. Conclusion It is therefore possible to generate a soluble recombinant HLA-DR*1101 backbone that is receptive for loading with different peptides to stain specific CD4+ T cells. As shown for other HLA-DR alleles, we confirm that not all the strategies to produce soluble HLA-DR*1101 multimers are equivalent.

  2. Antibody responses to an immunodominant nonstructural 1 synthetic peptide in patients with dengue fever and dengue hemorrhagic fever.

    Science.gov (United States)

    Huang, J H; Wey, J J; Sun, Y C; Chin, C; Chien, L J; Wu, Y C

    1999-01-01

    Two flaviviruses, dengue (DEN) virus and Japanese encephalitis (JE) virus, are important because of their global distribution and the frequency of epidemics in tropical and subtropical areas. To study the B-cell epitopes of nonstructural 1 (NS1) glycoprotein and anti-NS1 antibody response in DEN infection, a series of 15-mer synthetic peptides from the predicted B-cell linear epitopes of DEN-2 NS1 protein were prepared. Enzyme-linked immunosorbent assay (ELISA) was performed to analyze antibody responses to these peptides from sera of both DEN and JE patients. One peptide derived from DEN-2 NS1, D2 NS1-P1 (amino acids 1-15), was identified as the immunodominant epitope that reacted with sera from dengue fever (DF) patients but not JE patients. The isotype of D2 NS1-P1-specific antibodies was mainly immunoglobulin M (IgM) in all sera that tested positive. A specificity study demonstrated that sera from all four DEN types reacted with D2 NS1-P1. A dynamics study showed that specific antibodies to this peptide could be detected as early as 2 days after the onset of symptoms. We observed significant anti-D2 NS1-P1 antibody responses in 45% of patients with primary and secondary infections with DF or with dengue hemorrhagic fever. This is the first report demonstrating that significant anti-DEN NS1 antibodies can be induced in the sera of patients with primary DEN infection.

  3. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  4. Reproduction potentiated in nematodes (Caenorhabditis elegans) and guppy fish (Poecilia reticulata) by adding a synthetic peptide to their aqueous environment.

    Science.gov (United States)

    Davies, Keith G; Zimmerman, Brian; Dudley, Ed; Newton, Russell P; Hart, John E

    2015-03-01

    Ambient exposure to a short synthetic peptide has enhanced fecundity (number of offspring) in invertebrates and vertebrates, ostensibly by disinhibiting reproduction. In separate experiments, nematodes (Caenorhabditis elegans) and guppy fish (Poecilia reticulata) were exposed via their aqueous environment to a dissolved synthetic hexamer (6mer) peptide, IEPVFT (EPL036), at a concentration of 1 μmol l(-1). In the case of the worms, peptide was added to their aqueous buffer daily throughout the experiment (14 days); for the guppies, peptide administration was on the first 15 alternate days in a 50 week experiment. Fecundity rose by 79% among the worms. The number of descendants of the treated guppies was more than four times that of controls by week 26 (103 versus 25, including 72 juveniles versus 6), with 15.4% more estimated biomass in the test tank in total (i.e. including founders). It was deduced that treated females bred earlier, at a smaller size, and had larger brood sizes. The total number of fish in the control tank had caught up by termination, but biomass continued to lag the test tank. There were no overt signs of toxicity among either the worms or the fish. Bioinformatics has been unilluminating in explaining these results in terms, for example, of mimicry of an endogenous regulator. A mass spectrometric campaign to identify a receptor, using murine brain for expediency, proved inconclusive. Molecular modelling in silico indicated unexpectedly that the hexamer EPL036 might be acting as an antagonist, to pro-fecundity effect; that is, as a blocker of an inhibitor. This suggests that there awaits discovery an evolutionarily conserved reproductive inhibitor and its (anti-fecundity) receptor.

  5. Coiled-coil forming peptides for the induction of silver nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Božič Abram, Sabina [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Graduate School of Biomedicine, University of Ljubljana, Ljubljana 1000 (Slovenia); Aupič, Jana [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Doctoral Programme in Chemical Sciences, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana 1000 (Slovenia); Dražić, Goran [Laboratory for Materials Chemistry, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); Gradišar, Helena [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); EN-FIST, Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana 1000 (Slovenia); Jerala, Roman, E-mail: roman.jerala@ki.si [Department of Synthetic Biology and Immunology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana (Slovenia); EN-FIST, Centre of Excellence, Trg Osvobodilne fronte 13, Ljubljana 1000 (Slovenia)

    2016-04-08

    Biopolymers with defined sequence patterns offer an attractive alternative for the formation of silver nanoparticle (AgNP). A set of coiled-coil dimer forming peptides was tested for their AgNP formation ability. Seventeen of those peptides mediated the formation of AgNPs in aqueous solution at neutral pH, while the formation of a coiled-coil dimer inhibited the nanoparticle generation. A QSAR regression model on the relationship between sequence and function suggests that in this peptide type the patterns KXQQ and KXEE are favorable, whereas Ala residues appear to have an inhibitory effect. UV–VIS spectra of the obtained nanoparticles gave a peak at around 420 nm, typical for AgNPs in the size range around 40 nm, which was confirmed by dynamic light scattering and transmission electron microscopy. Peptide-induced AgNPs exhibited good antibacterial activity, even after a 15 min contact time, while they had low toxicity to human cells at the same concentrations. These results show that our designed peptides generate AgNPs with antibacterial activity at mild conditions and might be used for antibacterial coatings. - Highlights: • 17 of the 30 tested coiled-coil forming peptides induce AgNP formation. • Coiled-coil dimer formation suppresses AgNP generation of individual peptides. • Size of the peptide-induced silver nanoparticles is around 40 nm. • QSAR analysis points to the importance of KXQQ and KXEE motifs for AgNP generation. • Peptide-induced silver nanoparticles exhibit antibacterial activity.

  6. Understanding Peptide Oligomeric State in Langmuir Monolayers of Amphiphilic 3-Helix Bundle-Forming Peptide-PEG Conjugates

    Science.gov (United States)

    Shu, Jessica Y.; Xu, Ting

    2016-01-01

    Coiled-coil peptide–polymer conjugates are an emerging class of biomaterials. Fundamental understanding of the coiled-coil oligomeric state and assembly process of these hybrid building blocks is necessary to exert control over their assembly into well-defined structures. Here, we studied the effect of peptide structure and PEGylation on the self-assembly process and oligomeric state of a Langmuir monolayer of amphiphilic coiled-coil peptide–polymer conjugates using X-ray reflectivity (XR) and grazing-incidence X-ray diffraction (GIXD). Our results show that the oligomeric state of PEGylated amphiphiles based on 3-helix bundle-forming peptide is surface pressure dependent, a mixture of dimers and trimers was formed at intermediate surface pressure but transitions into trimers completely upon increasing surface pressure. Moreover, the interhelical distance within the coiled-coil bundle of 3-helix peptide-PEG conjugate amphiphiles was not perturbed under high surface pressure. Present studies provide valuable insights into the self-assembly process of hybrid peptide–polymer conjugates and guidance to develop biomaterials with controlled multivalency of ligand presentation. PMID:27784156

  7. Highly immunogenic and fully synthetic peptide-carrier constructs targetting GnRH

    DEFF Research Database (Denmark)

    Beekman, N.J.C.M.; Schaaper, W.M.M.; Turkstra, J.A.;

    1999-01-01

    using a tandem GnRH peptide as a branched polylysine construct, a lipo-thioester, a lipo-amide or a KLH conjugate in CFA, and the lipoamide peptide in an immuno-stimulating complex (ISCOM). We found the lipo-thioester and the branched polylysine constructs to be the most effective carrier molecules...... for the induction of antibodies against GnRH and immunocastration of pigs....

  8. Antibodies against a synthetic peptide identify the Epstein-Barr virus-determined nuclear antigen.

    OpenAIRE

    1984-01-01

    Five peptides corresponding to amino acid sequences predicted from all three reading frames of the nucleotide sequence of the third internal repeat array (IR3) of the Epstein-Barr virus (EBV) genome were synthesized chemically. All five peptides elicited antipeptide antibodies in rabbits. The antiserum raised against a 14-residue copolymer of glycine and alanine gave brilliant EBV-specific nuclear staining in the anticomplement immunofluorescence (ACIF) assay, in line with the original defini...

  9. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments.

    Science.gov (United States)

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika

    2007-02-23

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent.

  10. Synthetic peptide immunogens eliciting antibodies to Plasmodium falciparum sporozoite and merozoite surface antigens in H-2b and H-2k mice.

    Science.gov (United States)

    Rzepczyk, C M; Csurhes, P A; Lord, R; Matile, H

    1990-10-15

    Peptides representing conserved (MSA2/1A and MSA2/1B) and variant (MSA2/2, MSA2/6 and MSA2/7) regions of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum (FCQ-27/PNG isolate) were coupled to either peptide NP(NANP)5NA or peptide C(NANP)6 both of which contained the core sequence (NANP)n. The coupling was done via the N-terminus of one peptide and a cysteine residue on either terminus of the other. BL/10 (H-2b) and B10.BR (H-2k) mice were immunized with these MSA2-(NANP)n conjugates. The mice were also immunized with the unconjugated MSA2 peptides and with NP(NANP)5NA and C(NANP)6. Antibody responses were evaluated by 1) ELISA, in which the MSA2 peptides and C(NANP)6 were used as Ag; 2) immunofluorescence assays (IFAT) against intact sporozoites and merozoites; and 3) immunoblotting experiments against solubilized P. falciparum blood stage proteins. High titer antibodies to (NANP)n were elicited in both BL/10 and B10.BR mice after immunization with all the conjugates except MSA2/7-(NANP)n which gave only a very limited response in B10.BR mice. These antibodies recognized unfixed sporozoites. The conjugates also elicited antibodies to MSA2 as shown by ELISA, IFAT, and immunoblotting except for mice immunized with MSA2/1B-(NANP)n where an anti-MSA2 response was only detectable by immunoblotting. Immunization with unconjugated MSA2 peptides showed that MSA2/2 was immunogenic in both BL/10 and BR.10 mice, with MSA2/6 and MSA2/7 being immunogenic only in BL/10 mice. The antibodies elicited recognized both merozoites and the MSA2 protein. However, the antibody titers were lower overall than those seen when these peptides were used in the conjugated form. No anti-MSA2 antibodies were detected after immunization with MSA2/1A and MSA2/1B. Immunization of mice with the peptide NP(NANP)5NA produced antibodies in BL/10 (H-2b) mice only, and the immunogenicity of this preparation was poor. In contrast, C(NANP)6 produced a strong antibody response in both mouse strains

  11. Effects of mutations in de novo designed synthetic amphiphilic β-sheet peptides on self-assembly of fibrils.

    Science.gov (United States)

    Raz, Yoav; Rubinov, Boris; Matmor, Maayan; Rapaport, Hanna; Ashkenasy, Gonen; Miller, Yifat

    2013-07-25

    The self-assembly of two similar amphiphilic peptides into fibril structures is described. Molecular dynamic simulations show that both can organize similarly in a monolayer, but in the fibril bilayer, one prefers a single organization while the other forms two conformational variants. This assembly difference correlates well with our experimental results.

  12. Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Ruodan Xu

    2015-04-01

    Full Text Available Background/Aims: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. Methods: In the present study, using a transgenic murine embryonic stem (ES cell lineage expressing enhanced green fluorescent protein (EGFP under the control of α-myosin heavy chain (α-MHC promoter (pαMHC-EGFP, we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGLL, hNgf_C2, EnkaminE, Plannexin and C3 on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. Results: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. Conclusion: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to

  13. Photochemistry of free and bound Zn-chlorophyll analogues to synthetic peptides depend on the quinone and pH.

    Science.gov (United States)

    Razeghifard, Reza

    2015-11-01

    A synthetic peptide was used as a scaffold to bind Zn-Chlorophyll (ZnChl) analogues through histidine ligation to study their photochemistry in the presence of different type of quinones. The Chl analogues were chlorin e6 (Ce6), chlorin e6 trimethyl ester, pyropheophorbide a, and pheophorbide a while the quinones were PPBQ, DMBQ, NPHQ, DBTQ, DCBQ and PBQ. The binding of each ZnChl analogue to the peptide was verified by native gel electrophoresis. First the photo-stability of the ZnChl analogues were tested under continuous light. The ZnCe6 and ZnCe6TM analogues showed the least stability judged by the loss of optical signal intensity at their Qy band. The photoactivity of each ZnChl analogue was measured in the presence of each of the six quinones using time-resolved EPR spectroscopy. DMBQ was found to be the most efficient electron acceptor when all four ZnChl analogues were compared. The light-induced electron transfer between the ZnChl analogues complexed with the peptide and DMBQ were also measured using time-resolved EPR spectroscopy. The ZnCe6-peptide complex exhibited the highest photoactivity. The electron transfer in the complex was faster and the photoactivity yield was higher than those values obtained for free ZnCe6 and DMBQ. The fast phase of kinetics can be attributed to intra-protein electron transfer in the complex since it was not observed in the presence of DMBQ-glutathione adduct. Unlike free ZnCe6, the ZnCe6-peptide complex was robust and demonstrated very similar photoactivity efficiency in pH values 10, 8.0 and 5.0. The electron transfer kinetics were pH dependent and appeared to be modulated by the peptide charge and possibly fold. The charge recombination rate was slowed by an order of magnitude when the pH value was changed from 10.0 to 5.0. The implications of constructing the photoactive peptide complexes in terms of artificial photosynthesis are discussed.

  14. Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

    Science.gov (United States)

    Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

    2014-08-01

    Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma.

  15. Activity and biophysical inhibition resistance of a novel synthetic lung surfactant containing Super-Mini-B DATK peptide

    Directory of Open Access Journals (Sweden)

    Robert H. Notter

    2016-01-01

    Full Text Available Background/objectives. This study examines the surface activity, resistance to biophysical inhibition, and pulmonary efficacy of a synthetic lung surfactant containing glycerophospholipids combined with Super Mini-B (S-MB DATK, a novel and stable molecular mimic of lung surfactant protein (SP-B. The objective of the work is to test whether S-MB DATK synthetic surfactant has favorable biophysical and physiological activity for future use in treating surfactant deficiency or dysfunction in lung disease or injury.Methods. The structure of S-MB DATK peptide was analyzed by homology modeling and by FTIR spectroscopy. The in vitro surface activity and inhibition resistance of synthetic S-MB DATK surfactant was assessed in the presence and absence of albumin, lysophosphatidylcholine (lyso-PC, and free fatty acids (palmitoleic and oleic acid. Adsorption and dynamic surface tension lowering were measured with a stirred subphase dish apparatus and a pulsating bubble surfactometer (20 cycles/min, 50% area compression, 37 °C. In vivo pulmonary activity of S-MB DATK surfactant was measured in ventilated rabbits with surfactant deficiency/dysfunction induced by repeated lung lavages that resulted in arterial PO2 values <100 mmHg.Results. S-MB DATK surfactant had very high surface activity in all assessments. The preparation adsorbed rapidly to surface pressures of 46–48 mN/m at 37 °C (low equilibrium surface tensions of 22–24 mN/m, and reduced surface tension to <1 mN/m under dynamic compression on the pulsating bubble surfactometer. S-MB DATK surfactant showed a significant ability to resist inhibition by serum albumin, C16:0 lyso-PC, and free fatty acids, but surfactant inhibition was mitigated by increasing surfactant concentration. S-MB DATK synthetic surfactant quickly improved arterial oxygenation and lung compliance after intratracheal instillation to ventilated rabbits with severe surfactant deficiency.Conclusions. S-MB DATK is an active mimic

  16. Priming of Anti-Human Immunodeficiency Virus (HIV) CD8^+ Cytotoxic T Cells in vivo by Carrier-Free HIV Synthetic Peptides

    Science.gov (United States)

    Hart, Mary Kate; Weinhold, Kent J.; Scearce, Richard M.; Washburn, Eileen M.; Clark, Cynthia A.; Palker, Thomas J.; Haynes, Barton F.

    1991-11-01

    The generation of antiviral cytotoxic T lymphocytes (CTLs) is a critical component of the immune response to viral infections. A safe and nontoxic vaccine for AIDS would optimally use a carrier-free synthetic peptide immunogen containing only components of HIV necessary for induction of protective immune responses. We report that hybrid synthetic peptides containing either a HIV envelope gp120 T-cell determinant (T1) or the envelope gp41 fusion domain (F) N-terminal to HIV CTL determinants are capable of priming murine CD8^+, major histocompatibility complex class I-restricted anti-HIV CTLs in vivo. These data demonstrate that carrier-free, nonderivatized synthetic peptides can be used in vivo to induce anti-HIV CTL responses.

  17. The Establishment of Immunochemistry Test Based on a Synthetic Peptide Antibody for the Detection of a Porcine Circovirus-Like Virus P1

    Institute of Scientific and Technical Information of China (English)

    Libin WEN; Aihua MAO; Junming ZHOU; Lixin LU; Jianping XIE; Fengzhi WANG; Kongwang HE; Yanxiu NI; Xuehan ZHANG; Rongli GUO; Bin LI; Xiaomin WANG; Zhengyu YU

    2014-01-01

    Recently, a novel porcine circovirus-like virus P1 with a circular DNA genome of 0.648 kb was identified. P1 antigen was detected both in vitro and in vivo by synthetic peptide-derived polyclonal antibody-based immunochemistry. The designed peptides were synthesized by solid-phase technique, purified by high per-formance liquid chromatography, coupled to Keyhole limpet hemocyanin, and injected into rabbits to prepare polyclonal antibody. The emergence of positive cells revealed that synthetic peptide could elicit antibodies against P1 and viral protein could be synthesized. The polyclonal peptide antibodies described here was successfully ap-plied to immunochemical staining and proved helpful in diagnosing P1.

  18. Preparation and ectopic osteogenesis in vivo of scaffold based on mineralized recombinant human-like collagen loaded with synthetic BMP-2-derived peptide

    Energy Technology Data Exchange (ETDEWEB)

    Wu Bin; Zheng Qixin; Guo Xiaodong; Wu Yongchao [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Wang Yu; Cui Fuzai [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing 100084 (China)], E-mail: gxdwh@yahoo.com.cn

    2008-12-15

    The ideal bone graft material must be biocompatible, biodegradable, osteoconductive and osteoinductive. In this study, a new biomimetic scaffold based on mineralized recombinant collagen, nano-hydroxyapatite/recombinant human-like collagen/poly(lactic acid) (nHA/RHLC/PLA), was prepared and the synthetic P24 peptide derived from BMP-2 was introduced into the porous nHA/RHLC/PLA scaffold to improve its osteoinductive property. The nHA/RHLC/PLA implants loaded with 3 mg, 2 mg, 1 mg and 0 mg P24 peptide were implanted subcutaneously into rats. At the 4th, 8th and 12th weeks after implantation, the rats were sacrificed in batch and the samples were harvested. Their osteogenic capability was detected by CT scan and histological observation. The results indicated that the osteogenic capability of 3 mg, 2 mg and 1 mg of the P24 peptide was superior to the implants without the P24 peptide. There was no significant difference between implants with 3 mg and 2 mg P24 peptide, but the osteogenic capability of the two dosage groups was significantly better than that of the 1 mg group. It was concluded that BMP-2-derived peptide can increase the osteoinduction of nHA/RHLC/PLA scaffold and the P24 peptide induced new bone formation in a dose-dependent manner. The nHA/RHLC/PLA scaffold loaded with the synthetic BMP-2-derived peptide is a kind of ideal scaffold material for bone tissue engineering.

  19. Metalloprotease Peptide Inhibitors: A Semester-Long Organic Synthetic Research Project for the Introductory Laboratory Course

    Science.gov (United States)

    Pontrello, Jason K.

    2015-01-01

    A semester-long research project to synthesize unique compounds designed after published metalloprotease peptide inhibitors is presented. The research project encompasses a set of nine organic chemistry reactions traditionally taught in the second semester lab course, and the procedures are derived from scientific literature. The two principle…

  20. Nitrogen Forms in Synthetic Humic Acids Using Nitrogen—15 Nuclear Magnetic Resonance Technique

    Institute of Scientific and Technical Information of China (English)

    ZHUOSU-NENG; WENQI-XIAO

    1993-01-01

    15N-labelled phenolic polymers were synthesized by reactions of p-benzoquinone and 1,4-diphenol with 15N-labelled glycine and were studied by using 15N CP-MAS NMR technique in combination with chemical approaches.Results showed that the proportion of polymer nitrogen as N-phenyl amino acid N was not as great as expected,only accounting for 5%-15%;and most of N in polymers occurred in the forms of amide,pyrrole-and indole-like nitrogen,aliphatic amines and isonitrile.It seems that great differences existed between synthetic humic acids and soil humic acids in the type and distribution of nitrogen forms.

  1. Peptide and protein building blocks for synthetic biology: from programming biomolecules to self-organized biomolecular systems.

    Science.gov (United States)

    Bromley, Elizabeth H C; Channon, Kevin; Moutevelis, Efrosini; Woolfson, Derek N

    2008-01-18

    There are several approaches to creating synthetic-biological systems. Here, we describe a molecular-design approach. First, we lay out a possible synthetic-biology space, which we define with a plot of complexity of components versus divergence from nature. In this scheme, there are basic units, which range from natural amino acids to totally synthetic small molecules. These are linked together to form programmable tectons, for example, amphipathic alpha-helices. In turn, tectons can interact to give self-assembled units, which can combine and organize further to produce functional assemblies and systems. To illustrate one path through this vast landscape, we focus on protein engineering and design. We describe how, for certain protein-folding motifs, polypeptide chains can be instructed to fold. These folds can be combined to give structured complexes, and function can be incorporated through computational design. Finally, we describe how protein-based systems may be encapsulated to control and investigate their functions.

  2. The impact of synthetic analogs of histidine on copper(II) and nickel(II) coordination properties to an albumin-like peptide. Possible leads towards new metallodrugs.

    Science.gov (United States)

    Zawisza, Izabela; Mital, Mariusz; Polkowska-Nowakowska, Agnieszka; Bonna, Arkadiusz; Bal, Wojciech

    2014-10-01

    The purpose of our research was to obtain peptidomimetics possessing Cu(II) and Ni(II) binding properties, which would be useful for biomedical applications. In this context we used potentiometry, UV-VIS and CD spectroscopies to characterize the Cu(II) and Ni(II) binding properties of pentapeptide analogs of the N-terminal sequence of histatin 5. The peptides investigated had a general sequence DSXAK-am (am stands for C-terminal amide), with X including His and its three synthetic analogs, (4-thiazolyl)-L-alanine (1), (2-pyridyl)-L-alanine (2), and (pyrazol-1-yl)-L-alanine (3). The heterocyclic nitrogens present in these analogs were significantly more acidic than that of the His imidazole. We found that DSXAK-am peptides were able to bind Cu(II) and Ni(II) and form 4N complexes in a cooperative fashion, with similar affinities. These results indicate that acidic heterocyclic amino acids provide a viable alternative for histidine in peptidomimetics designed for metal ion binding.

  3. Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

    Directory of Open Access Journals (Sweden)

    Rina Ogawa

    Full Text Available We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA, but not to other phospholipids such as phosphatidylcholine (PC, phosphatidylethanolamine (PE, and phosphatidylserine (PS. A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide, was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

  4. Syntheses and Self-assembling Behaviors of Pentagonal Conjugates of Tryptophane Zipper-Forming Peptide

    Directory of Open Access Journals (Sweden)

    Nobuo Kimizuka

    2011-08-01

    Full Text Available Pentagonal conjugates of tryptophane zipper-forming peptide (CKTWTWTE with a pentaazacyclopentadecane core (Pentagonal-Gly-Trpzip and Pentagonal-Ala-Trpzip were synthesized and their self-assembling behaviors were investigated in water. Pentagonal-Gly-Trpzip self-assembled into nanofibers with the width of about 5 nm in neutral water (pH 7 via formation of tryptophane zipper, which irreversibly converted to nanoribbons by heating. In contrast, Pentagonal-Ala-Trpzip formed irregular aggregates in water.

  5. Atomic force microscopy of bacteria reveals the mechanobiology of pore forming peptide action.

    Science.gov (United States)

    Mularski, Anna; Wilksch, Jonathan J; Hanssen, Eric; Strugnell, Richard A; Separovic, Frances

    2016-06-01

    Time-resolved AFM images revealed that the antimicrobial peptide (AMP) caerin 1.1 caused localised defects in the cell walls of lysed Klebsiella pneumoniae cells, corroborating a pore-forming mechanism of action. The defects continued to grow during the AFM experiment, in corroboration with large holes that were visualised by scanning electron microscopy. Defects in cytoplasmic membranes were visualised by cryo-EM using the same peptide concentration as in the AFM experiments. At three times the minimum inhibitory concentration of caerin, 'pores' were apparent in the outer membrane. The capsule of K. pneumoniae AJ218 was unchanged by exposure to caerin, indicating that the ionic interaction of the positively charged peptide with the negatively charged capsular polysaccharide is not a critical component of AMP interaction with K. pneumoniae AJ218 cells. Further, the presence of a capsule confers no advantage to wild-type over capsule-deficient cells when exposed to the AMP caerin.

  6. Ribosome-independent biosynthesis of biologically active peptides: Application of synthetic biology to generate structural diversity.

    Science.gov (United States)

    Giessen, Tobias W; Marahiel, Mohamed A

    2012-07-16

    Peptide natural products continue to play an important role in modern medicine as last-resort treatments of many life-threatening diseases, as they display many interesting biological activities ranging from antibiotic to antineoplastic. A large fraction of these microbial natural products is assembled by ribosome-independent mechanisms. Progress in sequencing technology and the mechanistic understanding of secondary metabolite pathways has led to the discovery of many formerly cryptic natural products and a molecular understanding of their assembly. Those advances enable us to apply protein and metabolic engineering approaches towards the manipulation of biosynthetic pathways. In this review we discuss the application potential of both templated and non-templated pathways as well as chemoenzymatic strategies for the structural diversification and tailoring of peptide natural products. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Model prodrugs designed for the intestinal peptide transporter. A synthetic approach for coupling of hydroxy-containing compounds to dipeptides

    DEFF Research Database (Denmark)

    Friedrichsen, G M; Nielsen, C U; Steffansen, B

    2001-01-01

    The human peptide transporter, hPepT1, situated in the small intestine, may be exploited to increase absorption of drugs or model drugs by attaching them to a dipeptide, which is recognised by hPepT1. A synthetic protocol for this kind of model prodrugs was developed, in which model drugs...... intestine and be converted to the parent drug during or after transport into the blood circulation. Therefore, we investigated the influence of the electronegativity of the substituent in the 4-position of the phenyl ring on stability in aqueous solution at pH 6.0 and 7.4, corresponding to pH in jejunum...... and blood, respectively. In addition, the influence of the electronegativity of the substituent on stability upon storage was examined. Model prodrugs containing electron donating substituents in the 4-position of the phenyl ring decomposed upon storage, while model prodrugs containing no substituents...

  8. [Enhanced resistance to phytopathogenic bacteria in transgenic tobacco plants with synthetic gene of antimicrobial peptide cecropin P1].

    Science.gov (United States)

    Zakharchenko, N S; Rukavtsova, E B; Gudkov, A T; Bur'ianov, Ia I

    2005-11-01

    Plasmids with a synthetic gene of the mammalian antimicrobial peptide cecropin P1 (cecP1) controlled by the constitutive promoter 35S RNA of cauliflower mosaic virus were constructed. Agrobacterial transformation of tobacco plants was conducted using the obtained recombinant binary vector. The presence of gene cecP1 in the plant genome was confirmed by PCR. The expression of gene cecP1 in transgenic plants was shown by Northern blot analysis. The obtained transgenic plants exhibit enhanced resistance to phytopathogenic bacteria Pseudomonas syringae, P. marginata, and Erwinia carotovora. The ability of transgenic plants to express cecropin P1 was transmitted to the progeny. F1 and F2 plants had the normal phenotype (except for a changed coloration of flowers) and retained the ability to produce normal viable seeds upon self-pollination. Lines of F1 plants with Mendelian segregation of transgenic traits were selected.

  9. Quantitative matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis of synthetic polymers and peptides.

    Science.gov (United States)

    Hyzak, Lukas; Moos, Rebecca; von Rath, Friederike; Wulf, Volker; Wirtz, Michaela; Melchior, David; Kling, Hans-Willi; Köhler, Michael; Gäb, Siegmar; Schmitz, Oliver J

    2011-12-15

    Matrix-assisted laser desorption ionization (MALDI) is a very powerful and widely used mass spectrometric technique to ionize high molecular weight compounds. The most commonly used dried droplet (DD) technique can lead to a concentration distribution of the analyte on the target and is therefore often not suitable for reproducible analyses. We developed a new solvent-free deposition technique, called compressed sample (CS), to prevent the distribution of the analytes caused by the crystallization of the compounds. The CS technique presented in this work allows the quantitative analysis of synthetic polymers such as derivatized maltosides with correlation coefficients of 0.999 and peptides up to 3500 Da with correlation coefficients of at least 0.982 without the use of stable-isotope-labeled standards.

  10. Eight at one stroke - a synthetic tetra-disulfide peptide epitope.

    Science.gov (United States)

    Schrimpf, Andreas; Linne, Uwe; Geyer, Armin

    2017-02-13

    We have designed a cysteine-rich β-hairpin peptide which dimerises spontaneously to the antiparallel double β-hairpin motif C1-C12', C1'-C12, C5-C8, C5'-C8'-tricyclo-(CHWECCitGCRLVC)2. The highly regioselective oxidation of eight cysteines yields an intermolecular bi-disulfide 24mer hinge peptide from two individual 12mer β-hairpins, each rigidified by an additional intramolecular disulfide bond - all in all a tetra-disulfide. The reaction kinetics of air-oxidation were followed by HPLC and the constitutional isomer was identified by mass spectrometry. The hairpin conformation was characterised in detail by NMR spectroscopy and the opening angle of the antiparallel hinge was estimated from drift times obtained by ion-mobility spectrometry. Based on a set of investigated disulfide motifs, we are able to rationalise how the unbalanced number of bonded and non-bonded hydrogen pairs in a 12 mer hairpin causes their dimerisation. The unique dimeric bi-/tetra-disulfides provide systematic insights into β-hairpin formation. They can serve as a standalone structural element for the oligomerisation of peptide epitopes where structural diversity is generated from a minimal number of amino acids.

  11. Solution behavior of synthetic silk peptides and modified recombinant silk proteins

    Science.gov (United States)

    Foo, C. Wong Po; Bini, E.; Huang, J.; Lee, S. Y.; Kaplan, D. L.

    2006-02-01

    Spider dragline silk from Nephila clavipes possesses impressive mechanical properties derived in part from repetitive primary sequence containing polyalanine regions that self-assemble into crystalline β-sheets. In the present study, we have sought to understand more details of redox responses related to conformational transitions of modified silk peptides and a recombinant protein containing encoded methionine triggers. Regardless of the position of the methionine trigger relative to the polyalanine domain, chemical oxidation was rapid and slight increases in the α-helical structure and decreases in the β-sheet and random coil content were observed by CD and FTIR in the assembled silk-like peptides and the recombinant protein. CD results indicated that the decrease in β-sheet and random coil conformations, coupled with the increase in helical content during oxidation, occurred during the first 30 min of the reaction. No further conformational changes occurred after this time and the response was independent of methionine trigger location relative to the penta-alanine domain. These results were confirmed with fluorescence studies. The design, processing and utility of these modified redox triggered silk-like peptides and proteins suggest a range of potential utility, from biomaterials to engineered surface coatings with chemically alterable secondary structure and, thus, properties.

  12. Synthetic Peptides Derived from Bovine Lactoferricin Exhibit Antimicrobial Activity against E. coli ATCC 11775, S. maltophilia ATCC 13636 and S. enteritidis ATCC 13076

    Directory of Open Access Journals (Sweden)

    Nataly De Jesús Huertas Méndez

    2017-03-01

    Full Text Available Linear, dimeric, tetrameric, and cyclic peptides derived from lactoferricin B–containing non-natural amino acids and the RWQWR motif were synthesized, purified, and characterized using RP-HPLC, MALDI-TOF mass spectrometry, and circular dichroism. The antibacterial activity of peptides against Escherichia coli ATCC 11775, Stenotrophomonas maltophilia ATCC 13636, and Salmonella enteritidis ATCC 13076 was evaluated. The minimum inhibitory concentration (MIC and minimum bactericidal concentration (MBC were determined. The synthetic bovine lactoferricin exhibited antibacterial activity against E. coli ATCC 11775 and S. enteritidis ATCC 13076. The dimeric peptide (RRWQWR2K-Ahx exhibited the highest antibacterial activity against the tested bacterial strain. The monomeric, cyclic, tetrameric, and palindromic peptides containing the RWQWR motif exhibited high and specific activity against E. coli ATCC 11775. The results suggest that short peptides derived from lactoferricin B could be considered as potential candidates for the development of antibacterial agents against infections caused by E. coli.

  13. Identification of distinct physiochemical properties of toxic prefibrillar species formed by A{beta} peptide variants

    Energy Technology Data Exchange (ETDEWEB)

    Goeransson, Anna-Lena, E-mail: anngo@ifm.liu.se [Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linkoeping University (Sweden); Nilsson, K. Peter R., E-mail: petni@ifm.liu.se [Division of Organic Chemistry, Department of Physics, Chemistry and Biology, Linkoeping University (Sweden); Kagedal, Katarina, E-mail: katarina.kagedal@liu.se [Department of Clinical and Experimental Medicine, Linkoeping University (Sweden); Brorsson, Ann-Christin, E-mail: anki@ifm.liu.se [Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linkoeping University (Sweden)

    2012-04-20

    Highlights: Black-Right-Pointing-Pointer Identification of toxic prefibrillar A{beta} species. Black-Right-Pointing-Pointer Fluorescence measurements using a combined set of fluorophores. Black-Right-Pointing-Pointer Morphology studies using transmission electron microscopy. -- Abstract: The formation of amyloid-{beta} peptide (A{beta}) aggregates at an early stage during the self-assembly process is an important factor in the development of Alzheimer's disease. The toxic effect is believed to be exerted by prefibrillar species of A{beta}. It is therefore important to identify which prefibrillar species are toxic and characterize their distinct properties. In the present study, we investigated the in vitro aggregation behavior of A{beta}-derived peptides possessing different levels of neurotoxic activity, using fluorescence spectroscopy in combination with transmission electron microscopy. The toxicity of various A{beta} aggregates was assessed by using cultures of human neuroblastoma cells. Through combined use of the fluorescence probe 8-anilino-1-napthalenesulfonate (ANS) and the novel luminescent probe pentamer formyl thiophene acetic acid (p-FTAA), we were able to identify those A{beta} peptide-derived prefibrillar species which exhibited cellular toxicity. In particular, species, which formed early during the aggregation process and showed strong p-FTAA and ANS fluorescence, were the species that possessed toxic activities. Moreover, by manipulating the aggregation conditions, it was possible to change the capacity of the A{beta} peptide to form nontoxic versus toxic species.

  14. Genome-wide identification of genes conferring energy related resistance to a synthetic antimicrobial peptide (Bac8c.

    Directory of Open Access Journals (Sweden)

    Eileen C Spindler

    Full Text Available A fundamental issue in the design and development of antimicrobials is the lack of understanding of complex modes of action and how this complexity affects potential pathways for resistance evolution. Bac8c (RIWVIWRR-NH(2 is an 8 amino acid antimicrobial peptide (AMP that has been shown to have enhanced activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. We have previously demonstrated that Bac8c appears to interfere with multiple targets, at least in part through the disruption of cytoplasmic membrane related functions, and that resistance to this peptide does not easily develop using standard laboratory methods. Here, we applied a genomics approach, SCalar Analysis of Library Enrichement (SCALEs, to map the effect of gene overexpression onto Bac8c resistance in parallel for all genes and gene combinations (up to ∼ 10 adjacent genes in the E. coli genome (a total of ∼ 500,000 individual clones were mapped. Our efforts identified an elaborate network of genes for which overexpression leads to low-level resistance to Bac8c (including biofilm formation, multi-drug transporters, etc. This data was analyzed to provide insights into the complex relationships between mechanisms of action and potential routes by which resistance to this synthetic AMP can develop.

  15. Synthetic Growth Hormone-Releasing Peptides (GHRPs: A Historical Appraisal of the Evidences Supporting Their Cytoprotective Effects

    Directory of Open Access Journals (Sweden)

    Jorge Berlanga-Acosta

    2017-02-01

    Full Text Available Background: Growth hormone-releasing peptides (GHRPs constitute a group of small synthetic peptides that stimulate the growth hormone secretion and the downstream axis activity. Mounting evidences since the early 1980s delineated unexpected pharmacological cardioprotective and cytoprotective properties for the GHRPs. However, despite intense basic pharmacological research, alternatives to prevent cell and tissue demise before lethal insults have remained as an empty niche in the clinical armamentarium. Here, we have rigorously reviewed the investigational development of GHRPs and their clinical niching perspectives. Methodology: PubMed/MEDLINE databases, including original research and review articles, were explored. The search design was date escalated from 1980 and included articles in English only. Results and Conclusions: GHRPs bind to two different receptors (GHS-R1a and CD36, which redundantly or independently exert relevant biological effects. GHRPs’ binding to CD36 activates prosurvival pathways such as PI-3K/AKT1, thus reducing cellular death. Furthermore, GHRPs decrease reactive oxygen species (ROS spillover, enhance the antioxidant defenses, and reduce inflammation. These cytoprotective abilities have been revealed in cardiac, neuronal, gastrointestinal, and hepatic cells, representing a comprehensive spectrum of protection of parenchymal organs. Antifibrotic effects have been attributed to some of the GHRPs by counteracting fibrogenic cytokines. In addition, GHRP family members have shown a potent myotropic effect by promoting anabolia and inhibiting catabolia. Finally, GHRPs exhibit a broad safety profile in preclinical and clinical settings. Despite these fragmented lines incite to envision multiple pharmacological uses for GHRPs, especially as a myocardial reperfusion damage-attenuating candidate, this family of “drugable” peptides awaits for a definitive clinical niche.

  16. A synthetic peptide derived from the animo acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. Carlson; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1990-01-01

    textabstractSynthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP'2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific

  17. Inhibition of fibrin polymerization by synthetic peptides corresponding to Aalpha195-205 and gamma69-77 sites of fibrin molecule

    NARCIS (Netherlands)

    Pozniak, T.A.; Urvant, L.P.; Gritsenko, P.; Chernishov, V.I.; Pydiura, N.A.; Lugovskoi, E.V.; Komisarenko, S.V.

    2014-01-01

    Using the idea of "proline brackets" we have found four sites in fibrin amino acid sequence, and appropriate peptides were synthesized: gamma69NPDESSKPN77, Bbeta228QPDSSVKPY236, Bbeta455RPFFPQ460 and Aalpha195LPSRDRQHLPL205. Turbidity and electron-microscopy analyses have demonstrated that synthetic

  18. Induction of p53-Specific Immunity by a p53 Synthetic Long Peptide Vaccine in Patients Treated for Metastatic Colorectal Cancer

    NARCIS (Netherlands)

    Speetjens, Frank M.; Kuppen, PeterJ. K.; Welters, Marij. J. P.; Essahsah, Farah; van den Brink, Anne Marie E. G. Voet; Lantrua, M. Graziella Kallenberg; Valentijn, A. Rob P. M.; Oostendorp, Jaap; Fathers, Lorraine M.; Nijman, Hans W.; Drijfhout, Jan W.; van de Velde, Cornelis J. H.; Melief, Cornelis J. M.; van der Burg, Sjoerd H.

    2009-01-01

    Purpose: The tumor-associated self-antigen p53 is commonly overexpressed in cancer, including colorectal cancer, and can serve as a target for immunotherapy. The safety and immunogenicity of a p53 synthetic long peptide (p53-SLP) vaccine were investigated in patients treated for metastatic colorecta

  19. WT1 vaccination in AML and MDS: A pilot trial with synthetic analog peptides.

    Science.gov (United States)

    Brayer, Jason; Lancet, Jeffrey E; Powers, John; List, Alan; Balducci, Lodovico; Komrokji, Rami; Pinilla-Ibarz, Javier

    2015-07-01

    Peptide vaccines are capable of eliciting immune responses targeting tumor-associated antigens such as the Wilms' Tumor 1 (WT1) antigen, often overexpressed in myeloid malignancies. Here, we assessed the safety, tolerability, and immunogenicity of a polyvalent WT1 peptide vaccine. Individuals with WT1-positive acute myeloid leukemia (AML) in first (CR1) or second (CR2) remission or with higher-risk myelodysplastic syndrome (MDS) following at least 1 prior line of therapy were vaccinated with a mixture of peptides derived from the WT1 protein, with sargramostim injections before vaccination to amplify immunogenicity. Six vaccinations were delivered biweekly, continuing then monthly until patients received 12 vaccinations or showed disease relapse or progression. Therapeutic efficacy was evaluated by progression-free and overall survival. Immune responses were evaluated by delayed-type hypersensitivity testing and T-cell IFNγ ELISPOT at specified intervals. In 16 patients who received at least one vaccination, 10 completed the planned course of six vaccinations and six continued for up to six additional monthly vaccinations. Vaccinations were well tolerated, with no patients discontinuing due to toxicity. One of two patients with high-risk MDS experienced a prolonged decrease in transfusion dependence. Two of 14 AML patients demonstrated relapse-free survival >1 year. Both patients were in CR2 at time of vaccination, with duration of their remission exceeding duration of their first remission, suggesting a potential benefit. Our WT1 vaccine was well-tolerated. The clinical benefit that we observed in several patients suggests engagement of a protective immune response, indicating a need for further trials.

  20. Phosphorylated form of adrenocorticotropin and corticotropin-like intermediary lobe peptide in human tumors

    Energy Technology Data Exchange (ETDEWEB)

    Massias, J.F.; Hardouin, S.; Vieau, D.; Lenne, F.; Bertagna, X. (Univ Rene Descartes, Paris (France))

    1994-10-01

    Many peptides contribute to the heterogeneity of immunoreactive adrenocorticotropin (ACTH) in man. The use of a radioimmunoassay (RIA) specifically directed against the C-terminal end of ACTH allowed the precise study of the following four peptides: ACTH itself, corticotropin-like intermediary lobe peptide (CLIP) or ACTH and their phosphorylated forms on SeR[sup 31]. The authors have set up a high-performance liquid chromatography system that separates these four molecules in a single run, to establish their relative distributions in tumors responsible for Cushing's disease or for the ectopic ACTH syndrome, and to evaluate the possible interference of phospho-Ser[sup 31] on various RIA or immuno-radiometric assay (IRMA) recognition systems for ACTH. In this system, alkaline phosphatase treatment shifted the retention time of the phosphorylated peptides to that of their non-phosphorylated counterparts. In three tumors responsible for the ectopic ACTH syndrome, CLIP peptides were predominant in two and phosphorylated molecules represented between 22% and 50% of immuno-reactive materials. In five pituitary tumors responsible for Cushing's disease, ACTH peptides were predominant and the phosphorylated molecules varied between 35% and 75% in four of them. In the same tumor the ratios of phosphorylated to non-phosphorylated CLIP or ACTH were identical. The presence of phospho-Ser[sup 31] did not affect the recognition ability of two mid-ACTH and two C-terminal ACTH RIA's, nor of the ACTH IRMA. 15 refs., 5 figs., 2 tabs.

  1. Selective inhibition by a synthetic hirudin peptide of fibrin-dependent thrombosis in baboons

    Energy Technology Data Exchange (ETDEWEB)

    Cadroy, Y.; Hanson, S.R.; Harker, L.A. (Emory Univ., Atlanta, GA (United States)); Maraganore, J.M. (Biogen Inc., Cambridge, MA (United States))

    1991-02-15

    To determine the importance of the thrombin substrate recognition exosite for fibrinogen binding in the formation of both arterial and venous thrombi the authors evaluated the antithrombotic effects of the tyrosine-sulfated dodecapeptide from residues 53-64 of hirudin (H peptide) in a nonhuman primate model. This peptide was studied because it inhibits thrombin cleavages of fibrinogen by simple competition without blocking enzyme catalytic-site function. When an exteriorized arteriovenous access shunt model was used in baboons (Papio anubis), thrombus formation was induced by placing a thrombogenic device made of (i) a segment of tubing coated covalently with type I collagen, which generated platelet-rich thrombi under arterial flow conditions, and (ii) two subsequent annular regions of flow expansion that produced fibrin-rich thrombi typically associated with venous valves and veins. Thrombus formation was quantified by measurements of {sup 111}In-labeled platelet and {sup 125}I-labeled fibrinogen deposition in both arterial-flow and venous-flow portions of the device. These finding suggest that, by competitive inhibition of fibrinogen binding to thrombin, fibrin-rich venous-type thrombus formation may be selectively prevented. This strategy may be therapeutically attractive for preserving normal platelet function when conventional anticoagulant therapy is contraindicated.

  2. Synthetic surfactant containing SP-B and SP-C mimics is superior to single-peptide formulations in rabbits with chemical acute lung injury

    Directory of Open Access Journals (Sweden)

    Frans J. Walther

    2014-05-01

    Full Text Available Background. Chemical spills are on the rise and inhalation of toxic chemicals may induce chemical acute lung injury (ALI/acute respiratory distress syndrome (ARDS. Although the pathophysiology of ALI/ARDS is well understood, the absence of specific antidotes has limited the effectiveness of therapeutic interventions.Objectives. Surfactant inactivation and formation of free radicals are important pathways in (chemical ALI. We tested the potential of lipid mixtures with advanced surfactant protein B and C (SP-B and C mimics to improve oxygenation and lung compliance in rabbits with lavage- and chemical-induced ALI/ARDS.Methods. Ventilated young adult rabbits underwent repeated saline lung lavages or underwent intratracheal instillation of hydrochloric acid to induce ALI/ARDS. After establishment of respiratory failure rabbits were treated with a single intratracheal dose of 100 mg/kg of synthetic surfactant composed of 3% Super Mini-B (S-MB, a SP-B mimic, and/or SP-C33 UCLA, a SP-C mimic, in a lipid mixture (DPPC:POPC:POPG 5:3:2 by weight, the clinical surfactant Infasurf®, a bovine lung lavage extract with SP-B and C, or synthetic lipids alone. End-points consisted of arterial oxygenation, dynamic lung compliance, and protein and lipid content in bronchoalveolar lavage fluid. Potential mechanism of surfactant action for S-MB and SP-C33 UCLA were investigated with captive bubble surfactometry (CBS assays.Results. All three surfactant peptide/lipid mixtures and Infasurf equally lowered the minimum surface tension on CBS, and also improved oxygenation and lung compliance. In both animal models, the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA led to better arterial oxygenation and lung compliance than single peptide synthetic surfactants and Infasurf. Synthetic surfactants and Infasurf improved lung function further in lavage- than in chemical-induced respiratory failure, with the difference probably due to greater capillary

  3. Recombinant protein- and synthetic peptide-based immunoblot test for diagnosis of neurocysticercosis.

    Science.gov (United States)

    Noh, John; Rodriguez, Silvia; Lee, Yeuk-Mui; Handali, Sukwan; Gonzalez, Armando E; Gilman, Robert H; Tsang, Victor C W; Garcia, Hector H; Wilkins, Patricia P

    2014-05-01

    One of the most well-characterized tests for diagnosing neurocysticercosis (NCC) is the enzyme-linked immunoelectrotransfer blot (EITB) assay developed at the CDC, which uses lentil lectin-bound glycoproteins (LLGP) extracted from Taenia solium cysticerci. Although the test is very reliable, the purification process for the LLGP antigens has been difficult to transfer to other laboratories because of the need for expensive equipment and technical expertise. To develop a simpler assay, we previously purified and cloned the diagnostic glycoproteins in the LLGP fraction. In this study, we evaluated three representative recombinant or synthetic antigens from the LLGP fraction, individually and in different combinations, using an immunoblot assay (recombinant EITB). Using a panel of 249 confirmed NCC-positive and 401 negative blood serum samples, the sensitivity of the recombinant EITB assay was determined to be 99% and the specificity was 99% for diagnosing NCC. We also tested a panel of 239 confirmed NCC-positive serum samples in Lima, Peru, and found similar results. Overall, our data show that the performance characteristics of the recombinant EITB assay are comparable to those of the LLGP-EITB assay. This new recombinant- and synthetic antigen-based assay is sustainable and can be easily transferred to other laboratories in the United States and throughout the world.

  4. Peptides: A new class of anticancer drugs

    Directory of Open Access Journals (Sweden)

    Ryszard Smolarczyk

    2009-07-01

    Full Text Available Peptides are a novel class of anticancer agents embracing two distinct categories: natural antibacterial peptides, which are preferentially bound by cancer cells, and chemically synthesized peptides, which bind specifically to precise molecular targets located on the surface of tumor cells. Antibacterial peptides bind to both cell and mitochondrial membranes. Some of these peptides attach to the cell membrane, resulting in its disorganization. Other antibacterial peptides penetrate cancer cells without causing cell membrane damage, but they disrupt mitochondrial membranes. Thanks to phage and aptamer libraries, it has become possible to obtain synthetic peptides blocking or activating some target proteins found in cancer cells as well as in cells forming the tumor environment. These synthetic peptides can feature anti-angiogenic properties, block enzymes indispensable for sustained tumor growth, and reduce tumor ability to metastasize. In this review the properties of peptides belonging to both categories are discussed and attempts of their application for therapeutic purposes are outlined.

  5. Lsr2 of Mycobacterium leprae and Its Synthetic Peptides Elicit Restitution of T Cell Responses in Erythema Nodosum Leprosum and Reversal Reactions in Patients with Lepromatous Leprosy

    Science.gov (United States)

    Saini, Chaman; Prasad, H. K.; Rani, Rajni; Murtaza, A.; Misra, Namita; Shanker Narayan, N. P.

    2013-01-01

    The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742–752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs. PMID:23446220

  6. Bone induction by biomimetic PLGA copolymer loaded with a novel synthetic RADA16-P24 peptide in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Haitao; Hao, Shaofei [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Zheng, Qixin, E-mail: zheng-qx@163.com [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Li, Jingfeng [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Department of Orthopedics, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Zheng, Jin; Hu, Zhilei; Yang, Shuhua; Guo, Xiaodong [Department of Orthopaedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022 (China); Yang, Qin [Advanced Biomaterials and Tissue Engineering Center, Huazhong University of Science and Technology, Wuhan 430074 (China)

    2013-08-01

    Bone morphogenetic protein-2 (BMP-2) is a key bone morphogenetic protein, and poly(lactic-co-glycolic acid) (PLGA) has been widely used as scaffold for clinical use to carry treatment protein. In the previous studies, we have synthesized BMP-2-related peptide (P24) and found its capacity of inducing bone regeneration. In this research, we have synthesized a new amphiphilic peptide Ac-RADA RADA RADA RADA S[PO4]KIPKASSVPTELSAISTLYLDDD-CONH2 (RADA16-P24) with an assembly peptide RADA16-Ion the P24 item of BMP2 to form divalent ion-induced gelatin. Two methods of physisorption and chemical cross-linking were used to bind RADA16-P24 onto the surface of the copolymer PLGA to synthesize RADA16-P24–PLGA, and its capacity of attaching bone marrow stromal cells (BMSCs) was evaluated in vitro and inducing ectopic bone formation was examined in vivo. In vitro our results demonstrated that RADA16-P24–PLGA copolymer prepared by physisorbing or prepared by chemical cross-linking had a peptide binding rate of (2.0180 ± 0.5296)% or (10.0820 ± 0.8405)% respectively (P < 0.05). In addition the BMSCs proliferated vigorously in the RADA16-P24–PLGA biomaterials. Significantly the percentage of BMSCs attached to RADA16-P24–PLGA composite prepared by chemical cross-linking and physisorbing were (71.4 ± 7.5) % or (46.7 ± 5.8) % (P < 0.05). The in vivo study showed that RADA16-P24–PLGA chemical cross-linking could better induce ectopic bone formation compared with RADA16-P24–PLGA physisorbing and PLGA. It is concluded that the PLGA copolymer is a good RADA16-P24 carrier. This novel RADA16-P24–PLGA composite has strong osteogenic capability. - Highlights: • We have synthesized a new RADA16-P24 amphiphilic peptide. • It is an assembly peptide RADA16-Ion the P24 to form divalent ion-induced gelatin. • RADA16-P24/PLGA could better induce etopia osteogenesis compared with PLGA. • RADA16-P24–PLGA has strong osteogenic capability.

  7. Characterization of Protein and Peptide Binding to Nanogels Formed by Differently Charged Chitosan Derivatives

    Directory of Open Access Journals (Sweden)

    Anastasia Zubareva

    2013-07-01

    Full Text Available Chitosan (Chi is a natural biodegradable cationic polymer with remarkable potency as a vehicle for drug or vaccine delivery. Chi possesses multiple groups, which can be used both for Chi derivatization and for particle formation. The aim of this work was to produce stable nanosized range Chi gels (nanogels, NGs with different charge and to study the driving forces of complex formation between Chi NGs and proteins or peptides. Positively charged NGs of 150 nm in diameter were prepared from hexanoyl chitosan (HC by the ionotropic gelation method while negatively charged NGs of 190 nm were obtained from succinoyl Chi (SC by a Ca2+ coacervation approach. NGs were loaded with a panel of proteins or peptides with different weights and charges. We show that NGs preferentially formed complexes with oppositely charged molecules, especially peptides, as was demonstrated by gel-electrophoresis, confocal microscopy and HPLC. Complex formation was accompanied by a change in zeta-potential and decrease in size. We concluded that complex formation between Chi NGs and peptide/proteins is mediated mostly by electrostatic interactions.

  8. Direct quantitation of peptide mixtures without standards using clusters formed by electrospray ionization mass spectrometry.

    Science.gov (United States)

    Leib, Ryan D; Flick, Tawnya G; Williams, Evan R

    2009-05-15

    In electrospray ionization mass spectrometry, ion abundances depend on a number of different factors, including analyte surface activity, competition between analytes for charge, analyte concentration, as well as instrumental factors, including mass-dependent ion transmission and detection. Here, a novel method for obtaining quantitative information about solution-phase concentrations of peptide mixtures is described and demonstrated for five different peptide mixtures with relative concentrations ranging from 0.05% to 50%. In this method, the abundances of large clusters containing anywhere from 0 to 13 impurity molecules are measured and directly related to the relative solution-phase concentration of the peptides. For clusters containing approximately 15 or more peptides, the composition of the clusters approaches the statistical value indicating that these clusters are formed nonspecifically and that any differences in ion detection or ionization efficiency are negligible at these large cluster sizes. This method is accurate to within approximately 20% or better, even when the relative ion intensities of the protonated monomers can differ by over an order of magnitude compared to their solution-phase concentrations. Although less accurate than other quantitation methods that employ internal standards, this method does have the key advantages of speed, simplicity, and the ability to quantitate components in solution even when the identities of the components are unknown.

  9. Structure-Activity Relationship and Mode of Action of a Frog Secreted Antibacterial Peptide B1CTcu5 Using Synthetically and Modularly Modified or Deleted (SMMD) Peptides.

    Science.gov (United States)

    Abraham, Parvin; Sundaram, Anand; R, Asha; V, Reshmy; George, Sanil; Kumar, K Santhosh

    2015-01-01

    All life forms are equipped with rapidly acting, evolutionally conserved components of an innate immune defense system that consists of a group of unique and diverse molecules known as host defense peptides (HDPs). A Systematic and Modular Modification and Deletion (SMMD) approach was followed to analyse the structural requirement of B1CTcu5, a brevinin antibacterial peptide amide identified from the skin secretion of frog Clinotarsus curtipes, India, to show antibacterial activity and to explore the active core region. Seventeen SMMD-B1CTcu5 analogs were designed and synthesised by C and N-terminal amino acid substitution or deletion. Enhancement in cationicity by N-terminal Lys/Arg substitution or hydrophobicity by Trp substitution produced no drastic change in bactericidal nature against selected bacterial strains except S. aureus. But the sequential removal of N-terminal amino acids had a negative effect on bactericidal potency. Analog B1CTcu5-LIAG obtained by the removal of four N-terminal amino acids displayed bactericidal effect comparable to, or in excess of, the parent peptide with reduced hemolytic character. Its higher activity was well correlated with the improved inner membrane permeabilisation capacity. This region may act as the active core of B1CTcu5. Presence of C-terminal disulphide bond was not a necessary condition to display antibacterial activity but helped to promote hemolytic nature. Removal of the C-terminal rana box region drastically reduced antibacterial and hemolytic activity of the peptide, showing that this region is important for membrane targeting. The bactericidal potency of the D-peptide (DB1CTcu5) helped to rule out the stereospecific interaction with the bacterial membrane. Our data suggests that both the C and N-terminal regions are necessary for bactericidal activity, even though the active core region is located near the N-terminal of B1CTcu5. A judicious modification at the N-terminal region may produce a short SMMD analog

  10. Combinatorial Synthetic Peptide Vaccine Strategy Protects against Hypervirulent CovR/S Mutant Streptococci.

    Science.gov (United States)

    Pandey, Manisha; Mortensen, Rasmus; Calcutt, Ainslie; Powell, Jessica; Batzloff, Michael R; Dietrich, Jes; Good, Michael F

    2016-04-15

    Cluster of virulence responder/sensor (CovR/S) mutant group A streptococci (GAS) are serious human pathogens of multiple M protein strains that upregulate expression of virulence factors, including the IL-8 proteaseStreptococcus pyogenescell envelope proteinase (SpyCEP), thus blunting neutrophil-mediated killing and enabling ingress of bacteria from a superficial wound to deep tissue. We previously showed that a combination vaccine incorporating J8-DT (conserved peptide vaccine from the M protein) and a recombinant SpyCEP fragment protects against CovR/S mutants. To enhance the vaccine's safety profile, we identified a minimal epitope (S2) that was the target for anti-SpyCEP Abs that could protect IL-8 from SpyCEP-mediated proteolysis. Abs from healthy humans and from mice experimentally infected with GAS also recognized S2, albeit at low titers. Native SpyCEP may be poorly immunogenic (cryptic or subdominant), and it would be to the organism's advantage if the host did not induce a strong Ab response against it. However, S2 conjugated to diphtheria toxoid is highly immunogenic and induces Abs that recognize and neutralize SpyCEP. Hence, we describe a two-component peptide vaccine that induces Abs (anti-S2) that protect IL-8 from proteolysis and other Abs (anti-J8) that cause strain-independent killing in the presence of neutrophils. We show that either component alone is ineffectual in preventing skin infection and bacteremia due to CovR/S mutants but that the combination induces complete protection. This protection correlated with a significant influx of neutrophils to the infection site. The data strongly suggest that the lack of natural immunity to hypervirulent GAS strains in humans could be rectified by this combination vaccine.

  11. Removal of pyridine from liquid and gas phase by copper forms of natural and synthetic zeolites

    Energy Technology Data Exchange (ETDEWEB)

    Rehakova, Maria, E-mail: maria.rehakova@upjs.sk [Institute of Chemistry, Faculty of Science, P.J. Safarik University, 041 54 Kosice (Slovakia); Fortunova, Lubica [Institute of Chemistry, Faculty of Science, P.J. Safarik University, 041 54 Kosice (Slovakia); Bastl, Zdenek [J. Heyrovsky Institute of Physical Chemistry, ASCR, v.v.i., 18223 Prague 8 (Czech Republic); Nagyova, Stanislava [Department of Physics, Electrotechnical Faculty, Technical University, 042 00 Kosice (Slovakia); Dolinska, Silvia [Institute of Geotechnics, Slovak Academy of Sciences, 043 53 Kosice (Slovakia); Jorik, Vladimir [Department of Inorganic Chemistry, Faculty of Chemical and Food Technology, Slovak University of Technology, 81237 Bratislava (Slovakia); Jona, Eugen [Department of Chemistry and Technology of Inorganic Materials, Faculty of Industrial Technologies, Trencin University of Alexander Dubcek, 02032 Puchov (Slovakia)

    2011-02-15

    Zeoadsorbents on the basis of copper forms of synthetic zeolite ZSM5 and natural zeolite of the clinoptilolite type (CT) have been studied taking into account their environmental application in removing harmful pyridine (py) from liquid and gas phase. Sorption of pyridine by copper forms of zeolites (Cu-ZSM5 and Cu-CT) has been studied by CHN, X-ray photoelectron spectroscopy, X-ray powder diffractometry, FTIR spectroscopy, thermal analysis (TG, DTA and DTG) and analysis of the surface areas and the pore volumes by low-temperature adsorption of nitrogen. The results of thermal analyses of Cu-ZSM5, Cu-(py){sub x}ZSM5, Cu-CT and Cu-(py){sub x}CT zeolitic products with different composition (x depends on the experimental conditions of sorption of pyridine) clearly confirmed their different thermal properties as well as the sorption of pyridine. In the zeolitic pyridine containing samples the main part of the pyridine release process occurs at considerably higher temperatures than is the boiling point of pyridine, which proves strong bond and irreversibility of py-zeolite interaction. FTIR spectra of Cu-(py){sub x}zeolite samples showed well resolved bands of pyridine. The results of thermal analysis and FTIR spectroscopy are in a good agreement with the results of other used methods.

  12. Removal of pyridine from liquid and gas phase by copper forms of natural and synthetic zeolites.

    Science.gov (United States)

    Reháková, Mária; Fortunová, Lubica; Bastl, Zdeněk; Nagyová, Stanislava; Dolinská, Silvia; Jorík, Vladimír; Jóna, Eugen

    2011-02-15

    Zeoadsorbents on the basis of copper forms of synthetic zeolite ZSM5 and natural zeolite of the clinoptilolite type (CT) have been studied taking into account their environmental application in removing harmful pyridine (py) from liquid and gas phase. Sorption of pyridine by copper forms of zeolites (Cu-ZSM5 and Cu-CT) has been studied by CHN, X-ray photoelectron spectroscopy, X-ray powder diffractometry, FTIR spectroscopy, thermal analysis (TG, DTA and DTG) and analysis of the surface areas and the pore volumes by low-temperature adsorption of nitrogen. The results of thermal analyses of Cu-ZSM5, Cu-(py)(x)ZSM5, Cu-CT and Cu-(py)(x)CT zeolitic products with different composition (x depends on the experimental conditions of sorption of pyridine) clearly confirmed their different thermal properties as well as the sorption of pyridine. In the zeolitic pyridine containing samples the main part of the pyridine release process occurs at considerably higher temperatures than is the boiling point of pyridine, which proves strong bond and irreversibility of py-zeolite interaction. FTIR spectra of Cu-(py)(x)zeolite samples showed well resolved bands of pyridine. The results of thermal analysis and FTIR spectroscopy are in a good agreement with the results of other used methods.

  13. Discrimination between Fibrin and Fibrinogen by a Monoclonal Antibody against a Synthetic Peptide

    Science.gov (United States)

    Scheefers-Borchel, Ursula; Muller-Berghaus, Gert; Fuhge, Peter; Eberle, Reinhard; Heimburger, Nobert

    1985-10-01

    Circulating soluble fibrin, observed in the blood of patients with ongoing intravascular coagulation, is generated from the plasma protein fibrinogen by the limited proteolytic action of thrombin. We report the production of a monoclonal antibody that discriminates between fibrin and fibrinogen in blood. The synthetic hexapeptide Gly-Pro-Arg-Val-Val-Glu, representing the amino terminus of the α chain of human fibrin, was used as immunogen. This hexapeptide is located within the Aα chain of fibrinogen but becomes the amino terminus of the fibrin α chain, after fibrinopeptide A is removed by the action of thrombin, and thus becomes accessible for antibody binding. The monoclonal antibody we have prepared can discriminate between fibrin and fibrinogen and thus can be used in assay systems to quantitate soluble fibrin or, potentially, to image fibrin-rich thrombi.

  14. Side-chain interactions form late and cooperatively in the binding reaction between disordered peptides and PDZ domains

    DEFF Research Database (Denmark)

    Haq, S Raza; Chi, Celestine N; Bach, Anders

    2012-01-01

    used short peptides as a model system for intrinsically disordered proteins. Linear free-energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate...... governed by their association rate constants. Instead, we observe the opposite for peptide-PDZ interactions, namely that changes in Kd correlate with changes in koff....

  15. Organic solvent mediated self-association of an amyloid forming peptide from beta2-microglobulin: an atomic force microscopy study.

    Science.gov (United States)

    Chaudhary, Nitin; Singh, Shashi; Nagaraj, Ramakrishnan

    2008-01-01

    Human beta(2)-microglobulin (beta(2)m) forms amyloid fibrils in hemodialysis related amyloidosis. Peptides spanning the beta strands of beta(2)m have been shown to form amyloid fibrils in isolation. We have studied the self-association of a 13-residue peptide Ac-DWSFYLLYYTEFT-am (Pbeta(2)m) spanning one of the beta-strands of human beta(2)-microglobulin when dissolved in various organic solvents such as methanol (MeOH), trifluoroethanol (TFE), hexafluoroisopropanol (HFIP), and dimethylsulfoxide. We have observed that Pbeta(2)m forms amyloid fibrils when diluted from organic solvents into aqueous buffer at pH 7.0 as judged by increase in thioflavin T fluorescence. Fibril formation was observed to depend on the solvents in which peptide stock solutions were prepared. Circular dichroism spectra indicated propensity for helical conformation in MeOH, TFE, and HFIP. In buffer, beta-structure was observed irrespective of the solvent in which the peptide stock solutions were prepared. Atomic force microscopy images obtained by drying the peptide on mica from organic solvents indicated the ability of Pbeta(2)m to self-associate to form nonfibrillar structures. Morphology of the structures was dependent on the solvent in which the peptide was dissolved. Peptides that have the ability to self-associate such as amyloid-forming peptides would be attractive candidates for the generation of self-assembled structures with varying morphologies by appropriate choice of surfaces and solvents for dissolution. Copyright 2008 Wiley Periodicals, Inc.

  16. One-step affinity purification of recombinant urokinase-type plasminogen activator receptor using a synthetic peptide developed by combinatorial chemistry

    DEFF Research Database (Denmark)

    Jacobsen, B.; Gerdsvoll, H.; Funch, G.J.

    2007-01-01

    purification of a soluble, recombinant uPAR using the monoclonal antibody R2 or the peptide AE152 immobilized on Sepharose. The two affinity ligands perform equally well in purifying uPAR from Drosophila melanogaster Schneider 2 cell culture medium and yield products of comparable purity, activity...... purification of recombinant uPAR exploiting a high-affinity synthetic peptide antagonist (AE152). The corresponding parent peptide was originally identified in a random phage-display library and subsequently subjected to affinity maturation by combinatorial chemistry. This study compares the affinity......, and stability as judged by SDS-PAGE, size exclusion chromatography and surface plasmon resonance analysis. The general availability of peptide synthesis renders the present AE152-based affinity purification of uPAR more accessible than the traditional protein-based affinity purification strategies. In this way...

  17. Therapeutic genome mutagenesis using synthetic donor DNA and triplex-forming molecules.

    Science.gov (United States)

    Reza, Faisal; Glazer, Peter M

    2015-01-01

    Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is used as a template for recombination. Targeting the genome is similarly achieved by designing the sequence-specific mutagenic triplex-forming molecules to further recruit the recombination machinery thereby upregulating its activity with the recombinagenic donor DNA. This combination of extracellularly introduced, designed synthetic molecules and intercellularly ubiquitous, evolved natural machinery enables the mutagenesis of chromosomes and engineering of whole genomes with great fidelity while limiting nonspecific interactions. Herein, we demonstrate the harnessing of recombinagenic donor DNA and mutagenic triplex-forming molecular technology for potential therapeutic applications. These demonstrations involve, among others, utilizing this technology to correct genes so that they become physiologically functional, to induce dormant yet functional genes in place of non-functional counterparts, to place induced genes under regulatory elements, and to disrupt genes to abrogate a cellular vulnerability. Ancillary demonstrations of the design and synthesis of this recombinagenic and mutagenic molecular technology as well as their delivery and assayed interaction with duplex DNA reveal a potent technological platform for engineering specific changes into the living genome.

  18. Dual-function synthetic peptide derived from BMP4 for highly efficient tumor targeting and antiangiogenesis

    Directory of Open Access Journals (Sweden)

    Choi SH

    2016-09-01

    Full Text Available Suk Hyun Choi,1,* Jue Yeon Lee,2,* Jin Sook Suh,1 Yoon Shin Park,3 Chong Pyoung Chung,2 Yoon Jeong Park1 1Department of Dental Regenerative Biotechnology, Dental Research Institute, 2Central Research Institute, Nano Intelligent Biomedical Engineering Corporation (NIBEC, School of Dentistry, Seoul National University, Seoul, 3Department in Microbiology, School of Biological Sciences, College of Natural Sciences, Chungbuk National University, Cheongju, South Korea *These authors contributed equally to this work Abstract: Angiogenesis plays a critical role in the growth and metastasis of cancer, and growth factors released from cancer promote blood-vessel formation in the tumor microenvironment. The angiogenesis is accelerated via interactions of growth factors with the high-affinity receptors on cancer cells. In particular, heparan sulfate proteoglycans (HSPGs on the surface of cancer cells have been shown to be important in many aspects of determining a tumor’s phenotype and development. Specifically, the regulation of the interactions between HSPGs and growth factors results in changes in tumor progression. A peptide with heparin-binding (HBP activity has been developed and synthesized to inhibit tumor growth via the prevention of angiogenesis. We hypothesized that HBP could inhibit the interaction of growth factors and HSPGs on the surface of cancer cells, decrease paracrine signaling in endothelial cells (ECs, and finally decrease angiogenesis in the tumor microenvironment. In this study, we found that HBP had antiangiogenic effects in vitro and in vivo. The conditioned media obtained from a breast cancer cell line treated with HBP were used to culture human umbilical vein ECs (HUVECs to evaluate the antiangiogenic effect of HBP on ECs. HBP effectively inhibited the migration, invasion, and tube formation of HUVECs in vitro. In addition, the expressions of angiogenesis-mediating factors, including ERK, FAK, and Akt, were considerably

  19. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP) does not catalytically oxidize iron.

    Science.gov (United States)

    Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Hagen, Wilfred R

    2012-01-01

    The β-amyloid precursor protein (APP), which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II) binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III) product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II) just as in ferritin. We measured the ferroxidase activity indirectly (i) by the incorporation of the Fe(III) product of the ferroxidase reaction into transferrin and directly (ii) by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II) oxidation by molecular oxygen. Zn(II) binds to transferrin and diminishes its Fe(III) incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and - by implication - of APP should be re-evaluated.

  20. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP does not catalytically oxidize iron.

    Directory of Open Access Journals (Sweden)

    Kourosh Honarmand Ebrahimi

    Full Text Available The β-amyloid precursor protein (APP, which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II just as in ferritin. We measured the ferroxidase activity indirectly (i by the incorporation of the Fe(III product of the ferroxidase reaction into transferrin and directly (ii by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II oxidation by molecular oxygen. Zn(II binds to transferrin and diminishes its Fe(III incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and - by implication - of APP should be re-evaluated.

  1. Characterization of the conformational space of a triple-stranded beta-sheet forming peptide with molecular dynamics simulations

    NARCIS (Netherlands)

    Soto, P; Colombo, G

    2004-01-01

    Molecular dynamics (MD) simulations have been performed on a series of mutants of the 20 amino acid peptide Betanova in order to critically assess the ability of MD simulations to reproduce the folding and stability of small beta-sheet-forming peptides on currently accessible timescales. Simulations

  2. LC-MS analysis of the formed peptides from N-( O, O-diiso-propyl) phosphoryl aspartic acid

    Institute of Scientific and Technical Information of China (English)

    胡建军; 巨勇; 赵玉芬

    2000-01-01

    IC-ESI-MS method was used to analyze the formed di- and ri- peptide in the reaction system of N-(O, O-diisopropyl) phosphoryl aspartic acid and adenosine. Cluster ions of the peptides were given in the ESI-MS. The structures of these small peptides were confirmed by LC-MS-MS analysis. Compared wih the traditional HPLC-UV detection, this method showed good sensitivity and selectivity for peptide in the presence of compounds with strong UV ahsorption, such as nucleoside and nucleotide.

  3. Apoptosis Activation in Human Lung Cancer Cell Lines by a Novel Synthetic Peptide Derived from Conus californicus Venom

    Directory of Open Access Journals (Sweden)

    Irasema Oroz-Parra

    2016-02-01

    Full Text Available Lung cancer is one of the most common types of cancer in men and women and a leading cause of death worldwide resulting in more than one million deaths per year. The venom of marine snails Conus contains up to 200 pharmacologically active compounds that target several receptors in the cell membrane. Due to their diversity and specific binding properties, Conus toxins hold great potential as source of new drugs against cancer. We analyzed the cytotoxic effect of a 17-amino acid synthetic peptide (s-cal14.1a that is based on a native toxin (cal14.1a isolated from the sea snail Conus californicus. Cytotoxicity studies in four lung cancer cell lines were complemented with measurement of gene expression of apoptosis-related proteins Bcl-2, BAX and the pro-survival proteins NFκB-1 and COX-2, as well as quantification of caspase activity. Our results showed that H1299 and H1437 cell lines treated with s-call4.1a had decreased cell viability, activated caspases, and reduced expression of the pro-survival protein NFκB-1. To our knowledge, this is the first report describing activation of apoptosis in human lung cancer cell lines by s-cal14.1a and we offer insight into the possible mechanism of action.

  4. Effects of N-terminus modifications on the conformation and permeation activities of the synthetic peptide L1A.

    Science.gov (United States)

    Zanin, Luciana Puia Moro; de Araujo, Alexandre Suman; Juliano, Maria Aparecida; Casella, Tiago; Nogueira, Mara Correa Lelles; Ruggiero Neto, João

    2016-06-01

    We investigate the effect of the N-terminus modification of the L1A, a synthetic octadecapeptide, on its helical content, affinity and lytic action in model membranes and on its hemolytic and antibacterial activities. L1A and its acetylated analog displayed a selective antibacterial activity to Gram-negative bacteria without being hemolytic. The covalently linked 2-aminobezoic acid to the N-terminus impaired the antibacterial efficacy and increased hemolysis. Despite their lower net charge (+2), N-terminus modifications resulted in enhanced affinity and improved lytic efficiency in anionic vesicles. The analogs also showed higher helical content and consequently higher amphipathicity in these vesicles. The conformational analysis by molecular dynamics simulations in 30 % of TFE/water showed that the hydrophobic faces of the peptides are in close contact with CF3 groups of TFE while the hydrophilic faces with water molecules. Due to the loss of the amino charge, the N-termini of the analogs are buried in TFE molecules. The analysis of the pair distribution functions, obtained for the center of mass of the charged groups, has evidenced that the state of the N-terminus has influenced the possibility of different ion-pairing. The higher complexity of the bacterial cells compared with anionic vesicles hampers to establish correlations structure-function for the analogs.

  5. Alarin but not its alternative-splicing form, GALP (Galanin-like peptide) has antimicrobial activity

    Energy Technology Data Exchange (ETDEWEB)

    Wada, Akihiro, E-mail: a-wada@nagasaki-u.ac.jp [Department of Bacteriology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523 (Japan); Wong, Pooi-Fong [Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur (Malaysia); Hojo, Hironobu [Department of Applied Biochemistry, Institute of Glycoscience, Tokai University, Kanagawa 2591292 (Japan); Hasegawa, Makoto [Department of Bioscience, Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Shiga 5260829 (Japan); Ichinose, Akitoyo [Electron Microscopy Shop Central Laboratory, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523 (Japan); Llanes, Rafael [Institute Pedro Kouri, Havana (Cuba); Kubo, Yoshinao [Division of Cytokine Signaling, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 8528523 (Japan); Senba, Masachika [Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523 (Japan); Ichinose, Yoshio [Kenya Research Station, Institute of Tropical Medicine, Nagasaki University, Nagasaki 8528523 (Japan)

    2013-05-03

    Highlights: • Alarin inhibits the growth of E. coli but not S. aureus. • Alarin’s potency is comparable to LL-37 in inhibiting the growth of E. coli. • Alarin can cause bacterial membrane blebbing. • Alalin does not induce hemolysis on erythrocytes. -- Abstract: Alarin is an alternative-splicing form of GALP (galanin-like peptide). It shares only 5 conserved amino acids at the N-terminal region with GALP which is involved in a diverse range of normal brain functions. This study seeks to investigate whether alarin has additional functions due to its differences from GALP. Here, we have shown using a radial diffusion assay that alarin but not GALP inhibited the growth of Escherichia coli (strain ML-35). The conserved N-terminal region, however, remained essential for the antimicrobial activity of alarin as truncated peptides showed reduced killing effect. Moreover, alarin inhibited the growth of E. coli in a similar potency as human cathelicidin LL-37, a well-studied antimicrobial peptide. Electron microscopy further showed that alarin induced bacterial membrane blebbing but unlike LL-37, it did not cause hemolysis of erythrocytes. In addition, alarin is only active against the gram-negative bacteria, E. coli but not the gram-positive bacteria, Staphylococcus aureus. Thus, these data suggest that alarin has potentials as an antimicrobial and should be considered for the development in human therapeutics.

  6. A synthetic M protein peptide synergizes with a CXC chemokine protease to induce vaccine-mediated protection against virulent streptococcal pyoderma and bacteremia.

    Science.gov (United States)

    Pandey, Manisha; Langshaw, Emma; Hartas, Jon; Lam, Alfred; Batzloff, Michael R; Good, Michael F

    2015-06-15

    Infections caused by Streptococcus pyogenes (group A Streptococcus [GAS]) are highly prevalent in the tropics, in developing countries, and in the Indigenous populations of developed countries. These infections and their sequelae are responsible for almost 500,000 lives lost prematurely each year. A synthetic peptide vaccine (J8-DT) from the conserved region of the M protein has shown efficacy against disease that follows i.p. inoculation of bacteria. By developing a murine model for infection that closely mimics human skin infection, we show that the vaccine can protect against pyoderma and subsequent bacteremia caused by multiple GAS strains, including strains endemic in Aboriginal communities in the Northern Territory of Australia. However, the vaccine was ineffective against a hypervirulent cluster of virulence responder/sensor mutant GAS strain; this correlated with the strain's ability to degrade CXC chemokines, thereby preventing neutrophil chemotaxis. By combining J8-DT with an inactive form of the streptococcal CXC protease, S. pyogenes cell envelope proteinase, we developed a combination vaccine that is highly effective in blocking CXC chemokine degradation and permits opsonic Abs to kill the bacteria. Mice receiving the combination vaccine were strongly protected against pyoderma and bacteremia, as evidenced by a 100-1000-fold reduction in bacterial burden following challenge. To our knowledge, a vaccine requiring Abs to target two independent virulence factors of an organism is unique.

  7. Key peptide processing enzymes are expressed by a variant form of small-cell carcinoma of the lung.

    Science.gov (United States)

    North, W G; Du, J

    1998-01-01

    Small-cell carcinoma of the lung (SCCL) is a neuroendocrine tumor characterized by having the capacity to produce and secrete a number of small neuropeptides. These peptides serve the tumor as autocrine growth factors. SCCL is known to undergo a process of dedifferentiation to a variant (drug-resistant) form, and this process is associated with loss of marker enzymes such as neuron-specific enolase (NSE) and dopa decarboxylase (DDC). The current study was designed to discover if variant SCCL, represented by cell line NCI H82, retains some capacity to generate active neuropeptides (like vasopressin) from their precursors by continuing to express the three key classes of enzymes necessary for such conversions, namely prohormone convertases (PCs), carboxypeptidases (CPs), and peptidylglycine a-amidating monooxygenase (PAM). RT-PCR for mRNAs representing PC1, PC2, CPE, and PAM was performed on total RNA extracted from NCI H82. The primers selected for PCR and partial sequencing were synthetic 20, 21, 22, and 24 oligomers designed to yield products of 533, 880, 405, and 560 base pairs (bp) for PC1, PC2, CPE, and PAM, respectively. For the conditions used, we were able to demonstrate products for all four enzymes. Each of the four products generated were of the expected size. Cloning and sequencing of these products revealed that each had a structure identical to that published for the human form of the respective enzyme. Western analysis with antibodies against PC1, PC2, CPE, and PAM, provided evidence that mRNAs for the four enzymes are translated into proteins that could represent functional forms. Our findings therefore demonstrate that key enzymes involved in the generation of active neuropeptides, unlike the marker enzymes NSE and DDC, continue to be expressed by variant SCCL.

  8. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    DEFF Research Database (Denmark)

    Birkedal, Henrik; Nielsen, Peter E

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine...... PNA conjugated to the topoisomerase I inhibitor camptothecin was found to increase the frequency of repair domain mediated gene correctional events of the EGFP reporter in an in vitro HeLa cell nuclear extract assay, whereas PNA psoralen or chlorambucil conjugates both of which form covalent and also....... Consistent with the extract experiments, treatment with adduct forming PNA conjugates (psoralen and chlorambucil) resulted in a decrease in background correction frequencies in transiently transfected cells, whereas unmodified PNA or the PNA-camptothecin conjugate had little or no effect. These results...

  9. The human cathelicidin LL-37--A pore-forming antibacterial peptide and host-cell modulator.

    Science.gov (United States)

    Xhindoli, Daniela; Pacor, Sabrina; Benincasa, Monica; Scocchi, Marco; Gennaro, Renato; Tossi, Alessandro

    2016-03-01

    The human cathelicidin hCAP18/LL-37 has become a paradigm for the pleiotropic roles of peptides in host defence. It has a remarkably wide functional repertoire that includes direct antimicrobial activities against various types of microorganisms, the role of 'alarmin' that helps to orchestrate the immune response to infection, the capacity to locally modulate inflammation both enhancing it to aid in combating infection and limiting it to prevent damage to infected tissues, the promotion of angiogenesis and wound healing, and possibly also the elimination of abnormal cells. LL-37 manages to carry out all its reported activities with a small and simple, amphipathic, helical structure. In this review we consider how different aspects of its primary and secondary structures, as well as its marked tendency to form oligomers under physiological solution conditions and then bind to molecular surfaces as such, explain some of its cytotoxic and immunomodulatory effects. We consider its modes of interaction with bacterial membranes and capacity to act as a pore-forming toxin directed by our organism against bacterial cells, contrasting this with the mode of action of related peptides from other species. We also consider its different membrane-dependent effects on our own cells, which underlie many of its other activities in host defence. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale.

  10. Sequence selective recognition of double-stranded RNA using triple helix-forming peptide nucleic acids.

    Science.gov (United States)

    Zengeya, Thomas; Gupta, Pankaj; Rozners, Eriks

    2014-01-01

    Noncoding RNAs are attractive targets for molecular recognition because of the central role they play in gene expression. Since most noncoding RNAs are in a double-helical conformation, recognition of such structures is a formidable problem. Herein, we describe a method for sequence-selective recognition of biologically relevant double-helical RNA (illustrated on ribosomal A-site RNA) using peptide nucleic acids (PNA) that form a triple helix in the major grove of RNA under physiologically relevant conditions. Protocols for PNA preparation and binding studies using isothermal titration calorimetry are described in detail.

  11. An Efficient Synthetic Strategy for the Preparation of Nucleic Acid-Encoded Peptide and Protein Libraries for In Vitro Evolution Protocols

    Directory of Open Access Journals (Sweden)

    Peter A. Lohse

    2000-12-01

    Full Text Available We describe an improved synthetic strategy for the preparation of nucleic acid encoded peptide and protein libraries. A solid-phase format was used to prepare and purify a novel type of mRNA-template for in vitro mRNA-protein fusion synthesis. The present protocol simplifies and accelerates the preparation of fusion libraries and should prove most useful for in vitro protein evolution procedures which involve repetitive cycles of fusion library preparation and selection.

  12. Conserved molecular superlattices in a series of homologous synthetic mycobacterial cell-wall lipids forming interdigitated bilayers

    DEFF Research Database (Denmark)

    Martin-Bertelsen, Birte; Yaghmur, Anan; Franzyk, Henrik

    2016-01-01

    Synthetic analogues of the cell-wall lipid monomycoloyl glycerol (MMG) are promising as next-generation vaccine adjuvants. In the present study, the thermotropic phase behaviour of an array of synthetic MMG analogues was examined using simultaneous small- and wide-angle X-ray scattering under...... excess water conditions. The MMG analogues differed in the alkyl chain lengths and in the stereochemistry of the polar glycerol headgroup or of the lipid tails (native-like versus alternative compounds). All MMG analogues formed poorly hydrated lamellar phases at low temperatures and inverse hexagonal (H...

  13. In situ gel-forming AP-57 peptide delivery system for cutaneous wound healing.

    Science.gov (United States)

    Li, Xiaoling; Fan, Rangrang; Tong, Aiping; Yang, Meijia; Deng, Jiaojiao; Zhou, Liangxue; Zhang, Xiaoning; Guo, Gang

    2015-11-10

    In situ gel-forming system as local drug delivery system in dermal traumas has generated a great interest. Accumulating evidence shows that antimicrobial peptides play pivotal roles in the process of wound healing. Here in this study, to explore the potential application of antimicrobial peptide in wound healing, biodegradable poly(L-lactic acid)-Pluronic L35-poly(L-lactic acid) (PLLA-L35-PLLA) was developed at first. Then based on this polymer, an injectable in situ gel-forming system composed of human antimicrobial peptides 57 (AP-57) loaded nanoparticles and thermosensitive hydrogel was prepared and applied for cutaneous wound healing. AP-57 peptides were enclosed with biocompatible nanoparticles (AP-57-NPs) with high drug loading and encapsulation efficiency. AP-57-NPs were further encapsulated in a thermosensitive hydrogel (AP-57-NPs-H) to facilitate its application in cutaneous wound repair. As a result, AP-57-NPs-H released AP-57 in an extended period and exhibited quite low cytotoxicity and high anti-oxidant activity in vitro. Moreover, AP-57-NPs-H was free-flowing liquid at room temperature, and can form non-flowing gel without any crosslink agent upon applied on the wounds. In vivo wound healing assay using full-thickness dermal defect model of SD rats indicated that AP-57-NPs-H could significantly promote wound healing. At day 14 after operation, AP-57-NPs-H treated group showed nearly complete wound closure of 96.78 ± 3.12%, whereas NS, NPs-H and AP-57-NPs group recovered by about 68.78 ± 4.93%, 81.96 ± 3.26% and 87.80 ± 4.62%, respectively. Histopathological examination suggested that AP-57-NPs-H could promote cutaneous wound healing through enhancing granulation tissue formation, increasing collagen deposition and promoting angiogenesis in the wound tissue. Therefore, AP-57-NPs-H might have potential application in wound healing.

  14. Induction of neonatal lupus in pups of mice immunized with synthetic peptides derived from amino acid sequences of the serotoninergic 5-HT4 receptor.

    Science.gov (United States)

    Eftekhari, P; Roegel, J C; Lezoualc'h, F; Fischmeister, R; Imbs, J L; Hoebeke, J

    2001-02-01

    We have previously suggested that the recognition of a cross-reactive epitope on the 5-HT4 receptor and the 52-kDa SSA/Ro protein by serotonin-antagonizing autoantibodies could explain the electrophysiological symptoms of congenital heart block in neonatal lupus. To confirm this hypothesis, we immunized female mice with four synthetic peptides corresponding to the recognized epitopes. All mice developed anti-peptide antibodies, which cross-reacted with the Ro52 and 5-HT4 receptor peptides and recognized both cognate proteins. Peptide-immune mice were mated. The pups from mice immunized with the Ro52 peptides had no symptoms of neonatal lupus apart from bradycardia. However, pups from mice immunized with the 5-HT4 receptor peptides and bradycardia, atrioventricular block of type I or II, longer QT intervals, skin rashes and neuromotor problems. The 5-HT4 receptor was detectable in the different fetal tissues affected (heart, skin and brain) by immunohistochemistry. Hearts from diseased pups were less developed and showed disorganized myocardial hyperplasia, compared to the normal littermates. These results demonstrate that the serotoninergic 5-HT4 receptor is the antigenic target of physiopathological autoantibodies in neonatal lupus.

  15. Capability of Neutrophils to Form NETs Is Not Directly Influenced by a CMA-Targeting Peptide

    Science.gov (United States)

    Maueröder, Christian; Schall, Nicolas; Meyer, Frédéric; Mahajan, Aparna; Garnier, Benjamin; Hahn, Jonas; Kienhöfer, Deborah; Hoffmann, Markus H.; Muller, Sylviane

    2017-01-01

    During inflammatory reaction, neutrophils exhibit numerous cellular and immunological functions, notably the formation of neutrophil extracellular traps (NETs) and autophagy. NETs are composed of decondensed chromatin fibers coated with various antimicrobial molecules derived from neutrophil granules. NETs participate in antimicrobial defense and can also display detrimental roles and notably trigger some of the immune features of systemic lupus erythematosus (SLE) and other autoimmune diseases. Autophagy is a complex and finely regulated mechanism involved in the cell survival/death balance that may be connected to NET formation. To shed some light on the connection between autophagy and NET formation, we designed a number of experiments in human neutrophils and both in normal and lupus-prone MRL/lpr mice to determine whether the synthetic peptide P140, which is capable of selectively modulating chaperone-mediated autophagy (CMA) in lymphocytes, could alter NET formation. P140/Lupuzor™ is currently being evaluated in phase III clinical trials involving SLE patients. Overall our in vitro and in vivo studies established that P140 does not influence NET formation, cytokine/chemokine production, or CMA in neutrophils. Thus, the beneficial effect of P140/Lupuzor™ in SLE is apparently not directly related to modulation of neutrophil function. PMID:28191006

  16. Exploring the early steps of aggregation of amyloid-forming peptide KFFE

    Energy Technology Data Exchange (ETDEWEB)

    Wei Guanghong [Departement de Physique and Regroupement Quebecois sur les Materiaux de Pointe, Universite de Montreal, CP 6128, succursale centre-ville, Montreal, QC, H3C 3J7 (Canada); Mousseau, Normand [Departement de Physique and Regroupement Quebecois sur les Materiaux de Pointe, Universite de Montreal, CP 6128, succursale centre-ville, Montreal, QC, H3C 3J7 (Canada); Derreumaux, Philippe [Laboratoire de Biochimie, Theorique, UPR 9080 CNRS, IBPC, Universite Paris 7 Denis-Diderot, 13 rue Pierre et Marie Curie, 75005 Paris (France)

    2004-11-10

    It has been shown recently that even a tetrapeptide can form amyloid fibrils sharing all the characteristics of amyloid fibrils built from large proteins. Recent experimental studies also suggest that the toxicity observed in several neurodegenerative diseases, such as Alzheimer's disease and Creutzfeldt-Jakob disease, is not only related to the mature fibrils themselves, but also to the soluble oligomers formed early in the process of fibrillogenesis. This raises the interest in studying the early steps of the aggregation process. Although fibril formation follows the nucleation-condensation process, characterized by the presence of lag phase, the exact pathways remain to be determined. In this study, we used the activation-relaxation technique and a generic energy model to explore the process of self-assembly and the structures of the resulting aggregates of eight KFFE peptides. Our simulations show, starting from different states with a preformed antiparallel dimer, that eight chains can self-assemble to adopt, with various orientations, four possible distant oligomeric well-aligned structures of similar energy. Two of these structures show a double-layer {beta}-sheet organization, in agreement with the structure of amyloid fibrils as observed by x-ray diffraction; another two are mixtures of dimers and trimers. Our results also suggest that octamers are likely to be below the critical size for nucleation of amyloid fibrils for small peptides.

  17. Synthetic conantokin peptides potently inhibit N-methyl-D-aspartate receptor-mediated currents of retinal ganglion cells.

    Science.gov (United States)

    Huang, Luoxiu; Balsara, Rashna D; Castellino, Francis J

    2014-12-01

    Retinal ganglion cells (RGCs), which are the sole output neurons of the retina, express N-methyl-D-aspartate receptors (NMDARs), rendering these cells susceptible to glutamate excitotoxicity, with implications for loss of normal RGC excitatory responses in disorders such as glaucoma and diabetic retinopathy. Therefore, antagonists that inhibit NMDAR-mediated currents specifically by targeting the GluN2B component of the ion channel have the potential to serve as a basis for developing potential therapeutics. The roles of peptidic conantokins, which are potent brain neuronal NMDAR inhibitors, were studied. By using patch-clamp whole-cell analyses in dissociated RGCs and retinal whole-mount RGCs, we evaluated the effects of synthetic conantokin-G (conG) and conantokin-T (conT), which are small γ-carboxyglutamate-containing peptides, on NMDA-mediated excitatory responses in mouse RGCs. Both conG and conT inhibited the NMDA-mediated currents of dark-adapted dissociated and whole-mount RGCs in a dose-dependent, reversible, noncompetitive manner. Inhibition of NMDA-mediated steady-state currents by NMDAR nonsubunit-selective conT was approximately threefold greater than GluN2B-selective conG or ifenprodil, demonstrating its potential ability to inhibit both GluN2A- and GluN2B-containing ion channels in RGCs. Because the extent of inhibition of NMDA-evoked currents by conG and the pharmacologic GluN2B-selective inhibitor ifenprodil were similar (40-45%) to that of the GluN2A-selective antagonist NVP-AAM0077, we conclude that the levels of GluN2A and GluN2B subunits are similar in RGCs. These results provide a novel basis for developing effective neuroprotective agents to aid in the prevention of undesired glutamatergic excitotoxicity in neurodegenerative diseases of the retina and demonstrate functional assembly of NMDARs in RGCs.

  18. Large-scale purification of the synthetic peptide fragment 163-171 of human interleukin-beta by multi-dimensional displacement chromatography.

    Science.gov (United States)

    Viscomi, G C; Cardinali, C; Longobardi, M G; Verdini, A S

    1991-07-19

    Multi-dimensional chromatography has been used successfully in the displacement mode for the purification of the synthetic peptide H-Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-OH, the fragment 163-171 of human interleukin-beta. This peptide can mimic several of the in vivo and in vitro immunostimulatory activities of the entire protein, except for the inflammatory effect. A large-scale procedure has been developed to purify the synthetic peptide by reversed-phase (RP) and ion-exchange (IE) displacement chromatography (DC) in a single run without any pretreatment. Masses from 100 mg to about 35 g of the unpurified compounds synthesized by a solid-phase technique on a Merrifield-type resin and obtained by acidolytic cleavage from the solid support, can be purified in this way. In the RP-DC mode the carrier and the displacer were aqueous solutions of 0.1% trifluoroacetic acid and 50 mM benzyltributylammonium chloride, respectively, whereas in the IE-DC mode the carrier was water and the displacer 50 mM ammonium citrate solution. RP-DC and IE-DC were also performed in series by directing the effluent of the RP column onto the IE column. Peptide purities and recoveries greater than 96 and 90%, respectively, were obtained.

  19. Impaired hippocampal neuroligin-2 function by chronic stress or synthetic peptide treatment is linked to social deficits and increased aggression.

    Science.gov (United States)

    van der Kooij, Michael A; Fantin, Martina; Kraev, Igor; Korshunova, Irina; Grosse, Jocelyn; Zanoletti, Olivia; Guirado, Ramon; Garcia-Mompó, Clara; Nacher, Juan; Stewart, Michael G; Berezin, Vladimir; Sandi, Carmen

    2014-04-01

    Neuroligins (NLGNs) are cell adhesion molecules that are important for proper synaptic formation and functioning, and are critical regulators of the balance between neural excitation/inhibition (E/I). Mutations in NLGNs have been linked to psychiatric disorders in humans involving social dysfunction and are related to similar abnormalities in animal models. Chronic stress increases the likelihood for affective disorders and has been shown to induce changes in neural structure and function in different brain regions, with the hippocampus being highly vulnerable to stress. Previous studies have shown evidence of chronic stress-induced changes in the neural E/I balance in the hippocampus. Therefore, we hypothesized that chronic restraint stress would lead to reduced hippocampal NLGN-2 levels, in association with alterations in social behavior. We found that rats submitted to chronic restraint stress in adulthood display reduced sociability and increased aggression. This occurs along with a reduction of NLGN-2, but not NLGN-1 expression (as shown with western blot, immunohistochemistry, and electron microscopy analyses), throughout the hippocampus and detectable in different layers of the CA1, CA3, and DG subfields. Furthermore, using synthetic peptides that comprise sequences in either NLGN-1 (neurolide-1) or NLGN-2 (neurolide-2) involved in the interaction with their presynaptic partner neurexin (NRXN)-1, intra-hippocampal administration of neurolide-2 led also to reduced sociability and increased aggression. These results highlight hippocampal NLGN-2 as a key molecular substrate regulating social behaviors and underscore NLGNs as promising targets for the development of novel drugs for the treatment of dysfunctional social behaviors.

  20. Self-Assembling Peptides Form Immune Suppressive Amyloid Fibrils Effective in Autoimmune Encephalomyelitis.

    Science.gov (United States)

    Kurnellas, Michael P; Rothbard, Jonathan B; Steinman, Lawrence

    2015-01-01

    Amyloidogenic proteins have long been linked to neurodegenerative diseases. However, amyloid fibrils composed of six amino acids are protective in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). The reduction of pro-inflammatory cytokines, decrease in the number of inflammatory foci in the parenchyma and meninges of the brain and spinal cord, and amelioration of the neurological signs of EAE when amyloid fibril-forming hexapeptides are administered reveal that some fibrils provide benefit. The therapeutic activity of the amyloid fibrils arise from diverse pathways that include binding of pro-inflammatory mediators in the plasma, reduction of IL-6, TNF-α, and IFN-γ levels, and induction of type 1 interferon (IFN). Type 1 IFN has been used widely as a therapeutic agent for the treatment of MS and has been shown to be therapeutic in EAE with adoptive transfer of Th1 lymphocytes. However, type 1 IFN is known to exacerbate EAE with adoptive transfer of Th17 lymphocytes. Indeed, the amyloid fibril-forming peptide Tau 623-628 was therapeutic in Th1 adoptively transferred EAE, but ineffective in Th17 adoptively transferred EAE. However, the therapeutic effect of Tau 623-628 was restored in IFN-α/β receptor (IFNAR) knockout mice, indicating that other immune pathways independent of type 1 IFN induction play a role in the amelioration of EAE. Moreover, Amylin 28-33, a polar, non-ionizable peptide that does not form fibrils as rapidly as Tau 623-628, induces a small fraction of type 1 IFN compared to Tau 623-628 and is therapeutic in Th17 EAE. The diverse immunological pathways modulated by the self-assembling hexapeptides are under investigation with a goal to develop novel, safe, and potent therapeutics for neuroinflammation.

  1. Pore-forming bacterial toxins and antimicrobial peptides as modulators of ADAM function.

    Science.gov (United States)

    Reiss, Karina; Bhakdi, Sucharit

    2012-11-01

    Membrane-perturbating proteins and peptides are widespread agents in biology. Pore-forming bacterial toxins represent major virulence factors of pathogenic microorganisms. Membrane-damaging peptides constitute important antimicrobial effectors of innate immunity. Membrane perturbation can incur multiple responses in mammalian cells. The present discussion will focus on the interplay between membrane-damaging agents and the function of cell-bound metalloproteinases of the ADAM family. These transmembrane enzymes have emerged as the major proteinase family that mediate the proteolytic release of membrane-associated proteins, a process designated as "shedding". They liberate a large spectrum of functionally active molecules including inflammatory cytokines, growth factor receptors and cell adhesion molecules, thereby regulating such vital cellular functions as cell-cell adhesion, cell proliferation and cell migration. ADAM activation may constitute part of the cellular recovery machinery on the one hand, but likely also promotes inflammatory processes on the other. The mechanisms underlying ADAM activation and the functional consequences thereof are currently the subject of intensive research. Attention here is drawn to the possible involvement of purinergic receptors and ceramide generation in the context of ADAM activation following membrane perturbation by membrane-active agents.

  2. Influence of Amphibian Antimicrobial Peptides and Short Lipopeptides on Bacterial Biofilms Formed on Contact Lenses

    Directory of Open Access Journals (Sweden)

    Magdalena Maciejewska

    2016-10-01

    Full Text Available The widespread use of contact lenses is associated with several complications, including ocular biofilm-related infections. They are very difficult to manage with standard antimicrobial therapies, because bacterial growth in a biofilm is associated with an increased antibiotic resistance. The principal aim of this study was to evaluate the efficacy of antimicrobial peptides (AMPs in eradication of bacterial biofilms formed on commercially available contact lenses. AMPs were synthesized according to Fmoc/tBu chemistry using the solid-phase method. Minimum inhibitory concentration (MIC and minimum biofilm eradication concentration (MBEC of the compounds were determined. Anti-biofilm activity of the antimicrobial peptides determined at different temperatures (25 °C and 37 °C were compared with the effectiveness of commercially available contact lens solutions. All of the tested compounds exhibited stronger anti-biofilm properties as compared to those of the tested lens solutions. The strongest activity of AMPs was noticed against Gram-positive strains at a temperature of 25 °C. Conclusions: The results of our experiments encourage us toward further studies on AMPs and their potential application in the prophylaxis of contact lens-related eye infections.

  3. Studies on the intermolecular forces involved in the antibody-antigen interactions, using V3 synthetic peptides and sera from HIV1 seropositive patients.

    Science.gov (United States)

    Măgureanu, C G; Diaconu, C; Alexandrescu, R; Tirdei, G; Cernescu, C

    1994-01-01

    The nature of physical forces responsible for the antibody-antigen (Ab-Ag) reaction was analyzed in an original system, represented by synthetic peptides derived from the V3 consensus sequences of some HIV1 subtypes gp 120 and HIV1 positive human serum. For locating antigenic determines, flexibility, hydrophilicity and hydrophobicity profiles of the V3 peptides were analysed. The hydrophilicity indicates that V3 apex borders are involved in the first stage of the reaction. The flexibility and hydrophobicity suggest that the apex of the V3 loop (GPGR/Q) is involved in the stabilization of the complex by hydrophobic interactions. Further, we followed up the influence of the dielectric constant and of the pH upon the forces established between Ab and Ag. Modifications in the dielectric constant and pH reveal a significant contribution of electrostatic and van der Waals forces in securing the intermolecular complementarity. D2O produces the highest augmentation of the antibody affinity for the most hydrophilic peptides, while a very slight one was recorded for the most hydrophobic sequence. A high affinity of antibodies for the peptides MN, R and Z was registered at an acid pH, when their His residue was protonated. On the contrary, no influence was recorded in the case of the peptide A, which does not contain any His residue.

  4. Antioxidant activity of a novel synthetic hexa-peptide derived from an enzymatic hydrolysate of duck skin by-products.

    Science.gov (United States)

    Lee, Seung-Jae; Cheong, Sun Hee; Kim, Yon-Suk; Hwang, Jin-Woo; Kwon, Hyuck-Ju; Kang, Seo-Hee; Moon, Sang-Ho; Jeon, Byong-Tae; Park, Pyo-Jam

    2013-12-01

    A peptide was synthesized on the basis of our previous study from solid phase peptide synthesis using ASP48S (Peptron Inc.) and identified by the reverse phase high-performance liquid chromatography (HPLC) using a Vydac Everest C18 column. The molecular mass of the peptide found to be 693.90 Da, and the amino acid sequences of the peptide was Trp-Tyr-Pro-Ala-Ala-Pro. The purpose of this study was to evaluate antioxidant effects of the peptide by electron spin resonance (ESR) spectrometer, and on t-BHP-induced liver cells damage in Chang cells. The antioxidative activity of the peptide was evaluated by measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, alkyl and superoxide radical scavenging activity using an ESR spectrometer. The half maximal inhibitory concentration (IC50) value of the peptide for hydroxyl, DPPH, alkyl, and superoxide radical scavenging activity were 45.2, 18.5, 31.5, and 33.4 μM, respectively. In addition, the peptide inhibited productions of cell death against t-BHP-induced liver cell damage in Chang cells. It was presumed to be peptide involved in regulating the apoptosis-related gene expression in the cell environment. The present results indicate that the peptide substantially contributes to antioxidative properties in liver cells.

  5. HIV Tat protein and amyloid-β peptide form multifibrillar structures that cause neurotoxicity.

    Science.gov (United States)

    Hategan, Alina; Bianchet, Mario A; Steiner, Joseph; Karnaukhova, Elena; Masliah, Eliezer; Fields, Adam; Lee, Myoung-Hwa; Dickens, Alex M; Haughey, Norman; Dimitriadis, Emilios K; Nath, Avindra

    2017-02-20

    Deposition of amyloid-β plaques is increased in the brains of HIV-infected individuals, and the HIV transactivator of transcription (Tat) protein affects amyloidogenesis through several indirect mechanisms. Here, we investigated direct interactions between Tat and amyloid-β peptide. Our in vitro studies showed that in the presence of Tat, uniform amyloid fibrils become double twisted fibrils and further form populations of thick unstructured filaments and aggregates. Specifically, Tat binding to the exterior surfaces of the Aβ fibrils increases β-sheet formation and lateral aggregation into thick multifibrillar structures, thus producing fibers with increased rigidity and mechanical resistance. Furthermore, Tat and Aβ aggregates in complex synergistically induced neurotoxicity both in vitro and in animal models. Increased rigidity and mechanical resistance of the amyloid-β-Tat complexes coupled with stronger adhesion due to the presence of Tat in the fibrils may account for increased damage, potentially through pore formation in membranes.

  6. Qualitative computational bioanalytics: assembly of viral channel-forming peptides around mono and divalent ions.

    Science.gov (United States)

    Li, Li-Hua; Hsu, Hao-Jen; Fischer, Wolfgang B

    2013-12-06

    A fine-grained docking protocol was used to generate a bundle-like structure of the bitopic membrane protein Vpu from HIV-1. Vpu is a type I membrane protein with 81 amino acids. It is proposed that Vpu forms ion- and substrate-conducting bundles, which are located at the plasma membrane in the infected cell. The Vpu1-32 peptide that includes the transmembrane domain (TMD) is assembled into homo-pentameric bundles around prepositioned Na, K, Ca or Cl ions. For bundles with the lowest energy, the TMDs generate a hydrophobic pore. Bundles in which Ser-24 faces the pore have higher energy. The tilt of the helices in the lowest energy bundles is larger than bundles with serines facing the pore. Left-handed bundles are lowest in energy where the ions are located at the serines.

  7. Comparison of marmoset and human FSH using synthetic peptides of the β-subunit L2 loop region and anti-peptide antibodies.

    Science.gov (United States)

    Kutteyil, Susha S; Kulkarni, Bhalchandra J; Mojidra, Rahul; Joseph, Shaini; Pathak, Bhakti R; Mahale, Smita D

    2016-06-01

    Follicle stimulating hormone (FSH) is a glycoprotein hormone required for female and male gametogenesis in vertebrates. Common marmoset (Callithrix jacchus) is a New World primate monkey, used as animal model in biomedical research. Observations like, requirement of extremely high dose of human FSH in marmosets for superovulation compared to other primates and generation of antibodies in marmoset against human FSH after repeated superovulation cycles, point towards the possibility that FSH-FSH receptor (FSHR) interaction in marmosets might be different than in the humans. In this study we attempted to understand some of these structural differences using FSH peptides and anti-peptide antibody approach. Based on sequence alignment, in silico modeling and docking studies, L2 loop of FSH β-subunit (L2β) was found to be different between marmoset and human. Hence, peptides corresponding to region 32-50 of marmoset and human L2β loop were synthesized, purified and characterized. The peptides displayed dissimilarity in terms of molecular mass, predicted isoelectric point, predicted charge and in the ability to inhibit hormone-receptor interaction. Polyclonal antibodies generated against both the peptides were found to exhibit specific binding for the corresponding peptide and parent FSH in ELISA and Western blotting respectively and exhibited negligible reactivity to cross-species peptide and FSH in ELISA. The anti-peptide antibody against marmoset FSH was also able to detect native FSH in marmoset plasma samples and pituitary sections. In summary, the L2β loop of marmoset and human FSH has distinct receptor interaction ability and immunoreactivity indicating possibility of subtle conformational and biochemical differences between the two regions which may affect the FSH-FSHR interaction in these two primates. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  8. Fibrillar structures formed by covalently bound, short, β-stranded peptides on self-assembled monolayers.

    Science.gov (United States)

    Dugger, Jason W; Webb, Lauren J

    2015-03-24

    The ability to maintain or reproduce biomolecular structures on inorganic substrates has the potential to impact diverse fields such as sensing and molecular electronics, as well as the study of biological self-assembly and structure-function relationships. Because the structure and self-assembly of biomolecules are exquisitely sensitive to their local chemical and electrostatic environment, the goal of reproducing or mimicking biological function in an abiological environment, including at a surface, is challenging. However, simple and well-characterized chemical modifications of prepared surfaces can be used to tune surface chemistry, structure, electrostatics, and reactivity of inorganic materials to facilitate biofunctionalization and function. Here, we describe the covalent attachment of 13-residue β-stranded peptides containing alkyne groups to a flat gold surface functionalized with an azide-terminated self-assembled monolayer through a Huisgen cycloaddition, or "click", reaction. The chemical composition and structural morphology of these surfaces were characterized using X-ray photoelectron spectroscopy, grazing incidence angle reflection-absorption infrared spectroscopy, surface circular dichroism, and atomic force microscopy. The surface-bound β-strands self-assemble into antiparallel β-sheets to form fibrillar structures 24.9 ± 1.6 nm in diameter and 2.83 ± 0.74 nm in height on the reactive surface. The results herein provide a platform for studying and controlling the self-assembly process of biomolecules into larger supermolecular structures while allowing tunable control through chemical functionalization of the surface. Interest in the mechanisms of formation of fibrillar structures has most commonly been associated with neurodegenerative diseases, such as Alzheimer's and Parkinson's, but fibrils may actually represent the thermodynamic low-energy conformation of a much larger class of peptides and proteins. The protocol developed here is an

  9. Dodecamer d-AGATCTAGATCT and a homologous hairpin form triplex in the presence of peptide REWER.

    Directory of Open Access Journals (Sweden)

    Amrita Das

    Full Text Available We have designed a dodecamer d-AGATCTAGATCT (RY12 with alternate oligopurines and oligopyrimidines tracts and its homologous 28 bp hairpin oligomer (RY28 that forms a triple helix only in the presence of a pentapeptide REWER. An intermolecular triplex is formed by the single strand invasion of the RY28 duplex by RY12 in the presence of REWER. 5'- oligopurine end of RY12 binds to oligopurine sequence of RY28 in a parallel orientation and its oligopyrimidine stretch then changes strand and adopts an antiparallel orientation with the other strand of the duplex. Evidence for the formation of the triplex come from our studies of the UV melting curves, UV mixing curves, gel retardation assay, and chemical sequencing of 1∶1 mixture of dodecamer and hairpin oligonucleotides in the presence and absence of the peptide REWER. RY12 exists as a duplex that melts at 35°C. The hairpin (RY28 melts at 68°C. 1∶1 mixture of RY12 and RY28 in the absence of REWER gives a biphasic transition curve with thermodynamic properties corresponding to those of the melting of the duplex of RY12 and the hairpin RY28. However, the melting curve of this mixture is triphasic in the presence of the REWER; the thermodynamic parameters associated with the first phase (melting of the duplex of RY12, second phase (melting of the triplex and the third phase (melting of the hairpin show dependence on the molar ratio of peptide to oligonucleotides. Under appropriate conditions, gel retardation assay showed a shifted band that corresponds to a possible triplex. Chemical sequencing of KMnO4 and DEPC treated mixture of RY12, RY28 and REWER revealed the footprint of triplex.

  10. Highly efficient synthetic method onpyroacm resin using the boc SPPS protocol for C-terminal cysteine peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Juvekar, Vinayak; Kim, Kang Tae; Gong, Young Dae [Innovative Drug Library Research Center, Dept. of Chemistry, College of Science, Dongguk University, Seoul (Korea, Republic of)

    2017-01-15

    A very effective process on Pyroacm resin was developed for solid-phase peptide synthesis (SPPS) of C-terminal cysteine and cysteine ester peptides. The process uses cysteine side chain anchoring to the Pyroacm resin and the Boc protocol for SPPS. The Pyroacm resin showed remarkable stability under standard trifluoromethanesulfonic acid (TFMSA) cleavage condition. TFMSA cleavage of protecting groups generates a peptide-linked resin, which can be subjected to peptide modification reactions. Finally, the peptide can be cleaved from the resin using methoxycarbonylsulfenyl chloride. The utility of this protocol was demonstrated by its applications to the synthesis of model peptides, key intermediates in the preparation of natural products riparin 1.2 and a-factor.

  11. Vaccine development for allergen-specific immunotherapy based on recombinant allergens and synthetic allergen peptides: Lessons from the past and novel mechanisms of action for the future.

    Science.gov (United States)

    Valenta, Rudolf; Campana, Raffaela; Focke-Tejkl, Margit; Niederberger, Verena

    2016-02-01

    In the past, the development of more effective, safe, convenient, broadly applicable, and easy to manufacture vaccines for allergen-specific immunotherapy (AIT) has been limited by the poor quality of natural allergen extracts. Progress made in the field of molecular allergen characterization has now made it possible to produce defined vaccines for AIT and eventually for preventive allergy vaccination based on recombinant DNA technology and synthetic peptide chemistry. Here we review the characteristics of recombinant and synthetic allergy vaccines that have reached clinical evaluation and discuss how molecular vaccine approaches can make AIT more safe and effective and thus more convenient. Furthermore, we discuss how new technologies can facilitate the reproducible manufacturing of vaccines of pharmaceutical grade for inhalant, food, and venom allergens. Allergy vaccines in clinical trials based on recombinant allergens, recombinant allergen derivatives, and synthetic peptides allow us to target selectively different immune mechanisms, and certain of those show features that might make them applicable not only for therapeutic but also for prophylactic vaccination.

  12. Delivery of antisense oligonucleotide into cells using synthetic peptide; Gosei pepuchido wo mochiita anchisensu origonukureochido no saibounai donyu

    Energy Technology Data Exchange (ETDEWEB)

    Niidome, Takuro [Nagasaki University, Nagasaki (Japan). Dept. of Applied Chemistry

    1999-12-16

    Much attention has been attracted to the antisense oligonucleotide as a novel nucleic acid medicine. However, many problems to be solved such as delivery system in vivo and permeation through cell membrane are pointed out. In this study, we found out that some cationic peptides were useful as an oligonucleotide-carrier molecule into cells. Furthermore, to develop a cell specific gene delivery system using the cationic peptide, we modified the peptides with several galactose residues. As a result, the modified peptides showed high transfer efficiencies into hepatoma cells, and then, it was clear that the internalization into cells was mediated by asialoglycoprotein receptor on hepatoma cell. (author)

  13. Antimicrobial peptide (Cn-AMP2) from liquid endosperm of Cocos nucifera forms amyloid-like fibrillar structure.

    Science.gov (United States)

    Gour, Shalini; Kaushik, Vibha; Kumar, Vijay; Bhat, Priyanka; Yadav, Subhash C; Yadav, Jay K

    2016-04-01

    Cn-AMP2 is an antimicrobial peptide derived from liquid endosperm of coconut (Cocos nucifera). It consists of 11 amino acid residues and predicted to have high propensity for β-sheet formation that disposes this peptide to be amyloidogenic. In the present study, we have examined the amyloidogenic propensities of Cn-AMP2 in silico and then tested the predictions under in vitro conditions. The in silico study revealed that the peptide possesses high amyloidogenic propensity comparable with Aβ. Upon solubilisation and agitation in aqueous buffer, Cn-AMP2 forms visible aggregates that display bathochromic shift in the Congo red absorbance spectra, strong increase in thioflavin T fluorescence and fibrillar morphology under transmission electron microscopy. All these properties are typical of an amyloid fibril derived from various proteins/peptides including Aβ.

  14. Sequence analysis of peptides with biological activities using electrospray-Fourier trans- form ion cyclotron resonance mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The mass spectra of five peptides with biological activities are reported. All mass spectra were recorded using a 4.7-T Fourier transform ion cyclotron resonance mass spectrometer equipped with an external electrospray source. The accurate molecular weights for the five peptides prepared by solid phase synthesis were measured as 1765.9013, 1063.5420, 1092.5254, 820.3804 and 1078.5193, respectively. All the data were obtained with the external calibration. Differences between observed and theoretical monoisotopic molecular weights were in the (0.2-1.0)×10-6 range. The complete primary sequence for the five polypep-tides were determined using the method of in-source electro-spray ionization/collision induced dissociation (ESI/CID). All the intact y series ions and b series ions were obtained from various peptides respectively, thus determining the sequences of the five polypeptides. We found that the measured accura-te molecular mass of sample 4 was not in agreement with that expected from the planned synthetic peptide. The se-quences of sample 4 were determined through analysis. The corresponding accurate masses of b series ions and y series ions were gained, which proved that it was correct to re-determine the sequences.

  15. Synthetic Cationic Peptide IDR-1002 Provides Protection against Bacterial Infections through Chemokine Induction and Enhanced Leukocyte Recruitment

    DEFF Research Database (Denmark)

    Nijnik, Anastasia; Madera, Laurence; Ma, Shuhua

    2010-01-01

    With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobia...

  16. Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity.

    Science.gov (United States)

    Björling, E; Broliden, K; Bernardi, D; Utter, G; Thorstensson, R; Chiodi, F; Norrby, E

    1991-01-01

    Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys. Images PMID:2068087

  17. A synthetic peptide shows retro- and anterograde neuronal transport before disrupting the chemosensation of plant-pathogenic nematodes.

    Directory of Open Access Journals (Sweden)

    Dong Wang

    Full Text Available Cyst nematodes are a group of plant pathogens each with a defined host range that cause major losses to crops including potato, soybean and sugar beet. The infective mobile stage hatches from dormant eggs and moves a short distance through the soil to plant roots, which it then invades. A novel strategy for control has recently been proposed in which the plant is able to secrete a peptide which disorientates the infective stage and prevents invasion of the pathogen. This study provides indirect evidence to support the mechanism by which one such peptide disrupts chemosensory function in nematodes. The peptide is a disulphide-constrained 7-mer with the amino acid sequence CTTMHPRLC that binds to nicotinic acetylcholine receptors. A fluorescently tagged version of this peptide with both epifluorescent and confocal microscopy was used to demonstrate that retrograde transport occurs from an aqueous environment along bare-ending primary cilia of chemoreceptive sensilla. The peptide is transported to the cell bodies of these neurons and on to a limited number of other neurons to which they connect. It appears to be localised in both neuronal processes and organelles adjacent to nuclei of some neurons suggesting it could be transported through the Golgi apparatus. The peptide takes 2.5 h to reach the neuronal cell bodies. Comparative studies established that similar but less abundant uptake occurs for Caenorhabditis elegans along its well studied dye-filling chemoreceptive neurons. Incubation in peptide solution or root-exudate from transgenic plants that secrete the peptide disrupted normal orientation of infective cyst nematodes to host root diffusate. The peptide probably undergoes transport along the dye-filling non-cholinergic chemoreceptive neurons to their synapses where it is taken up by the interneurons to which they connect. Coordinated responses to chemoreception are disrupted when the sub-set of cholinergic interneurons secrete the peptide

  18. A synthetic peptide shows retro- and anterograde neuronal transport before disrupting the chemosensation of plant-pathogenic nematodes.

    Science.gov (United States)

    Wang, Dong; Jones, Laura M; Urwin, Peter E; Atkinson, Howard J

    2011-03-07

    Cyst nematodes are a group of plant pathogens each with a defined host range that cause major losses to crops including potato, soybean and sugar beet. The infective mobile stage hatches from dormant eggs and moves a short distance through the soil to plant roots, which it then invades. A novel strategy for control has recently been proposed in which the plant is able to secrete a peptide which disorientates the infective stage and prevents invasion of the pathogen. This study provides indirect evidence to support the mechanism by which one such peptide disrupts chemosensory function in nematodes. The peptide is a disulphide-constrained 7-mer with the amino acid sequence CTTMHPRLC that binds to nicotinic acetylcholine receptors. A fluorescently tagged version of this peptide with both epifluorescent and confocal microscopy was used to demonstrate that retrograde transport occurs from an aqueous environment along bare-ending primary cilia of chemoreceptive sensilla. The peptide is transported to the cell bodies of these neurons and on to a limited number of other neurons to which they connect. It appears to be localised in both neuronal processes and organelles adjacent to nuclei of some neurons suggesting it could be transported through the Golgi apparatus. The peptide takes 2.5 h to reach the neuronal cell bodies. Comparative studies established that similar but less abundant uptake occurs for Caenorhabditis elegans along its well studied dye-filling chemoreceptive neurons. Incubation in peptide solution or root-exudate from transgenic plants that secrete the peptide disrupted normal orientation of infective cyst nematodes to host root diffusate. The peptide probably undergoes transport along the dye-filling non-cholinergic chemoreceptive neurons to their synapses where it is taken up by the interneurons to which they connect. Coordinated responses to chemoreception are disrupted when the sub-set of cholinergic interneurons secrete the peptide at synapses that

  19. Flow, form, and function: Distinguishing eco-hydraulic controls with relevance beyond the stream reach using synthetic channel morphologies

    Science.gov (United States)

    Lane, Belize; Pasternack, Gregory; Sandoval-Solis, Samuel

    2017-04-01

    Rivers are highly complex, dynamic systems that support numerous ecosystem functions including transporting sediment, modulating biogeochemical processes, and regulating habitat availability for native species. The extent and timing of these functions is largely controlled by the interplay of hydrologic dynamics (i.e., flow) and the shape and structure of the river channel (i.e., form). In spite of this, the majority of river restoration studies are limited to the influence of flow on ecosystem function without regard for the role of channel form in modulating eco-hydraulic response. The few studies that have effectively examined the flow-form interface highlight the scientific and management value of such analyses, but are highly resource intensive. This study represents a first attempt to apply synthetic channel design to the evaluation of river flow-form-function linkages, with the aim of improving basic understanding of how the interplay between flow and form affects ecosystem functions across a range of regionally-significant flows and forms with minimal resource requirements. Archetypal Mediterranean-montane channel types were used to guide the design of 3D synthetic morphologies. These morphologies were then used to quantify 2D eco-hydraulic response to different channel configurations under select hydrologic scenarios (distinguished by alteration and water year type). The eco-hydraulic performance of alternative flow-form settings, based on spatiotemporal patterns of depth and velocity, was evaluated with respect to a suite of river ecosystem functions related to geomorphic diversity, aquatic habitat, and riparian habitat. The methods described herein provide a potential design and inventory tool for quantifying river ecosystem functions and management trade-offs of alternative flow-form combinations with minimal resource and data requirements. While addressing specific scientific questions of interest for Mediterranean-montane rivers, the general framework

  20. Full protection in mink against mink enteritis virus with new generation canine parvovirus vaccines based on synthetic peptide or recombinant protein

    DEFF Research Database (Denmark)

    Langeveld, J. P.; Kamstrup, Søren; Uttenthal, Åse

    1995-01-01

    Two recently developed vaccines—one based on synthetic peptide and one based on recombinant capsid protein—fully protected dogs against heavy experimental canine parvovirus (CPV) infection. The high sequence homology (>98%) and antigenic similarity between CPV and mink enteritis virus (MEV), feline...... panleukopenia virus, and raccoon parvovirus, suggest that both vaccines could protect mink, cats and raccoons against these respective host range variants. This was tested in mink and turned out to be the case. The two vaccines were fully protective and as effective as a conventional commercial vaccine based...

  1. A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions

    OpenAIRE

    de Vos van Steenwijk, Peggy J.; Ramwadhdoebe, Tamara H.; Löwik, Margriet J. G.; van der Minne, Caroline E.; Berends-van der Meer, Dorien M A; Fathers, Lorraine M; Valentijn, A. Rob P. M.; Oostendorp, Jaap; Fleuren, Gert Jan; Hellebrekers, Bart W. J.; Welters, Marij J. P.; van Poelgeest, Mariette I.; Melief, Cornelis J. M.; Kenter, Gemma G; van der Burg, Sjoerd H.

    2012-01-01

    The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping long-peptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16+ high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre- and po...

  2. Where Does the Electron Go? Stable and Metastable Peptide Cation Radicals Formed by Electron Transfer

    Science.gov (United States)

    Pepin, Robert; Layton, Erik D.; Liu, Yang; Afonso, Carlos; Tureček, František

    2017-01-01

    Electron transfer to doubly and triply charged heptapeptide ions containing polar residues Arg, Lys, and Asp in combination with nonpolar Gly, Ala, and Pro or Leu generates stable and metastable charge-reduced ions, (M + 2H)+●, in addition to standard electron-transfer dissociation (ETD) fragment ions. The metastable (M + 2H)+● ions spontaneously dissociate upon resonant ejection from the linear ion trap, giving irregularly shaped peaks with offset m/ z values. The fractions of stable and metastable (M + 2H)+● ions and their mass shifts depend on the presence of Pro-4 and Leu-4 residues in the peptides, with the Pro-4 sequences giving larger fractions of the stable ions while showing smaller mass shifts for the metastables. Conversion of the Asp and C-terminal carboxyl groups to methyl esters further lowers the charge-reduced ion stability. Collisional activation and photodissociation at 355 nm of mass-selected (M + 2H)+● results in different dissociations that give sequence specific MS3 spectra. With a single exception of charge-reduced (LKGLADR + 2H)+●, the MS3 spectra do not produce ETD sequence fragments of the c and z type. Hence, these (M + 2H)+● ions are covalent radicals, not ion-molecule complexes, undergoing dramatically different dissociations in the ground and excited electronic states. The increased stability of the Pro-4 containing (M + 2H)+● ions is attributed to radicals formed by opening of the Pro ring and undergoing further stabilization by hydrogen atom migrations. UV-VIS photodissociation action spectroscopy and time-dependent density functional theory calculations are used in a case in point study of the stable (LKGPADR + 2H)+● ion produced by ETD. In contrast to singly-reduced peptide ions, doubly reduced (M + 3H)+ ions are stable only when formed from the Pro-4 precursors and show all characteristics of even electron ions regarding no photon absorption at 355 nm or ion-molecule reactions, and exhibiting proton driven

  3. A randomized placebo-controlled phase Ia malaria vaccine trial of two virosome-formulated synthetic peptides in healthy adult volunteers.

    Directory of Open Access Journals (Sweden)

    Blaise Genton

    Full Text Available BACKGROUND AND OBJECTIVES: Influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. The aim of the study was to proof the concept that virosomes can also be used to elicit high titers of antibodies against synthetic peptides. The specific objective was to demonstrate the safety and immunogenicity of two virosome-formulated P. falciparum protein derived synthetic peptide antigens given in two different doses alone or in combination. METHODOLOGY/PRINCIPAL FINDINGS: The design was a single blind, randomized, placebo controlled, dose-escalating study involving 46 healthy Caucasian volunteers aged 18-45 years. Five groups of 8 subjects received virosomal formulations containing 10 microg or 50 microg of AMA 49-CPE, an apical membrane antigen-1 (AMA-1 derived synthetic phospatidylethanolamine (PE-peptide conjugate or 10 ug or 50 ug of UK39, a circumsporozoite protein (CSP derived synthetic PE-peptide conjugate or 50 ug of both antigens each. A control group of 6 subjects received unmodified virosomes. Virosomal formulations of the antigens (designated PEV301 and PEV302 for the AMA-1 and the CSP virosomal vaccine, respectively or unmodified virosomes were injected i. m. on days 0, 60 and 180. In terms of safety, no serious or severe adverse events (AEs related to the vaccine were observed. 11/46 study participants reported 16 vaccine related local AEs. Of these 16 events, all being pain, 4 occurred after the 1(st, 7 after the 2(nd and 5 after the 3(rd vaccination. 6 systemic AEs probably related to the study vaccine were reported after the 1(st injection, 10 after the 2(nd and 6 after the 3(rd. Generally, no difference in the distribution of the systemic AEs between either the doses applied (10 respectively 50 microg or the synthetic antigen vaccines (PEV301 and PEV302 used for immunization was found. In terms of immunogenicity, both PEV301 and PEV302

  4. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    Science.gov (United States)

    Laman, J D; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J P; Claassen, E; Boersma, W J

    1992-03-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross

  5. Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients.

    Science.gov (United States)

    de la Fuente-Núñez, César; Mansour, Sarah C; Wang, Zhejun; Jiang, Lucy; Breidenstein, Elena B M; Elliott, Melissa; Reffuveille, Fany; Speert, David P; Reckseidler-Zenteno, Shauna L; Shen, Ya; Haapasalo, Markus; Hancock, Robert E W

    2014-01-01

    Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.

  6. Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

    Directory of Open Access Journals (Sweden)

    César de la Fuente-Núñez

    2014-10-01

    Full Text Available Cystic fibrosis (CF patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1 and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α production by human peripheral blood mononuclear cells (PBMC and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.

  7. Peptide ligands specific to the oxidized form of escherichia coli thioredoxin.

    Energy Technology Data Exchange (ETDEWEB)

    Scholle, M. D.; Banach, B. S.; Hamdan, S. M.; Richardson, C. C.; Kay, B. K.; Biosciences Division; Amunix, Inc.; Univ. of Illinois at Chicago; Harvard Medical School

    2008-11-01

    Thioredoxin (Trx) is a highly conserved redox protein involved in several essential cellular processes. In this study, our goal was to isolate peptide ligands to Escherichia coli Trx that mimic protein-protein interactions, specifically the T7 polymerase-Trx interaction. To do this, we subjected Trx to affinity selection against a panel of linear and cysteine-constrained peptides using M13 phage display. A novel cyclized conserved peptide sequence, with a motif of C(D/N/S/T/G)D(S/T)-hydrophobic-C-X-hydrophobic-P, was isolated to Trx. These peptides bound specifically to the E. coli Trx when compared to the human and spirulina homologs. An alanine substitution of the active site cysteines (CGPC) resulted in a significant loss of peptide binding affinity to the Cys-32 mutant. The peptides were also characterized in the context of Trx's role as a processivity factor of the T7 DNA polymerase (gp5). As the interaction between gp5 and Trx normally takes place under reducing conditions, which might interfere with the conformation of the disulfide-bridged peptides, we made use of a 22 residue deletion mutant of gp5 in the thioredoxin binding domain (gp5{Delta}22) that bypassed the requirements of reducing conditions to interact with Trx. A competition study revealed that the peptide selectively inhibits the interaction of gp5{Delta}22 with Trx, under oxidizing conditions, with an IC50 of {approx} 10 {micro}M.

  8. Three homologous subunits form a high affinity peptide-gated ion channel in Hydra

    DEFF Research Database (Denmark)

    Dürrnagel, Stefan; Kuhn, Anne; Tsiairis, Charisios D

    2010-01-01

    properties, like a low Na(+) selectivity and a low amiloride affinity, that are different from other channels of the DEG/ENaC gene family, suggesting that a component of the native Hydra channel might still be lacking. Here, we report the cloning of a new ion channel subunit from Hydra, HyNaC5. The new......Recently, three ion channel subunits of the degenerin (DEG)/epithelial Na(+) channel (ENaC) gene family have been cloned from the freshwater polyp Hydra magnipapillata, the Hydra Na(+) channels (HyNaCs) 2-4. Two of them, HyNaC2 and HyNaC3, co-assemble to form an ion channel that is gated...... by the neuropeptides Hydra-RFamides I and II. The HyNaC2/3 channel is so far the only cloned ionotropic receptor from cnidarians and, together with the related ionotropic receptor FMRFamide-activated Na(+) channel (FaNaC) from snails, the only known peptide-gated ionotropic receptor. The HyNaC2/3 channel has pore...

  9. Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

    Directory of Open Access Journals (Sweden)

    Alfred T Welzel

    Full Text Available Soluble non-fibrillar assemblies of amyloid-beta (Aβ and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD. Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted.

  10. Human Anti-Aβ IgGs Target Conformational Epitopes on Synthetic Dimer Assemblies and the AD Brain-Derived Peptide

    Science.gov (United States)

    Welzel, Alfred T.; Williams, Angela D.; McWilliams-Koeppen, Helen P.; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A.; Ehrlich, Hartmut J.; Schwarz, Hans P.; Walsh, Dominic M.; Solomon, Alan; O’Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer’s disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ’s conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody’s nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody’s lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted. PMID:23209707

  11. Human anti-Aβ IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

    Science.gov (United States)

    Welzel, Alfred T; Williams, Angela D; McWilliams-Koeppen, Helen P; Acero, Luis; Weber, Alfred; Blinder, Veronika; Mably, Alex; Bunk, Sebastian; Hermann, Corinna; Farrell, Michael A; Ehrlich, Hartmut J; Schwarz, Hans P; Walsh, Dominic M; Solomon, Alan; O'Nuallain, Brian

    2012-01-01

    Soluble non-fibrillar assemblies of amyloid-beta (Aβ) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aβ immunotherapy is a promising and advanced therapeutic strategy, but the precise Aβ species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aβ conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aβ dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aβ extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aβ's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aβ monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aβ NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aβ NAbs are warranted.

  12. Inhibition of iodine-125-labeled human follitropin binding to testicular receptor by epidermal growth factor and synthetic peptides

    Energy Technology Data Exchange (ETDEWEB)

    Sluss, P.M.; Krystek, S.R. Jr.; Andersen, T.T.; Melson, B.E.; Huston, J.S.; Ridge, R.; Reichert, L.E. Jr.

    1986-05-06

    Two tetrapeptide sequence homologies between mouse epidermal growth factor precursor (mEGFP) and human follitropin (FSH) were revealed by a computer program that identifies identical residues among polypeptide sequences. The two tetrapeptides, Lys-Thr-Cys-Thr (KTCT) and Thr-Arg-Asp-Leu (TRDL), are present in the hormone-specific beta subunit of FSH from all species studied. These tetrapeptides are not present in the alpha subunit, which is common to all pituitary glycoprotein hormones. Both tetrapeptides are also found in mEGFP, and one tetrapeptide, TRDL, is located within the 53-residue form of mEGF purified from mouse submaxillary glands. Computer-generated hydropathy profiles predicted that both tetrapeptides are located in hydrophilic portions of the FSH beta subunit and that TRDL is in a hydrophilic portion of commercially available mEGF. Therefore, the tetrapeptides might be accessible to receptor binding sites for FSH. We report that mEGF inhibits binding of /sup 125/I-labeled human FSH to receptors in testis by 50% (I50) at a concentration of 1.8 X 10(-5) M. No binding inhibition was observed by GnRH or arginine-vasopressin at 10(-4) M, neither of which contain the tetrapeptide sequences. FSH beta subunit, which contains both tetrapeptides, also inhibited binding (I50 = 9 X 10(-8) M) of /sup 125/I-labeled human FSH to testis receptor. Thus, it appears that FSH beta subunit and mEGF are capable of inhibiting binding of FSH to testicular FSH receptors, presumably through interactions that include the homologous tetrapeptides. This presumption was supported by the observation that the synthetic tetrapeptides (KTCT or TRDL) were also active in inhibiting binding of /sup 125/I-labeled human FSH to testis receptor.

  13. 钛表面人工肽抑制格登链球菌生长的实验研究%Effects of the synthetic peptide coatings inhibiting the growth of Streptococcus gordonii on titanium surface

    Institute of Scientific and Technical Information of China (English)

    马勍; 张溪; 张旭; 孙迎春; 高平

    2016-01-01

    目的:检测自主设计合成的人工肽在钛表面的吸附情况及吸附后的抗菌效果,为种植体抗菌的实验研究提供新的思路。方法使用ExPASy ProtParam、ProtScale软件、圆二色光谱和Zeta电位仪等,分析或测定人工肽与抗菌肽的各项理化性质及结构特征。利用常温孵育的方法将人工肽锚定于钛表面。使用X射线光电子能谱与原子力显微镜检测人工肽在钛表面的吸附。通过共聚焦激光显微镜观察锚定于钛表面的人工肽对格登链球菌的抑菌作用。使用透射电镜检测游离人工肽与抗菌肽对格登链球菌结构的破坏作用。结果自主设计合成的人工肽仍具有抗菌肽发挥抗菌作用所需的各项理化性质及结构特征。在孵育于5 g/L人工肽的PBS溶液中的钛片表面即可检测到人工肽的吸附,且该试件对格登链球菌的存活及黏附均有一定的抑制效果。结论自主设计合成的人工肽实现了在钛表面吸附并抑制格登链球菌生长的设想。%Objective To detect the independently designed synthetic peptide adsorbed to the titanium surface and its inhibitory effect on streptococcus gordonii, and to provide a new means for antibiosis reseach on oral implants. Methods The physical and chemical properties of the synthetic peptide and antimicrobial peptide were measured by ExPASy Prot⁃Param tool, ProtScale analysis, circular dichroism and Zeta potential instrument. The synthetic peptide was anchored on the surface of the titanium specimen through incubation at room temperature. The adsorption of the synthetic peptide to the titani⁃um surface was examined by X-ray photoelectron spectroscopy (XPS) and the atomic force microscope (AFM). The inhibitory effect on streptococcus gordonii of the synthetic peptide fixed on the titanium surface was viewed by confocal laser scanning microscopy (CLSM). The destructive effects of the synthetic peptide and the antimicrobial peptide on

  14. Assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide

    Science.gov (United States)

    Chen, Yu-Shiun; Hung, Yao-Ching; Lin, Wei-Hsu; Huang, Guewha Steven

    2010-05-01

    To assess the ability of gold nanoparticles (GNPs) to act as a size-dependent carrier, a synthetic peptide resembling foot-and-mouth disease virus (FMDV) protein was conjugated to GNPs ranging from 2 to 50 nm in diameter (2, 5, 8, 12, 17, 37, and 50 nm). An extra cysteine was added to the C-terminus of the FMDV peptide (pFMDV) to ensure maximal conjugation to the GNPs, which have a high affinity for sulfhydryl groups. The resultant pFMDV-GNP conjugates were then injected into BALB/c mice. Immunization with pFMDV-keyhole limpet hemocyanin (pFMDV-KLH) conjugate was also performed as a control. Blood was obtained from the mice after 4, 6, 8, and 10 weeks and antibody titers against both pFMDV and the carriers were measured. For the pFMDV-GNP immunization, specific antibodies against the synthetic peptide were detected in the sera of mice injected with 2, 5, 8, 12, and 17 nm pFMDV-GNP conjugates. Maximal antibody binding was noted for GNPs of diameter 8-17 nm. The pFMDV-GNPs induced a three-fold increase in the antibody response compared to the response to pFMDV-KLH. However, sera from either immunized mouse group did not exhibit an antibody response to GNPs, while the sera from pFMDV-KLH-immunized mice presented high levels of binding activity against KLH. Additionally, the uptake of pFMDV-GNP in the spleen was examined by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscopy (TEM). The quantity of GNPs that accumulated in the spleen correlated to the magnitude of the immune response induced by pFMDV-GNP. In conclusion, we demonstrated the size-dependent immunogenic properties of pFMDV-GNP conjugates. Furthermore, we established that GNPs ranging from 8 to 17 nm in diameter may be ideal for eliciting a focused antibody response against a synthetic pFMDV peptide.

  15. Assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yu-Shiun [Department of Materials Science and Engineering, National Chiao Tung University, 1001 University Road, EE137, Hsinchu 300, Taiwan (China); Hung, Yao-Ching [Department of Obstetrics and Gynecology, School of Medicine, China Medical University and Hospital, 91 Hsueh-Shih Road, Taichung 40402, Taiwan (China); Lin, Wei-Hsu [Institute of Nanotechnology, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan (China); Huang, Guewha Steven, E-mail: gstevehuang@mail.nctu.edu.tw [Department of Materials Science and Engineering, Institute of Nanotechnology, National Chiao Tung University, 1001 University Road, Hsinchu 300, Taiwan, Republic of China (China)

    2010-05-14

    To assess the ability of gold nanoparticles (GNPs) to act as a size-dependent carrier, a synthetic peptide resembling foot-and-mouth disease virus (FMDV) protein was conjugated to GNPs ranging from 2 to 50 nm in diameter (2, 5, 8, 12, 17, 37, and 50 nm). An extra cysteine was added to the C-terminus of the FMDV peptide (pFMDV) to ensure maximal conjugation to the GNPs, which have a high affinity for sulfhydryl groups. The resultant pFMDV-GNP conjugates were then injected into BALB/c mice. Immunization with pFMDV-keyhole limpet hemocyanin (pFMDV-KLH) conjugate was also performed as a control. Blood was obtained from the mice after 4, 6, 8, and 10 weeks and antibody titers against both pFMDV and the carriers were measured. For the pFMDV-GNP immunization, specific antibodies against the synthetic peptide were detected in the sera of mice injected with 2, 5, 8, 12, and 17 nm pFMDV-GNP conjugates. Maximal antibody binding was noted for GNPs of diameter 8-17 nm. The pFMDV-GNPs induced a three-fold increase in the antibody response compared to the response to pFMDV-KLH. However, sera from either immunized mouse group did not exhibit an antibody response to GNPs, while the sera from pFMDV-KLH-immunized mice presented high levels of binding activity against KLH. Additionally, the uptake of pFMDV-GNP in the spleen was examined by inductively coupled plasma mass spectroscopy (ICP-MS) and transmission electron microscopy (TEM). The quantity of GNPs that accumulated in the spleen correlated to the magnitude of the immune response induced by pFMDV-GNP. In conclusion, we demonstrated the size-dependent immunogenic properties of pFMDV-GNP conjugates. Furthermore, we established that GNPs ranging from 8 to 17 nm in diameter may be ideal for eliciting a focused antibody response against a synthetic pFMDV peptide.

  16. Simulations of the pore structures for a M2G1yR derived channel forming peptide in membrane

    Science.gov (United States)

    Al-Rawi, Ahlam N.; Al-Rawi, Asma; Chen, Jianhan; Herrera, Alvaro; Tomich, John; Rahman, Talat S.

    2008-03-01

    In an effort to develop a peptide-based compound suitable for clinical use as a channel replacement therapeutic for treating channelopathies such as cystic fibrosis, we present a reductionist model that appears to capture many of the biophysical properties of an intact ion channel using short channel-forming peptides. We have developed two anion selective channel-forming peptides with near native and altered properties from the peptides derived from the glycine receptor: NK4-M2GlyR-p22 WT (KKKKPAR-VGLGITTVLTMTTQS) and NK4-M2GlyR-p22 S22W (KKKKPARVGLGITTVLTMTTQW), respectively. Starting with the two structures determined by solution multidimensional NMR (800 MHz) in SDS, we used CHARMM and NAMD to perform molecular dynamics simulations on the monomers. Using the existing experimental data, we then built an initial 5- helix assembly by altering the tilted angle, rotational angle and pore radius. We investigated the impact of the single mutation at position 22 on the structure and dynamics of the pore formed in a membrane build in a hydrated POPC lipid bilayer. Probable structures for both assemblies are presented.

  17. [3H]Azidodantrolene photoaffinity labeling, synthetic domain peptides and monoclonal antibody reactivity identify the dantrolene binding sequence on RyR1

    Energy Technology Data Exchange (ETDEWEB)

    Paul-Pletzer, Kalanethee; Yamamoto, Takeshi; Bhat, Manju B.; Ma, Jianjie; Ikemoto, Noriaki; Jimenez, Leslie S.; Morimoto, Hiromi; Williams, Philip G.; Parness, Jerome

    2002-06-14

    Dantrolene is a drug that suppresses intracellular Ca2+ release from sarcoplasmic reticulum in normal skeletal muscle and is used as a therapeutic agent in individuals susceptible to malignant hyperthermia. Though its precise mechanism of action has not been elucidated, we have identified the N-terminal region (amino acids 1-1400) of the skeletal muscle isoform of the ryanodine receptor (RyR1), the primary Ca2+ release channel in sarcoplasmic reticulum, as a molecular target for dantrolene using the photoaffinity analog [3H]azidodantrolene(1). Here, we demonstrate that heterologously expressed RyR1 retains its capacity to be specifically labeled with [3H]azidodantrolene,indicating that muscle specific factors are not required for this ligand-receptor interaction. Synthetic domain peptides of RyR1, previously shown to affect RyR1 function in vitro and in vivo, were exploited as potential drug binding site mimics and used in photoaffinity labeling experiments. Only DP1 and DP1-2, peptide s containing the amino acid sequence corresponding to RyR1 residues 590-609, were specifically labeled by [3H]azidodantrolene. A monoclonal anti-RyR1 antibody which recognizes RyR1 and its 1400 amino acid N-terminal fragment, recognizes DP1 and DP1-2 in both Western blots and immunoprecipitation assays, and specifically inhibits [3H]azidodantrolene photolabeling of RyR1 and its N-terminal fragment in sarcoplasmic reticulum. Our results indicate that synthetic domain peptides can mimic a native, ligand binding conformation in vitro, and that the dantrolene binding site and the epitope for the monoclonal antibody on RyR1 are equivalent and composed of amino-acids 590-609.

  18. Similar patterns of rDNA evolution in synthetic and recently formed natural populations of Tragopogon (Asteraceae allotetraploids

    Directory of Open Access Journals (Sweden)

    Soltis Pamela S

    2010-09-01

    Full Text Available Abstract Background Tragopogon mirus and T. miscellus are allotetraploids (2n = 24 that formed repeatedly during the past 80 years in eastern Washington and adjacent Idaho (USA following the introduction of the diploids T. dubius, T. porrifolius, and T. pratensis (2n = 12 from Europe. In most natural populations of T. mirus and T. miscellus, there are far fewer 35S rRNA genes (rDNA of T. dubius than there are of the other diploid parent (T. porrifolius or T. pratensis. We studied the inheritance of parental rDNA loci in allotetraploids resynthesized from diploid accessions. We investigate the dynamics and directionality of these rDNA losses, as well as the contribution of gene copy number variation in the parental diploids to rDNA variation in the derived tetraploids. Results Using Southern blot hybridization and fluorescent in situ hybridization (FISH, we analyzed copy numbers and distribution of these highly reiterated genes in seven lines of synthetic T. mirus (110 individuals and four lines of synthetic T. miscellus (71 individuals. Variation among diploid parents accounted for most of the observed gene imbalances detected in F1 hybrids but cannot explain frequent deviations from repeat additivity seen in the allotetraploid lines. Polyploid lineages involving the same diploid parents differed in rDNA genotype, indicating that conditions immediately following genome doubling are crucial for rDNA changes. About 19% of the resynthesized allotetraploid individuals had equal rDNA contributions from the diploid parents, 74% were skewed towards either T. porrifolius or T. pratensis-type units, and only 7% had more rDNA copies of T. dubius-origin compared to the other two parents. Similar genotype frequencies were observed among natural populations. Despite directional reduction of units, the additivity of 35S rDNA locus number is maintained in 82% of the synthetic lines and in all natural allotetraploids. Conclusions Uniparental reductions of

  19. Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.

    Science.gov (United States)

    Oehlke, Johannes; Wallukat, Gerd; Wolf, Yvonne; Ehrlich, Angelika; Wiesner, Burkhard; Berger, Hartmut; Bienert, Michael

    2004-07-01

    In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.

  20. Synthetic peptide, Ala-Arg-Glu-Gly-Glu-Met, abolishes pro-proliferative and anti-apoptotic effects of high glucose in vascular smooth muscle cells.

    Science.gov (United States)

    Cao, Xiaozhou; Lyu, Yi; Ning, Junyu; Tang, Xiaozhi; Shen, Xinchun

    2017-02-11

    Apoptosis plays a critical role in normal vascular development and atherosclerosis. However, high glucose has been reported to generate a certain level of ROS that can inhibit vascular smooth muscle cell (VSMC) apoptosis, with the underlying mechanism remaining unclear. In this study, a synthetic peptide AREGEM (Ala-Arg-Glu-Gly-Glu-Met) exhibited antioxidative effects and was used to investigate its function in VSMCs during hyperglycaemia. MTT assay results demonstrated that AREGEM significantly attenuated high glucose-induced VSMCs proliferation. Flow cytometry displayed that high glucose levels inhibited cell apoptosis, whereas this effect was attenuated by pre-incubation with AREGEM. In addition, the 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescent probe assay further demonstrated that AREGEM reduced intracellular ROS accumulation in VSMCs. Furthermore, this peptide was able to prevent the decrease of caspase-3 activity and the increase of the ratio of Bcl-2/Bax protein in VSMCs exposed to high glucose. These findings demonstrated that AREGEM is able to abolish the effects of high glucose in VSMCs; therefore, this peptide can be a potential candidate to develop a novel strategy for curing diabetic related diseases.

  1. Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

    Science.gov (United States)

    Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

    2001-01-01

    We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

  2. Evaluation of a synthetic peptide from the Taenia saginata 18kDa surface/secreted oncospheral adhesion protein for serological diagnosis of bovine cysticercosis.

    Science.gov (United States)

    Guimarães-Peixoto, Rafaella Paola Meneguete; Pinto, Paulo Sérgio Arruda; Santos, Marcus Rebouças; Polêto, Marcelo Depólo; Silva, Letícia Ferreira; Silva-Júnior, Abelardo

    2016-12-01

    Bovine cysticercosis is a zoonotic infection widely spread throughout Brazil, creating a burden on hygiene maintenance and the economy. Diagnosis of cysticercosis usually relies on post mortem inspection of carcasses in slaughterhouses. This detection method provides only low sensitivity. Recent advancements have improved the performance of serologic tests, such as ELISA, providing greater sensitivity and specificity. The objective of the current study was to identify and evaluate a synthetic peptide derived from the Taenia saginata 18kDa oncospheric surface protein for the diagnosis of bovine cysticercosis in ELISA. Test performance of the identified peptide was compared to an ELISA based on a heterologous crude Taenia crassiceps antigen (Tcra), widely used for the sero-diagnosis of bovine cysticercosis. Based on the primary sequence of an in silico structural model of the 18kDa protein, an epitope region designated EP1 was selected (46-WDTKDMAGYGVKKIEV-61). The peptide derived from this region yielded 91.6% (CI=80-96%) sensitivity and 90% (CI=82-95%) specificity when used in an ELISA, whereas the crude antigen yielded 70% (CI=56-8%) sensitivity and 82% (CI=73-89%) specificity. Thus, we conclude that EP1 has higher diagnostic potential for detecting bovine cysticercosis than the crude antigen Tcra.

  3. Modelling the chemistry of star-forming filaments - II. Testing filament characteristics with synthetic observations

    Science.gov (United States)

    Seifried, D.; Sánchez-Monge, Á.; Suri, S.; Walch, S.

    2017-06-01

    We present synthetic continuum and 13CO and C18O line emission observations of dense and cold filaments. The filaments are dynamically evolved using 3D-magnetohydrodynamic simulations that include one of the largest on-the-fly chemical networks used to date, which models the detailed evolution of H2 and CO. We investigate the reliability of observable properties, in particular filament mass and width, under different simulation conditions like magnetic field orientation and cosmic ray ionization rate. We find that filament widths of ˜0.1 pc can be probed with both line and continuum emission observations with a high accuracy (deviations ≤20 per cent). However, the width of more narrow filaments can be significantly overestimated by up to a factor of a few. Masses obtained via the dust emission are accurate within a few per cent whereas the masses inferred from molecular line emission observations deviate from the actual mass by up to a factor of 10 and show large differences for different J transitions. The inaccurate estimate of filament masses and widths of narrow filaments using molecular line observations can be attributed to (i) the non-isothermal state of the filaments, (ii) optical depth effects and (iii) the subthermally excited state of CO, while inclination effects and opacity correction only influence the obtained masses and widths by less than 50 per cent. Both, mass and width estimates, can be improved by using two isotopes to correct for the optical depth. Since gas and dust temperatures generally differ (by up to 25 K), the filaments appear more gravitationally unstable if the (too low) dust temperature is used for the stability analysis.

  4. Use of the cell wall precursor lipid II by a pore-forming peptide antibiotic

    NARCIS (Netherlands)

    Breukink, E; Wiedemann, [No Value; van Kraaij, C; Kuipers, OP; Sahl, HG; de Kruijff, B; Wiedemann, I.

    1999-01-01

    Resistance to antibiotics is increasing in some groups of clinically important pathogens. For instance, high vancomycin resistance has emerged in enterococci. Promising alternative antibiotics are the peptide antibiotics, abundant in host defense systems, which kill their targets by permeabilizing t

  5. Antifungal Activity of a Synthetic Cationic Peptide against the Plant Pathogens Colletotrichum graminicola and Three Fusarium Species.

    Science.gov (United States)

    Johnson, Eric T; Evans, Kervin O; Dowd, Patrick F

    2015-09-01

    A small cationic peptide (JH8944) was tested for activity against a number of pathogens of agricultural crops. JH8944 inhibited conidium growth in most of the tested plant pathogens with a dose of 50 μg/ml, although one isolate of Fusarium oxysporum was inhibited at 5 μg/ml of JH8944. Most conidia of Fusarium graminearum were killed within 6 hours of treatment with 50 μg/ml of JH8944. Germinating F. graminearum conidia required 238 μg/ml of JH8944 for 90% growth inhibition. The peptide did not cause any damage to tissues surrounding maize leaf punctures when tested at a higher concentration of 250 μg/ml even after 3 days. Liposomes consisting of phosphatidylglycerol were susceptible to leakage after treatment with 25 and 50 μg/ml of JH8944. These experiments suggest this peptide destroys fungal membrane integrity and could be utilized for control of crop fungal pathogens.

  6. Sterically locked synthetic bilin derivatives and phytochrome Agp1 from Agrobacterium tumefaciens form photoinsensitive Pr- and Pfr-like adducts.

    Science.gov (United States)

    Inomata, Katsuhiko; Hammam, Mostafa A S; Kinoshita, Hideki; Murata, Yasue; Khawn, Htoi; Noack, Steffi; Michael, Norbert; Lamparter, Tilman

    2005-07-01

    Phytochrome photoreceptors undergo reversible photoconversion between the red-absorbing form, Pr, and the far-red-absorbing form, Pfr. The first step in the conversion from Pr to Pfr is a Z to E isomerization around the C15=C16 double bond of the bilin chromophore. We prepared four synthetic biliverdin (BV) derivatives in which rings C and D are sterically locked by cyclizing with an additional carbon chain. In these chromophores, which are termed 15Za, 15Zs, 15Ea, and 15Es, the C15=C16 double bond is in either the Z or E configuration and the C14-C15 single bond in either the syn or anti conformation. The chromophores were assembled with Agrobacterium phytochrome Agp1, which incorporates BV as natural chromophore. All locked BV derivatives bound covalently to the protein and formed adducts with characteristic spectral properties. The 15Za adduct was spectrally similar to the Pr form and the 15Ea adduct similar to the Pfr form of the BV adduct. Thus, the chromophore of Agp1 adopts a C15=C16 Z configuration and a C14-C15 anti conformation in the Pr form and a C15=C16 E configuration and a C14-C15 anti conformation in the Pfr form. Both the 15Zs and the 15Es adducts absorbed only in the blue region of the visible spectra. All chromophore adducts were analyzed by size exclusion chromatography and histidine kinase activity to probe for protein conformation. In either case, the 15Za adduct behaved like the Pr and the 15Ea adduct like the Pfr form of Agp1. Replacing the natural chromophore by a locked 15Ea derivative can thus bring phytochrome holoprotein in the Pfr form in darkness. In this way, physiological action of Pfr can be studied in vivo and separated from Pr/Pfr cycling and other light effects.

  7. Direct Quantitation of Peptide Mixtures without Standards using Clusters Formed by Electrospray Ionization Mass Spectrometry

    OpenAIRE

    Leib, Ryan D.; Flick, Tawnya G.; Williams, Evan R.

    2009-01-01

    In electrospray ionization mass spectrometry, ion abundances depend on a number of different factors, including analyte surface activity, competition between analytes for charge, analyte concentration, as well as instrumental factors, including mass-dependent ion transmission and detection. Here, a novel method for obtaining quantitative information about solution-phase concentrations of peptide mixtures is described and demonstrated for five different peptide mixtures with relative concentra...

  8. Specific adducts formed through a radical reaction between peptides and contact allergenic hydroperoxides.

    Science.gov (United States)

    Redeby, Theres; Nilsson, Ulrika; Altamore, Timothy M; Ilag, Leopold; Ambrosi, Annalisa; Broo, Kerstin; Börje, Anna; Karlberg, Ann-Therese

    2010-01-01

    The first step in the development of contact allergy (allergic contact dermatitis) includes the penetration of an allergy-causing chemical (hapten) into the skin, where it binds to macromolecules such as proteins. The protein-hapten adduct is then recognized by the immune system as foreign to the body. For hydroperoxides, no relevant hapten target proteins or protein-hapten adducts have so far been identified. In this work, bovine insulin and human angiotensin I were used as model peptides to investigate the haptenation mechanism of three hydroperoxide haptens: (5R)-5-isopropenyl-2-methyl-2-cyclohexene-1-hydroperoxide (Lim-2-OOH), cumene hydroperoxide (CumOOH), and 1-(1-hydroperoxy-1-methylethyl) cyclohexene (CycHexOOH). These hydroperoxides are expected to react via a radical mechanism, for which 5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (Fe(III)TPPCl) was used as a radical initiator. The reactions were carried out in 1:1 ethanol/10 mM ammonium acetate buffer pH 7.4, for 3 h at 37 degrees C, and the reaction products were either enzymatically digested or analyzed directly by MALDI/TOF-MS, HPLC/MS/MS, and 2D gel electrophoresis. Both hydroperoxide-specific and unspecific reaction products were detected, but only in the presence of the iron catalyst. In the absence of catalyst, the hydroperoxides remained unreacted. This suggests that the hydroperoxides can enter into the skin and remain inert until activated. Through the detection of a Lim-2-OOH adduct bound at the first histidine (of two) of angiotensin I, it was confirmed that hydroperoxides have the potential to form specific antigens in contact allergy.

  9. 2-Pyrrolinodoxorubicin and its peptide-vectorized form bypass multidrug resistance.

    Science.gov (United States)

    Castex, Cédric; Merida, Peggy; Blanc, Emmanuelle; Clair, Philippe; Rees, Anthony R; Temsamani, Jamal

    2004-07-01

    A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein, which is capable of lowering intracellular drug concentrations. In the present study, we tested the capability of 2-pyrrolinodoxorubicin (p-DOX), a highly potent derivative of DOX, to bypass multidrug resistance. The accumulation, intracellular distribution and cytotoxicity of p-DOX were tested in two cell lines (K562 and A2780) and their DOX-resistant counterparts (K562/ADR and A2780/ADR). Cellular accumulation and cytotoxicity were dramatically lowered for DOX in resistant cell lines, in comparison with non-resistant cells. In contrast, cellular accumulation, intracellular distribution and cytotoxicity of p-DOX were independent of the nature of the cell lines. The p-DOX showed potent dose-dependent inhibition of cell growth against resistant cells as compared with DOX. After treatment of resistant cells with verapamil, the intracellular levels of DOX were markedly increased and consequent cytotoxicity improved. In contrast, treatment of resistant cells with verapamil did not cause any further enhancement of cell uptake or an increase in the cytotoxic effect of the derivative p-DOX, indicating that the compound bypasses the P-glycoprotein. Finally, we show that vectorization of p-DOX by a peptide vector (SynB3) which has been shown to enhance the brain uptake of DOX and to decrease its heart accumulation does not affect this property. These results indicate that p-DOX and its vectorized form are potent and effective in overcoming multidrug resistance.

  10. Ion channel stability and hydrogen bonding. Molecular modelling of channels formed by synthetic alamethicin analogues.

    Science.gov (United States)

    Breed, J; Kerr, I D; Molle, G; Duclohier, H; Sansom, M S

    1997-12-04

    Several analogues of the channel-forming peptaibol alamethicin have been demonstrated to exhibit faster switching between channel substates than does unmodified alamethicin. Molecular modelling studies are used to explore the possible molecular basis of these differences. Models of channels formed by alamethicin analogues were generated by restrained molecular dynamics in vacuo and refined by short molecular dynamics simulations with water molecules within and at either mouth of the channel. A decrease in backbone solvation was found to correlate with a decrease in open channel stability between alamethicin and an analogue in which all alpha-amino-isobutyric acid residues of alamethicin were replaced by leucine. A decrease in the extent of hydrogen-bonding at residue 7 correlates with lower open channel stabilities of analogues in which the glutamine at position 7 was replaced by smaller polar sidechains. These two observations indicate the importance of alamethicin/water H-bonds in stabilizing the open channel.

  11. Polymer-Peptide Nanoparticles: Synthesis and Characterization

    Science.gov (United States)

    Dong, He; Shu, Jessica Y.; Xu, Ting

    2010-03-01

    Conjugation of synthetic polymers to peptides offers an efficient way to produce novel supramolecular structures. Herein, we report an attempt to prepare synthetic micellar nanoparticles using amphiphilic peptide-polymer conjugates as molecular building blocks. Spherical nanoparticles were formed upon dissolution of peptides in PBS buffer through the segregation of hydrophobic and hydrophilic segments. Both molecular and nano- structures were thoroughly investigated by a variety of biophysical techniques, including circular dichroism (CD), dynamic light scattering (DLS), size exclusion chromatography (SEC), transmission electron microscopy (TEM) and small angle X-ray scattering (SAXS). The results demonstrate that structural properties of these biohybrid materials depend on both the geometry of the hydrophobic domain and the size of synthetic polymers. Given the diversity of functional peptide sequences, hydrophilic polymers and hydrophobic moieties, these materials would be expected to self-assemble into various types of nanostructures to cover a wide range of biological applications.

  12. pH-dependent solution structure and activity of a reduced form of the host-defense peptide myticin C (Myt C) from the mussel Mytilus galloprovincialis.

    Science.gov (United States)

    Martinez-Lopez, Alicia; Encinar, Jose Antonio; Medina-Gali, Regla Maria; Balseiro, Pablo; Garcia-Valtanen, Pablo; Figueras, Antonio; Novoa, Beatriz; Estepa, Amparo

    2013-07-04

    Myticin C (Myt C) is a highly variable host-defense peptide (HDP) associated to the immune response in the mediterranean mussel (Mytilus galloprovincialis), which has shown to be active across species due to its strong antiviral activity against a fish rhabdovirus found in fish cells overexpressing this HDP. However, the potential antimicrobial properties of any synthetic analogue of Myt C has not yet been analysed. Thus, in this work we have synthesised the sequence of the mature peptide of Myt C variant c and analysed the structure activity relationships of its reduced (non-oxidized) form (red-MytCc). In contrast to results previously reported for oxidized isoforms of mussel myticins, red-MytCc was not active against bacteria at physiological pH and showed a moderate antiviral activity against the viral haemorrhagic septicaemia (VHS) rhabdovirus. However, its chemotactic properties remained active. Structure/function studies in neutral and acid environments by means of infrared spectroscopy indicated that the structure of red-MytCc is pH dependent, with acid media increasing its alpha-helical content. Furthermore, red-MytCc was able to efficiently aggregate artificial phospholipid membranes at low pH, as well as to inhibit the Escherichia coli growth, suggesting that this activity is attributable to its more structured form in an acidic environment. All together, these results highlight the dynamic and environmentally sensitive behavior of red-Myt C in solution, and provide important insights into Myt C structure/activity relationships and the requirements to exert its antimicrobial/immunomodulatory activities. On the other hand, the pH-dependent direct antimicrobial activity of Myt C suggests that this HDP may be a suitable template for the development of antimicrobial agents that would function selectively in specific pH environments, which are sorely needed in this "antibiotic-resistance era".

  13. Humoral and cellular immune responses to synthetic peptides of the Leishmania donovani kinetoplastid membrane protein-11

    DEFF Research Database (Denmark)

    Jensen, A T; Gasim, S; Ismail, A

    1998-01-01

    Native kinetoplastid membrane protein-11 (KMP-11), purified from crude extracts of Leishmania donovani parasites, activates T cells from individuals who have recovered from visceral leishmaniasis. In this work we used three 38-mer peptides spanning the amino acid sequence of the L. donovani KMP-11...... as solid-phase ligands in enzyme-linked immunosorbent assays (ELISAs) and as stimulating antigens in lymphoproliferative assays in order to evaluate humoral and cellular immune responses to well-defined sequences of the protein. Antibody reactivity against the three peptides was measured in plasma from 63...... Sudanese visceral leishmaniasis patients (VL) and the percentage of patients with anti-KMP-11 antibodies in ELISA were 37% (KMP-11-1), 30% (KMP-11-2) and 58% (KMP-11-3). The fraction of VL patients with measurable antibody reactivity in one or more of the three ELISAs was 79%. Cross-reactivity to the KMP...

  14. Mimicking of Chondrocyte Microenvironment Using In Situ Forming Dendritic Polyglycerol Sulfate-Based Synthetic Polyanionic Hydrogels.

    Science.gov (United States)

    Dey, Pradip; Schneider, Tobias; Chiappisi, Leonardo; Gradzielski, Michael; Schulze-Tanzil, Gundula; Haag, Rainer

    2016-04-01

    A stable polymeric network that mimics the highly polyanionic extracellular cartilage matrix still remains a great challenge. The main aim of this study is to present the synthesis of dendritic polyglycerol sulfate (dPGS)-based in situ forming hydrogels using strain promoted azide-alkyne cycloaddition reactions. A real time rheological study has been used to characterize the hydrogel properties. The viability of encapsulated human chondrocytes in the different hydrogels are monitored using live-dead staining. Furthermore, type I and II collagen gene have been analyzed. Hydrogels with elastic moduli ranging from 1 to 5 kPa have been prepared by varying the dPGS amount. The chondrocyte viability in dPGS hydrogels is found to be higher than in pure PEG and alginate-based hydrogels after 21 d. The higher cell viability in the dPGS engineered hydrogels can be explained by the fact that dPGS can interact with different proteins responsible for cell growth and proliferation.

  15. Evidence that a synthetic amyloid-ß oligomer-binding peptide (ABP) targets amyloid-ß deposits in transgenic mouse brain and human Alzheimer's disease brain.

    Science.gov (United States)

    Chakravarthy, Balu; Ito, Shingo; Atkinson, Trevor; Gaudet, Chantal; Ménard, Michel; Brown, Leslie; Whitfield, James

    2014-03-14

    The synthetic ~5 kDa ABP (amyloid-ß binding peptide) consists of a region of the 228 kDa human pericentrioloar material-1 (PCM-1) protein that selectively and avidly binds in vitro Aβ1-42 oligomers, believed to be key co-drivers of Alzheimer's disease (AD), but not monomers (Chakravarthy et al., (2013) [3]). ABP also prevents Aß1-42 from triggering the apoptotic death of cultured human SHSY5Y neuroblasts, likely by sequestering Aß oligomers, suggesting that it might be a potential AD therapeutic. Here we support this possibility by showing that ABP also recognizes and binds Aβ1-42 aggregates in sections of cortices and hippocampi from brains of AD transgenic mice and human AD patients. More importantly, ABP targets Aβ1-42 aggregates when microinjected into the hippocampi of the brains of live AD transgenic mice.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed...... in several kinds of cancer, in particular, ovarian, glioblastomas, and breast cancer, but not in normal tissue. The library consisted of six individual positional sublibraries in the format, H-O(1-6)XXXXX-NH(2), O being one of the 19 proteinogenic amino acids (cysteine omitted) and X an equimolar mixture...... of these. The library consisted of 114 mixtures in total. Using a biotin-streptavidin assay, the binding of each sublibrary to NR6M, NR6W-A, and NR6 cells was tested. These cells express EGFRvIII, EGFR, and neither of the receptors, respectively. The result from each sublibrary was examined to identify...

  17. Antimicrobial Activity and Stability of Short and Long Based Arachnid Synthetic Peptides in the Presence of Commercial Antibiotics

    Directory of Open Access Journals (Sweden)

    Ivan Arenas

    2016-02-01

    Full Text Available Four antimicrobial peptides (AMPs named Pin2[G], Pin2[14], P18K and FA1 were chemically synthesized and purified. The four peptides were evaluated in the presence of eight commercial antibiotics against four microorganisms of medical importance: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. The commercial antibiotics used were amoxicillin, azithromycin, ceftriaxone, gentamicin, levofloxacin, sulfamethoxazole, trimethoprim and vancomycin. The best AMP against P. aeruginosa was the peptide FA1, and the best AMP against S. aureus was Pin2[G]. Both FA1 and Pin2[G] were efficient against E. coli, but they were not effective against K. pneumoniae. As K. pneumoniae was resistant to most of the commercial antibiotics, combinations of the AMPs FA1 and Pin2[G] were prepared with these antibiotics. According to the fractional inhibitory concentration (FIC index, the best antimicrobial combinations were obtained with concomitant applications of mixtures of FA1 with levofloxacin and sulfamethoxazole. However, combinations of FA1 or Pin2[G] with other antibiotics showed that total inhibitory effect of the combinations were greater than the sum of the individual effects of either the antimicrobial peptide or the antibiotic. We also evaluated the stability of the AMPs. The AMP Pin2[G] manifested the best performance in saline buffer, in supernatants of bacterial growth and in human blood plasma. Nevertheless, all AMPs were cleaved using endoproteolytic enzymes. These data show advantages and disadvantages of AMPs for potential clinical treatments of bacterial infections, using them in conjunction with commercial antibiotics.

  18. Antifungal Activity of a Synthetic Cationic Peptide against the Plant Pathogens Colletotrichum graminicola and Three Fusarium Species

    Directory of Open Access Journals (Sweden)

    Eric T. Johnson

    2015-09-01

    Full Text Available A small cationic peptide (JH8944 was tested for activity against a number of pathogens of agricultural crops. JH8944 inhibited conidium growth in most of the tested plant pathogens with a dose of 50 μg/ml, although one isolate of Fusarium oxysporum was inhibited at 5 μg/ml of JH8944. Most conidia of Fusarium graminearum were killed within 6 hours of treatment with 50 μg/ml of JH8944. Germinating F. graminearum conidia required 238 μg/ml of JH8944 for 90% growth inhibition. The peptide did not cause any damage to tissues surrounding maize leaf punctures when tested at a higher concentration of 250 μg/ml even after 3 days. Liposomes consisting of phosphatidylglycerol were susceptible to leakage after treatment with 25 and 50 μg/ml of JH8944. These experiments suggest this peptide destroys fungal membrane integrity and could be utilized for control of crop fungal pathogens.

  19. Scaffold-forming and Adhesive Contributions of Synthetic Laminin-binding Proteins to Basement Membrane Assembly.

    Science.gov (United States)

    McKee, Karen K; Capizzi, Stephanie; Yurchenco, Peter D

    2009-03-27

    Laminins that possess three short arms contribute to basement membrane assembly by anchoring to cell surfaces, polymerizing, and binding to nidogen and collagen IV. Although laminins containing the alpha4 and alpha5 subunits are expressed in alpha2-deficient congenital muscular dystrophy, they may be ineffective substitutes because they bind weakly to cell surfaces and/or because they lack the third arm needed for polymerization. We asked whether linker proteins engineered to bind to deficient laminins that provide such missing activities would promote basement membrane assembly in a Schwann cell model. A chimeric fusion protein (alphaLNNd) that adds a short arm terminus to laminin through the nidogen binding locus was generated and compared with the dystrophy-ameliorating protein miniagrin (mAgrin) that binds to the laminin coiled-coil dystroglycan and sulfatides. alphaLNNd was found to mediate laminin binding to collagen IV, to bind to galactosyl sulfatide, and to selectively convert alpha-short arm deletion-mutant laminins LmDeltaalphaLN and LmDeltaalphaLN-L4b into polymerizing laminins. This protein enabled polymerization-deficient laminin but not an adhesion-deficient laminin lacking LG domains (LmDeltaLG) to assemble an extracellular matrix on Schwann cell surfaces. mAgrin, on the other hand, enabled LmDeltaLG to form an extracellular matrix on cell surfaces without increasing accumulation of non-polymerizing laminins. These gain-of-function studies reveal distinct polymerization and anchorage contributions to basement membrane assembly in which the three different LN domains mediate the former, and the LG domains provide primary anchorage with secondary contributions from the alphaLN domain. These findings may be relevant for an understanding of the pathogenesis and treatment of laminin deficiency states.

  20. Characterization of biofilm-like structures formed by Pseudomonas aeruginosa in a synthetic mucus medium

    Directory of Open Access Journals (Sweden)

    Haley Cecily L

    2012-08-01

    Full Text Available Abstract Background The accumulation of thick stagnant mucus provides a suitable environment for the growth of Pseudomonas aeruginosa and Staphylococcus aureus within the lung alveoli of cystic fibrosis (CF patients. These infections cause significant lung damage, leading to respiratory failure and death. In an artificial mucin containing medium ASM+, P. aeruginosa forms structures that resemble typical biofilms but are not attached to any surface. We refer to these structures as biofilm like structures (BLS. Using ASM+ in a static microtiter plate culture system, we examined the roles of mucin, extracellular DNA, environmental oxygen (EO2, and quorum sensing (QS in the development of biofilm-like structures (BLS by P. aeruginosa; and the effect of EO2 and P. aeruginosa on S. aureus BLS. Results Under 20% EO2, P. aeruginosa strain PAO1 produced BLS that resemble typical biofilms but are confined to the ASM+ and not attached to the surface. Levels of mucin and extracellular DNA within the ASM+ were optimized to produce robust well developed BLS. At 10% EO2, PAO1 produced thicker, more developed BLS, while under 0% EO2, BLS production was diminished. In contrast, the S. aureus strain AH133 produced well-developed BLS only under 20% EO2. In PAO1, loss of the QS system genes rhlI and rhlR affected the formation of BLS in ASM+ in terms of both structure and architecture. Whether co-inoculated into ASM+ with AH133, or added to established AH133 BLS, PAO1 eliminated AH133 within 48–56 h. Conclusions The thick, viscous ASM+, which contains mucin and extracellular DNA levels similar to those found in the CF lung, supports the formation of biofilm-like structures similar to the aggregates described within CF airways. Alterations in environmental conditions or in the QS genes of P. aeruginosa, as occurs naturally during the progression of CF lung infection, affect the architecture and quantitative structural features of these BLS. Thus, ASM+ provides an

  1. Synthetic antimicrobial β-peptide in dual-treatment with fluconazole or ketoconazole enhances the in vitro inhibition of planktonic and biofilm Candida albicans.

    Science.gov (United States)

    Mora-Navarro, Camilo; Caraballo-León, Jean; Torres-Lugo, Madeline; Ortiz-Bermúdez, Patricia

    2015-12-01

    Fungal infections are a pressing concern for human health worldwide, particularly for immunocompromised individuals. Current challenges such as the elevated toxicity of common antifungal drugs and the emerging resistance towards these could be overcome by multidrug therapy. Natural antimicrobial peptides, AMPs, in combination with other antifungal agents are a promising avenue to address the prevailing challenges. However, they possess limited biostability and susceptibility to proteases, which has significantly hampered their development as antifungal therapies. β-peptides are synthetic materials designed to mimic AMPs while allowing high tunability and increased biostability. In this work, we report for the first time the inhibition achieved in Candida albicans when treated with a mixture of a β-peptide model and fluconazole or ketoconazole. This combination treatment enhanced the biological activity of these azoles in planktonic and biofilm Candida, and also in a fluconazole-resistant strain. Furthermore, the in vitro cytotoxicity of the dual treatment was evaluated towards the human hepatoma cell line, HepG2, a widely used model derived from liver tissue, which is primarily affected by azoles. Analyses based on the LA-based method and the mass-action law principle, using a microtiter checkerboard approach, revealed synergism of the combination treatment in the inhibition of planktonic C. albicans. The dual treatment proved to be fungicidal at 48 and 72 h. Interestingly, it was also found that the viability of HepG2 was not significantly affected by the dual treatments. Finally, a remarkable enhancement in the inhibition of the highly azole-resistant biofilms and fluconazole resistant C. albicans strain was obtained.

  2. Synthetic peptides from two Pf sporozoite invasion-associated proteins specifically interact with HeLa and HepG2 cells.

    Science.gov (United States)

    Arévalo-Pinzón, Gabriela; Curtidor, Hernando; Muñoz, Marina; Patarroyo, Manuel A; Patarroyo, Manuel E

    2011-09-01

    Two recently described molecules have been associated with sporozoite traversal ability and hepatocyte entry: sporozoite invasion-associated proteins (SIAP)-1 and -2. The HeLa and HepG2 cell binding ability of synthetic peptides spanning the whole SIAP-1 and -2 sequences has been studied in the search for identifying these proteins' functionally active specific regions. Twelve HepG-2 and seventeen HeLa cell high-activity binding peptides (HABPs) have been identified in SIAP-1, 8 of them having high specific binding affinity for both cell lines. Four HepG2 HABPs and two HeLa HABPs have been identified in SIAP-2, one of them interacting with both HeLa and HepG2 cells. SIAP-1 and SIAP-2 HABPs bound specifically and saturably to heparin sulfate and chondroitin sulfate-type membrane receptors on host cells. Circular dichroism assays have shown high α-helix content in SIAP-1 and SIAP-2 HABP secondary structure. Immunofluorescence analysis has revealed that specific peptides against SIAP proteins are highly immunogenic in mice and that anti-SIAP-1 and -2 antibodies recognize the native protein in Plasmodium falciparum sporozoites. Polymorphism studies have shown that a most SIAP-1 and -2 HABPs are conserved among P. falciparum strains. Our results have suggested that SIAP-1 and -2 participate in host-pathogen interactions during cell-traversal and hepatocyte invasion and highlighted the relevance of the ongoing identification and study of potentially new molecules when designing a fully protective antimalarial vaccine.

  3. A synthetic peptide corresponding to the C-terminal 25 residues of phage MS2 coded lysis protein dissipates the protonmotive force in Escherichia coli membrane vesicles by generating hydrophilic pores

    NARCIS (Netherlands)

    Goessens, Wil H.F.; Driessen, Arnold J.M.; Wilschut, Jan; Duin, Jan van

    1988-01-01

    The RNA phage MS2 encodes a protein, 75 amino acids long, that is necessary and sufficient for lysis of the host cell. DNA deletion analysis has shown that the lytic activity is confined to the C-terminal half of the protein. We have examined the effects of a synthetic peptide, covering the C-termin

  4. Absolute quantification of norovirus capsid protein in food, water, and soil using synthetic peptides with electrospray and MALDI mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Hartmann, Erica M. [Center for Environmental Security and Security Defense Systems Initiative, The Biodesign Institute, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287-5904 (United States); Colquhoun, David R.; Schwab, Kellogg J. [Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (United States); Halden, Rolf U., E-mail: halden@asu.edu [Center for Environmental Security and Security Defense Systems Initiative, The Biodesign Institute, Arizona State University, 781 E. Terrace Mall, Tempe, AZ 85287-5904 (United States); Department of Environmental Health Sciences, The Johns Hopkins University, Bloomberg School of Public Health, 615 N. Wolfe St., Baltimore, MD 21205 (United States)

    2015-04-09

    Highlights: • Mass spectrometry-based methods for norovirus quantification are developed. • Absolute quantification is achieved using internal heavy isotope-labeled standards. • A single labeled peptide serves in two distinct detection strategies. • These methods are validated for food, water, and soil analysis. • MS-based detection limits are lowered by two orders of magnitude. - Abstract: Norovirus infections are one of the most prominent public health problems of microbial origin in the U.S. and other industrialized countries. Surveillance is necessary to prevent secondary infection, confirm successful cleanup after outbreaks, and track the causative agent. Quantitative mass spectrometry, based on absolute quantitation with stable-isotope labeled peptides, is a promising tool for norovirus monitoring because of its speed, sensitivity, and robustness in the face of environmental inhibitors. In the current study, we present two new methods for the detection of the norovirus genogroup I capsid protein using electrospray and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The peptide TLDPIEVPLEDVR was used to quantify norovirus-like particles down to 500 attomoles with electrospray and 100 attomoles with MALDI. With MALDI, we also demonstrate a detection limit of 1 femtomole and a quantitative dynamic range of 5 orders of magnitude in the presence of an environmental matrix effect. Due to the rapid processing time and applicability to a wide range of environmental sample types (bacterial lysate, produce, milk, soil, and groundwater), mass spectrometry-based absolute quantitation has a strong potential for use in public health and environmental sciences.

  5. Simulations of the Pore Structures for a M2GlyR Derived Channel Forming Peptide in Different Membrane Environments

    Science.gov (United States)

    Al-Rawi, A.; Herrera, A.; Tomich, J.; Rahman, T.

    2007-03-01

    As part of an effort to develop a peptide-based compound suitable for clinical use as a channel replacement therapeutic for treating channelopathies such as cystic fibrosis, we present a reductionist model that appears to grasp the characteristics of ion channeling peptides. In particular we present the observed changes in the functional characteristics of NK4-M2GlyR p22 (KKKKPARVGLGITTVLTMTTQS), a M2 GlyR derived channel forming peptide. Starting with a structure determined by multidimensional NMR (800 MHz) in SDS, a potential from CHARMM force-field was used to relax the structure of NK4-M2GlyR p22. Following the relaxation, numerous pore structures were generated for the symmetric five-helix assembly with geometries varying from cylindrical to conical. As it is difficult a priori to assign accurately the orientation of the hydrophilic portion of M2GlyR derived amphipath towards the inside of the pore, we tilted and rotated the helical structure by five different angles about the backbone axis before forming the pore. Energy minimization of the channel was performed in vacuum, in phosphotidylcholine (POPC) membrane, and 60% POPC 30% phosphotidylethanolamine (POPE) in order to determine the effect of the environment surrounding on the structure on its energy minimization. We will present the various pore assemblies, in the different membrane environments, used to predict the most probably membrane bound structure.

  6. Rational design of fiber forming supramolecular structures

    Science.gov (United States)

    Wang, Benjamin K; Kanahara, Satoko M

    2016-01-01

    Recent strides in the development of multifunctional synthetic biomimetic materials through the self-assembly of multi-domain peptides and proteins over the past decade have been realized. Such engineered systems have wide-ranging application in bioengineering and medicine. This review focuses on fundamental fiber forming α-helical coiled-coil peptides, peptide amphiphiles, and amyloid-based self-assembling peptides; followed by higher order collagen- and elastin-mimetic peptides with an emphasis on chemical / biological characterization and biomimicry. PMID:27022140

  7. Identification of an immunodominant epitope in glycoproteins B and G of herpes simplex viruses (HSVs) using synthetic peptides as antigens in assay of antibodies to HSV in herpes simplex encephalitis patients.

    Science.gov (United States)

    Bhullar, S S; Chandak, N H; Baheti, N N; Purohit, H J; Taori, G M; Daginawala, H F; Kashyap, R S

    2014-01-01

    Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

  8. Conserved Molecular Superlattices in a Series of Homologous Synthetic Mycobacterial Cell-Wall Lipids Forming Interdigitated Bilayers.

    Science.gov (United States)

    Martin-Bertelsen, Birte; Yaghmur, Anan; Franzyk, Henrik; Justesen, Sarah; Kirkensgaard, Jacob J K; Foged, Camilla

    2016-12-06

    Synthetic analogues of the cell-wall lipid monomycoloyl glycerol (MMG) are promising as next-generation vaccine adjuvants. In the present study, the thermotropic phase behavior of an array of synthetic MMG analogues was examined by using simultaneous small- and wide-angle X-ray scattering under excess water conditions. The MMG analogues differed in the alkyl chain lengths and in the stereochemistry of the polar glycerol headgroup or of the lipid tails (native-like versus alternative compounds). All MMG analogues formed poorly hydrated lamellar phases at low temperatures and inverse hexagonal (H2) phases at higher temperatures prior to melting. MMG analogues with a native-like lipid acid configuration self-assembled into noninterdigitated bilayers whereas the analogues displaying an alternative lipid acid configuration formed interdigitated bilayers in a subgel (Lc') state. This is in contrast to previously described interdigitated phases for other lipids, which are usually in a gel (Lβ) state. All investigated MMG analogues displayed an abrupt direct temperature-induced phase transition from Lc' to H2. This transition is ultimately driven by the lipid chain melting and the accompanying change in molecular shape. No intermediate structures were found, but the entire array of MMG analogues displayed phase coexistence during the lamellar to H2 transition. The structural data also showed that the headgroups of the MMG analogues adopting the alternative lipid acid configuration were ordered and formed a two-dimensional molecular superlattice, which was conserved regardless of the lipid tail length. To our knowledge, the MMG analogues with an alternative lipid acid configuration represent the first example of a lipid system showing both interdigitation and superlattice formation, and as such could serve as an interesting model system for future studies. The MMG analogues are also relevant from a subunit vaccine perspective because they are well-tolerated and display

  9. WKYMVm-induced activation of formyl peptide receptor 2 stimulates ischemic neovasculogenesis by promoting homing of endothelial colony-forming cells.

    Science.gov (United States)

    Heo, Soon Chul; Kwon, Yang Woo; Jang, Il Ho; Jeong, Geun Ok; Yoon, Jung Won; Kim, Chi Dae; Kwon, Sang Mo; Bae, Yoe-Sik; Kim, Jae Ho

    2014-03-01

    Endothelial colony-forming cells (ECFCs) are recruited to the sites of ischemic injury in order to contribute to neovascularization and repair of injured tissues. However, therapeutic potential of ECFCs is limited due to low homing and engraftment efficiency of transplanted ECFCs. The G-protein-coupled formyl peptide receptor (FPR) 2 has been implicated in regulation of inflammation and angiogenesis, while the role of FPR2 in homing and engraftment of ECFCs and neovascularization in ischemic tissues has not been fully defined. This study was undertaken to investigate the effects of WKYMVm, a selective FPR2 agonist isolated by screening synthetic peptide libraries, on homing ability of ECFCs and vascular regeneration of ischemic tissues. WKYMVm stimulated chemotactic migration, angiogenesis, and proliferation ability of human ECFCs in vitro. Small interfering RNA-mediated silencing of FPR2, but not FPR3, abrogated WKYMVm-induced migration and angiogenesis of ECFCs. Intramuscular injection of WKYMVm resulted in attenuation of severe hind limb ischemia and promoted neovascularization in ischemic limb. ECFCs transplanted via tail vein into nude mice were incorporated into capillary vessels in the ischemic hind limb, resulting in augmented neovascularization and improved ischemic limb salvage. Intramuscular injection of WKYMVm promoted homing of exogenously administered ECFCs to the ischemic limb and ECFC-mediated vascular regeneration. Silencing of FPR2 expression in ECFCs resulted in abrogation of WKYMVm-induced in vivo homing of exogenously transplanted ECFCs to the ischemic limb, neovascularization, and ischemic limb salvage. These results suggest that WKYMVm promotes repair of ischemic tissues by stimulating homing of ECFCs and neovascularization via a FPR2-dependent mechanism. © AlphaMed Press.

  10. Insights from Synthetic Star-forming Regions - III. Calibration of Measurement Techniques of Star-formation Rates

    CERN Document Server

    Koepferl, Christine M; Dale, James E

    2016-01-01

    Through an extensive set of realistic synthetic observations (produced in Paper I), we assess in this part of the paper series (Paper III) how the choice of observational techniques affects the measurement of star-formation rates (SFRs) in star-forming regions. We test the accuracy of commonly used techniques and construct new methods to extract the SFR, so that these findings can be applied to measure the SFR in real regions throughout the Milky Way. We investigate diffuse infrared SFR tracers such as those using 24 {\\mu}m, 70 {\\mu}m and total infrared emission, which have been previously calibrated for global galaxy scales. We set up a toy model of a galaxy and show that the infrared emission is consistent with the intrinsic SFR using extra-galactic calibrated laws (although the consistency does not prove their reliability). For local scales, we show that these techniques produce completely unreliable results for single star-forming regions, which are governed by different characteristic timescales. We show...

  11. Chemical Methods for Peptide and Protein Production

    Directory of Open Access Journals (Sweden)

    Istvan Toth

    2013-04-01

    Full Text Available Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported a-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

  12. Skin scarification with Plasmodium falciparum peptide vaccine using synthetic TLR agonists as adjuvants elicits malaria sporozoite neutralizing immunity

    Science.gov (United States)

    Mitchell, Robert A.; Altszuler, Rita; Frevert, Ute; Nardin, Elizabeth H.

    2016-01-01

    Malaria eradication will require a combination of vector control, chemotherapy and an easily administered vaccine. Sterile immunity can be elicited in humans by immunization with sporozoites, the infective stage injected by bite of the mosquito vector, however, whole parasite vaccines present formidable logistical challenges for production, storage and administration. The “gold standard” for infectious disease eradiation, the Smallpox Eradication Programme, utilized mass immunization using the skin scarification (SS) route. SS may more closely mimic the natural route of malaria infection initiated by sporozoites injected by mosquito bite which elicits both neutralizing antibodies and protective cell mediated immunity. We investigated the potential of SS immunization using a malaria repeat peptide containing a protective B cell epitope of Plasmodium falciparum, the most lethal human species, and delivery vehicles containing TLR agonists as adjuvants. In a murine model, SS immunization with peptide in combination with TLR-7/8 and -9 agonists elicited high levels of systemic sporozoite neutralizing antibody, Th1- type CD4+ T cells and resistance to challenge by bites of infected mosquitoes. SS provides the potential to elicit humoral immunity to target Plasmodium at multiple stages of its complex life cycle. PMID:27624667

  13. Inhibition of human spermatozoa-zona pellucida binding by a combinatorially derived peptide from a synthetic target.

    Science.gov (United States)

    Pieczenik, George; Garrisi, John; Cohen, Jacques

    2006-09-01

    Intact zona-free human oocytes were screened using a combinatorial peptide library selection protocol. Pieczenik Peptide Sequence 1 (PPS1) HEHRKRG binds human spermatozoa. A complementary and unique binding sequence HNSSLSPLATPA (PPS2) was developed from the first PPS1 ligand that binds to the human zona pellucida or oolemma. Cytoplasm-free zonae from unfertilized eggs were obtained and used as an assay system to test the effects of exposure to these two ligands. Spermatozoa were inserted into evacuated zonae and their behaviour and binding activity were assessed at regular intervals. The behaviour of spermatozoa exposed to PPS1 and unlabelled spermatozoa injected into unexposed zonae was similar as far as binding was concerned (50 and 54% binding), but PPS1 exposed spermatozoa had higher motility and displacement, marked by their escape from the zona pellucida. Zonae exposed to PPS2 inhibited the interaction between injected spermatozoa and the inside of the zona when compared with controls (8.3 and 53.8% attached respectively, P movie sequence taken approximately 30 min after injection of spermatozoa into empty human zonae pellucidae shows behaviour of non-manipulated spermatozoa into zonae not exposed or exposed to ligand. This may be purchased for viewing on the Internet at www.rbmonline.com/Article/2159 (free to web subscribers).

  14. Conserved synthetic peptides from the hemagglutinin of influenza viruses induce broad humoral and T-cell responses in a pig model.

    Directory of Open Access Journals (Sweden)

    Júlia Vergara-Alert

    Full Text Available Outbreaks involving either H5N1 or H1N1 influenza viruses (IV have recently become an increasing threat to cause potential pandemics. Pigs have an important role in this aspect. As reflected in the 2009 human H1N1 pandemia, they may act as a vehicle for mixing and generating new assortments of viruses potentially pathogenic to animals and humans. Lack of universal vaccines against the highly variable influenza virus forces scientists to continuously design vaccines à la carte, which is an expensive and risky practice overall when dealing with virulent strains. Therefore, we focused our efforts on developing a broadly protective influenza vaccine based on the Informational Spectrum Method (ISM. This theoretical prediction allows the selection of highly conserved peptide sequences from within the hemagglutinin subunit 1 protein (HA1 from either H5 or H1 viruses which are located in the flanking region of the HA binding site and with the potential to elicit broader immune responses than conventional vaccines. Confirming the theoretical predictions, immunization of conventional farm pigs with the synthetic peptides induced humoral responses in every single pig. The fact that the induced antibodies were able to recognize in vitro heterologous influenza viruses such as the pandemic H1N1 virus (pH1N1, two swine influenza field isolates (SwH1N1 and SwH3N2 and a H5N1 highly pathogenic avian virus, confirm the broad recognition of the antibodies induced. Unexpectedly, all pigs also showed T-cell responses that not only recognized the specific peptides, but also the pH1N1 virus. Finally, a partial effect on the kinetics of virus clearance was observed after the intranasal infection with the pH1N1 virus, setting forth the groundwork for the design of peptide-based vaccines against influenza viruses. Further insights into the understanding of the mechanisms involved in the protection afforded will be necessary to optimize future vaccine formulations.

  15. Self-assembly of amyloid-forming peptides by molecular dynamics simulations.

    Science.gov (United States)

    Wei, Guanghong; Song, Wei; Derreumaux, Philippe; Mousseau, Normand

    2008-05-01

    Protein aggregation is associated with many neurodegenerative diseases. Understanding the aggregation mechanisms is a fundamental step in order to design rational drugs interfering with the toxic intermediates. This self-assembly process is however difficult to observe experimentally, which gives simulations an important role in resolving this problem. This study shows how we can proceed to gain knowledge about the first steps of aggregation. We first start by characterizing the free energy surface of the Abeta (16-22) dimer, a well-studied system numerically, using molecular dynamics simulations with OPEP coarse-grained force field. We then turn to the study of the NHVTLSQ peptide in 4-mers and 16-mers, extracting information on the onset of aggregation. In particular, the simulations indicate that the peptides are mostly random coil at room temperature, but can visit diverse amyloid-competent topologies, albeit with a low probability. The fact that the 16-mers constantly move from one structure to another is consistent with the long lag phase measured experimentally, but the rare critical steps leading to the rapid formation of amyloid fibrils still remain to be determined.

  16. Hepatitis C Virus Subtype 3a Envelope Protein 1 Binding with Human Leukocyte Antigen Class I Types of Pakistani Population: Candidate Epitopes for Synthetic Peptide Vaccine

    Directory of Open Access Journals (Sweden)

    Hamid Nawaz-Tipu

    2015-10-01

    Full Text Available The object of this cross sectional study was to determine the HCV subtype 3a envelope protein binding affinity with Human Leukocyte Antigen. Envelope 1 (E1 protein is one of the structural proteins responsible for entering the cells through the receptors. The binding affinity of E1 protein epitopes to the selected Human Leukocyte Antigen (HLA class I alleles was investigated using the computer-based tools. These prediction tools were also used to design the synthetic vaccine’s candidate epitopes and to identify the individuals/populations who are likely to be responder to those vaccines.The mean frequency of HLA I antigens in Pakistani population was calculated. Threealleles each from HLA A and B were selected. E1 protein sequence extracted from HCV 3a isolates was retrieved and twenty-four sequences of it were selected. NetMHCcons 1.0 server was used to determine the binding affinities of HLA alleles to the epitope sequences of 10 amino acids in length.A02, A03, A11, A24, A33, B08, B13, B15, B35 and B40 were the first five antigens moreprevalent in Pakistan each from HLA A and HLA B.. We did not find any binding affinity between HLA A*201, B*1501 and B*4001 and epitopes from E1 sequences in a threshold of50 nM. Totally five various epitopes derived from different isolates were characterized.The prediction of HLA-E1 epitope specific bindings and the forthcoming response can be a useful bioinformatics tool to uncover the right synthetic peptides for vaccine design purposes.

  17. Hepatitis C Virus Subtype 3a Envelope Protein 1 Binding with Human Leukocyte Antigen Class I Types of Pakistani Population: Candidate Epitopes for Synthetic Peptide Vaccine.

    Science.gov (United States)

    Nawaz-Tipu, Hamid

    2015-10-01

    The object of this cross sectional study was to determine the HCV subtype 3a envelope protein binding affinity with Human Leukocyte Antigen. Envelope 1 (E1) protein is one of the structural proteins responsible for entering the cells through the receptors. The binding affinity of E1 protein epitopes to the selected Human Leukocyte Antigen (HLA) class I alleles was investigated using the computer-based tools. These prediction tools were also used to design the synthetic vaccine's candidate epitopes and to identify the individuals/populations who are likely to be responder to those vaccines.The mean frequency of HLA I antigens in Pakistani population was calculated. Three alleles each from HLA A and B were selected. E1 protein sequence extracted from HCV 3a isolates was retrieved and twenty-four sequences of it were selected. NetMHCcons 1.0 server was used to determine the binding affinities of HLA alleles to the epitope sequences of 10 amino acids in length.A02, A03, A11, A24, A33, B08, B13, B15, B35 and B40 were the first five antigens more prevalent in Pakistan each from HLA A and HLA B.. We did not find any binding affinity between HLA A*201, B*1501 and B*4001 and epitopes from E1 sequences in a threshold of 50 nM. Totally five various epitopes derived from different isolates were characterized.The prediction of HLA-E1 epitope specific bindings and the forthcoming response can be a useful bioinformatics tool to uncover the right synthetic peptides for vaccine design purposes.

  18. Preparation and characterization of polysulfone affinity membranes bearing a synthetic peptide ligand for the separation of murine immunoglobulins.

    Science.gov (United States)

    Boi, Cristiana; Algeri, Cristian; Sarti, Giulio C

    2008-01-01

    Affinity membranes have been prepared by immobilizing D-PAM, a synthetic ligand that exhibits affinity for the Fc portion of antibodies, onto poliethersulfone microporous membranes. The ligand density has been measured and the ligand utilization was evaluated and compared with literature data available for chromatographic beads. The resulting new affinity membranes have been experimentally characterized and tested by using pure murine IgG solutions and mouse serum. Equilibrium and kinetic parameters have been obtained in batch experiments using pure protein solutions. The highest binding capacity measured for murine IgG was 45 microg/cm(2) obtained at 1.2 mg/mL protein concentration at equilibrium, while the maximum static binding capacity calculated with the Langmuir model was 81 microg/cm(2). The adsorption of murine IgG on the affinity membranes was described using different isotherms: Freundlich and Temkin models have been considered and critically compared with the Langmuir adsorption model. A dynamic binding capacity of 21 microg/cm(2) was obtained by feeding a solution of 0.3 mg/mL of murine IgG, confirming the results obtained in batch experiments at the same concentration. The affinity membranes considered are endowed with good binding capacity for murine IgG and good selectivity for immunoglobulins and can be considered for the capturing step of an antibody production process.

  19. Determination of Free-Form and Peptide Bound Pyrraline in the Commercial Drinks Enriched with Different Protein Hydrolysates

    Directory of Open Access Journals (Sweden)

    Zhili Liang

    2016-07-01

    Full Text Available Pyrraline, a causative factor for the recent epidemics of diabetes and cardiovascular disease, is also employed as an indicator to evaluate heat damage and formation of advanced glycation end-products (AGEs in foods. Peptide-enriched drinks (PEDs are broadly consumed worldwide due to rapid rate of absorption and perceived health effects. It can be hypothesized that PED is an important source of pyrraline, especially peptide bound pyrraline (Pep-Pyr. In this study we determined free-form pyrraline (Free-Pyr and Pep-Pyr in drinks enriched with whey protein hydrolysate (WPH, soy protein hydrolysate (SPH and collagen protein hydrolysate (CPH. A detection method was developed using ultrahigh-performance liquid chromatography with UV-visible detector coupled with tandem mass spectrometry after solid-phase extraction (SPE. The SPE led to excellent recovery rates ranging between 93.2% and 98.5% and a high reproducibility with relative standard deviations (RSD of <5%. The limits of detection and quantification obtained were 30.4 and 70.3 ng/mL, respectively. Pep-Pyr was identified as the most abundant form (above 96 percent of total pyrraline, whereas Free-Pyr was present in a small proportion (less than four percent of total pyrraline. The results indicate that PED is an important extrinsic source of pyrraline, especially Pep-Pyr. As compared with CPH- and SPH-enriched drinks, WPH-enriched drinks contained high content of Pep-Pyr. The Pep-Pyr content is associated with the distribution of peptide lengths and the amino acid compositions of protein in PEDs.

  20. A synthetic antimicrobial peptide BTD-S expressed in Arabidopsis thaliana confers enhanced resistance to Verticillium dahliae.

    Science.gov (United States)

    Li, Feng; Shen, Hao; Wang, Ming; Fan, Kai; Bibi, Noreen; Ni, Mi; Yuan, Shuna; Wang, Xuede

    2016-08-01

    BTD-S is a synthetic non-cyclic θ-defensin derivative which was previously designed in our laboratory based on baboon θ-defensins (BTDs). It shows robust antimicrobial activity against economically important phytopathogen, Verticillium dahliae. Here, we deduced the coding nucleotide sequence of BTD-S and introduced the gene into wild-type (ecotype Columbia-0) Arabidopsis thaliana plants. Results demonstrated that BTD-S-transgenic lines displayed in bioassays inhibitory effects on the growth of V. dahliae in vivo and in vitro. Based on symptom severity, enhanced resistance was found in a survey of BTD-S-transgenic lines. Besides, crude protein extracts from root tissues of BTD-S-transformed plants significantly restricted the growth of fungal hyphae and the germination of conidia. Also, fungal biomass over time determined by real-time PCR demonstrated the overgrowth of V. dahliae in wild-type plants 2-3 weeks after inoculation, while almost no fungal DNA was detected in aerial tissues of their transgenic progenitors. The result suggested that fungus failed to invade and progress acropetally up to establish a systemic infection in BTD-S-transgenic plants. Moreover, the assessment of basal defense responses was performed in the leaves of WT and BTD-S-transgenic plants. The mitigated oxidative stress and low antioxidase level in BTD-S-transgenic plants revealed that BTD-S acts via permeabilizing target microbial membranes, which is in a category different from hypersensitive response-dependent defense. Taken together, our results demonstrate that BTD-S is a promising gene to be explored for transgenic engineering for plant protection against Verticillium wilt.

  1. Using Triple Helix Forming Peptide Nucleic Acids for Sequence-selective Recognition of Double-stranded RNA

    Science.gov (United States)

    Hnedzko, Dziyana; Cheruiyot, Samwel K.; Rozners, Eriks

    2014-01-01

    Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple helix forming peptide nucleic acids (PNAs) that bind in the major grove of RNA helix. The strategy developed uses chemically modified nucleobases, such as 2-aminopyridine (M) that enables strong triple helical binding at physiologically relevant conditions, and 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E) that enable recognition of isolated pyrimidines in the purine rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid-phase synthesis and HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included. PMID:25199637

  2. EFFECT OF NATURAL AND SYNTHETIC POLYMER ON RELEASE OF KETOTIFEN FUMARATE MATRIX TABLETS: A SUSTAINED RELEASE DOSAGE FORM

    Directory of Open Access Journals (Sweden)

    Md. M. Rahman*, A. B. Ripon Khalipha , Md. A. K. Azad , MD. Z. Faruki , A. K. Chaurasiya and H. Hossain

    2013-04-01

    Full Text Available ABSTRACT: With the blend of Methocel K15, a synthetic polymer and xanthan gum, a natural polymer (3:1 was used in the formulation of matrix tablets to find out the effect of natural polymer in the sustained release dosage form. Direct compression process was applied for the preparation of Ketotifen fumarate tablets. The dissolution profiles were carried out by USP apparatus 2 (paddle at 50 rpm in 500 ml 0.1 N HCl and distilled water. For interpreting the results a one way analysis of variance (ANOVA was exploited. Statistically significant differences were found among the drug release profile from different matrices. At a higher polymeric content (60% of the total tablet weight, drug release from the combination of Methocel K15M and xanthan gum (3:1 was slower. On the contrary, at a lower polymeric level (30% of the total tablet weight; the rate of drug release was prominent. The best-fit release kinetics was accomplished with the Higuchi model followed by the zero-order plot, Korsmeyer and Hixson Crowell equations. One formulation showed drug release is more controlled. The data obtained proved that the formulations are useful for a sustained release of ketotifen fumarate. From these formulations corresponded more controlled of the drug release by the higher polymeric level of methocel K15M & xanthan gum and vice versa. The extended release of the model drug found from the higher proportion of methocel K15M and xanthan gum. As a result, the frequency of administration of such type of drug reduced.

  3. [EFFECT OF SYNTHETIC PEPTIDES ON AGING OF PATIENTS WITH CHRONIC POLYMORBIDITY AND ORGANIC BRAIN SYNDROME OF THE CENTRAL NERVOUS SYSTEM IN REMISSION].

    Science.gov (United States)

    Meshchaninov, V N; Tkachenko, E L; Zharkov, S V; Gavrilov, I V; Katyreva, Iu E

    2015-01-01

    We've estimated the cellular and metabolic part of geroprophylactic effects of short synthetic tripeptides vesugen and pinealon for correction of the biological age. 32 people (18 men, 12 women) aged 41-83 years with polymorbidity and the organic brain syndrome in remission participated in the study. The preparations of "Pinealon" and "Vesugen" have had the significant anabolic effect. They have improved the activity of the Central nervous system and other vital organs, which slows the rate of aging by biological age indicators. Vesugen has demonstrated more visible geroprophylactic effect than Pinealon. At the same time we've found the prooxidant activity through chemiluminescence. Decrease of markers CD34+ positive hematopoietic polypotent cells in blood has shown significant inhibition of hemopoiesis. Apparently, the cells have not been involved in the adaptive reactions. Pinealon and Vesugen haven't affected the degree of chromatin condensation, so they are safe on nuclear genetic level. This property should be studied in future. In geriatric practice, we recommend to apply the peptides Pinealon and Vesugen as geroprotectors anabolic neuroprotective and no antioxidant type for reducing the rate of aging in patients with the organic brain syndrome vascular and/or traumatic genesis.

  4. Ir-Uv Double Resonance Spectroscopy of a Cold Protonated Fibril-Forming Peptide: NNQQNY\\cdotH+

    Science.gov (United States)

    DeBlase, Andrew F.; Harrilal, Christopher P.; Walsh, Patrick S.; McLuckey, Scott A.; Zwier, Timothy S.

    2016-06-01

    Protein aggregation to form amyloid-like fibrils is a purported molecular manifestation that leads to Alzheimer's, Huntington's, and other neurodegenerative diseases. The propensity for a protein to aggregate is often driven by the presence of glutamine (Q) and asparagine (N) rich tracts within the primary sequence. For example, Eisenberg and coworkers [Nature 2006, 435, 773] have shown by X-ray crystallography that the peptides NNQQNY and GNNQQNY aggregate into a parallel β-sheet configuration with side chains that intercalate into a "steric zipper". These sequences are commonly found at the N-terminus of the prion-determining domain in the yeast protein Sup35, a typical fibril-forming protein. Herein, we invoke recent advances in cold ion spectroscopy to explore the nascent conformational preferences of the protonated peptides that are generated by electrospray ionization. Towards this aim, we have used UV and IR spectroscopy to record conformation-specific photofragment action spectra of the NNQQNY monomer cryogenically cooled in an octopole ion trap. This short peptide contains 20 hydride stretch oscillators, leading to a rich infrared spectrum with at least 18 resolved transitions in the 2800-3800 cm-1 region. The infrared spectrum suggests the presence of both a free acid OH moiety and an H-bonded tyrosine OH group. We compare our results with resonant ion dip infrared spectra (RIDIRS) of the acyl/NH-benzyl capped neutral glutamine amino acid and its corresponding dipeptide: Ac-Q-NHBn and Ac-QQ-NHBn, respectively. These comparisons bring empirical insight to the NH stretching region of the spectrum, which contains contributions from free and singly H-bonded NH2 side-chain groups, and from peptide backbone amide NH groups. We further compare our spectrum to harmonic calculations at the M05-2X/6-31+G* level of theory, which were performed on low energy structures obtained from Monte Carlo conformational searches using the Amber* and OPLS force fields to assess

  5. Constitutively active form of natriuretic peptide receptor 2 ameliorates experimental pulmonary arterial hypertension.

    Science.gov (United States)

    Nawa, Nobutoshi; Ishida, Hidekazu; Katsuragi, Shinichi; Baden, Hiroki; Takahashi, Kunihiko; Higeno, Ryota; Torigoe, Fumiko; Mihara, Seiko; Narita, Jun; Miura, Kohji; Nakamura, Kazufumi; Kogaki, Shigetoyo; Ozono, Keiichi

    2016-01-01

    We recently found a constitutively active mutant of natriuretic peptide receptor 2 (caNPR2; V883M), which synthesizes larger amounts of cyclic guanosine monophosphate (cGMP) intracellularly without any ligand stimulation than existing drugs. The aim of this study was to investigate the therapeutic effects of gene transduction using caNPR2 for pulmonary arterial hypertension (PAH). In vitro gene transduction into human pulmonary arterial smooth muscle cells using Sendai virus (SeV) vectors carrying caNPR2 induced 10,000-fold increases in the synthesis of cGMP without ligand stimulation, and the proliferation of caNPR2-expressing cells was significantly attenuated. The PAH model rats generated by hypoxia and the administration of SU5416 were then treated with SeV vectors through a direct injection into the left pulmonary artery. Right ventricular systolic pressure was significantly decreased 2 weeks after the treatment, while systemic blood pressure remained unchanged. Histological analyses revealed that the medial wall thickness and occlusion rate of pulmonary arterioles were significantly improved in caNPR2-treated lungs. Neither the systemic integration of virus vectors nor side effects were observed. The massive stimulation of cGMP synthesis by gene therapy with caNPR2 was safe and effective in a PAH rat model and, thus, has potential as a novel therapy for patients with severe progressive PAH.

  6. Marine sponge cyclic peptide theonellamide A disrupts lipid bilayer integrity without forming distinct membrane pores.

    Science.gov (United States)

    Espiritu, Rafael Atillo; Cornelio, Kimberly; Kinoshita, Masanao; Matsumori, Nobuaki; Murata, Michio; Nishimura, Shinichi; Kakeya, Hideaki; Yoshida, Minoru; Matsunaga, Shigeki

    2016-06-01

    Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides derived from the marine sponge Theonella sp. These peptides specifically bind to 3β-hydroxysterols, resulting in 1,3-β-D-glucan overproduction and membrane damage in yeasts. The inclusion of cholesterol or ergosterol in phosphatidylcholine membranes significantly enhanced the membrane affinity of theonellamide A (TNM-A) because of its direct interaction with 3β-hydroxyl groups of sterols. To better understand TNM-induced membrane alterations, we investigated the effects of TNM-A on liposome morphology. (31)P nuclear magnetic resonance (NMR) and dynamic light scattering (DLS) measurements revealed that the premixing of TNM-A with lipids induced smaller vesicle formation. When giant unilamellar vesicles were incubated with exogenously added TNM-A, confocal micrographs showed dynamic changes in membrane morphology, which were more frequently observed in cholesterol-containing than sterol-free liposomes. In conjunction with our previous data, these results suggest that the membrane action of TNM-A proceeds in two steps: 1) TNM-A binds to the membrane surface through direct interaction with sterols and 2) accumulated TNM-A modifies the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption.

  7. Detection of purine cytosine permease of S. cerevisiae: use of antibodies against a synthetic peptide corresponding to a predicted sequence in the N-terminal domain of the protein.

    Science.gov (United States)

    Grandier-Vazeille, X; Neaud, V; Geoffre, S

    1993-12-15

    A synthetic peptide, selected in the predicted N-terminal amino-acid sequence of the purine cytosine permease (gene FCY2), linked to albumins proved a remarkably good immunogen in rabbits. In ELISA, sera reacted with the synthetic peptide and with specific proteins of plasma-membrane-enriched fractions of mutant Saccharomyces cerevisiae pAB strains (amplified FCY2 gene) with high titers and high avidity. Western blots of plasma membrane proteins of pAB strain probed with antisera showed two bands: a major (45 kDa) and minor band (50 kDa). On the contrary, plasma-membrane-enriched fractions of mutant S. cerevisiae pJDB strain (deficient in FCY2 gene) gave no signal when probed in the same conditions. These results demonstrate the specificity of the antisera and also suggest that the 45 kDa and 50 kDa proteins are both products of the FCY2 gene.

  8. Dengue fever virus and Japanese encephalitis virus synthetic peptides, with motifs to fit HLA class I haplotypes prevalent in human populations in endemic regions, can be used for application to skin Langerhans cells to prime antiviral CD8+ cytotoxic T cells (CTLs)--a novel approach to the protection of humans.

    Science.gov (United States)

    Becker, Y

    1994-09-01

    Flaviviruses were reported to induce CD8+ cytotoxic T cells in infected individuals, indicating that nonapeptides, proteolytic cleavage products of the viral precursor protein, enter the endoplasmic reticulum in infected cells and interact with HLA class I molecules. The assembled HLA class I molecules are transported to the plasma membrane and prime CD8+ T cells. Current knowledge of the interaction of viral peptides with HLA molecules is reviewed. Based on this review, an idea is presented to use synthetic flavivirus peptides with an amino acid motif to fit with the HLA class I peptide binding group of HLA haplotypes prevalent in a given population in an endemic area. These synthetic viral peptides may be introduced into the human skin using a lotion containing the peptides ("Peplotion") together with substances capable of enhancing the penetration of these peptides into the skin to reach Langerhans cells. The peptide-treated Langerhans cells, professional antigen-presenting cells, may bind the synthetic viral peptides by their HLA class I peptide-binding grooves. Antigens carrying Langerhans cells are able to migrate and induce the cellular immune response in the lymph nodes. This approach to the priming of antiviral CD8+ cytotoxic T cells may provide cellular immune protection from flavivirus infection without inducing the humoral immune response, which can lead to the shock syndrome in Dengue fever patients. To be able to develop anti-Dengue virus synthetic peptides for populations with different HLA class I haplotypes, it is necessary to develop computational studies to design HLA class I Dengue virus synthetic peptides with motifs to fit the HLA haplotypes of the population living in an endemic region for Dengue fever. Experiments to study Dengue virus and Japanese encephalitis peptides vaccines and their effectiveness in protection against Dengue fever and Japanese encephalitis are needed. The development of human antiviral vaccines for application of viral

  9. Rabbit IgG directed to a synthetic C-terminal peptide of the major grass pollen allergen Lol p I inhibits human basophil histamine release induced by natural Lol p I.

    Science.gov (United States)

    van Ree, R; Aalberse, R C

    1995-03-01

    The potential role of allergen-specific IgG antibodies as 'blocking' antibodies in allergen-induced human basophil histamine release was investigated. This was studied in a model with the major grass pollen allergen Lol p I and polyclonal rabbit antisera directed against this allergen and against a synthetic peptide of its C terminus. When allergen and antibodies were allowed to preincubate, Lol p I induced histamine release was inhibited up to 85% by the antiserum against Lol p I. By omitting preincubation, and thereby more closely mimicking an in vivo situation, up to 55% inhibition was realized. This indicates that allergen-specific IgG can act as 'blocking' antibody without preincubation. Immunization of rabbits with a synthetic C-terminal peptide of Lol p I resulted in antibodies reactive with natural Lol p I. Despite their 100-fold lower avidity for Lol p I (as compared with antinatural Lol p I), these antibodies had the capacity to inhibit Lol p I induced histamine release for > 90% (up to 50% without preincubation). This indicates that it is possible to block histamine release induced by a major allergen with low-avidity IgG antibodies directed against a minor proportion of the allergen (25 amino acids). IgE antibodies from the donors studied were unreactive with this synthetic peptide, indicating that for blocking activity identical epitope specificity of IgE and IgG is not essential. This opens interesting perspectives for application of synthetic peptides in immunotherapy, distinct from their effects on T cell reactivity.

  10. Tapasin discriminates peptide-human leukocyte antigen-A*02:01 complexes formed with natural ligands

    DEFF Research Database (Denmark)

    Røder, Gustav Andreas; Geironson, Linda; Rasmussen, Michael

    2011-01-01

    A plethora of peptides are generated intracellularly, and most peptide-human leukocyte antigen (HLA)-I interactions are of a transient, unproductive nature. Without a quality control mechanism, the HLA-I system would be stressed by futile attempts to present peptides not sufficient for the stable...... according to the identity of the peptide. The facilitation was also specific for the identity of the HLA-I heavy chain, where it correlated to established tapasin dependence hierarchies. Two large sets of HLA-A*02:01 binding peptides, one extracted from natural HLA-I ligands from the SYFPEITHI database...... functionally discriminate the selected SYFPEITHI peptides from the other peptide binders with high sensitivity and specificity. We suggest that this HLA-I- and peptide-specific function, together with the functions exerted by the more C-terminal parts of tapasin, are major features of tapasin-mediated HLA...

  11. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  12. Coupling Optical and Electrical Measurements in Artificial Membranes: Lateral Diffusion of Lipids and Channel Forming Peptides in Planar Bilayers

    Directory of Open Access Journals (Sweden)

    Duclohier H

    1998-01-01

    Full Text Available Planar lipid bilayers (PLB were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc., presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K+ channel was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

  13. 合成肽抗原在戊型肝炎病毒感染诊断中的应用%Serodiagnosis of Hepatitis E Virus Infection by ELISA Based on Synthetic Peptide Antigens

    Institute of Scientific and Technical Information of China (English)

    周小林; 刘云霞; 崔梅萍; 谷娟娟; 张伟; 孙兰英

    2001-01-01

    An ELISA for the detection of anti-HEV using synthetic peptideantigens was developed. The synthetic antigens were encoded by OFR2 and OFR3 genes of HEV. The purpose of this study was to determine the applicability of the synthetic antigens in the serodiagnosis of hepatitis E. The anti-HEV detection using synthetic antigens was carried out in 47 healthy subjects and 89 patients with acute or chronic viral hepatitis. The results showed that the positive rate of anti-HEV-IgG in healthy subjects was 4.2%(2/47), and no IgM antibody to HEV was found. The positive rates of IgG and IgM antibodies to HEV in the hepatitis patients were 8.9% and 10% respectively. In addition, we compared the detecting efficacy of the synthetic antigens with that of the market reagent in 57 serum samples, the total coincident rate was 87.7% (50/57). All of the results accorded with the literatures reported. This study suggests that the ELISA based on the synthetic peptide antigens was specific, sensitive and convenient in diagnosis of HEV infection, it can be widely used in both clinical and epidemiological reseaches.

  14. Effect of syntheticpeptide oligomers and fluorinated solvents on Kv1.3 channel properties and membrane conductance.

    Directory of Open Access Journals (Sweden)

    Maria I Lioudyno

    Full Text Available The impact of synthetic amyloid β (1-42 (Aβ(1-42 oligomers on biophysical properties of voltage-gated potassium channels Kv 1.3 and lipid bilayer membranes (BLMs was quantified for protocols using hexafluoroisopropanol (HFIP or sodium hydroxide (NaOH as solvents prior to initiating the oligomer formation. Regardless of the solvent used Aβ(1-42 samples contained oligomers that reacted with the conformation-specific antibodies A11 and OC and had similar size distributions as determined by dynamic light scattering. Patch-clamp recordings of the potassium currents showed that synthetic Aβ(1-42 oligomers accelerate the activation and inactivation kinetics of Kv 1.3 current with no significant effect on current amplitude. In contrast to oligomeric samples, freshly prepared, presumably monomeric, Aβ(1-42 solutions had no effect on Kv 1.3 channel properties. Aβ(1-42 oligomers had no effect on the steady-state current (at -80 mV recorded from Kv 1.3-expressing cells but increased the conductance of artificial BLMs in a dose-dependent fashion. Formation of amyloid channels, however, was not observed due to conditions of the experiments. To exclude the effects of HFIP (used to dissolve lyophilized Aβ(1-42 peptide, and trifluoroacetic acid (TFA (used during Aβ(1-42 synthesis, we determined concentrations of these fluorinated compounds in the stock Aβ(1-42 solutions by (19F NMR. After extensive evaporation, the concentration of HFIP in the 100× stock Aβ(1-42 solutions was ∼1.7 μM. The concentration of residual TFA in the 70× stock Aβ(1-42 solutions was ∼20 μM. Even at the stock concentrations neither HFIP nor TFA alone had any effect on potassium currents or BLMs. The Aβ(1-42 oligomers prepared with HFIP as solvent, however, were more potent in the electrophysiological tests, suggesting that fluorinated compounds, such as HFIP or structurally-related inhalational anesthetics, may affect Aβ(1-42 aggregation and potentially enhance ability

  15. Effect of syntheticpeptide oligomers and fluorinated solvents on Kv1.3 channel properties and membrane conductance.

    Science.gov (United States)

    Lioudyno, Maria I; Broccio, Matteo; Sokolov, Yuri; Rasool, Suhail; Wu, Jessica; Alkire, Michael T; Liu, Virginia; Kozak, J Ashot; Dennison, Philip R; Glabe, Charles G; Lösche, Mathias; Hall, James E

    2012-01-01

    The impact of synthetic amyloid β (1-42) (Aβ(1-42)) oligomers on biophysical properties of voltage-gated potassium channels Kv 1.3 and lipid bilayer membranes (BLMs) was quantified for protocols using hexafluoroisopropanol (HFIP) or sodium hydroxide (NaOH) as solvents prior to initiating the oligomer formation. Regardless of the solvent used Aβ(1-42) samples contained oligomers that reacted with the conformation-specific antibodies A11 and OC and had similar size distributions as determined by dynamic light scattering. Patch-clamp recordings of the potassium currents showed that synthetic Aβ(1-42) oligomers accelerate the activation and inactivation kinetics of Kv 1.3 current with no significant effect on current amplitude. In contrast to oligomeric samples, freshly prepared, presumably monomeric, Aβ(1-42) solutions had no effect on Kv 1.3 channel properties. Aβ(1-42) oligomers had no effect on the steady-state current (at -80 mV) recorded from Kv 1.3-expressing cells but increased the conductance of artificial BLMs in a dose-dependent fashion. Formation of amyloid channels, however, was not observed due to conditions of the experiments. To exclude the effects of HFIP (used to dissolve lyophilized Aβ(1-42) peptide), and trifluoroacetic acid (TFA) (used during Aβ(1-42) synthesis), we determined concentrations of these fluorinated compounds in the stock Aβ(1-42) solutions by (19)F NMR. After extensive evaporation, the concentration of HFIP in the 100× stock Aβ(1-42) solutions was ∼1.7 μM. The concentration of residual TFA in the 70× stock Aβ(1-42) solutions was ∼20 μM. Even at the stock concentrations neither HFIP nor TFA alone had any effect on potassium currents or BLMs. The Aβ(1-42) oligomers prepared with HFIP as solvent, however, were more potent in the electrophysiological tests, suggesting that fluorinated compounds, such as HFIP or structurally-related inhalational anesthetics, may affect Aβ(1-42) aggregation and potentially enhance

  16. Creating functional peptide architectures at interfaces

    Science.gov (United States)

    Tirrell, Matthew

    2001-03-01

    Short peptide sequences, derived from whole proteins, can be useful synthetic agents for conferring a specific biological function to a material surface. Their ability to do this depends on delivering them to the surface in a biologically recognizable form, that is in a spatial configuration that is not too different from that adopted by the peptide in the whole protein. Most functional proteins have secondary and tertiary levels of structure that are essential to their activities; peptides have simpler but no less important structures. In our work, we have focussed on peptides derived from extracellular matrix proteins. We have found that attaching synthetic lipid tails to peptides fragments gives them two very useful properties for surface modification. The hydrophobic tails give rise to a self-assembly capacity enabling these molecules to organize into membrane, monolayer and bilayer structures. Less expected is that this level of self-assembly induces a second level in the peptide headgroup. Peptides from alpha-helical and triple-helical regions of protein are induced by the lipid tails to form protein-like secondary structures and therefore to have more effective biological activity.

  17. Polymorphism of amyloid fibrils formed by a peptide from the yeast prion protein Sup35: AFM and Tip-Enhanced Raman Scattering studies

    Energy Technology Data Exchange (ETDEWEB)

    Krasnoslobodtsev, Alexey V., E-mail: akrasnos@unomaha.edu [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States); Department of Physics, University of Nebraska Omaha, Omaha, NE 68182 (United States); Deckert-Gaudig, Tanja [IPHT-Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Zhang, Yuliang [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States); Deckert, Volker [IPHT-Leibniz Institute of Photonic Technology, Albert-Einstein-Str. 9, D-07745 Jena (Germany); Institute for Physical Chemistry and Abbe Center of Photonics, University of Jena, Helmholtzweg 4, D-07743 Jena (Germany); Lyubchenko, Yuri L., E-mail: ylyubchenko@unmc.edu [Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198 (United States)

    2016-06-15

    Aggregation of prion proteins is the cause of various prion related diseases. The infectious form of prions, amyloid aggregates, exist as multiple strains. The strains are thought to represent structurally different prion protein molecules packed into amyloid aggregates, but the knowledge on the structure of different types of aggregates is limited. Here we report on the use of AFM (Atomic Force Microscopy) and TERS (Tip-Enhanced Raman Scattering) to study morphological heterogeneity and access underlying conformational features of individual amyloid aggregates. Using AFM we identified the morphology of amyloid fibrils formed by the peptide (CGNNQQNY) from the yeast prion protein Sup35 that is critically involved in the aggregation of the full protein. TERS results demonstrate that morphologically different amyloid fibrils are composed of a distinct set of conformations. Fibrils formed at pH 5.6 are composed of a mixture of peptide conformations (β-sheets, random coil and α-helix) while fibrils formed in pH~2 solution primarily have β-sheets. Additionally, peak positions in the amide III region of the TERS spectra suggested that peptides have parallel arrangement of β-sheets for pH~2 fibrils and antiparallel arrangement for fibrils formed at pH 5.6. We also developed a methodology for detailed analysis of the peptide secondary structure by correlating intensity changes of Raman bands in different regions of TERS spectra. Such correlation established that structural composition of peptides is highly localized with large contribution of unordered secondary structures on a fibrillar surface. - Highlights: • Amyloid polymorphs were characterized by AFM and TERS. • A mixture of peptide secondary structures in fibrils were identified using TERS. • TERS recognizes packing arrangement (parallel versus antiparallel) of peptides. • TERS is a powerful tool for high resolution structural analysis of fibrils.

  18. A placebo-controlled randomized HPV16 synthetic long-peptide vaccination study in women with high-grade cervical squamous intraepithelial lesions.

    Science.gov (United States)

    de Vos van Steenwijk, Peggy J; Ramwadhdoebe, Tamara H; Löwik, Margriet J G; van der Minne, Caroline E; Berends-van der Meer, Dorien M A; Fathers, Lorraine M; Valentijn, A Rob P M; Oostendorp, Jaap; Fleuren, Gert Jan; Hellebrekers, Bart W J; Welters, Marij J P; van Poelgeest, Mariette I; Melief, Cornelis J M; Kenter, Gemma G; van der Burg, Sjoerd H

    2012-09-01

    The aim of this study was to investigate the capacity of an HPV16 E6/E7 synthetic overlapping long-peptide vaccine to stimulate the HPV16-specific T-cell response, to enhance the infiltration of HPV16-specific type 1 T cells into the lesions of patients with HPV16+ high-grade cervical squamous intraepithelial lesion (HSIL) and HPV clearance. This was a placebo-controlled randomized phase II study in patients with HPV16-positive HSIL. HPV16-specific T-cell responses were determined pre- and post-vaccination by ELISPOT, proliferation assay and cytokine assays in PBMC and HSIL-infiltrating lymphocytes, and delayed-type hypersensitivity skin tests. Motivational problems of this patient group to postpone treatment of their premalignant lesions affected the inclusion rates and caused the study to stop prematurely. Of the accrued patients, 4 received a placebo and 5 received 1-2 vaccinations. Side effects mainly were flu-like symptoms and injection site reactions. A strong HPV-specific IFNγ-associated T-cell response was detected by ELISPOT in all vaccinated patients. The outcome of the skin tests correlated well with the ELISPOT analysis. The cytokine profile associated with HPV16-specific proliferation varied from robust type 1 to dominant type 2 responses. No conclusions could be drawn on vaccine-enhanced T-cell infiltration of the lesion, and there was no HPV clearance at the time of LEEP excision. Thus, vaccination of HSIL patients results in increased HPV16-specific T-cell immunity. Further development of this type of treatment relies on the ability to motivate patients and in the reduction in the side effects.

  19. Alzheimer amyloid beta-peptide A-beta25-35 blocks adenylate cyclase-mediated forms of hippocampal long-term potentiation.

    Science.gov (United States)

    Bisel, Blaine E; Henkins, Kristen M; Parfitt, Karen D

    2007-02-01

    Progressive memory loss and deposition of amyloid beta (Abeta) peptides throughout cortical regions are hallmarks of Alzheimer's disease (AD). Several studies in mice and rats have shown that overexpression of amyloid precursor protein (APP) or pretreatment with Abeta peptide fragments results in the inhibition of hippocampal long-term potentiation (LTP) as well as impairments in learning and memory of hippocampal-dependent tasks. For these studies we have investigated the effects of the Abeta(25-35) peptide fragment on LTP induced by adenylate cyclase stimulation followed immediately by application of Mg(++)-free aCSF ("chemLTP"). Treatment of young adult slices with the Abeta(25-35) peptide had no significant effect on basal synaptic transmission in area CA1, but treatment with the peptide for 20 min before inducing chemLTP with isoproterenol (ISO; 1 microM) or forskolin (FSK;10 microM) + Mg(++)-free aCSF resulted in complete blockade of LTP. In contrast, normal ISO-chemLTP was observed after treatment with the control peptide Abeta(35-25). The ability of the Abeta(25-35) peptide fragment to block this and other forms of synaptic plasticity may help elucidate the mechanisms underlying hippocampal deficits observed in animal models of AD and/or AD individuals.

  20. Programmed Nanomaterial Assemblies in Large Scales: Applications of Synthetic and Genetically- Engineered Peptides to Bridge Nano-Assemblies and Macro-Assemblies

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Hiroshi

    2014-09-09

    Work is reported in these areas: Large-scale & reconfigurable 3D structures of precise nanoparticle assemblies in self-assembled collagen peptide grids; Binary QD-Au NP 3D superlattices assembled with collagen-like peptides and energy transfer between QD and Au NP in 3D peptide frameworks; Catalytic peptides discovered by new hydrogel-based combinatorial phage display approach and their enzyme-mimicking 2D assembly; New autonomous motors of metal-organic frameworks (MOFs) powered by reorganization of self-assembled peptides at interfaces; Biomimetic assembly of proteins into microcapsules on oil-in-water droplets with structural reinforcement via biomolecular recognition-based cross-linking of surface peptides; and Biomimetic fabrication of strong freestanding genetically-engineered collagen peptide films reinforced by quantum dot joints. We gained the broad knowledge about biomimetic material assembly from nanoscale to microscale ranges by coassembling peptides and NPs via biomolecular recognition. We discovered: Genetically-engineered collagen-like peptides can be self-assembled with Au NPs to generate 3D superlattices in large volumes (> μm{sup 3}); The assembly of the 3D peptide-Au NP superstructures is dynamic and the interparticle distance changes with assembly time as the reconfiguration of structure is triggered by pH change; QDs/NPs can be assembled with the peptide frameworks to generate 3D superlattices and these QDs/NPs can be electronically coupled for the efficient energy transfer; The controlled assembly of catalytic peptides mimicking the catalytic pocket of enzymes can catalyze chemical reactions with high selectivity; and, For the bacteria-mimicking swimmer fabrication, peptide-MOF superlattices can power translational and propellant motions by the reconfiguration of peptide assembly at the MOF-liquid interface.

  1. Using triple-helix-forming Peptide nucleic acids for sequence-selective recognition of double-stranded RNA.

    Science.gov (United States)

    Hnedzko, Dziyana; Cheruiyot, Samwel K; Rozners, Eriks

    2014-09-08

    Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple-helix-forming peptide nucleic acids (PNAs) that bind in the major grove of the RNA helix. The strategy developed uses chemically modified nucleobases, such as 2-aminopyridine (M), which enables strong triple-helical binding under physiologically relevant conditions, and 2-pyrimidinone (P) and 3-oxo-2,3-dihydropyridazine (E), which enable recognition of isolated pyrimidines in the purine-rich strand of the RNA duplex. Detailed protocols for preparation of modified PNA monomers, solid-phase synthesis, HPLC purification of PNA oligomers, and measuring dsRNA binding affinity using isothermal titration calorimetry are included.

  2. Synthetic peptides corresponding to the four P regions of Electrophorus electricus Na+ channel: interaction with and organization in model phospholipid membranes.

    Science.gov (United States)

    Pouny, Y; Shai, Y

    1995-06-13

    The hydropathy plot of the alpha subunit of the voltage-gated Na+ channel reveals four homologous repeats, each of which is homologous to Shaker type K+ channel monomer and contains six putative transmembrane segments and a hydrophobic segment within the loop connecting transmembrane segments S5 and S6. Current models predict that the four homologous segments [designated H5 or P regions (PR)] from the S5-S6 loop of each repeat lie in the aqueous pore. Peptides corresponding to the P regions of the four domains of the Electrophorus electricus (eel) Na+ channel (25-36 aa long, designated as PR-I, PR-II, PR-III, and PR-IV) and a 23-mer preceding PR-II (designated pre-PR-II) were synthesized and fluorescently labeled. The segments were then structurally and functionally characterized for their interaction with phospholipid membranes. Although the sequences of the four P regions are significantly different, they all bind to zwitterionic phospholipid membranes with similar partition coefficients (approximately 10(4) M-1). The pre-PR-II does not bind membranes at all. Resonance energy transfer measurements, between donor/acceptor-labeled pairs of peptides, revealed that besides the PR-I/PR-III pair, all other pairs form heteroaggregates but do not coassemble with unrelated membrane-bound peptide. Circular dichroism (CD) spectroscopy revealed that PR-I, PR-II, and PR-III adopt similar partial alpha-helical structures (approximately 30%) in 40% trifluoroethanol and in solutions of 1% sodium dodecylsulfate (SDS). The PR-IV (36 aa) adopts approximately 18% alpha-helical structure, and pre-PR-II gives a low CD signal. These findings are in line with proposed models in which the P regions are packed in close proximity in the lumen of the hydrophobic core of the channel. Furthermore, the finding that the PRs adopt similar partial alpha-helical structures in two different hydrophobic environments might suggest that partial alpha-helical structures also exist in the native channel

  3. A synthetic mechano-growth factor E peptide promotes rat tenocyte migration by lessening cell stiffness and increasing F-actin formation via the FAK-ERK1/2 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Bingyu [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Luo, Qing, E-mail: qing.luo@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Mao, Xinjian [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Xu, Baiyao [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Yang, Li [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Ju, Yang [Department of Mechanical Science and Engineering, Nagoya University, Nagoya 464-8603 (Japan); Song, Guanbin, E-mail: song@cqu.edu.cn [Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-03-10

    Tendon injuries are common in sports and are frequent reasons for orthopedic consultations. The management of damaged tendons is one of the most challenging problems in orthopedics. Mechano-growth factor (MGF), a recently discovered growth repair factor, plays positive roles in tissue repair through the improvement of cell proliferation and migration and the protection of cells against injury-induced apoptosis. However, it remains unclear whether MGF has the potential to accelerate tendon repair. We used a scratch wound assay in this study to demonstrate that MGF-C25E (a synthetic mechano-growth factor E peptide) promotes the migration of rat tenocytes and that this promotion is accompanied by an elevation in the expression of the following signaling molecules: focal adhesion kinase (FAK) and extracellular signal regulated kinase1/2 (ERK1/2). Inhibitors of the FAK and ERK1/2 pathways inhibited the MGF-C25E-induced tenocyte migration, indicating that MGF-C25E promotes tenocyte migration through the FAK-ERK1/2 signaling pathway. The analysis of the mechanical properties showed that the Young's modulus of tenocytes was decreased through treatment of MGF-C25E, and an obvious formation of pseudopodia and F-actin was observed in MGF-C25E-treated tenocytes. The inhibition of the FAK or ERK1/2 signals restored the decrease in Young's modulus and inhibited the formation of pseudopodia and F-actin. Overall, our study demonstrated that MGF-C25E promotes rat tenocyte migration by lessening cell stiffness and increasing pseudopodia formation via the FAK-ERK1/2 signaling pathway. - Highlights: • Mechano-growth factor E peptide (MGF-C25E) promotes migration of rat tenocytes. • MGF-C25E activates the FAK-ERK1/2 pathway in rat tenocytes. • MGF-C25E induces the actin remodeling and the formation of pseudopodia, and decreases the stiffness in rat tenocytes. • MGF-C25E promotes tenocyte migration via altering stiffness and forming pseudopodia by the activation of the

  4. Click chemistry in peptide-based drug design.

    Science.gov (United States)

    Li, Huiyuan; Aneja, Rachna; Chaiken, Irwin

    2013-08-16

    Click chemistry is an efficient and chemoselective synthetic method for coupling molecular fragments under mild reaction conditions. Since the advent in 2001 of methods to improve stereochemical conservation, the click chemistry approach has been broadly used to construct diverse chemotypes in both chemical and biological fields. In this review, we discuss the application of click chemistry in peptide-based drug design. We highlight how triazoles formed by click reactions have been used for mimicking peptide and disulfide bonds, building secondary structural components of peptides, linking functional groups together, and bioconjugation. The progress made in this field opens the way for synthetic approaches to convert peptides with promising functional leads into structure-minimized and more stable forms.

  5. Click Chemistry in Peptide-Based Drug Design

    Directory of Open Access Journals (Sweden)

    Irwin Chaiken

    2013-08-01

    Full Text Available Click chemistry is an efficient and chemoselective synthetic method for coupling molecular fragments under mild reaction conditions. Since the advent in 2001 of methods to improve stereochemical conservation, the click chemistry approach has been broadly used to construct diverse chemotypes in both chemical and biological fields. In this review, we discuss the application of click chemistry in peptide-based drug design. We highlight how triazoles formed by click reactions have been used for mimicking peptide and disulfide bonds, building secondary structural components of peptides, linking functional groups together, and bioconjugation. The progress made in this field opens the way for synthetic approaches to convert peptides with promising functional leads into structure-minimized and more stable forms.

  6. Treatment of mice with the suppressor of cytokine signaling-1 mimetic peptide, tyrosine kinase inhibitor peptide, prevents development of the acute form of experimental allergic encephalomyelitis and induces stable remission in the chronic relapsing/remitting form.

    Science.gov (United States)

    Mujtaba, Mustafa G; Flowers, Lawrence O; Patel, Chintak B; Patel, Ravi A; Haider, Mohammad I; Johnson, Howard M

    2005-10-15

    We have previously characterized a novel tyrosine kinase inhibitor peptide (Tkip) that is a mimetic of suppressor of cytokine signaling 1 (SOCS-1) and inhibits JAK2 phosphorylation of the transcription factor STAT1alpha. We show in this study that Tkip protects mice against experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis. Mice are immunized with myelin basic protein (MBP) for induction of disease. Tkip (63 mug) administered every other day suppressed the development of acute EAE in 75% of New Zealand White (NZW) mice. Furthermore, Tkip completely protected SJL/J mice, which where induced to get the relapsing/remitting form of EAE, against relapses compared with control groups in which >70% of the mice relapsed after primary incidence of disease. Protection of mice by Tkip was similar to that seen with the type I IFN, IFN-tau. Protection of mice correlated with lower MBP Ab titers in Tkip-treated groups as well as suppression of MBP-induced proliferation of splenocytes taken from EAE-afflicted mice. Cessation of Tkip and IFN-tau administration resulted in SJL/J mice relapsing back into disease. Prolonged treatment of mice with Tkip produced no evidence of cellular toxicity or weight loss. Consistent with its JAK2 inhibitory function, Tkip also inhibited the activity of the inflammatory cytokine TNF-alpha, which uses the STAT1alpha transcription factor. The data presented in this study show that Tkip, like the type I IFN, IFN-tau, inhibits both the autoreactive cellular and humoral responses in EAE and ameliorates both the acute and chronic relapsing/remitting forms of EAE.

  7. The effect of vasoactive intestinal peptide on development of form deprivation myopia in the chick: a pharmacological and immunocytochemical study.

    Science.gov (United States)

    Seltner, R L; Stell, W K

    1995-05-01

    The role of vasoactive intestinal peptide (VIP) in the development of form deprivation myopia (FDM) was examined. Daily intravitreal injection of porcine VIP reduced, but did not eliminate FDM at a maximal daily dose of 1 x 10(-5) mol/injection. A VIP analogue reported to be relatively hydrolysis-resistant in vivo, had no effect on development of FDM at any dose tested. Two VIP antagonists completely abolished FDM. The one reported to be selective for central nervous system VIP receptors was 100 times more potent than one reported to be selective for peripheral nervous system receptors (ED50 = 2 x 10(-10) and 2 x 10(-8) mol/injection respectively). By immunofluorescence using antiserum to porcine VIP, VIP-like immunoreactivity was localized to a subset of amacrine cells (AC) and in three parallel layers in the inner plexiform layer (IPL) (10%, 40% and 70% of IPL thickness from the AC layer). Immunoreactive nerve fibres were also seen in the choroid, the ciliary body and the iris. These results suggest that VIP may play a role in both normal development of the refractive properties of the eye, and in the development of FDM.

  8. Chromatofocusing of peptides and proteins using linear pH gradients formed on strong ion-exchange adsorbents.

    Science.gov (United States)

    Kang, Xuezhen; Frey, Douglas D

    2004-08-05

    Although it is commonly believed that a column packing used for chromatofocusing must have an "even" buffering capacity in order to produce a linear pH gradient, it is demonstrated here that linear pH gradients suitable for chromatofocusing can be produced on a column packing having a minimal buffering capacity. In particular, if either a strong-acid cation-exchange column packing or a strong-base anion-exchange column packing is presaturated with either a weak acid titrated with a strong base, or a weak base titrated with a strong acid, respectively, to the initial pH, then a linear or nearly linear pH gradient can be formed using a polyampholyte elution buffer by taking advantage of the presence of small quantities of weak-acid or weak-base functional groups that generally exist on these types of column packings. Experimental and theoretical studies are used to demonstrate that such systems have potential advantages over traditional chromatofocusing methods in terms of the speed of the separation, the resolution achieved, and the range of applications possible. Among other techniques described, a method for separating tryptic peptides using chromatofocusing and a strong-acid cation-exchange column packing is demonstrated to be a useful alternative to capillary isoelectric focusing and ion-exchange chromatography using a salt gradient for this purpose.

  9. Screening of TACE Peptide Inhibitors from Phage Display Peptide Library

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To obtain the recombinant tumor necrosis factor-α converting enzyme (TACE) ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RTPCR, and the expression plasmids were constructed by inserting T800 and T1300 into plasmid pET28a and pET-28c respectively. The recombinant T800 and T1300 were induced by IPTG, and SDSPAGE and Western blotting analysis results revealed that T800 and T1300 were highly expressed in the form of inclusion body. After Ni2+-NTA resin affinity chromatography, the recombinant proteins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence (TRWLVYFSRPYLVAT) was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3 %. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE.

  10. Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase

    NARCIS (Netherlands)

    Wu, Bian; Wijma, Hein J.; Song, Lu; Rozeboom, Henriette J.; Poloni, Claudia; Tian, Yue; Arif, Muhammad I.; Nuijens, Timo; Quaedflieg, Peter J. L. M.; Szymanski, Wiktor; Feringa, Ben L.; Janssen, Dick B.

    2016-01-01

    The properties of synthetic peptides, including potency, stability, and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we explore

  11. In vitro and in vivo studies for assessing the immune response and protection-inducing ability conferred by Fasciola hepatica-derived synthetic peptides containing B- and T-cell epitopes.

    Directory of Open Access Journals (Sweden)

    Jose Rojas-Caraballo

    Full Text Available Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (p<0.05 and a mixed Th1/Th2/Th17/Treg immune response, according to IFN-γ, IL-4, IL-17 and IL-10 levels, accompanied by increased CD62L+ T-cell populations. A high level of protection was obtained in mice vaccinated with peptides B2, B5, B6 and T15 formulated in the ADAD vaccination system with the AA0029 immunomodulator. The bioinformatics approach used in the present study led to the identification of seven peptides as vaccine candidates against the infection caused by Fasciola hepatica (a liver-fluke trematode. However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance.

  12. Insights from Synthetic Star-forming Regions: II. Verifying Dust Surface Density, Dust Temperature & Gas Mass Measurements with Modified Blackbody Fitting

    CERN Document Server

    Koepferl, Christine M; Dale, James E

    2016-01-01

    We use a large data-set of realistic synthetic observations (PaperI) to assess how observational techniques affect the measurement of physical properties of star-forming regions. In this paper (PaperII), we explore the reliability of the measured total gas mass, dust surface density and dust temperature maps derived from modified blackbody fitting of synthetic Herschel observations. We found from our pixel-by-pixel analysis of the measured dust surface density and dust temperature a worrisome error spread especially close to star-formation sites and low-density regions, where for those "contaminated" pixels the surface densities can be under/overestimated by up to three orders of magnitude. In light of this, we recommend to treat the pixel-based results from this technique with caution in regions with active star formation. In regions of high background typical in the inner Galactic plane, we are not able to recover reliable surface density maps of individual synthetic regions, since low-mass regions are lost...

  13. In vitro and in vivo studies for assessing the immune response and protection-inducing ability conferred by Fasciola hepatica-derived synthetic peptides containing B- and T-cell epitopes.

    Science.gov (United States)

    Rojas-Caraballo, Jose; López-Abán, Julio; Pérez del Villar, Luis; Vizcaíno, Carolina; Vicente, Belén; Fernández-Soto, Pedro; del Olmo, Esther; Patarroyo, Manuel Alfonso; Muro, Antonio

    2014-01-01

    Fasciolosis is considered the most widespread trematode disease affecting grazing animals around the world; it is currently recognised by the World Health Organisation as an emergent human pathogen. Triclabendazole is still the most effective drug against this disease; however, resistant strains have appeared and developing an effective vaccine against this disease has increasingly become a priority. Several bioinformatics tools were here used for predicting B- and T-cell epitopes according to the available data for Fasciola hepatica protein amino acid sequences. BALB/c mice were immunised with the synthetic peptides by using the ADAD vaccination system and several immune response parameters were measured (antibody titres, cytokine levels, T-cell populations) to evaluate their ability to elicit an immune response. Based on the immunogenicity results so obtained, seven peptides were selected to assess their protection-inducing ability against experimental infection with F. hepatica metacercariae. Twenty-four B- or T-epitope-containing peptides were predicted and chemically synthesised. Immunisation of mice with peptides so-called B1, B2, B5, B6, T14, T15 and T16 induced high levels of total IgG, IgG1 and IgG2a (pFasciola hepatica (a liver-fluke trematode). However, vaccine efficacy must be evaluated in other host species, including those having veterinary importance.

  14. Use of synthetic peptides to represent surface-exposed epitopes defined by neutralizing dengue complex- and flavivirus group-reactive monoclonal antibodies on the native dengue type-2 virus envelope glycoprotein.

    Science.gov (United States)

    Falconar, Andrew K I

    2008-07-01

    The reactions of neutralizing monoclonal antibodies (mAbs) that defined dengue virus (DENV) complex, flavivirus subgroup or group neutralizing epitopes were tested against synthetic peptide sequences from domains I, II and III of the envelope (E) glycoproteins of different DENV-2 genotypes/strains. The DENV complex-reactive mAb identified the surface-exposed 304-GKFKV/IVKEIA-313 peptides and the DENV complex-conserved 393-KKGSSIGQ/KM-401 peptides in domain III, which were located adjacently in the native glycoprotein. Both flavivirus group-reactive mAbs reacted most strongly with fusion sequence peptides from domain II when they contained a cysteine (C) by glycine (G) substitution (underlined) (101-WGNGGGLFG-109) to represent the native rotated C side chain. The 393-401 sequence represents a newly identified epitope, present as a highly flexible coil located between the 385 and 393 cell-binding sequence and the 401 and 413 sequence involved in the E glycoprotein homo-trimer formation. The 101-109 sequence containing 105-C by G substitution and the 393-401 sequence are good candidates for diagnostic assays and cross-protection experiments.

  15. Functional and topological studies with Trp-containing analogs of the peptide StII(1-30) derived from the N-terminus of the pore forming toxin sticholysin II: contribution to understand its orientation in membrane.

    Science.gov (United States)

    Ros, Uris; Souto, Ana Lucia C F; de Oliveira, Felipe J; Crusca, Edson; Pazos, Fabiola; Cilli, Eduardo M; Lanio, Maria E; Schreier, Shirley; Alvarez, Carlos

    2013-07-01

    Sticholysin II (St II) is the most potent cytolysin produced by the sea anemone Stichodactyla helianthus, exerting hemolytic activity via pore formation in membranes. The toxin's N-terminus contains an amphipathic α-helix that is very likely involved in pore formation. We have previously demonstrated that the synthetic peptide StII(1-30) encompassing the 1-30 segment of St II forms pores of similar radius to that of the protein (around 1 nm), being a good model of toxin functionality. Here we have studied the functional and conformational properties of fluorescent analogs of StII(1-30) in lipid membranes. The analogs were obtained by replacing Leu residues at positions 2, 12, 17, and 24 with the intrinsically fluorescent amino acid Trp (StII(1-30L2W), StII(1-30L12W), StII(1-30L17W), or StII(1-30L24W), respectively). The exchange by Trp did not significantly modify the activity and conformation of the parent peptide. The blue-shift and intensity enhancement of fluorescence in the presence of membrane indicated that Trp at position 2 is more deeply buried in the hydrophobic region of the bilayer. These experiments, as well as assays with water-soluble or spin-labeled lipid-soluble fluorescence quenchers suggest an orientation of StII(1-30) with its N-terminus oriented towards the hydrophobic core of the bilayer while the rest of the peptide is more exposed to the aqueous environment, as hypothesized for sticholysins.

  16. Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342

    Directory of Open Access Journals (Sweden)

    Atreya Chintamani

    2009-07-01

    Full Text Available Abstract Background Previous reports of site-directed deletion analysis on gamma (γ-phage lysin protein (PlyG have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199 abrogates its binding activity specific to the cell wall of Bacillus anthracis. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for B. anthracis. Furthermore, by using B. anthracis (Sterne, 34F2, an animal vaccine and B. cereus-4342, a γ-phage susceptible rare strain as surrogates of B. anthracis, development of proof-of-concepts for B. anthracis are feasible. Results Using four different methods, we evaluated six synthetic peptides representing the putative binding motif including flanking sequences (PlyG-P1 through P6 for the bacterial cell wall binding capacity. Our analysis identified PlyG-P1, PlyG-P3 and PlyG-P5 to have binding capability to both B. anthracis (Sterne, 34F2 and B. cereus-4342. The peptides however did not bind to B. cereus-11778, B. thuringiensis, and B. cereus-10876 suggesting their specificity for B. anthracis-Sterne and B. cereus-4342. PlyG-P3 in combination with fluorescent light microscopy detected even a single bacterium in plasma spiked with the bacteria. Conclusion Overall, these studies illustrate that the short 10-amino acid sequence 'LKMTADFILQ' in fact is a stand-alone bacterial cell wall-binding motif of PlyG. In principle, synthetic peptides PlyG-P1, PlyG-P3 and PlyG-P5, especially PlyG-P3 coupled with Qdot-nanocrystals are useful as high-sensitivity bio-probes in developing detection technologies for B. anthracis.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    A cornerstone of mass spectrometry based proteomics is to relate with high statistical significance experimentally obtained tandem mass spectrometry (MS/MS) data to peptide sequences from a protein database. Most sequence specific fragment ions in MS/MS spectra are represented by a subset...... of complementary ion pairs. Here, we investigated the reliabilities of complementary ion pairs formed in CAD and CAD/ETD MS/MS and developed a reliability-based approach of intensification of ion signals of complementary pairs prior to database searching. In a large-scale proteomics experiment using high...... by deisotoping/deconvolution of CAD MS/MS spectra. A novel approach for extracting sequence-specific fragment ions of co-isolated peptides was developed based on the complementarity rules. This technique demonstrated an impressive gain of 42.4% more peptide identifications as compared with the use of the initial...

  18. Comparison of the amyloid pore forming properties of rat and human Alzheimer’s beta-amyloid peptide 1-42: Calcium imaging data

    Directory of Open Access Journals (Sweden)

    Coralie Di Scala

    2016-03-01

    Full Text Available The data here consists of calcium imaging of human neuroblastoma SH-SY5Y cells treated with the calcium-sensitive dye Fluo-4AM and then incubated with nanomolar concentrations of either human or rat Alzheimer’s β-amyloid peptide Aβ1-42. These data are both of a qualitative (fluorescence micrographs and semi-quantitative nature (estimation of intracellular calcium concentrations of cells probed by Aβ1-42 peptides vs. control untreated cells. Since rat Aβ1-42 differs from its human counterpart at only three amino acid positions, this comparative study is a good assessment of the specificity of the amyloid pore forming assay. The interpretation of this dataset is presented in the accompanying study “Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides” [1].

  19. Constraining cyclic peptides to mimic protein structure motifs

    DEFF Research Database (Denmark)

    Hill, Timothy A.; Shepherd, Nicholas E.; Diness, Frederik;

    2014-01-01

    Many proteins exert their biological activities through small exposed surface regions called epitopes that are folded peptides of well-defined three-dimensional structures. Short synthetic peptide sequences corresponding to these bioactive protein surfaces do not form thermodynamically stable...... protein-like structures in water. However, short peptides can be induced to fold into protein-like bioactive conformations (strands, helices, turns) by cyclization, in conjunction with the use of other molecular constraints, that helps to fine-tune three-dimensional structure. Such constrained cyclic...... peptides can have protein-like biological activities and potencies, enabling their uses as biological probes and leads to therapeutics, diagnostics and vaccines. This Review highlights examples of cyclic peptides that mimic three-dimensional structures of strand, turn or helical segments of peptides...

  20. Gas-phase reactivity of peptide thiyl (RS•), perthiyl (RSS•), and sulfinyl (RSO•) radical ions formed from atmospheric pressure ion/radical reactions.

    Science.gov (United States)

    Tan, Lei; Xia, Yu

    2013-04-01

    In this study, we demonstrated the formation of gas-phase peptide perthiyl (RSS•) and thiyl (RS•) radical ions besides sulfinyl radical (RSO•) ions from atmospheric pressure (AP) ion/radical reactions of peptides containing inter-chain disulfide bonds. The identity of perthiyl radical was verified from characteristic 65 Da (•SSH) loss in collision-induced dissociation (CID). This signature loss was further used to assess the purity of peptide perthiyl radical ions formed from AP ion/radical reactions. Ion/molecule reactions combined with CID were carried out to confirm the formation of thiyl radical. Transmission mode ion/molecule reactions in collision cell (q2) were developed as a fast means to estimate the population of peptide thiyl radical ions. The reactivity of peptide thiyl, perthiyl, and sulfinyl radical ions was evaluated based on ion/molecule reactions toward organic disulfides, allyl iodide, organic thiol, and oxygen, which followed in order of thiyl (RS•) > perthiyl (RSS•) > sulfinyl (RSO•). The gas-phase reactivity of these three types of sulfur-based radicals is consistent with literature reports from solution studies.

  1. 两种合成抗菌肽的结构及抗菌作用机理%Solution structure and antibacterial mechanism of two synthetic antimicrobial peptides

    Institute of Scientific and Technical Information of China (English)

    杨林; 范美华; 刘雪珠; 武梅; 石戈; 廖智

    2011-01-01

    Mytilin-derived-peptide-1 (MDP-1) and mytilin-derived-peptide-2 (MDP-2) are two truncated decapeptides with reversed sequence synthesized corresponding to the residues 20-29 of mytilin-1 (GenBank Accession No. FJ973154) from M. Coruscus. The objective of this study is to characterize the structural basis of these two peptides for their antimicrobial activities and functional differences, and to investigate the inhibitory mechanism of MDPs on Escherichia coli and Sarcina lutea. The structures of MDP-1 and MDP-2 in solution were determined by 'H 2D NMR methods; the antibactericidal effects of MDPs on E. Coli and S. Lutea were observed by transmitted electron microscopy (TEM). Both MDP-1 and MDP-2 have a well-defined loop structure stabilized by two additional disulfide bridges, which resemble the-hairpin structure of mytilin-1 model. The surface profile of MDPs' structureswas characterized by protruding charged residues surrounded by hydrophobic residues. TEM analysis showed that MDPs destroyed cytoplasmic membrane and cell wall of bacteria and the interface between the cell wall and membrane was blurred. Furthermore, some holes were observed in treated bacteria, which resulted in cell death. Structural comparison between MDP-1 and MDP-2 shows that the distribution of positively charged amino acids on the loop of MDPs is topologically different significantly, which might be the reason why MDP-2 has higher activity than MDP-1. Furthermore, TEM results suggested that the bactericidal mechanisms of MDPs against E. Coli and S. Lutea were similar. Both MDP-1 and MDP-2 could attach to the negatively charged bacterial wall by positively charged amino acid residues and destroy the bacteria membrane in a pore-forming manner, thus cause the contents of the cells to release and eventually cell death.%为深入了解两种新型人工抗菌肽mytilin-derived-peptide-1 (MDP-1)和mytilin-derived-peptide-2 (MDP-2)的溶液结构和抗菌机理并探讨两种抗菌肽之间活性

  2. A water-forming NADH oxidase from Lactobacillus pentosus and its potential application in the regeneration of synthetic biomimetic cofactors

    Directory of Open Access Journals (Sweden)

    Claudia eNowak

    2015-09-01

    Full Text Available The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox. Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13 % FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyse the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as a by-product.

  3. A water-forming NADH oxidase from Lactobacillus pentosus suitable for the regeneration of synthetic biomimetic cofactors.

    Science.gov (United States)

    Nowak, Claudia; Beer, Barbara; Pick, André; Roth, Teresa; Lommes, Petra; Sieber, Volker

    2015-01-01

    The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

  4. Bactericidal effect of bovine lactoferrin and synthetic peptide lactoferrin chimera in Streptococcus pneumoniae and the decrease in luxS gene expression by lactoferrin

    NARCIS (Netherlands)

    N. León-Sicairos; U.A. Angulo-Zamudio; J.E. Vidal; C.A. López-Torres; J.G.M. Bolscher; K. Nazmi; R. Reyes-Cortes; M. Reyes-López; M. de la Garza; A. Canizalez-Román

    2014-01-01

    Streptococcus pneumoniae (pneumococcus) is responsible for nearly one million child deaths annually. Pneumococcus causes infections such as pneumonia, otitis media, meningitis, and sepsis. The human immune system includes antibacterial peptides and proteins such as lactoferrin (LF), but its activity

  5. Surfactant treatment before first breath for respiratory distress syndrome in preterm lambs: comparison of a peptide-containing synthetic lung surfactant with porcine-derived surfactant

    Directory of Open Access Journals (Sweden)

    van Zyl JM

    2013-08-01

    Full Text Available Johann M van Zyl,1 Johan Smith2 1Division of Pharmacology, Department of Medicine, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa; 2Department of Paediatrics and Child Health, Tygerberg Children's Hospital, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa Background: In a recent study utilizing a saline-lavaged adult rabbit model, we described a significant improvement in systemic oxygenation and pulmonary shunt after the instillation of a novel synthetic peptide-containing surfactant, Synsurf. Respiratory distress syndrome in the preterm lamb more closely resembles that of the human infant, as their blood gas, pH values, and lung mechanics deteriorate dramatically from birth despite ventilator support. Moreover, premature lambs have lungs which are mechanically unstable, with the advantage of being able to measure multiple variables over extended periods. Our objective in this study was to investigate if Synsurf leads to improved systemic oxygenation, lung mechanics, and histology in comparison to the commercially available porcine-derived lung surfactant Curosurf® when administered before first breath in a preterm lamb model. Materials and methods: A Cesarean section was performed under general anesthesia on 18 time-dated pregnant Dohne Merino ewes at 129–130 days gestation. The premature lambs were delivered and ventilated with an expiratory tidal volume of 6–8 mL/kg for the first 30 minutes and thereafter at 8–10 mL/kg. In a randomized controlled trial, the two surfactants tested were Synsurf and Curosurf®, both at a dose of 100 mg/kg phospholipids (1,2-dipalmitoyl-L-α-phosphatidylcholine; 90% in Synsurf, 40% in Curosurf®. A control group of animals was treated with normal saline. Measurements of physiological variables, blood gases, and lung mechanics were made before and after surfactant and saline replacement and at 15, 30, 45, 60, 90, 120, 180

  6. Experimental evidence for the ancestry of allotetraploid Trifolium repens and creation of synthetic forms with value for plant breeding

    Directory of Open Access Journals (Sweden)

    Williams Warren M

    2012-04-01

    Full Text Available Abstract Background White clover (Trifolium repens is a ubiquitous weed of the temperate world that through use of improved cultivars has also become the most important legume of grazed pastures world-wide. It has long been suspected to be allotetraploid, but the diploid ancestral species have remained elusive. Putative diploid ancestors were indicated by DNA sequence phylogeny to be T. pallescens and T. occidentale. Here, we use further DNA evidence as well as a combination of molecular cytogenetics (FISH and GISH and experimental hybridization to test the hypothesis that white clover originated as a hybrid between T. pallescens and T. occidentale. Results T. pallescens plants were identified with chloroplast trnL intron DNA sequences identical to those of white clover. Similarly, T. occidentale plants with nuclear ITS sequences identical to white clover were also identified. Reciprocal GISH experiments, alternately using labeled genomic DNA probes from each of the putative ancestral species on the same white clover cells, showed that half of the chromosomes hybridized with each probe. F1 hybrids were generated by embryo rescue and these showed strong interspecific chromosome pairing and produced a significant frequency of unreduced gametes, indicating the likely mode of polyploidization. The F1 hybrids are inter-fertile with white clover and function as synthetic white clovers, a valuable new resource for the re-incorporation of ancestral genomes into modern white clover for future plant breeding. Conclusions Evidence from DNA sequence analyses, molecular cytogenetics, interspecific hybridization and breeding experiments supports the hypothesis that a diploid alpine species (T. pallescens hybridized with a diploid coastal species (T. occidentale to generate tetraploid T. repens. The coming together of these two narrowly adapted species (one alpine and the other maritime, along with allotetraploidy, has led to a transgressive hybrid with a

  7. Syntheses of C-peptides and human proinsulin.

    Science.gov (United States)

    Yanaihara, N; Yanaihara, C; Sakagami, M; Sakura, N; Hashimoto, T; Nishida, T

    1978-01-01

    Syntheses of human, dog, rat, and duck C-peptides and their analogues and preliminary results on the total synthesis of human proinsulin are described. In the syntheses of the C-peptides, chain elongation was performed exclusively by the azide-fragment condensation method in solution. The synthetic human, dog, rat, and duck C-peptides and their analogues were proved to be homogeneous by several analytic means. With these synthetic peptides, radioimmunoassay systems for dog, rat, and duck C-peptides were developed. For the total synthesis of human proinsulin, 10 protected peptide hydrazides were prepared, and the linearly protected hexaoctacontapeptide having the proposed sequence of human proinsulin was constructed by the azide-fragment condensation method in solution starting from the C-terminal undecapeptide (HP 75-86). After deblocking of the alpha-amino protection, the partially protected hexaoctacontapeptide was treated with sodium in liquid ammonia. The ensuing sulfhydryl form was converted to the S-sulfonate form, which was reduced and then air-oxidized. The oxidized material was purified by gel filtration on Sephadex G-50 (fine) followed by ion-exchange chromatography on DEAE-cellulose. The cross-reactivity in the insulin radioimmunoassay of the ensuing product was 62.5 per cent of porcine proinsulin on a weight basis at B/Bo = 60 per cent. Acid hydrolysis and amino acid analysis of this product gave the theoretically expected ratios. In addition, this peptide, as well as the S-sulfonate form of the hexaoctacontapeptide, showed displacement curves superimposable on that of synthetic human C-peptide on an equimolar basis in the human C-peptide radioimmunoassay (antiserum 527). These results confirm the synthesis of human proinsulin.

  8. Structure-Activity Relationship of Synthetic Variants of the Milk-Derived Antimicrobial Peptide αs2-Casein f(183–207)

    Science.gov (United States)

    Begley, Máire; Clifford, Tanya; Deasy, Thérèse; Considine, Kiera; Hill, Colin

    2013-01-01

    Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183–207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183–207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193–203) and αs2-casein f(197–207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193–207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments. PMID:23793637

  9. Sensibilidad in vitro de micobacterias a dos péptidos sintéticos híbridos con actividad antimicrobiana In-vitro activity of two hybrid synthetic peptides having antimicrobial activity against mycobacteria

    Directory of Open Access Journals (Sweden)

    E. Zerbini

    2006-12-01

    Full Text Available El aumento de aislamientos clínicos de Mycobacterium tuberculosis resistentes a las drogas esenciales y de casos de micobacteriosis diseminadas debidas al complejo Mycobacterium avium hacen necesario investigar nuevos agentes antimicobacterianos. Los péptidos antimicrobianos son un nuevo grupo de antibióticos que poseen un mecanismo de acción particular. Algunos de ellos, como la cecropina y la melitina, han sido aislados de insectos y han demostrado buena actividad in vitro contra bacterias gram positivas y gram negativas. Híbridos sintéticos de esos péptidos han presentado mayor actividad que los péptidos individuales. En este trabajo se evaluó la actividad in vitro de dos péptidos híbridos sintéticos de melitina y cecropina contra M. tuberculosis, complejo M. avium, Mycobacterium fortuitum y Mycobacterium smegmatis. Se determinó la concentración inhibitoria mínima empleando la técnica de macrodilución en caldo. Luego se estableció la concentración bactericida mínima en medio Lowenstein Jensen. Los péptidos evaluados mostraron ser activos in vitro contra M. smegmatis, mientras que no presentaron ninguna actividad contra las otras micobacterias estudiadas.The increase in both Mycobacterium tuberculosis human clinical isolates resistant to the essential drugs and cases of disseminated micobacteriosis due to Mycobacterium avium Complex, underlines the need to investigate new antimicobacterial agents. The antimicrobial peptides are a new group of active antibiotics with a particular mechanism of action. Some of them, like cecropin and melittin, isolated from insects, have demonstrated good in vitro activity against Gram-positive and Gram-negative bacteria. Synthetic hybrids of those peptides have been more active than individual peptides. In this study, the in vitro activity of two hybrid synthetic peptides from melittin and cecropin against M. tuberculosis, M. avium Complex, Mycobacterium fortuitum and Mycobacterium smegmatis

  10. LF-15 & T7, synthetic peptides derived from tumstatin, attenuate aspects of airway remodelling in a murine model of chronic OVA-induced allergic airway disease.

    Directory of Open Access Journals (Sweden)

    Karryn T Grafton

    Full Text Available BACKGROUND: Tumstatin is a segment of the collagen-IV protein that is markedly reduced in the airways of asthmatics. Tumstatin can play an important role in the development of airway remodelling associated with asthma due to its anti-angiogenic properties. This study assessed the anti-angiogenic properties of smaller peptides derived from tumstatin, which contain the interface tumstatin uses to interact with the αVβ3 integrin. METHODS: Primary human lung endothelial cells were exposed to the LF-15, T3 and T7 tumstatin-derived peptides and assessed for cell viability and tube formation in vitro. The impact of the anti-angiogenic properties on airways hyperresponsiveness (AHR was then examined using a murine model of chronic OVA-induced allergic airways disease. RESULTS: The LF-15 and T7 peptides significantly reduced endothelial cell viability and attenuated tube formation in vitro. Mice exposed to OVA+ LF-15 or OVA+T7 also had reduced total lung vascularity and AHR was attenuated compared to mice exposed to OVA alone. T3 peptides reduced cell viability but had no effect on any other parameters. CONCLUSION: The LF-15 and T7 peptides may be appropriate candidates for use as novel pharmacotherapies due to their small size and anti-angiogenic properties observed in vitro and in vivo.

  11. Insights on the mechanism of thioredoxin reductase inhibition by gold N-heterocyclic carbene compounds using the synthetic linear selenocysteine containing C-terminal peptide hTrxR(488-499): an ESI-MS investigation.

    Science.gov (United States)

    Pratesi, Alessandro; Gabbiani, Chiara; Michelucci, Elena; Ginanneschi, Mauro; Papini, Anna Maria; Rubbiani, Riccardo; Ott, Ingo; Messori, Luigi

    2014-07-01

    Gold-based drugs typically behave as strong inhibitors of the enzyme thioredoxin reductase (hTrxR), possibly as the consequence of direct Gold(I) coordination to its active site selenocysteine. To gain a deeper insight into the molecular basis of enzyme inhibition and prove gold-selenocysteine coordination, the reactions of three parent Gold(I) NHC compounds with the synthetic C-terminal dodecapeptide of hTrxR containing Selenocysteine at position 498, were investigated by electrospray ionization mass spectrometry (ESI-MS). Formation of 1:1 Gold-peptide adducts, though in highly different amounts, was demonstrated in all cases. In these adducts the same [Au-NHC](+) moiety is always associated to the intact peptide. Afterward, tandem MS experiments, conducted on a specific Gold-peptide complex, pointed out that Gold is coordinated to the selenolate group. The relatively large strength of the Gold-selenolate coordinative bond well accounts for potent enzyme inhibition typically afforded by these Gold(I) compounds. In a selected case, the time course of enzyme inhibition was explored. Interestingly, enzyme inhibition turned out to show up very quickly and reached its maximum just few minutes after mixing. Overall, the present results offer some clear insight into the process of thioredoxin reductase inhibition by Gold-based compounds. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. An extended CCR5 ECL2 peptide forms a helix that binds HIV-1 gp120 through non-specific hydrophobic interactions.

    Science.gov (United States)

    Abayev, Meital; Moseri, Adi; Tchaicheeyan, Oren; Kessler, Naama; Arshava, Boris; Naider, Fred; Scherf, Tali; Anglister, Jacob

    2015-05-01

    C-C chemokine receptor 5 (CCR5) serves as a co-receptor for HIV-1. The CCR5 N-terminal segment, the second extracellular loop (ECL2) and the transmembrane helices have been implicated in binding the envelope glycoprotein gp120. Peptides corresponding to the sequence of the putative ECL2 as well as peptides containing extracellular loops 1 and 3 (ECL1 and ECL3) were found to inhibit HIV-1 infection. The aromatic residues in the C-terminal half of an ECL2 peptide were shown to interact with gp120. In the present study, we found that, in aqueous buffer, the segment Q188-Q194 in an elongated ECL2 peptide (R168-K197) forms an amphiphilic helix, which corresponds to the beginning of the fifth transmembrane helix in the crystal structure of CCR5. Two-dimensional saturation transfer difference NMR spectroscopy and dynamic filtering studies revealed involvement of Y187, F189, W190 and F193 of the helical segment in the interaction with gp120. The crystal structure of CCR5 shows that the aromatic side chains of F189, W190 and F193 point away from the binding pocket and interact with the membrane or with an adjacent CCR5 molecule, and therefore could not interact with gp120 in the intact CCR5 receptor. We conclude that these three aromatic residues of ECL2 peptides interact with gp120 through hydrophobic interactions that are not representative of the interactions of the intact CCR5 receptor. The HIV-1 inhibition by ECL2 peptides, as well as by ECL1 and ECL3 peptides and peptides corresponding to ECL2 of CXCR4, which serves as an alternative HIV-1 co-receptor, suggests that there is a hydrophobic surface in the envelope spike that could be a target for HIV-1 entry inhibitors. The structures and NMR data of ECL2S (Q186-T195) were deposited under Protein Data Bank ID 2mzx and BioMagResBank ID 25505. © 2015 FEBS.

  13. Structural Characterization of Peptide Antibodies

    DEFF Research Database (Denmark)

    Chailyan, Anna; Marcatili, Paolo

    2015-01-01

    The role of proteins as very effective immunogens for the generation of antibodies is indisputable. Nevertheless, cases in which protein usage for antibody production is not feasible or convenient compelled the creation of a powerful alternative consisting of synthetic peptides. Synthetic peptides...... can be modified to obtain desired properties or conformation, tagged for purification, isotopically labeled for protein quantitation or conjugated to immunogens for antibody production. The antibodies that bind to these peptides represent an invaluable tool for biological research and discovery...

  14. A comparative immunocytochemical study using an antiserum against a synthetic analogue of the corpora cardiaca peptide Pea-CAH-I (MI, neurohormone D) of Periplaneta americana.

    Science.gov (United States)

    Eckert, M; Gabriel, J; Birkenbeil, H; Greiner, G; Rapus, J; Gäde, G

    1996-06-01

    An antiserum against the octapeptide Pea-CAH-I, a member of the adipokinetic hormone/red pigment-concentrating hormone family, has been produced for immunocytochemical staining in insects and various other invertebrate species. The anti-Pea-CAH-I serum stains the glandular corpora cardiaca cells of those insect species that synthesize identical or structurally similar peptides. In the corpora cardiaca of species producing peptides with a different C-terminus, these cells remain unstained. Pea-CAH-I-like immunoreactivity has also been found in neurons of the central nervous system of all invertebrate orders studied. The antiserum recognizes the C-terminal sequence Pro-Asn-Trp-NH2 of the Pea-CAH-I molecule as established by enzyme immunoassay. The widespread Pea-CAH-I-like immunoreactivity in all nervous systems of the studied animals probably does not reflect the presence of Pea-CAH-I but the occurrence of peptides carrying similar epitopes.

  15. Simultaneous determination of propranolol and amiloride in synthetic binary mixtures and pharmaceutical dosage forms by synchronous fluorescence spectroscopy: a multivariate approach

    Science.gov (United States)

    Divya, O.; Shinde, Mandakini

    2013-07-01

    A multivariate calibration model for the simultaneous estimation of propranolol (PRO) and amiloride (AMI) using synchronous fluorescence spectroscopic data has been presented in this paper. Two multivariate techniques, PCR (Principal Component Regression) and PLSR (Partial Least Square Regression), have been successfully applied for the simultaneous determination of AMI and PRO in synthetic binary mixtures and pharmaceutical dosage forms. The SF spectra of AMI and PRO (calibration mixtures) were recorded at several concentrations within their linear range between wavelengths of 310 and 500 nm at an interval of 1 nm. Calibration models were constructed using 32 samples and validated by varying the concentrations of AMI and PRO in the calibration range. The results indicated that the model developed was very robust and able to efficiently analyze the mixtures with low RMSEP values.

  16. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, R.D.; Fieles, W.E.; Schotland, D.L.; Hogue-Angeletti, R.; Barchi, R.L.

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies. The membrane topology of the peptide recognized by this antiserum was proved in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% right-side-out as determined by (/sup 3/H)saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. The data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by the anitserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.

  17. Integrated automation for continuous high-throughput synthetic chromosome assembly and transformation to identify improved yeast strains for industrial production of peptide sweetener brazzein

    Science.gov (United States)

    Production and recycling of recombinant sweetener peptides in industrial biorefineries involves the evaluation of large numbers of genes and proteins. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly synthesize, clone, and express heterologous gene ope...

  18. Vaccination with synthetic analog peptides derived from WT1 oncoprotein induces T-cell responses in patients with complete remission from acute myeloid leukemia.

    Science.gov (United States)

    Maslak, Peter G; Dao, Tao; Krug, Lee M; Chanel, Suzanne; Korontsvit, Tatyana; Zakhaleva, Victoria; Zhang, Ronghua; Wolchok, Jedd D; Yuan, Jianda; Pinilla-Ibarz, Javier; Berman, Ellin; Weiss, Mark; Jurcic, Joseph; Frattini, Mark G; Scheinberg, David A

    2010-07-15

    A pilot study was undertaken to assess the safety, activity, and immunogenicity of a polyvalent Wilms tumor gene 1 (WT1) peptide vaccine in patients with acute myeloid leukemia in complete remission but with molecular evidence of WT1 transcript. Patients received 6 vaccinations with 4 WT1 peptides (200 microg each) plus immune adjuvants over 12 weeks. Immune responses were evaluated by delayed-type hypersensitivity, CD4+ T-cell proliferation, CD3+ T-cell interferon-gamma release, and WT1 peptide tetramer staining. Of the 9 evaluable patients, 7 completed 6 vaccinations and WT1-specific T-cell responses were noted in 7 of 8 patients. Three patients who were HLA-A0201-positive showed significant increase in interferon-gamma-secreting cells and frequency of WT1 tetramer-positive CD8+ T cells. Three patients developed a delayed hypersensitivity reaction after vaccination. Definite related toxicities were minimal. With a mean follow-up of 30 plus or minus 8 months after diagnosis, median disease-free survival has not been reached. These preliminary data suggest that this polyvalent WT1 peptide vaccine can be administered safely to patients with a resulting immune response. Further studies are needed to establish the role of vaccination as viable postremission therapy for acute myeloid leukemia.

  19. Synthetic peptides containing B- and T-cell epitope of dengue virus-2 E domain III provoked B- and T-cell responses.

    Science.gov (United States)

    Li, Shanfeng; Peng, Liang; Zhao, Wei; Zhong, Hua; Zhang, Fuchun; Yan, Ziqiang; Cao, Hong

    2011-05-09

    Our previous work applied a combination of bioinformatics approaches and in vitro assays to identify the dengue-2 virus (DENV-2)-specific B- and T-cell epitopes. In this report, we first evaluated the antigenicity of both B- and T-cell epitopes reacting with different sera against DENV-2 by ELISA as well as the ability of T-cell epitope to activate CD4(+) T-cell producing IFN-γ using ELISPOT, which showed a specific reactivity between either B- or T-cell epitope and DENV-2 antisera, and a significant increase of IFN-γ producing cells in DENV-2 infected mice. Then, a multi-epitope peptide containing the above B-, T-cell epitopes of envelope domain III (EDIII) of DENV-2 and pan-DR epitope (PADRE) was bioinformatically designed and synthesized. The verification of its immunogenicity and protective effect was performed in in vitro and in vivo experiments. The results showed that a high level of antibody in mice elicited by the multi-epitope peptide was detected by ELISA and the anti-peptide sera binding to the vero cells infected with DEN-2 was observed with immunofluorescence test. More importantly, the peptide could induce lymphoproliferation in vitro and a predominant Th1 type of immune response was examined by flow cytometry. We also found that the virus replication in the mice vaccinated with the multi-epitope peptide was obviously less than that of the control groups. These results may provide some important information for the development of dengue vaccine.

  20. A synthetic form of frizzled 8-associated antiproliferative factor enhances p53 stability through USP2a and MDM2.

    Directory of Open Access Journals (Sweden)

    Jayoung Kim

    Full Text Available Frizzled 8-associated Antiproliferative Factor (APF is a sialoglycopeptide urinary biomarker of interstitial cystitis/painful bladder syndrome (IC/PBS, a chronic condition of unknown etiology with variable symptoms that generally include pelvic and/or perineal pain, urinary frequency, and urgency. We previously reported that native human APF suppresses the proliferation of normal bladder epithelial cells through a mechanism that involves increased levels of p53. The goal of this study was to delineate the regulatory mechanism whereby p53 expression is regulated by APF. Two APF-responsive cell lines (T24 bladder carcinoma cells and the immortalized human bladder epithelial cell line, TRT-HU1 were treated with asialo-APF (as-APF, a chemically synthesized form of APF. Biochemical analysis revealed that as-APF increased p53 levels in two ways: by decreasing ubiquitin specific protease 2a (USP2a expression leading to enhanced ubiquitination of murine double minute 2 E3 ubiquitin ligase (MDM2, and by suppressing association of p53 with MDM2, thus impairing p53 ubiquitination. Biological responses to as-APF were suppressed by increased expression of wild type, but not mutant USP2a, which enhanced cell growth via upregulation of a cell cycle mediator, cyclin D1, at both transcription and protein levels. Consistent with this, gene silencing of USP2a with siRNA arrested cell proliferation. Our findings suggest that APF upregulates cellular p53 levels via functional attenuation of the USP2a-MDM2 pathway, resulting in p53 accumulation and growth arrest. These data also imply that targeting USP2a, MDM2, p53 and/or complex formation by these molecules may be relevant in the development of novel therapeutic approaches to IC/PBS.

  1. Comparative study of the neuroprotective and nootropic activities of the carboxylate and amide forms of the HLDF-6 peptide in animal models of Alzheimer's disease.

    Science.gov (United States)

    Bogachouk, Anna P; Storozheva, Zinaida I; Solovjeva, Olga A; Sherstnev, Vyacheslav V; Zolotarev, Yury A; Azev, Vyacheslav N; Rodionov, Igor L; Surina, Elena A; Lipkin, Valery M

    2016-01-01

    A comparative study of the neuroprotective and nootropic activities of two pharmaceutical substances, the HLDF-6 peptide (HLDF-6-OH) and its amide form (HLDF-6-NH2), was conducted. The study was performed in male rats using two models of a neurodegenerative disorder. Cognitive deficit in rats was induced by injection of the beta-amyloid fragment 25-35 (βA 25-35) into the giant-cell nucleus basalis of Meynert or by coinjection of βA 25-35 and ibotenic acid into the hippocampus. To evaluate cognitive functions in animals, three tests were used: the novel object recognition test, the conditioned passive avoidance task and the Morris maze. Comparative analysis of the data demonstrated that the neuroprotective activity of HLDF-6-NH2, evaluated by improvement of cognitive functions in animals, surpassed that of the native HLDF-6-OH peptide. The greater cognitive/ behavioral effects can be attributed to improved kinetic properties of the amide form of the peptide, such as the character of biodegradation and the half-life time. The effects of HLDF-6-NH2 are comparable to, or exceed, those of the reference compounds. Importantly, HLDF-6-NH2 exerts its effects at much lower doses than the reference compounds.

  2. Detection of selective cationic amphipatic antibacterial peptides by Hidden Markov models.

    Science.gov (United States)

    Polanco, Carlos; Samaniego, Jose L

    2009-01-01

    Antibacterial peptides are researched mainly for the potential benefit they have in a variety of socially relevant diseases, used by the host to protect itself from different types of pathogenic bacteria. We used the mathematical-computational method known as Hidden Markov models (HMMs) in targeting a subset of antibacterial peptides named Selective Cationic Amphipatic Antibacterial Peptides (SCAAPs). The main difference in the implementation of HMMs was focused on the detection of SCAAP using principally five physical-chemical properties for each candidate SCAAPs, instead of using the statistical information about the amino acids which form a peptide. By this method a cluster of antibacterial peptides was detected and as a result the following were found: 9 SCAAPs, 6 synthetic antibacterial peptides that belong to a subregion of Cecropin A and Magainin 2, and 19 peptides from the Cecropin A family. A scoring function was developed using HMMs as its core, uniquely employing information accessible from the databases.

  3. Ni(II)-NTA modified poly(ethylene imine) glycopolymers: physicochemical properties and first in vitro study of polyplexes formed with HIV-derived peptides.

    Science.gov (United States)

    Hauptmann, Nicole; Pion, Marjorie; Muñoz-Fernández, María-Ángeles; Komber, Hartmut; Werner, Carsten; Voit, Brigitte; Appelhans, Dietmar

    2013-05-01

    Alternative delivery entities are desirable in immunotherapies in which polyplexes are widely formed by electrostatic interactions to induce cellular uptake processes for bioactive molecules. In our study, biocompatible Ni(II)-nitrilo(triacetic acid)-modified poly(ethylene imine)-maltose (Ni-NTA-DG) is realized and evaluated as complexation agent against His-tagged peptides using fluorescence polarization and dynamic light scattering. The polyplexes are stable until a pH of 6.5-6.0, and also up to 50 mM of imidazole. A first uptake approach shows that polyplexes lead to an increase in peptide uptake in monocyte-derived immature dendritic cells. In summary, Ni-NTA-DG represents a promising (delivery) platform for forthcoming in vitro applications. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.

    Science.gov (United States)

    Gordon, R D; Fieles, W E; Schotland, D L; Hogue-Angeletti, R; Barchi, R L

    1987-01-01

    A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies; noninnervated membrane was not labeled, consistent with the known distribution of sodium channels in this tissue. The membrane topology of the peptide recognized by this antiserum was probed in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% "right-side-out" as determined by [3H]saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. Gold particles identifying the antibody were found almost exclusively associated with the cytoplasmic surface of the innervated membrane. Our data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by our antiserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure. Images PMID:2432607

  5. Peptide identification

    Science.gov (United States)

    Jarman, Kristin H [Richland, WA; Cannon, William R [Richland, WA; Jarman, Kenneth D [Richland, WA; Heredia-Langner, Alejandro [Richland, WA

    2011-07-12

    Peptides are identified from a list of candidates using collision-induced dissociation tandem mass spectrometry data. A probabilistic model for the occurrence of spectral peaks corresponding to frequently observed partial peptide fragment ions is applied. As part of the identification procedure, a probability score is produced that indicates the likelihood of any given candidate being the correct match. The statistical significance of the score is known without necessarily having reference to the actual identity of the peptide. In one form of the invention, a genetic algorithm is applied to candidate peptides using an objective function that takes into account the number of shifted peaks appearing in the candidate spectrum relative to the test spectrum.

  6. Natriuretic Peptides, Diagnostic and Prognostic Biomarkers

    OpenAIRE

    Rutten, Joost

    2010-01-01

    textabstractIn humans, the natriuretic peptide family consists of three different types of peptides: atrial natriuretic peptide (synonym: atrial natriuretic factor), B-type natriuretic peptide (synonym: brain natriuretic peptide) and C-natriuretic peptide.1 Atrial natriuretic peptide (ANP) was the fi rst natriuretic peptide to be discovered and in humans ANP is predominantly formed in the cardiomyocytes of the atria.2 B-type natriuretic peptide (BNP) was fi rst discovered in porcine brain hen...

  7. 猪口蹄疫O型合成肽疫苗及其主要特点%Synthetic peptide vaccine of O-type foot-and-mouth disease of swine and its main characteristic

    Institute of Scientific and Technical Information of China (English)

    任巧玲; 邢宝松; 郭红霞

    2014-01-01

    At present, foot-and-mouth disease is one of the animal diseases which seriously endan-ger Chinese pig industry,,and vaccination is an important prevention methord for this disease. New-ly developed Synthetic peptide vaccine of O-type foot-and-mouth disease in swine has aroused great attention for its high immunogenicity, good biological safety, differentiate infection from vacci-nation, and so on. Foot-and-mouth disease virus, the antigenic epitope of type O foot-and-mouth disease virus, and the research status of synthetic peptide vaccine of type O foot-and-mouth dis-ease of swine and its main characteristic are discussed in this article In order to provide refer-ences for the promotion and the application of this vaccination.%口蹄疫是当前严重危害我国养猪业的疾病之一,长期以来免疫接种是我国预防该病的重要措施。近年来研制出的猪口蹄疫O型合成肽疫苗以其免疫原性高、生物安全性好、可有效区分免疫动物和感染动物等优点引起了人们的高度重视。本文主要介绍了口蹄疫病毒、O型口蹄疫病毒的抗原位点和猪口蹄疫O型合成肽疫苗的研究概况及其主要特点,旨在为猪口蹄疫O型合成肽疫苗的推广应用提供参考。

  8. Priming immunization against cholera toxin and E. coli heat-labile toxin by a cholera toxin short peptide-beta-galactosidase hybrid synthesized in E. coli.

    OpenAIRE

    Jacob, C O; Leitner, M.; Zamir, A.; Salomon, D.; Arnon, R

    1985-01-01

    A synthetic oligodeoxynucleotide encoding for a small peptide was employed for the expression of this peptide in a form suitable for immunization. The encoded peptide, namely, the region 50-64 of the B subunit of cholera toxin (CTP3), had previously been identified as a relevant epitope of cholera toxin. Thus, multiple immunizations with its conjugate to a protein carrier led to an efficient neutralizing response against native cholera toxin. Immunization with the resulting fusion protein of ...

  9. Combining UV photodissociation with electron transfer for peptide structure analysis.

    Science.gov (United States)

    Shaffer, Christopher J; Marek, Ales; Pepin, Robert; Slovakova, Kristina; Turecek, Frantisek

    2015-03-01

    The combination of near-UV photodissociation with electron transfer and collisional activation provides a new tool for structure investigation of isolated peptide ions and reactive intermediates. Two new types of pulse experiments are reported. In the first one called UV/Vis photodissociation-electron transfer dissociation (UVPD-ETD), diazirine-labeled peptide ions are shown to undergo photodissociation in the gas phase to form new covalent bonds, guided by the ion conformation, and the products are analyzed by electron transfer dissociation. In the second experiment, called ETD-UVPD wherein synthetic labels are not necessary, electron transfer forms new cation-peptide radical chromophores that absorb at 355 nm and undergo specific backbone photodissociation reactions. The new method is applied to distinguish isomeric ions produced by ETD of arginine containing peptides. Copyright © 2015 John Wiley & Sons, Ltd.

  10. A novel approach for predicting acyl glucuronide reactivity via Schiff base formation: development of rapidly formed peptide adducts for LC/MS/MS measurements.

    Science.gov (United States)

    Wang, Jianyao; Davis, Margaret; Li, Fangbiao; Azam, Farooq; Scatina, JoAnn; Talaat, Rasmy

    2004-09-01

    A novel technique to study the reactivity of acyl glucuronide metabolites to protein has been developed and is described herein. Considered here are acyl glucuronide metabolites, which have undergone the rearrangement of the glucuronic acid moiety at physiological temperature and pH. The investigation of the reactivity of these electrophilic metabolites was carried out by measuring the rate of reaction of rearranged AG metabolites in forming the corresponding acyl glucuronide-peptide adduct in the presence of Lys-Phe. This differs from the parallel technique used in forming AG adducts of proteins that have been previously reported. In the study described here, the Schiff base adduct, diclofenac acyl glucuronide-Lys-Phe product, was generated and structurally elucidated by liquid chromatography tandem mass spectrometry (LC/MS/MS) analysis. The product structure was proved to be a Schiff base adduct by chemical derivatization by nucleophilic addition of HCN and chemical reduction with NaCNBH(3), followed by LC/MS/MS analysis. It is proposed here that the degree of reactivity of acyl glucuronides as measured by covalent binding to protein is proportional to the amount of its peptide adduct generated with the peptide technique described. The application of this technique to the assessment of the degree of reactivity of acyl glucuronide metabolites was validated by developing a reactivity rank of seven carboxylic acid-containing drugs. Consistency was achieved between the ranking of reactivity in the peptide technique for these seven compounds and the rankings found in the literature. In addition, a correlation (R(2) = 0.95) was revealed between the formation of a peptide adduct and the rearrangement rate of the primary acyl glucuronide of seven tested compounds. A structure effect on the degree of reactivity has demonstrated the rate order: acetic acid > propionic acid > benzoic acid derivatives. A rational explanation of this order was proposed, based on the inherent

  11. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    DEFF Research Database (Denmark)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...

  12. Toxoplasma gondii-Derived Synthetic Peptides Containing B- and T-Cell Epitopes from GRA2 Protein Are Able to Enhance Mice Survival in a Model of Experimental Toxoplasmosis

    Science.gov (United States)

    Bastos, Luciana M.; Macêdo, Arlindo G.; Silva, Murilo V.; Santiago, Fernanda M.; Ramos, Eliezer L. P.; Santos, Fabiana A. A.; Pirovani, Carlos P.; Goulart, Luiz R.; Mineo, Tiago W. P.; Mineo, José R.

    2016-01-01

    Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2) is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN), as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b), mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-α and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii. PMID:27313992

  13. Toxoplasma gondii-derived synthetic peptides containing B- and T-cell epitopes from GRA2 protein are able to enhance mice survival in a model of experimental toxoplasmosis

    Directory of Open Access Journals (Sweden)

    Luciana Machado Bastos

    2016-06-01

    Full Text Available Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2 is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN, as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b, mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-alpha and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

  14. Citrullinated peptides in the diagnosis of rheumatoid arthritis.

    Science.gov (United States)

    Gómara, María J; Haro, Isabel

    2013-01-01

    Antibodies directed against citrullinated proteins and peptides (ACPAs) are the most specific serological markers available for diagnosing rheumatoid arthritis (RA). ACPAs may be detected several years before symptoms of RA appear, and their presence at disease onset is a good predictor of the development of erosive joint lesions. RA patients can be classified into two major groups: those who have ACPAs and those who do not. The presence of ACPAs at early stages of RA predicts the development of earlier and more widespread joint erosions, and low remission rates.Synthetic peptides can replace cognate proteins in solid-phase assays for specific autoantibody recognition in RA patients. The use of synthetic peptides instead of proteins represents an advantage in terms of the reproducibility of such immunoassays. Proteins also contain non-citrullinated epitopes that are recognized by non-RA sera and this could reduce the specificity of the test. The use of synthetic citrullinated peptides gives absolute control over the exact epitopes presented. Furthermore, it is difficult to prepare sufficient amounts of high-quality antigenic proteins with a well-defined degree of citrullination. Synthetic citrullinated peptides, in contrast, are easily obtained in a pure form with a well-defined chemical structure and the epitopes can be precisely oriented in the plate by covalent binding of the peptides.Chimeric peptides bearing different citrullinated protein domains have recently been used in the design of RA diagnosis systems. The results of the application of those systems indicate that more than one serological test is required to classify RA patients based on the presence or absence of ACPAs. Each of the target molecules reported (fibrin, vimentin and filaggrin) helps to identify a particular subset of RA patients.

  15. Synthetic LDL as targeted drug delivery vehicle

    Science.gov (United States)

    Forte, Trudy M.; Nikanjam, Mina

    2012-08-28

    The present invention provides a synthetic LDL nanoparticle comprising a lipid moiety and a synthetic chimeric peptide so as to be capable of binding the LDL receptor. The synthetic LDL nanoparticle of the present invention is capable of incorporating and targeting therapeutics to cells expressing the LDL receptor for diseases associated with the expression of the LDL receptor such as central nervous system diseases. The invention further provides methods of using such synthetic LDL nanoparticles.

  16. Prediction of the effect on antihyperglycaemic action of sitagliptin by plasma active form glucagon-like peptide-1

    Institute of Scientific and Technical Information of China (English)

    Akifumi; Kushiyama; Takako; Kikuchi; Kentaro; Tanaka; Tazu; Tahara; Toshiko; Takao; Yukiko; Onishi; Yoko; Yoshida; Shoji; Kawazu; Yasuhiko; Iwamoto

    2016-01-01

    AIM: To investigate whether active glucagon-like peptide-1(GLP-1) is a prediction Factor of Effect of sitagliptin on patients with type 2 diabetes mellitus(GLP-1 FEST:UMIN000010645). METHODS: Seventy-six patients with type 2 diabetes, who had insufficient glycemic control [Hemoglobin A1c(Hb A1c) ≥ 7%] in spite of treatment with metformin and/or sulfonylurea, were included in the investigation. Patients were divided into three groups by tertiles of fasting plasma active GLP-1 level, before the administration of 50 mg sitagliptin. RESULTS: At baseline, body mass index, serum UA, insulin and HOMA-IR were higher in the high active GLP-1 group than in the other two groups. The high active GLP-1 group did not show any decline of Hb A1c(7.6% ± 1.4% to 7.5% ± 1.5%), whereas the middle and low groups indicated significant decline of Hb A1c(7.4 ± 0.7 to 6.8 ± 0.6 and 7.4 ± 1.2 to 6.9 ± 1.3, respectively) during six months. Only the low and middle groups showed a significant increment of active GLP-1, C-peptide level, a decreased log and proinsulin/insulin ratio after administration. In logistic analysis, the low or middle group is a significantexplanatory variable for an Hb A1 c decrease of ≥ 0.5%, and its odds ratio is 4.5(1.40-17.6)(P = 0.01) against the high active GLP-1 group. This remains independent when adjusted for Hb A1 c level before administration, patients’ medical history, medications, insulin secretion and insulin resistance.CONCLUSION: Plasma fasting active GLP-1 is an independent predictive marker for the efficacy of dipeptidyl peptidase 4 inhibitor sitagliptin.

  17. Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses.

    Science.gov (United States)

    Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Keller, Ulrich Auf dem; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

    2016-01-01

    Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with

  18. Development of SI-traceable C-peptide certified reference material NMIJ CRM 6901-a using isotope-dilution mass spectrometry-based amino acid analyses.

    Science.gov (United States)

    Kinumi, Tomoya; Goto, Mari; Eyama, Sakae; Kato, Megumi; Kasama, Takeshi; Takatsu, Akiko

    2012-07-01

    A certified reference material (CRM) is a higher-order calibration material used to enable a traceable analysis. This paper describes the development of a C-peptide CRM (NMIJ CRM 6901-a) by the National Metrology Institute of Japan using two independent methods for amino acid analysis based on isotope-dilution mass spectrometry. C-peptide is a 31-mer peptide that is utilized for the evaluation of β-cell function in the pancreas in clinical testing. This CRM is a lyophilized synthetic peptide having the human C-peptide sequence, and contains deamidated and pyroglutamylated forms of C-peptide. By adding water (1.00 ± 0.01) g into the vial containing the CRM, the C-peptide solution in 10 mM phosphate buffer saline (pH 6.6) is reconstituted. We assigned two certified values that represent the concentrations of total C-peptide (mixture of C-peptide, deamidated C-peptide, and pyroglutamylated C-peptide) and C-peptide. The certified concentration of total C-peptide was determined by two amino acid analyses using pre-column derivatization liquid chromatography-mass spectrometry and hydrophilic chromatography-mass spectrometry following acid hydrolysis. The certified concentration of C-peptide was determined by multiplying the concentration of total C-peptide by the ratio of the relative area of C-peptide to that of the total C-peptide measured by liquid chromatography. The certified value of C-peptide (80.7 ± 5.0) mg/L represents the concentration of the specific entity of C-peptide; on the other hand, the certified value of total C-peptide, (81.7 ± 5.1) mg/L can be used for analyses that does not differentiate deamidated and pyroglutamylated C-peptide from C-peptide itself, such as amino acid analyses and immunochemical assays.

  19. Purified high molecular weight synthetic Aβ(1-42) and biological Aβ oligomers are equipotent in rapidly inducing MTT formazan exocytosis.

    Science.gov (United States)

    Weidner, Adam M; Housley, Molly; Murphy, M Paul; Levine, Harry

    2011-06-15

    Synthetic soluble Aβ oligomers are often used as a surrogate for biologic material in a number of model systems. We compared the activity of Aβ oligomers (synthetic and cell culture media derived) on the human SH-SY5Y neuroblastoma and C2C12 mouse myoblast cell lines in a novel, modified MTT assay. Separating oligomers from monomeric peptide by size exclusion chromatography produced effects at peptide concentrations approaching physiologic levels (10-100 nM). Purified oligomers, but not monomers or fibrils, elicited an increase of a detergent-insoluble form of MTT formazan within 2h as opposed to a control toxin (H(2)O(2)). This effect was comparable for biological and synthetic peptide in both cell types. Monomeric Aβ attenuated the effect of soluble oligomers. This study suggests that the activities of biological and synthetic oligomers are indistinguishable during early stages of Aβ oligomer-cell interaction.

  20. Isolation and molecular characterization of porcine calcitonin gene-related peptide (CGRP) and its endocrine effects in the porcine pancreas

    DEFF Research Database (Denmark)

    Rasmussen, T N; Bersani, M; Schmidt, P;

    1998-01-01

    The aim of this study was to investigate the possible role of porcine calcitonin gene-related peptide (CGRP) in the regulation of the endocrine porcine pancreas. Initially, we isolated and purified CGRP from extracts of porcine adrenal glands and pancreases. A single molecular form of the peptide...... was found in the two tissues. The adrenal peptide was sequenced and found to differ from human alpha-CGRP at six positions and from human beta-CGRP at three positions. By immunohistochemistry, CGRP was found in nerve fibers in the pancreatic ganglia. A synthetic replica of the porcine peptide was infused...

  1. Inhibitory effect of the carnosine-gallic acid synthetic peptide on MMP-2 and MMP-9 in human fibrosarcoma HT1080 cells.

    Science.gov (United States)

    Kim, Sung-Rae; Eom, Tae-Kil; Byun, Hee-Guk

    2014-09-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix components and play important roles in a variety of biological and pathological processes such as malignant tumor metastasis and invasion. In this study, we constructed carnosine-gallic acid peptide (CGP) to identify a better MMP inhibitor than carnosine. The inhibitory effects of CGP on MMP-2 and MMP-9 were investigated in the human fibrosarcoma (HT1080) cell line. As a result, CGP significantly decreased MMP-2 and MMP-9 expression levels without a cytotoxic effect. Moreover, CGP may inhibit migration and invasion in HT1080 cells through the urokinase plasminogen activator (uPA)-uPA receptor signaling pathways to inhibit MMP-2 and MMP-9. Based on these results, it appears that CGP may play an important role in preventing and treating several MMP-2 and MMP-9-mediated health problems such as metastasis.

  2. Synthetic heparin-binding factor analogs

    Science.gov (United States)

    Pena, Louis A.; Zamora, Paul O.; Lin, Xinhua; Glass, John D.

    2010-04-20

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain, and preferably two peptide chains branched from a dipeptide branch moiety composed of two trifunctional amino acid residues, which peptide chain or chains bind a heparin-binding growth factor receptor and are covalently bound to a non-signaling peptide that includes a heparin-binding domain, preferably by a linker, which may be a hydrophobic linker. The synthetic heparin-binding growth factor analogs are useful as pharmaceutical agents, soluble biologics or as surface coatings for medical devices.

  3. Facile synthesis of 1-alkoxy-1H-benzo- and 7-azabenzotriazoles from peptide coupling agents, mechanistic studies, and synthetic applications

    Directory of Open Access Journals (Sweden)

    Mahesh K. Lakshman

    2014-08-01

    Full Text Available (1H-Benzo[d][1,2,3]triazol-1-yloxytris(dimethylaminophosphonium hexafluorophosphate (BOP, 1H-benzo[d][1,2,3]triazol-1-yl 4-methylbenzenesulfonate (Bt-OTs, and 3H-[1,2,3]triazolo[4,5-b]pyridine-3-yl 4-methylbenzenesulfonate (At-OTs are classically utilized in peptide synthesis for amide-bond formation. However, a previously undescribed reaction of these compounds with alcohols in the presence of a base, leads to 1-alkoxy-1H-benzo- (Bt-OR and 7-azabenzotriazoles (At-OR. Although BOP undergoes reactions with alcohols to furnish 1-alkoxy-1H-benzotriazoles, Bt-OTs proved to be superior. Both, primary and secondary alcohols undergo reaction under generally mild reaction conditions. Correspondingly, 1-alkoxy-1H-7-azabenzotriazoles were synthesized from At-OTs. Mechanistically, there are three pathways by which these peptide-coupling agents can react with alcohols. From 31P{1H}, [18O]-labeling, and other chemical experiments, phosphonium and tosylate derivatives of alcohols seem to be intermediates. These then react with BtO− and AtO− produced in situ. In order to demonstrate broader utility, this novel reaction has been used to prepare a series of acyclic nucleoside-like compounds. Because BtO− is a nucleofuge, several Bt-OCH2Ar substrates have been evaluated in nucleophilic substitution reactions. Finally, the possible formation of Pd π–allyl complexes by departure of BtO− has been queried. Thus, alpha-allylation of three cyclic ketones was evaluated with 1-(cinnamyloxy-1H-benzo[d][1,2,3]triazole, via in situ formation of pyrrolidine enamines and Pd catalysis.

  4. Membrane-targeted self-assembling cyclic peptide nanotubes.

    Science.gov (United States)

    Rodríguez-Vázquez, Nuria; Ozores, H Lionel; Guerra, Arcadio; González-Freire, Eva; Fuertes, Alberto; Panciera, Michele; Priegue, Juan M; Outeiral, Juan; Montenegro, Javier; Garcia-Fandino, Rebeca; Amorin, Manuel; Granja, Juan R

    2014-01-01

    Peptide nanotubes are novel supramolecular nanobiomaterials that have a tubular structure. The stacking of cyclic components is one of the most promising strategies amongst the methods described in recent years for the preparation of nanotubes. This strategy allows precise control of the nanotube surface properties and the dimensions of the tube diameter. In addition, the incorporation of 3- aminocycloalkanecarboxylic acid residues in the nanotube-forming peptides allows control of the internal properties of the supramolecular tube. The research aimed at the application of membrane-interacting self-assembled cyclic peptide nanotubes (SCPNs) is summarized in this review. The cyclic peptides are designed to interact with phospholipid bilayers to induce nanotube formation. The properties and orientation of the nanotube can be tuned by tailoring the peptide sequence. Hydrophobic peptides form transmembrane pores with a hydrophilic orifice, the nature of which has been exploited to transport ions and small molecules efficiently. These synthetic ion channels are selective for alkali metal ions (Na(+), K(+) or Cs(+)) over divalent cations (Ca(2+)) or anions (Cl(-)). Unfortunately, selectivity was not achieved within the series of alkali metal ions, for which ion transport rates followed the diffusion rates in water. Amphipathic peptides form nanotubes that lie parallel to the membrane. Interestingly, nanotube formation takes place preferentially on the surface of bacterial membranes, thus making these materials suitable for the development of new antimicrobial agents.

  5. In vitro and in vivo antimicrobial activity of a synthetic peptide derived from the C-terminal region of human chemokine CCL13 against Pseudomonas aeruginosa.

    Science.gov (United States)

    Cossio-Ayala, Mayte; Domínguez-López, Mariana; Mendez-Enriquez, Erika; Portillo-Téllez, María Del Carmen; García-Hernández, Enrique

    2017-08-01

    Chemokines are important mediators of immunological responses during inflammation and under steady-state conditions. In addition to regulating cell migration, some chemotactic cytokines have direct effects on bacteria. Here, we characterized the antibacterial ability of the synthetic oligopeptide CCL1357-75, which corresponds to the carboxyl-terminal region of the human chemokine CCL13. In vitro measurements indicated that CCL1357-75 disrupts the cell membrane of Pseudomonas aeruginosa through a mechanism coupled to an unordered-helicoidal conformational transition. In a murine pneumonic model, CCL1357-75 improved mouse survival and bacterial clearance and decreased neutrophil recruitment, proinflammatory cytokines and lung pathology compared with that observed in untreated infected animals. Overall, our study supports the ability of chemokines and/or chemokine-derived oligopeptides to act as direct defense agents against pathogenic bacteria and suggests their potential use as alternative antibiotics. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Development of new peptide synthetic method of enzyme using the extraction reactivity; Chushutsu hanno wo mochiita shiki pepuchido koso goseiho no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    Hirata, Makoto [Oita University, Oita (Japan)

    1999-03-05

    Recently, taste and bioactivation of large number of oligopeptide become clear, and the development of the efficient synthetic method becomes the urgency. In the production process by conventional enzyme reaction which combined the crystallization, because the solubility of the product to the water which is reaction solvent is low, the yield remained at about 60%, and the problem of reaction inhibition of the product by the crystal had also been indicated. In the enzyme synthesis of the aspartame in which he is the representative oligopeptide, it aimed at the establishment of the new synthesis method which can improve yield and reaction rate, while the segregation enzyme was continuously utilized. In this synthetic method, supply of organic solvent which dissolved the substrate, extraction of the substrate from organic solvent to water phase, synthesis reaction by the segregation enzyme in water phase, extraction of the aspartame which is a product from water phase to organic solvent progress, and they continuously progress by one complete mixing reactor. The process which controlled these speeds and yields was quantitatively analyzed, and material balance style considering substrate, enzyme and mass transfer of the product and enzyme reaction speed was deduced. The optimum operating condition for improving yield and productivity of the purpose product using this solution was examined, and optimum supply concentration and agitation speed of aspartic acid which was a substrate were started, and the optimum operating condition which realizes the improvement in high yield and productivity over 90% of the aspartame was clarified. Like this, it is that this research adopts features of liquid Citrus nobilis two-phase partition for the enzyme synthesis of the aspartame, and it is considered that there is a value, because it is the creative research which verified that the productivity can be greatly improved by the utilization of the chemical-engineering technique, and

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

    DEFF Research Database (Denmark)

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

    2002-01-01

    -I restricted porcine CD8(+) T-cell epitope currently known is a 9-residue peptide from the polyprotein of CSFV (J. Gen. Virol, 76 (1995) 3039). Based on results with the CSFV epitope and two porcine haplotypes (H4 and H7), the in vitro refold assay appeared able to discriminate between peptide-free and peptide...

  8. Immunotherapy with Allergen Peptides

    OpenAIRE

    Larché Mark

    2007-01-01

    Specific allergen immunotherapy (SIT) is disease-modifying and efficacious. However, the use of whole allergen preparations is associated with frequent allergic adverse events during treatment. Many novel approaches are being designed to reduce the allergenicity of immunotherapy preparations whilst maintaining immunogenicity. One approach is the use of short synthetic peptides which representing dominant T cell epitopes of the allergen. Short peptides exhibit markedly reduced capacity to cro...

  9. Production and characterization of peptide antibodies

    DEFF Research Database (Denmark)

    Trier, Nicole Hartwig; Hansen, Paul Robert; Houen, Gunnar

    2012-01-01

    Proteins are effective immunogens for generation of antibodies. However, occasionally the native protein is known but not available for antibody production. In such cases synthetic peptides derived from the native protein are good alternatives for antibody production. These peptide antibodies...... are powerful tools in experimental biology and are easily produced to any peptide of choice. A widely used approach for production of peptide antibodies is to immunize animals with a synthetic peptide coupled to a carrier protein. Very important is the selection of the synthetic peptide, where factors...... such as structure, accessibility and amino acid composition are crucial. Since small peptides tend not to be immunogenic, it may be necessary to conjugate them to carrier proteins in order to enhance immune presentation. Several strategies for conjugation of peptide-carriers applied for immunization exist...

  10. Chemical synthetic biology: a mini-review

    Directory of Open Access Journals (Sweden)

    Cristiano eChiarabelli

    2013-09-01

    Full Text Available Chemical synthetic biology (CSB is a branch of synthetic biology (SB oriented towards the synthesis of chemical structures alternative to those present in nature. Whereas SB combines biology and engineering with the aim of synthesizing biological structures or life forms that do not exist in nature – often based on genome manipulation, CSB uses and assembles biological parts, synthetic or not, to create new and alternative structures. A short epistemological note will introduce the theoretical concepts related to these fields, whereas the text will be largely devoted to introduce and comment two main projects of CSB, carried out in our laboratory in the recent years.The Never Born Biopolymers (NBB project deals with the construction and the screening of RNA and peptide sequences that are not present in nature, whereas the Minimal Cell project focuses on the construction of semi-synthetic compartments (usually liposomes containing the minimal and sufficient number of components to perform the basic function of a biological cell.These two topics are extremely important for both the general understanding of biology in terms of function, organization and development, and for applied biotechnology.

  11. The synthetic Plasmodium falciparum circumsporozoite peptide PfCS102 as a malaria vaccine candidate: a randomized controlled phase I trial.

    Directory of Open Access Journals (Sweden)

    Régine Audran

    Full Text Available BACKGROUND: Fully efficient vaccines against malaria pre-erythrocytic stage are still lacking. The objective of this dose/adjuvant-finding study was to evaluate the safety, reactogenicity and immunogenicity of a vaccine candidate based on a peptide spanning the C-terminal region of Plasmodium falciparum circumsporozoite protein (PfCS102 in malaria naive adults. METHODOLOGY AND PRINCIPAL FINDINGS: Thirty-six healthy malaria-naive adults were randomly distributed into three dose blocks (10, 30 and 100 microg and vaccinated with PfCS102 in combination with either Montanide ISA 720 or GSK proprietary Adjuvant System AS02A at days 0, 60, and 180. Primary end-point (safety and reactogenicity was based on the frequency of adverse events (AE and of abnormal biological safety tests; secondary-end point (immunogenicity on P. falciparum specific cell-mediated immunity and antibody response before and after immunization. The two adjuvant formulations were well tolerated and their safety profile was good. Most AEs were local and, when systemic, involved mainly fatigue and headache. Half the volunteers in AS02A groups experienced severe AEs (mainly erythema. After the third injection, 34 of 35 volunteers developed anti-PfCS102 and anti-sporozoite antibodies, and 28 of 35 demonstrated T-cell proliferative responses and IFN-gamma production. Five of 22 HLA-A2 and HLA-A3 volunteers displayed PfCS102 specific IFN-gamma secreting CD8(+ T cell responses. Responses were only marginally boosted after the 3(rd vaccination and remained stable for 6 months. For both adjuvants, the dose of 10 microg was less immunogenic in comparison to 30 and 100 microg that induced similar responses. AS02A formulations with 30 microg or 100 microg PfCS102 induced about 10-folds higher antibody and IFN-gamma responses than Montanide formulations. CONCLUSIONS/SIGNIFICANCE: PfCS102 peptide was safe and highly immunogenic, allowing the design of more advanced trials to test its potential

  12. Synthetic peptides of actin-tropomyosin binding region of troponin I and heat shock protein 20 modulate the relaxation process of skinned preparations of taenia caeci from guinea pig.

    Science.gov (United States)

    Yoshino, Yasumasa; Sakurai, Wataru; Morimoto, Sachio; Watanabe, Masaru

    2005-12-01

    To explore the possible role of the thin filament-linked regulation of cross-bridge cycling in living smooth muscle contraction, we studied the effects of TnIp and HSP20p, a synthetic peptide originating from an actin tropomyosin binding region of rabbit cardiac troponin I (residues 136-147; GKFKRPTLRRVR), and that of human heat shock protein 20 (residues 110-121; GFVAREFHRRYR) on the relaxation of skinned (cell membrane ilized) preparations from guinea pig taenia caeci. An active stress of the skinned preparations, resulting from actin-myosin interaction, rapidly decayed following Ca(2+) removal (relaxation). TnIp accelerated the initial rapid phase and slowed the following slow phase of the relaxation. On the other hand, HSP20p only slowed the whole process of the relaxation. The relaxation time courses were well fitted in a double exponential manner, and the double exponential decay of the stress could be explained as a portion of fast-detaching cross bridges not to dissociate rapidly by Ca(2+) removal, but to transfer to latch bridges dissociating very slowly. Our present results suggested that (i) TnIp and HSP20p accelerated transferring from fast-detaching cross bridges to slow-detaching (latch) bridges, and (ii) TnIp accelerated dissociation of the fast-detaching cross bridges and the latch bridges, while HSP20p slowed dissociation the fast-detaching cross bridges. Since TnIp and HSP20p are thought to bind to actin and tropomyosin, but not to myosin, we concluded that through thin-filament-dependent mechanisms these peptides regulated the formation and/or deformation of latch bridges in smooth muscle. The thin-filament-dependent regulation might physiologically control the stress maintenance and relaxation in smooth muscle cells.

  13. Defensins and cystein rich peptides: two types of antimicrobial peptides in marine molluscs

    Directory of Open Access Journals (Sweden)

    G Arenas Díaz

    2010-06-01

    Full Text Available This review focuses on defensins and cystein rich peptides, which are the most abundant natural antimicrobial peptides (AMPs described in molluscs. These are compact peptides, 3-5 kDa in molecular mass, cationic and amphipatic; the presence of at least six cysteine residues forming three or four disulfide bridges is their prime structural characteristic. A 3-D structural characterization of these molecules has been included in recent investigations, using currently-available techniques. AMPs have been purified from hemocytes, epithelial tissue and plasma as well as cloned and chemically synthesized. Their antibacterial activity against Gram-positive and Gram-negative bacteria and fungi has been shown; only a synthetic mytilin fragment has displayed activity against viruses.

  14. Rapid Optimization of Mcl-1 Inhibitors using Stapled Peptide Libraries Including Non-Natural Side Chains.

    Science.gov (United States)

    Rezaei Araghi, Raheleh; Ryan, Jeremy A; Letai, Anthony; Keating, Amy E

    2016-05-20

    Alpha helices form a critical part of the binding interface for many protein-protein interactions, and chemically stabilized synthetic helical peptides can be effective inhibitors of such helix-mediated complexes. In particular, hydrocarbon stapling of peptides to generate constrained helices can improve binding affinity and other peptide properties, but determining the best stapled peptide variant often requires laborious trial and error. Here, we describe the rapid discovery and optimization of a stapled-helix peptide that binds to Mcl-1, an antiapoptotic protein that is overexpressed in many chemoresistant cancers. To accelerate discovery, we developed a peptide library synthesis and screening scheme capable of identifying subtle affinity differences among Mcl-1-binding stapled peptides. We used our method to sample combinations of non-natural amino-acid substitutions that we introduced into Mcl-1 inhibitors in the context of a fixed helix-stabilizing hydrocarbon staple that increased peptide helical content and reduced proteolysis. Peptides discovered in our screen contained surprising substitutions at sites that are conserved in natural binding partners. Library-identified peptide M3d is the most potent molecule yet tested for selectively triggering mitochondrial permeabilization in Mcl-1 dependent cell lines. Our library approach for optimizing helical peptide inhibitors can be readily applied to the study of other biomedically important targets.

  15. Bone induction by biomimetic PLGA-(PEG-ASP)n copolymer loaded with a novel synthetic BMP-2-related peptide in vitro and in vivo.

    Science.gov (United States)

    Lin, Zhen-Yu; Duan, Zhi-Xia; Guo, Xiao-Dong; Li, Jing-Feng; Lu, Hong-Wei; Zheng, Qi-Xin; Quan, Da-Ping; Yang, Shu-Hua

    2010-06-01

    BMP-2 is one of the most important growth factors of bone regeneration. Polylactide-co-glycolic acid (PLGA), which is used as a biodegradable scaffold for delivering therapeutic agents, has been intensively investigated. In previous studies, we synthesized a novel BMP-2-related peptide (designated P24) and found that it could enhance the osteoblastic differentiation of bone marrow stromal cells (BMSCs). The objective of this study was to construct a biomimetic composite by incorporating P24 into a modified PLGA-(PEG-ASP)n copolymer to promote bone formation. In vitro, our results demonstrated that PLGA-(PEG-ASP)n scaffolds were shown to be an efficient system for sustained release of P24. Significantly more BMSCs attached to the P24/PLGA-(PEG-ASP)n and PLGA-(PEG-ASP)n membranes than to PLGA, and the cells in the two groups subsequently proliferated more vigorously than those in the PLGA group. The expression of osteogenic markers in P24/PLGA-(PEG-ASP)n group was stronger than that in the PLGA-(PEG-ASP)n and PLGA groups. Radiographic and histological examination, Western blotting and RT-PCR showed that P24/PLGA-(PEG-ASP)n scaffold could induce more effective ectopic bone formation in vivo, as compared with PLGA-(PEG-ASP)n or gelatin sponge alone. It is concluded that the PLGA-(PEG-ASP)n copolymer is a good P24 carrier and can serve as a good scaffold for controlled release of P24. This novel P24/PLGA-(PEG-ASP)n composite promises to be an excellent biomaterial for inducing bone regeneration. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  16. Synthetic heparin-binding growth factor analogs

    Science.gov (United States)

    Pena, Louis A.; Zamora, Paul; Lin, Xinhua; Glass, John D.

    2007-01-23

    The invention provides synthetic heparin-binding growth factor analogs having at least one peptide chain that binds a heparin-binding growth factor receptor, covalently bound to a hydrophobic linker, which is in turn covalently bound to a non-signaling peptide that includes a heparin-binding domain. The synthetic heparin-binding growth factor analogs are useful as soluble biologics or as surface coatings for medical devices.

  17. Venom neutralization by purified bioactive molecules: Synthetic peptide derivatives of the endogenous PLA(2) inhibitory protein PIP (a mini-review).

    Science.gov (United States)

    Thwin, Maung-Maung; Samy, Ramar Perumal; Satyanarayanajois, Seetharama D; Gopalakrishnakone, Ponnampalam

    2010-12-15

    Envenomation due to snakebite constitutes a significant public health problem in tropical and subtropical countries. Antivenom therapy is still the mainstay of treatment for snake envenomation, and yet despite recent research focused on the prospects of using antivenom adjuncts to aid in serotherapy, no new products have emerged so far for therapeutic use. Various methodologies including molecular biology, crystallography, functional and morphological approaches, etc., are employed in the search for such inhibitors with a view to generate molecules that can stop partially or completely the activities of toxic phospholipase A(2) (PLA(2)) and snake venom metalloproteinase (SvMPs) enzymes at the molecular level. Herein, both natural and synthetic inhibitors derived from a variety of sources including medicinal plants, mammals, marine animals, fungi, bacteria, and from the venom and blood of snakes have been briefly reviewed. Attention has been focused on the snake serum-based phospholipase A(2) inhibitors (PLIs), particularly on the PLI derived from python snake serum (PIP), highlighting the potential of the natural product, PIP, or possible derivatives of it, as a complementary treatment to serotherapy against the inflammation and/or muscle-damaging activity of snake venoms. The data indicate a more efficient pathway for inhibition and blocking the activity of PLA(2)s and matrix metalloproteinases (MMPs), thus representing a feasible complementary treatment for snakebites. Such information may be helpful for interfering on the biological processes that these molecules are involved in human inflammatory-related diseases, and also for the development of new drugs for treatment of snake envenomation.

  18. 人工合成多肽FGL对神经细胞模型PC12细胞增殖与凋亡的影响%Effects of synthetic peptides FG loop on PC12 cells proliferation and apoptosis

    Institute of Scientific and Technical Information of China (English)

    付洪龙; 马学晓; 于腾波; 陈伯华; 李宁

    2011-01-01

    背景:FGL是NCAM的核心活性多肽片段,可直接作用于成纤维细胞生长因子受体1,激活NCAM的信号传导途径.目的:观察FGL人工合成多肽联合培养对PC12细胞增殖和凋亡的作用.方法:将培养的PC12细胞分为对照组和实验组,实验组预先加入1%的FGL多肽溶液.分别于培养1,3,5,7,9 d采用细胞计数试剂8法检测细胞增殖情况.将PC12细胞分为正常组、实验组和损伤组,损伤组加入H2O2刺激16 h.实验组加入H2O2与FGL人工合成多肽刺激16 h,流式细胞仪检测细胞凋亡,荧光定量PCR法检测PC12中的核转录因子κB mRNA表达.结果与结论:FGL人工合成多肽与PC12复合培养细胞生长良好,可明显促进PC12细胞的活性并且减低PC12 细胞凋亡并可明显降低凋亡模型中PC12细胞核转录因子κB基因的表达.说明FGL多肽可以明显促进PC12细胞增殖,并可以抑制PC12细胞凋亡.%BACKGROUND: FG loop (FGL) is a core active peptide fragment of neural cell adhesion molecule (NCAM), which can directly act on fibroblast growth factor receptor 1 (FGFR1) to activate NCAM signal pathway.OBJECTIVE: To observe the effects of synthetic peptides FGL on PC12 cells proliferation and apoptosis.METHODS: ①PC12 cells proliferation and apoptosis: The cultured PC12 cells were divided into control group and experiment group. The experimental group was added with 1% FGL peptide solution. The control group was pre-coated with poly-lysine plates. The cells were cultured 1, 3, 5, 7, 9 d respectively to detect cell proliferation by using Cell Counting Kit-8. ②PC12 apoptosis and nuclear factor kappa B mRNA detection: The PC12 cells were divided into normal group, experimental group and injury group. H2O2 was added into the injury group for 16 hours stimulation. In the experimental group, H2O2 and FGL were used for 16 hours stimulation. The cell apoptosis were detected by flow cytometry; mRNA expression of nuclear factor kappa B was detected by quantitative

  19. De novo sequencing of two novel peptides homologous to calcitonin-like peptides, from skin secretion of the Chinese Frog, Odorrana schmackeri

    Directory of Open Access Journals (Sweden)

    Geisa P.C. Evaristo

    2015-09-01

    Full Text Available An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues or CGRPs (37 amino acids and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.

  20. Synthesis of N-glyoxylyl peptides and their in vitro evaluation as HIV-1 protease inhibitors.

    Science.gov (United States)

    Qasmi, D; de Rosny, E; René, L; Badet, B; Vergely, I; Boggetto, N; Reboud-Ravaux, M

    1997-04-01

    A series of novel synthetic peptides containing an N-terminal glyoxylyl function (CHOCO-) have been tested as inhibitors of HIV-1 protease. The N-glyoxylyl peptide CHOCO-Pro-Ile-Val-NH2, which fulfills the specificity requirements of the MA/CA protease cleavage site together with the criteria of transition state analogue of the catalyzed reaction, was found to be a moderate competitive inhibitor although favorable interactions were visualized between its hydrated form and the catalytic aspartates using molecular modeling. Increasing the length of the peptide sequence led to compounds acting only as substrates.

  1. Generation of novel bone forming cells (monoosteophils from the cathelicidin-derived peptide LL-37 treated monocytes.

    Directory of Open Access Journals (Sweden)

    Zhifang Zhang

    Full Text Available BACKGROUND: Bone generation and maintenance involve osteoblasts, osteoclasts, and osteocytes which originate from unique precursors and rely on key growth factors for differentiation. However, an incomplete understanding of bone forming cells during wound healing has led to an unfilled clinical need such as nonunion of bone fractures. Since circulating monocytes are often recruited to sites of injury and may differentiate into various cell types including osteoclasts, we investigated the possibility that circulating monocytes in the context of tissue injury may also contribute to bone repair. In particular, we hypothesized that LL-37 (produced from hCAP-18, cathelicidin, which recruits circulating monocytes during injury, may play a role in bone repair. METHODS AND FINDINGS: Treatment of monocytes from blood with LL-37 for 6 days resulted in their differentiation to large adherent cells. Growth of LL-37-differentiated monocytes on osteologic discs reveals bone-like nodule formation by scanning electron microscopy (SEM. In vivo transplantation studies in NOD/SCID mice show that LL-37-differentiated monocytes form bone-like structures similar to endochondral bone formation. Importantly, LL-37-differentiated monocytes are distinct from conventional monocyte-derived osteoclasts, macrophages, and dendritic cells and do not express markers of the mesenchymal stem cells (MSC lineage, distinguishing them from the conventional precursors of osteoblasts. Furthermore, LL-37 differentiated monocytes express intracellular proteins of both the osteoblast and osteoclast lineage including osteocalcin (OC, osteonectin (ON, bone sialoprotein II (BSP II, osteopontin (OP, RANK, RANKL, MMP-9, tartrate resistant acid phosphatase (TRAP, and cathepsin K (CK. CONCLUSION: Blood derived monocytes treated with LL-37 can be differentiated into a novel bone forming cell that functions both in vitro and in vivo. We propose the name monoosteophil to indicate their monocyte

  2. Topical peptides as cosmeceuticals

    Directory of Open Access Journals (Sweden)

    Varadraj Vasant Pai

    2017-01-01

    Full Text Available Peptides are known to have diverse biological roles, most prominently as signaling/regulatory molecules in a broad variety of physiological processes including defense, immunity, stress, growth, homeostasis and reproduction. These aspects have been used in the field of dermatology and cosmetology to produce short, stable and synthetic peptides for extracellular matrix synthesis, pigmentation, innate immunity and inflammation. The evolution of peptides over the century, which started with the discovery of penicillin, has now extended to their usage as cosmeceuticals in recent years. Cosmeceutical peptides may act as signal modulators of the extracellular matrix component, as structural peptides, carrier peptides and neurotransmitter function modulators. Transdermal delivery of peptides can be made more effective by penetration enhancers, chemical modification or encapsulation of peptides. The advantages of using peptides as cosmeceuticals include their involvement in many physiological functions of the skin, their selectivity, their lack of immunogenicity and absence of premarket regulatory requirements for their use. However, there are disadvantages: clinical evidence for efficacy is often weak, absorption may be poor due to low lipophilicity, high molecular weight and binding to other ingredients, and prices can be quite high.

  3. Racemization of the Succinimide Intermediate Formed in Proteins and Peptides: A Computational Study of the Mechanism Catalyzed by Dihydrogen Phosphate Ion

    Directory of Open Access Journals (Sweden)

    Ohgi Takahashi

    2016-10-01

    Full Text Available In proteins and peptides, d-aspartic acid (d-Asp and d-β-Asp residues can be spontaneously formed via racemization of the succinimide intermediate formed from l-Asp and l-asparagine (l-Asn residues. These biologically uncommon amino acid residues are known to have relevance to aging and pathologies. Although nonenzymatic, the succinimide racemization will not occur without a catalyst at room or biological temperature. In the present study, we computationally investigated the mechanism of succinimide racemization catalyzed by dihydrogen phosphate ion, H2PO4−, by B3LYP/6-31+G(d,p density functional theory calculations, using a model compound in which an aminosuccinyl (Asu residue is capped with acetyl (Ace and NCH3 (Nme groups on the N- and C-termini, respectively (Ace–Asu–Nme. It was shown that an H2PO4− ion can catalyze the enolization of the Hα–Cα–C=O portion of the Asu residue by acting as a proton-transfer mediator. The resulting complex between the enol form and H2PO4− corresponds to a very flat intermediate region on the potential energy surface lying between the initial reactant complex and its mirror-image geometry. The calculated activation barrier (18.8 kcal·mol−1 after corrections for the zero-point energy and the Gibbs energy of hydration for the enolization was consistent with the experimental activation energies of Asp racemization.

  4. Racemization of the Succinimide Intermediate Formed in Proteins and Peptides: A Computational Study of the Mechanism Catalyzed by Dihydrogen Phosphate Ion

    Science.gov (United States)

    Takahashi, Ohgi; Kirikoshi, Ryota; Manabe, Noriyoshi

    2016-01-01

    In proteins and peptides, d-aspartic acid (d-Asp) and d-β-Asp residues can be spontaneously formed via racemization of the succinimide intermediate formed from l-Asp and l-asparagine (l-Asn) residues. These biologically uncommon amino acid residues are known to have relevance to aging and pathologies. Although nonenzymatic, the succinimide racemization will not occur without a catalyst at room or biological temperature. In the present study, we computationally investigated the mechanism of succinimide racemization catalyzed by dihydrogen phosphate ion, H2PO4−, by B3LYP/6-31+G(d,p) density functional theory calculations, using a model compound in which an aminosuccinyl (Asu) residue is capped with acetyl (Ace) and NCH3 (Nme) groups on the N- and C-termini, respectively (Ace–Asu–Nme). It was shown that an H2PO4− ion can catalyze the enolization of the Hα–Cα–C=O portion of the Asu residue by acting as a proton-transfer mediator. The resulting complex between the enol form and H2PO4− corresponds to a very flat intermediate region on the potential energy surface lying between the initial reactant complex and its mirror-image geometry. The calculated activation barrier (18.8 kcal·mol−1 after corrections for the zero-point energy and the Gibbs energy of hydration) for the enolization was consistent with the experimental activation energies of Asp racemization. PMID:27735868

  5. Collagen mimetic peptide discs promote assembly of a broad range of natural protein fibers through hydrophobic interactions.

    Science.gov (United States)

    McGuinness, Kenneth; Nanda, Vikas

    2017-07-19

    Collagen mimetic peptides that alone formed two-dimensional nanoscale discs driven by hydrophobic interactions were shown in electron microscopy studies to also co-assemble with natural fibrous proteins to produce discs-on-a-string (DoS) nanostructures. In most cases, peptide discs also facilitated bundling of the protein fibers. This provides insight into how synthetic and natural proteins may be combined to develop multicomponent, multi-dimensional architectures at the nanoscale.

  6. [Peptide synthesis aiming at elucidation and creation of protein functions].

    Science.gov (United States)

    Futaki, S

    1998-11-01

    The recent development of molecular biology has been elucidating outlines of the cross-talk of biomolecules. The understanding of the function of these biomolecules from the viewpoint of chemistry is now demanded not only for the understanding of biological systems but also for the creation of novel functional molecules. Here two topics are described about peptide synthesis aiming at the elucidation and the creation of protein functions. The first topic is the development of approaches for the synthesis of Tyr (SO3H)-containing peptides. Tyrosine sulfation is one of the most popular protein post-translational modifications. Synthetic peptides are of great help for the elucidation of the biological significance of tyrosine sulfation. We have developed two approaches for the efficient synthesis of tyrosine sulfate [Tyr (SO3H)]-containing peptides. The first approach employs a dimethylformamide-sulfur trioxide (DMF-SO3) complex as a sulfating agent and safety-catch protecting groups for the selective sulfation of tyrosine in the presence of serine. The second approach employs the direct introduction of Tyr(SO3H) into the peptide chain in the form of Fmoc-Tyr(SO3Na) followed by deprotection at 4 degrees C in trifluoroacetic acid. These approaches were successfully applied for the synthesis of cholecystokinin (CCK)-related peptides. The second topic deals with new approaches for the creation of artificial proteins through assembling alpha-helical peptides via selective disulfide or thioether formation. Approaches to assemble individual peptide segments on a peptide template were also developed. Four peptides corresponding to the transmembrane segments of the sodium channel (S4 in repeat I-IV) were assembled on a peptide template to give a protein having ion channel activity with rectification.

  7. A synthetic peptide derived from human immunodeficiency virus type 1 gp120 downregulates the expression and function of chemokine receptors CCR5 and CXCR4 in monocytes by activating the 7-transmembrane G-protein-coupled receptor FPRL1/LXA4R.

    Science.gov (United States)

    Deng, X; Ueda, H; Su, S B; Gong, W; Dunlop, N M; Gao, J L; Murphy, P M; Wang, J M

    1999-08-15

    Because envelope gp120 of various strains of human immunodeficiency virus type 1 (HIV-1) downregulates the expression and function of a variety of chemoattractant receptors through a process of heterologous desensitization, we investigated whether epitopes derived from gp120 could mimic the effect. A synthetic peptide domain, designated F peptide, corresponding to amino acid residues 414-434 in the V4-C4 region of gp120 of the HIV-1 Bru strain, potently reduced monocyte binding and chemotaxis response to macrophage inflammatory protein 1beta (MIP-1beta) and stromal cell-derived factor 1alpha (SDF-1alpha), chemokines that use the receptors CCR5 and CXCR4, respectively. Further study showed that F peptide by itself is an inducer of chemotaxis and calcium mobilization in human monocytes and neutrophils. In cross-desensitization experiments, among the numerous chemoattractants tested, only the bacterial chemotactic peptide fMLF, when used at high concentrations, partially attenuated calcium mobilization induced by F peptide in phagocytes, suggesting that this peptide domain might share a 7-transmembrane, G-protein-coupled receptor with fMLF. By using cells transfected with cDNAs encoding receptors that interact with fMLF, we found that F peptide uses an fMLF receptor variant, FPRL1, as a functional receptor. The activation of monocytes by F peptide resulted in downregulation of the cell surface expression of CCR5 and CXCR4 in a protein kinase C-dependent manner. These results demonstrate that activation of FPRL1 on human moncytes by a peptide domain derived from HIV-1 gp120 could lead to desensitization of cell response to other chemoattractants. This may explain, at least in part, the initial activation of innate immune responses in HIV-1-infected patients followed by immune suppression.

  8. A multiscale approach to characterize the early aggregation steps of the amyloid-forming peptide GNNQQNY from the yeast prion sup-35.

    Science.gov (United States)

    Nasica-Labouze, Jessica; Meli, Massimiliano; Derreumaux, Philippe; Colombo, Giorgio; Mousseau, Normand

    2011-05-01

    The self-organization of peptides into amyloidogenic oligomers is one of the key events for a wide range of molecular and degenerative diseases. Atomic-resolution characterization of the mechanisms responsible for the aggregation process and the resulting structures is thus a necessary step to improve our understanding of the determinants of these pathologies. To address this issue, we combine the accelerated sampling properties of replica exchange molecular dynamics simulations based on the OPEP coarse-grained potential with the atomic resolution description of interactions provided by all-atom MD simulations, and investigate the oligomerization process of the GNNQQNY for three system sizes: 3-mers, 12-mers and 20-mers. Results for our integrated simulations show a rich variety of structural arrangements for aggregates of all sizes. Elongated fibril-like structures can form transiently in the 20-mer case, but they are not stable and easily interconvert in more globular and disordered forms. Our extensive characterization of the intermediate structures and their physico-chemical determinants points to a high degree of polymorphism for the GNNQQNY sequence that can be reflected at the macroscopic scale. Detailed mechanisms and structures that underlie amyloid aggregation are also provided.

  9. A multiscale approach to characterize the early aggregation steps of the amyloid-forming peptide GNNQQNY from the yeast prion sup-35.

    Directory of Open Access Journals (Sweden)

    Jessica Nasica-Labouze

    2011-05-01

    Full Text Available The self-organization of peptides into amyloidogenic oligomers is one of the key events for a wide range of molecular and degenerative diseases. Atomic-resolution characterization of the mechanisms responsible for the aggregation process and the resulting structures is thus a necessary step to improve our understanding of the determinants of these pathologies. To address this issue, we combine the accelerated sampling properties of replica exchange molecular dynamics simulations based on the OPEP coarse-grained potential with the atomic resolution description of interactions provided by all-atom MD simulations, and investigate the oligomerization process of the GNNQQNY for three system sizes: 3-mers, 12-mers and 20-mers. Results for our integrated simulations show a rich variety of structural arrangements for aggregates of all sizes. Elongated fibril-like structures can form transiently in the 20-mer case, but they are not stable and easily interconvert in more globular and disordered forms. Our extensive characterization of the intermediate structures and their physico-chemical determinants points to a high degree of polymorphism for the GNNQQNY sequence that can be reflected at the macroscopic scale. Detailed mechanisms and structures that underlie amyloid aggregation are also provided.

  10. Synthetic Covalently Linked Dimeric Form of H2 Relaxin Retains Native RXFP1 Activity and Has Improved In Vitro Serum Stability

    Directory of Open Access Journals (Sweden)

    Vinojini B. Nair

    2015-01-01

    Full Text Available Human (H2 relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG7 linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin.

  11. Micropipette Technique Study of Natural and Synthetic Lung Surfactants at the Air-Water Interface: Presence of a SP-B Analog Peptide Promotes Membrane Aggregation, Formation of Tightly Stacked Lamellae, and Growth of Myelin Figures.

    Science.gov (United States)

    Parra, Elisa; Kinoshita, Koji; Needham, David

    2016-10-03

    The present study is a microscopic interfacial characterization of a series of lung surfactant materials performed with the micropipette technique. The advantages of this technique include the measurement of equilibrium and dynamic surface tensions while acquiring structural and dynamic information at microscopic air-water interfaces in real time and upon compression. Here, we characterized a series of animal-derived and synthetic lung surfactant formulations, including native surfactant obtained from porcine lungs (NS); the commercial Curosurf, Infasurf, and Survanta; and a synthetic Super Mini-B (SMB)-containing formulation. It was observed that the presence of the natural hydrophobic proteins and, more strikingly, the peptide SMB, promoted vesicle condensation as thick membrane stacks beneath the interface. Only in the presence of SMB, these stacks underwent spontaneous structural transformations, consisting of the nucleation and growth of microtubes and in some cases their subsequent coiling into helices. The dimensions of these tubes (2-15 μm diameter) and their linear (2-3 μm/s) and volumetric growth rates (20-30 μm(3)/s) were quantified, and no specific effects were found on them for increasing SMB concentrations from 0.1 to 4%. Nevertheless, a direct correlation between the number of tubes and SMB contents was found, suggesting that SMB molecules are the promoters of tube nucleation in these membranes. A detailed analysis of the tube formation process was performed following previous models for the growth of myelin figures, proposing a combined mechanism between dehydration-rehydration of the lipid bilayers and induction of mechanical defects by SMB that would act as nucleation sites for the tubes. The formation of tubes was also observed in Infasurf, and in NS only after subsequent expansion and compression but neither in the other clinical surfactants nor in protein-free preparations. Finally, the connection between this data and the observations from

  12. The Role of Antimicrobial Peptides in Preventing Multidrug-Resistant Bacterial Infections and Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Kyung-Soo Hahm

    2011-09-01

    Full Text Available Over the last decade, decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. Furthermore, biofilms, which are microbial communities that cause serious chronic infections and dental plaque, form environments that enhance antimicrobial resistance. As a result, there is a continuous search to overcome or control such problems, which has resulted in antimicrobial peptides being considered as an alternative to conventional drugs. Antimicrobial peptides are ancient host defense effector molecules in living organisms. These peptides have been identified in diverse organisms and synthetically developed by using peptidomimic techniques. This review was conducted to demonstrate the mode of action by which antimicrobial peptides combat multidrug-resistant bacteria and prevent biofilm formation and to introduce clinical uses of these compounds for chronic disease, medical devices, and oral health. In addition, combinations of antimicrobial peptides and conventional drugs were considered due to their synergetic effects and low cost for therapeutic treatment.

  13. Structural mimics of viruses through peptide/DNA co-assembly.

    Science.gov (United States)

    Ni, Rong; Chau, Ying

    2014-12-31

    A synthetic mimic of viral structure has been constructed by the synergistic co-assembly of a 16-amino acid peptide and plasmid DNA. The rational design of this short peptide, including segments for binding DNA and forming β-sheet, is inspired by viral capsid protein. The resulting nanostructures, which we term nanococoons, appear as ellipsoids of virus-like dimension (65 × 47 nm) and display repeating stripes of ∼4 nm wide. We propose that the co-assembly process involves DNA as a template to assist the organization of peptide strands by electrostatic interaction, while the bilayer β-sheets and their lateral association stabilize the peptide "capsid" and organize the DNA within. The hierarchy affords an extremely stable structure, protecting peptide and DNA against enzymatic digestion. It opens a new and facile avenue to fabricate viral alternatives with diverse functions.

  14. Antimicrobial effect of synthetic antimicrobial peptide on Candida Albicans in vitro%人工合成抗菌肽对白色念珠菌体外抑制作用的初步研究

    Institute of Scientific and Technical Information of China (English)

    孙倩; 李茵; 李金陆; 杨圣辉; 郑东翔

    2011-01-01

    Objective To evaluate the inhibiting effects of synthetic antimicrobial peptide (AMPs) on Candida albicans in vitro. Methods The effect of AMPs on Candida albicans was evaluated by K-B method. The inhibition of different dose of AMPs on Candida albibans was also detected by measuring the diameter of the inhibiting zones of Candida albicans. The minimal inhibitory concentration of AMP on Candida albicans was examined by cylinder-plate method. Results The AMPs had significant inhibiting effect on the Candida albicans. The paper with AMPs at concentrations of 307.2,153.6,76.8,38.4,19.2 and 9.6μg lead to the different diameter of inhibiting zone at 57.89 ± 1.36,37.44 ± 1.24,35.89 ± 1.69,30 ± 5.48,16.44 ± 1.01 and 10.11 ± 0.60mm. The diameter of inhibiting zone showed positive correlation with the dose of AMPs(P <0.01 ). The minimal inhibitory concentration of AMPs on Candida albicans was 128μg/ml. Conclusion AMPs had inhibiting effect on Candida albicans and the inhibition was in a dose-dependent manner.%目的 体外观察人工合成抗菌肽(synthetic antimicrobial peptide,AMPs)对白色念珠菌的抑制作用.方法 人工合成抗菌七肽并制备成含不同药物剂量的药敏纸片,通过纸片扩散法观察AMPs体外对白色念珠菌的抑制作用,测量含不同剂量AMPs的药敏纸片对白色念珠菌产生抑菌环的直径.同时应用杯碟法评价AMPs对白色念珠菌的最小抑菌浓度.结果 AMPs体外可对白色念珠菌产生抑制作用.含AMPs 307.2、153.6、76.8、38.4、19.2、9.6μg的药敏纸片对白色念珠菌产生的抑菌环直径分别为57.89±1.36、37.44±1.24、35.89±1.69、30±5.48、16.44 4-1.01、10.11±0.60mm,且抑菌环直径与药物剂量间具有正相关性(r=1,P<0.01).杯碟法显示AMPs对白色念珠菌的最小抑菌浓度为128μg/ml.结论 AMPs对白色念珠菌具有一定抑制作用,此种抑制作用随药物剂量的增加而增强.

  15. Amyloid-β(25-35) peptides aggregate into cross-β sheets in unsaturated anionic lipid membranes at high peptide concentrations.

    Science.gov (United States)

    Tang, Jennifer; Alsop, Richard J; Backholm, Matilda; Dies, Hannah; Shi, An-Chang; Rheinstädter, Maikel C

    2016-04-07

    One of the hallmarks of Alzheimer's disease is the formation of protein plaques in the brain, which mainly consist of amyloid-β peptides of different lengths. While the role of these plaques in the pathology of the disease is not clear, the mechanism behind peptide aggregation is a topic of intense research and discussion. Because of their simplicity, synthetic membranes are promising model systems to identify the elementary processes involved. We prepared unsaturated zwitterionic/anionic lipid membranes made of 1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine (DMPS) at concentrations of POPC/3 mol% DMPS containing 0 mol%, 3 mol%, 10 mol%, and 20 mol% amyloid-β25-35 peptides. Membrane-embedded peptide clusters were observed at peptide concentrations of 10 and 20 mol% with a typical cluster size of ∼11 μm. Cluster density increased with peptide concentration from 59 (±3) clusters per mm(2) to 920 (±64) clusters per mm(2), respectively. While monomeric peptides take an α-helical state when embedded in lipid bilayers at low peptide concentrations, the peptides in peptide clusters were found to form cross-β sheets and showed the characteristic pattern in X-ray experiments. The presence of the peptides was accompanied by an elastic distortion of the bilayers, which can induce a long range interaction between the peptides. The experimentally observed cluster patterns agree well with Monte Carlo simulations of long-range interacting peptides. This interaction may be the fundamental process behind cross-β sheet formation in membranes and these sheets may serve as seeds for further growth into amyloid fibrils.

  16. Clinical Research of Synthetic Antibacterial Peptide in Antibacterial Properties of Oral Bacteria%人工合成抗菌肽对口腔细菌抗菌性能的临床研究

    Institute of Scientific and Technical Information of China (English)

    刘晓丹

    2016-01-01

    菌肽对口腔细菌的抗菌性能不同,Temporin-1CEa的最小抑菌浓度较小,且抑菌效果佳。%Objective To investigate synthetic antibacterial peptide in antibacterial properties of oral bacteria.Methods 9 kinds of synthetic antibacterial peptide(Brevinin,chensinin-1,chensinin-1b,L-K5V1,L-K6,L-K6V1, L-K6V2 and temporin lcea,Temporin-1CEb)and 6 kinds of oral bacteria(Candida albicans,Fusobacterium nucleatum, Lactobacilus acidophilus,Streptococcus mutans,Streptococcus salivarius,Streptococcus sanguinis)were used to vitro experiment.Synthetic antibacterial peptide in antibacterial properties of oral bacteria was analyzed.Results Chensinin-1b,L-K5V1,L-K6,L-K6V1,L-K6V2,Temporin-1CEa had high inhibitory rate with Candida albicans.Brevinin, chensinin-1b,L-K5V1,L-K6,L-K6V1,L-K6V2,Temporin-1CEa,Temporin-1CEb had high inhibitory rate with Fusobacterium nucleatum,Streptococcus mutans,Streptococcus salivarius,Streptococcus sanguinis.Brevinin,chensinin-1,chensinin-1b, L-K5V1,L-K6,L-K6V1,L-K6V2,Temporin-1CEa,Temporin-1CEb had high inhibitory rate with Lactobacilus acidophilus. Minimum inhibitory concentrations of Brevinin:Streptococcus sanguinis>Fusobacterium nucleatum>Streptococcus salivarius>Streptococcus mutans>Lactobacilus acidophilus.Minimum inhibitory concentrations of chensinin-1: Lactobacilus acidophilus.Minimum inhibitory concentrations of chensinin-1b:Lactobacilus acidophilus>Streptococcus salivarius>Streptococcus sanguinis>Fusobacterium nucleatum>Candida albicans>Streptococcus mutans. Minimum inhibitory concentrations of L-K5V1:Lactobacilus acidophilus>Fusobacterium nucleatum>Streptococcus mutans>Candida albicans>Streptococcus salivarius>Streptococcus sanguinis.Minimum inhibitory concentrations of L-K6:Streptococcus salivarius>Streptococcus sanguinis>Candida albicans>Streptococcus mutans>Fusobacterium nucleatum>Lactobacilus acidophilus.Minimum inhibitory concentrations of L-K6V1:Lactobacilus acidophilus>Streptococcus sanguinis>Streptococcus salivarius

  17. Review: Production and functionality of active peptides from milk.

    Science.gov (United States)

    Muro Urista, C; Álvarez Fernández, R; Riera Rodriguez, F; Arana Cuenca, A; Téllez Jurado, A

    2011-08-01

    In recent years, research on the production of active peptides obtained from milk and their potential functionality has grown, to a great extent. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions, and they may ultimately have an influence on health. Individual proteins of casein or milk-derived products such as cheese and yogurt have been used as a protein source to study the isolation and activity of peptides with several applications. Currently, the milk whey waste obtained in the production of cheese also represents a protein source from which active peptides could be isolated with potential industrial applications. The active properties of milk peptides and the results found with regard to their physiological effects have led to the classification of peptides as belonging to the group of ingredients of protein nature, appropriate for use in functional foods or pharmaceutical formulations. In this study, the main peptides obtained from milk protein and the past research studies about its production and biological activities will be explained. Second, an analysis will be made on the methods to determinate the biological activities, the separation of bioactive peptides and its structure identification. All of these form the base required to obtain synthetic peptides. Finally, we explain the experimental animal and human trials done in the past years. Nevertheless, more research is required on the design and implementation of equipment for the industrial production and separation of peptides. In addition, different authors suggest that more emphasis should therefore be given to preclinical studies, proving that results are consistent and that effects are demonstrated repeatedly by several research human groups.

  18. Antimicrobial Properties of α-MSH and Related Synthetic Melanocortins

    Directory of Open Access Journals (Sweden)

    A. Catania

    2006-01-01

    Full Text Available The natural antimicrobial peptides are ancient host defense effector molecules, present in organisms across the evolutionary spectrum. Several properties of α-melanocyte stimulating hormone (α-MSH suggested that it could be a natural antimicrobial peptide. α-MSH is a primordial peptide that appeared during the Paleozoic era, long before adaptive immunity developed and, like natural antimicrobial molecules, is produced by barrier epithelia, immunocytes, and within the central nervous system. α-MSH was discovered to have antimicrobial activity against two representative pathogens, Staphylococcus aureus and Candida albicans. The candidacidal influences of α-MSH appeared to be mediated by increases in cell cyclic adenosine monophosphate (cAMP. The cAMP-inducing capacity of α-MSH likely interferes with the yeast's own regulatory mechanisms of this essential signaling pathway. It is remarkable that this mechanism of action in yeast mimics the influences of α-MSH in mammalian cells in which the peptide binds to G-protein-linked melanocortin receptors, activates adenylyl cyclase, and increases cAMP. When considering that most of the natural antimicrobial peptides enhance the local inflammatory reaction, the anti-inflammatory and antipyretic effects of α-MSH confer unique properties to this molecule relative to other natural antimicrobial molecules. Synthetic derivatives, chemically stable and resistant to enzymatic degradation, could form the basis for novel therapies that combine anti-inflammatory and antimicrobial properties.

  19. The membranotropic activity of N-terminal peptides from the pore-forming proteins sticholysin I and II is modulated by hydrophobic and electrostatic interactions as well as lipid composition

    Indian Academy of Sciences (India)

    Uris Ros; Lohans Pedrera; Daylín Díaz; Juan C De Karam; Tatiane P Sudbrack; Pedro A Valiente; Diana Martínez; Eduardo M Cilli; Fabiola Pazos; Rosangela Itri; Maria E Lanio; Shirley Schreier; Carlos Álvarez

    2011-12-01

    The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1–31 of St I (StI1-31) or 1–30 of St II (StII1-30) and demonstrated that StII1-30 promotes erythrocyte lysis to a higher extent than StI1-31. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1–10 hydrophobic sequence in StII1-30 for displaying higher binding and activity, in spite of both peptides’ abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities.

  20. Liquid-phase synthesis of bridged peptides using olefin metathesis of a protected peptide with a long aliphatic chain anchor.

    Science.gov (United States)

    Aihara, Keisuke; Komiya, Chiaki; Shigenaga, Akira; Inokuma, Tsubasa; Takahashi, Daisuke; Otaka, Akira

    2015-02-01

    Bridged peptides including stapled peptides are attractive tools for regulating protein-protein interactions (PPIs). An effective synthetic methodology in a heterogeneous system for the preparation of these peptides using olefin metathesis and hydrogenation of protected peptides with a long aliphatic chain anchor is reported.

  1. Solvent exposure of Tyr10 as a probe of structural differences between monomeric and aggregated forms of the amyloid-β peptide

    Science.gov (United States)

    Aran Terol, Pablo; Kumita, Janet R.; Hook, Sharon C.; Dobson, Christopher M.; Esbjörner, Elin K.

    2015-01-01

    Aggregation of amyloid-β (Aβ) peptides is a characteristic pathological feature of Alzheimer's disease. We have exploited the relationship between solvent exposure and intrinsic fluorescence of a single tyrosine residue, Tyr10, in the Aβ sequence to probe structural features of the monomeric, oligomeric and fibrillar forms of the 42-residue Aβ1-42. By monitoring the quenching of Tyr10 fluorescence upon addition of water-soluble acrylamide, we show that in Aβ1-42 oligomers this residue is solvent-exposed to a similar extent to that found in the unfolded monomer. By contrast, Tyr10 is significantly shielded from acrylamide quenching in Aβ1-42 fibrils, consistent with its proximity to the fibrillar cross-β core. Furthermore, circular dichroism measurements reveal that Aβ1-42 oligomers have a considerably lower β-sheet content than the Aβ1-42 fibrils, indicative of a less ordered molecular arrangement in the former. Taken together these findings suggest significant differences in the structural assembly of oligomers and fibrils that are consistent with differences in their biological effects. PMID:26551456

  2. Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2

    DEFF Research Database (Denmark)

    Blume, N; Madsen, O D; Kofod, Hans;

    1990-01-01

    Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did n...

  3. Antimicrobial peptides in human sepsis

    Directory of Open Access Journals (Sweden)

    Lukas eMartin

    2015-08-01

    Full Text Available Nearly 100 years ago, antimicrobial peptides (AMPs were identified as an important part of innate immunity. They exist in species from bacteria to mammals and can be isolated in body fluids and on surfaces constitutively or induced by inflammation. Defensins have anti-bacterial effects against Gram-positive and Gram-negative bacteria as well as anti-viral and anti-yeast effects. Human neutrophil peptides (HNP 1-3 and human beta-defensins (HBDs 1-3 are some of the most important defensins in humans. Recent studies have demonstrated higher levels of HNP -1-3 and HBD-2 in sepsis. The bactericidal/permeability increasing protein (BPI attenuates local inflammatory response and decreases systemic toxicity of endotoxins. Moreover, BPI might reflect the severity of organ dysfunction in sepsis. Elevated plasma lactoferrin is detected in patients with organ failure. HNP-1-3, lactoferrin, BPI and heparin-binding protein (HBP are increased in sepsis. Human lactoferrin peptide 1-11 (hLF 1-11 possesses antimicrobial activity and modulates inflammation. The recombinant form of lactoferrin (talactoferrin alpha, TLF has been shown to decrease mortality in critically ill patients. A phase II/III study with TLF in sepsis did not confirm this result. The growing number of multiresistant bacteria is an ongoing problem in sepsis therapy. Furthermore, antibiotics are known to promote the liberation of pro-inflammatory cell components and thus augment the severity of sepsis. Compared to antibiotics, AMPs kill bacteria but also neutralize pathogenic factors such as lipopolysaccharide (LPS. The obstacle to applying naturally occurring AMPs is their high nephro- and neurotoxicity. Therefore, the challenge is to develop peptides to treat septic patients effectively without causing harm. This overview focuses on natural and synthetic AMPs in human and experimental sepsis and their potential to provide significant improvements in the treatment of critically ill with severe

  4. Synthetic environments

    Science.gov (United States)

    Lukes, George E.; Cain, Joel M.

    1996-02-01

    The Advanced Distributed Simulation (ADS) Synthetic Environments Program seeks to create robust virtual worlds from operational terrain and environmental data sources of sufficient fidelity and currency to interact with the real world. While some applications can be met by direct exploitation of standard digital terrain data, more demanding applications -- particularly those support operations 'close to the ground' -- are well-served by emerging capabilities for 'value-adding' by the user working with controlled imagery. For users to rigorously refine and exploit controlled imagery within functionally different workstations they must have a shared framework to allow interoperability within and between these environments in terms of passing image and object coordinates and other information using a variety of validated sensor models. The Synthetic Environments Program is now being expanded to address rapid construction of virtual worlds with research initiatives in digital mapping, softcopy workstations, and cartographic image understanding. The Synthetic Environments Program is also participating in a joint initiative for a sensor model applications programer's interface (API) to ensure that a common controlled imagery exploitation framework is available to all researchers, developers and users. This presentation provides an introduction to ADS and the associated requirements for synthetic environments to support synthetic theaters of war. It provides a technical rationale for exploring applications of image understanding technology to automated cartography in support of ADS and related programs benefitting from automated analysis of mapping, earth resources and reconnaissance imagery. And it provides an overview and status of the joint initiative for a sensor model API.

  5. Advances in Synthetic Peptides Reagent Discovery

    Science.gov (United States)

    2013-07-01

    chemical or biological in nature, in air, food or water supplies is of critical to ensure the highest degree of survivability across the spectrum of...to promote specific and high affinity binding. Longer incubations may result in nonspecific attachment, such as early biofilm formation. Because

  6. Immunotherapy with Allergen Peptides

    Directory of Open Access Journals (Sweden)

    Larché Mark

    2007-06-01

    Full Text Available Specific allergen immunotherapy (SIT is disease-modifying and efficacious. However, the use of whole allergen preparations is associated with frequent allergic adverse events during treatment. Many novel approaches are being designed to reduce the allergenicity of immunotherapy preparations whilst maintaining immunogenicity. One approach is the use of short synthetic peptides which representing dominant T cell epitopes of the allergen. Short peptides exhibit markedly reduced capacity to cross link IgE and activate mast cells and basophils, due to lack of tertiary structure. Murine pre-clinical studies have established the feasibility of this approach and clinical studies are currently in progress in both allergic and autoimmune diseases.

  7. Self-assembly of a nine-residue amyloid-forming peptide fragment of SARS corona virus E-protein: mechanism of self aggregation and amyloid-inhibition of hIAPP.

    Science.gov (United States)

    Ghosh, Anirban; Pithadia, Amit S; Bhat, Jyotsna; Bera, Supriyo; Midya, Anupam; Fierke, Carol A; Ramamoorthy, Ayyalusamy; Bhunia, Anirban

    2015-04-01

    Molecular self-assembly, a phenomenon widely observed in nature, has been exploited through organic molecules, proteins, DNA, and peptides to study complex biological systems. These self-assembly systems may also be used in understanding the molecular and structural biology which can inspire the design and synthesis of increasingly complex biomaterials. Specifically, use of these building blocks to investigate protein folding and misfolding has been of particular value since it can provide tremendous insights into peptide aggregation related to a variety of protein misfolding diseases, or amyloid diseases (e.g., Alzheimer's disease, Parkinson's disease, type-II diabetes). Herein, the self-assembly of TK9, a nine-residue peptide of the extra membrane C-terminal tail of the SARS corona virus envelope, and its variants were characterized through biophysical, spectroscopic, and simulated studies, and it was confirmed that the structure of these peptides influences their aggregation propensity, hence, mimicking amyloid proteins. TK9, which forms a beta-sheet rich fibril, contains a key sequence motif that may be critical for beta-sheet formation, thus making it an interesting system to study amyloid fibrillation. TK9 aggregates were further examined through simulations to evaluate the possible intra- and interpeptide interactions at the molecular level. These self-assembly peptides can also serve as amyloid inhibitors through hydrophobic and electrophilic recognition interactions. Our results show that TK9 inhibits the fibrillation of hIAPP, a 37 amino acid peptide implicated in the pathology of type-II diabetes. Thus, biophysical and NMR experimental results have revealed a molecular level understanding of peptide folding events, as well as the inhibition of amyloid-protein aggregation are reported.

  8. Synthetic foldamers.

    Science.gov (United States)

    Guichard, Gilles; Huc, Ivan

    2011-06-07

    Foldamers are artificial folded molecular architectures inspired by the structures and functions of biopolymers. This highlight focuses on important developments concerning foldamers produced by chemical synthesis and on the perspectives that these new self-organized molecular scaffolds offer. Progress in the field has led to synthetic objects that resemble small proteins in terms of size and complexity yet that may not contain any α-amino acids. Foldamers have introduced new tools and concepts to develop biologically active substances, synthetic receptors and novel materials.

  9. Synthetic Phage for Tissue Regeneration

    Directory of Open Access Journals (Sweden)

    So Young Yoo

    2014-01-01

    Full Text Available Controlling structural organization and signaling motif display is of great importance to design the functional tissue regenerating materials. Synthetic phage, genetically engineered M13 bacteriophage has been recently introduced as novel tissue regeneration materials to display a high density of cell-signaling peptides on their major coat proteins for tissue regeneration purposes. Structural advantages of their long-rod shape and monodispersity can be taken together to construct nanofibrous scaffolds which support cell proliferation and differentiation as well as direct orientation of their growth in two or three dimensions. This review demonstrated how functional synthetic phage is designed and subsequently utilized for tissue regeneration that offers potential cell therapy.

  10. Peptide conjugation: before or after nanoparticle formation?

    Science.gov (United States)

    Valetti, Sabrina; Mura, Simona; Noiray, Magali; Arpicco, Silvia; Dosio, Franco; Vergnaud, Juliette; Desmaële, Didier; Stella, Barbara; Couvreur, Patrick

    2014-11-19

    We report herein a detailed study concerning the impact of different bioconjugation and nanoformulation strategies on the in vitro targeting ability of peptide-decorated squalenoyl gemcitabine (SQdFdC) nanoparticles (NPs). NPs have been functionalized with the CKAAKN peptide, previously identified as an efficient homing device within the pancreatic pathological microenvironment. Two approaches have been followed: (i) either the CKAAKN peptide was directly conjugated at the surface of preformed SQdFdC nanoparticles (conjugation after NP formation) or (ii) it was first reacted with a maleimide squalenoyl derivative before the resulting bioconjugate was co-nanoprecipitated with SQdFdC to form the peptide-decorated NPs (conjugation before NP formation). NPs were characterized with respect to mean diameter, zeta potential, and stability over time. Then, their specific interaction with the sFRP-4 protein was evaluated by surface plasmon resonance. Although both synthetic strategies allowed us to formulate NPs able to interact with the corresponding receptor, enhanced target binding and better specific avidity were observed with CKAAKN-NPs functionalized before NP formation. These NPs displayed the highest cell uptake and cytotoxicity in an in vitro model of human MIA Paca-2 pancreatic cancer cells.

  11. Synthetic Astrobiology

    Science.gov (United States)

    Rothschild, Lynn J.

    2016-01-01

    Synthetic biology - the design and construction of new biological parts and systems and the redesign of existing ones for useful purposes - has the potential to transform fields from pharmaceuticals to fuels. Our lab has focused on the potential of synthetic biology to revolutionize all three major parts of astrobiology: Where do we come from? Where are we going? and Are we alone? For the first and third, synthetic biology is allowing us to answer whether the evolutionary narrative that has played out on planet earth is likely to have been unique or universal. For example, in our lab we are re-evolving the biosynthetic pathways of amino acids in order to understand potential capabilities of an early organism with a limited repertoire of amino acids and developing techniques for the recovery of metals from spent electronics on other planetary bodies. And what about the limits for life? Can we create organisms that expand the envelope for life? In the future synthetic biology will play an increasing role in human activities both on earth, in fields as diverse as human health and the industrial production of novel bio-composites. Beyond earth, we will rely increasingly on biologically-provided life support, as we have throughout our evolutionary history. In order to do this, the field will build on two of the great contributions of astrobiology: studies of the origin of life and life in extreme environments.

  12. Synthetic Astrobiology

    Science.gov (United States)

    Rothschild, Lynn J.

    2015-01-01

    Synthetic biology - the design and construction of new biological parts and systems and the redesign of existing ones for useful purposes - has the potential to transform fields from pharmaceuticals to fuels. Our lab has focused on the potential of synthetic biology to revolutionize all three major parts of astrobiology: Where do we come from? Where are we going? and Are we alone? For the first and third, synthetic biology is allowing us to answer whether the evolutionary narrative that has played out on planet earth is likely to have been unique or universal. For example, in our lab we are re-evolving the biosynthetic pathways of amino acids in order to understand potential capabilities of an early organism with a limited repertoire of amino acids and developing techniques for the recovery of metals from spent electronics on other planetary bodies. In the future synthetic biology will play an increasing role in human activities both on earth, in fields as diverse as human health and the industrial production of novel bio-composites. Beyond earth, we will rely increasingly on biologically-provided life support, as we have throughout our evolutionary history. In order to do this, the field will build on two of the great contributions of astrobiology: studies of the origin of life and life in extreme environments.

  13. Epitope mapping of the NS4 and NS5 gene products of hepatitis C virus and the use of a chimeric NS4-NS5 synthetic peptide for serodiagnosis.

    Science.gov (United States)

    Rosa, C; Osborne, S; Garetto, F; Griva, S; Rivella, A; Calabresi, G; Guaschino, R; Bonelli, F

    1995-10-01

    Specific domains of the NS4 and NS5 gene products of hepatitis C virus have been identified using hydrophilicity profiles for the prediction of potential immunogenic regions, and epitope scanning techniques. Peptides synthesised on the basis of such data show excellent reactivity in the ELISA format. Introduction of a glycine-glycine spacer between two peptides (NS4-12 and NS5-44) to give a single chimeric peptides does not appear to impair immunoreactivity. An ELISA based on the chimeric peptide and a Core-NS3 recombinant protein correctly diagnoses a cohort of haemodialysed patients, three commercial HCV panels and the sera of a negative control population.

  14. Perspectives and Peptides of the Next Generation

    Science.gov (United States)

    Brogden, Kim A.

    Shortly after their discovery, antimicrobial peptides from prokaryotes and eukaryotes were recognized as the next potential generation of pharmaceuticals to treat antibiotic-resistant bacterial infections and septic shock, to preserve food, or to sanitize surfaces. Initial research focused on identifying the spectrum of antimicrobial agents, determining the range of antimicrobial activities against bacterial, fungal, and viral pathogens, and assessing the antimicrobial activity of synthetic peptides versus their natural counterparts. Subsequent research then focused on the mechanisms of antimicrobial peptide activity in model membrane systems not only to identify the mechanisms of antimicrobial peptide activity in microorganisms but also to discern differences in cytotoxicity for prokaryotic and eukaryotic cells. Recent, contemporary work now focuses on current and future efforts to construct hybrid peptides, peptide congeners, stabilized peptides, peptide conjugates, and immobilized peptides for unique and specific applications to control the growth of microorganisms in vitro and in vivo.

  15. A Convenient Synthetic Method of Metal Dendritic Porphyrins

    Institute of Scientific and Technical Information of China (English)

    Wen Bin CUI; Jie ZHOU; Lei CHEN; Xiao Bin DENG; Chun GUO

    2006-01-01

    A convenient synthetic method of metal dendritic porphyrins through the convergent synthetic strategy is described. The porphyrin core were linked with the synthetic fragments by forming ether or ester bonds to give five target compounds were prepared.

  16. Peptide binding specificity of the chaperone calreticulin

    DEFF Research Database (Denmark)

    Sandhu, N.; Duus, K.; Jorgensen, C.S.;

    2007-01-01

    Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length and composit......Calreticulin is a molecular chaperone with specificity for polypeptides and N-linked monoglucosylated glycans. In order to determine the specificity of polypeptide binding, the interaction of calreticulin with polypeptides was investigated using synthetic peptides of different length...... and composition. A large set of available synthetic peptides (n=127) was tested for binding to calreticulin and the results analysed by multivariate data analysis. The parameter that correlated best with binding was hydrophobicity while beta-turn potential disfavoured binding. Only hydrophobic peptides longer...... a peptide-binding specificity for hydrophobic sequences and delineate the fine specificity of calreticulin for hydrophobic amino acid residues....

  17. A synthetic amino acid residue containing a new oligopeptide-based photosensitive fluorescent organogel.

    Science.gov (United States)

    Maiti, Dibakar Kumar; Banerjee, Arindam

    2013-01-01

    A synthetic amino acid (with a stilbene residue in the main chain) containing a tripeptide-based organogelator has been discovered. This peptide-based synthetic molecule 1 self-assembles in various organic solvents to form an organogel. The gel has been thoroughly characterized by using various microscopic techniques including field-emission scanning electron microscopy (FESEM), atomic force microscopy (AFM), X-ray diffraction (XRD), UV-visible and fluorescence spectroscopy, and rheology. Morphological investigations using FESEM and AFM show a nanofibrillar network structure. Interestingly, the organogel is photoresponsive and a gel-sol transition occurred by irradiating the gel with UV light of 365 nm for 2 h as shown by the UV and fluorescence study. This photoresponsive fluorescent gel holds promise for new peptide-based soft materials with interesting applications.

  18. Improvement of an enzyme-linked immunosorbent assay for equine herpesvirus type 4 by using a synthetic-peptide 24-mer repeat sequence of glycoprotein G as an antigen.

    Science.gov (United States)

    Bannai, Hiroshi; Nemoto, Manabu; Tsujimura, Koji; Yamanaka, Takashi; Maeda, Ken; Kondo, Takashi

    2016-02-01

    To increase the sensitivity of an enzyme-linked immunosorbent assay (ELISA) for equine herpesvirus type 4 (EHV-4) that uses a 12-mer peptide of glycoprotein G (gG4-12-mer: MKNNPIYSEGSL) [4], we used a longer peptide consisting of a 24-mer repeat sequence (gG4-24-mer: MKNNPIYSEGSLMLNVQHDDSIHT) as an antigen. Sera of horses experimentally infected with EHV-4 reacted much more strongly to the gG4-24-mer peptide than to the gG4-12-mer peptide. We used peptide ELISAs to test paired sera from horses naturally infected with EHV-4 (n=40). gG4-24-mer ELISA detected 37 positive samples (92.5%), whereas gG4-12-mer ELISA detected only 28 (70.0%). gG4-24-mer ELISA was much more sensitive than gG4-12-mer ELISA.

  19. Identification and characterization of novel host defense peptides from the skin secretion of the fungoid frog, Hydrophylax bahuvistara (Anura: Ranidae).

    Science.gov (United States)

    Vineeth Kumar, Thundi Parambil Vasanth Kumar; Asha, Radhamony; Shyla, Gopal; George, Sanil

    2017-01-10

    Two novel peptides (brevinin1 HYba1 and brevinin1 HYba2) were identified from the skin secretion of the frog Hydrophylax bahuvistara, endemic to Western Ghats, India, and their amino acid sequences were confirmed using cDNA cloning and LC/MS/MS. Antibacterial, hemolytic, and cytotoxic activities of brevinin1 peptides and their synthetic analogs (amidated C-terminus) were investigated and compared. All the peptides except the acidic forms showed antibacterial activity against all tested Gram-positive and Gram-negative bacteria. They exhibited low hemolysis on human erythrocytes and showed potent cytotoxic activity against Hep 3B cancer cell line. Upon amidation, the peptides showed increased activity against the tested microbes without altering their hemolytic and cytotoxic properties. The study also emphasizes the need for screening endemic amphibian fauna of Western Ghats, as a potential source of host defense peptides with possible therapeutic applications in the future.

  20. Synthetic chromosomes.

    Science.gov (United States)

    Schindler, Daniel; Waldminghaus, Torsten

    2015-11-01

    What a living organism looks like and how it works and what are its components-all this is encoded on DNA, the genetic blueprint. Consequently, the way to change an organism is to change its genetic information. Since the first pieces of recombinant DNA have been used to transform cells in the 1970s, this approach has been enormously extended. Bigger and bigger parts of the genetic information have been exchanged or added over the years. Now we are at a point where the construction of entire chromosomes becomes a reachable goal and first examples appear. This development leads to fundamental new questions, for example, about what is possible and desirable to build or what construction rules one needs to follow when building synthetic chromosomes. Here we review the recent progress in the field, discuss current challenges and speculate on the appearance of future synthetic chromosomes.

  1. Mammalian peptide isomerase: platypus-type activity is present in mouse heart.

    Science.gov (United States)

    Koh, Jennifer M S; Chow, Stephanie J P; Crossett, Ben; Kuchel, Philip W

    2010-06-01

    Male platypus (Ornithorhynchus anatinus) venom has a peptidyl aminoacyl L/D-isomerase (hereafter called peptide isomerase) that converts the second amino acid residue in from the N-terminus from the L- to the D-form, and vice versa. A reversed-phase high-performance liquid chromatography (RP-HPLC) assay has been developed to monitor the interconversion using synthetic hexapeptides derived from defensin-like peptide-2 (DLP-2) and DLP-4 as substrates. It was hypothesised that animals other than the platypus would have peptide isomerase with the same substrate specificity. Accordingly, eight mouse tissues were tested and heart was shown to have the activity. This is notable for being the first evidence of a peptide isomerase being present in a higher mammal and heralds finding the activity in man.

  2. Synthetic River Valleys

    Science.gov (United States)

    Brown, R.; Pasternack, G. B.

    2011-12-01

    The description of fluvial form has evolved from anecdotal descriptions to artistic renderings to 2D plots of cross section or longitudinal profiles and more recently 3D digital models. Synthetic river valleys, artificial 3D topographic models of river topography, have a plethora of potential applications in fluvial geomorphology, and the earth sciences in general, as well as in computer science and ecology. Synthetic river channels have existed implicitly since approximately the 1970s and can be simulated from a variety of approaches spanning the artistic and numerical. An objective method of synthesizing 3D stream topography based on reach scale attributes would be valuable for sizing 3D flumes in the physical and numerical realms, as initial input topography for morphodynamic models, stream restoration design, historical reconstruction, and mechanistic testing of interactions of channel geometric elements. Quite simply - simulation of synthetic channel geometry of prescribed conditions can allow systematic evaluation of the dominant relationships between river flow and geometry. A new model, the control curve method, is presented that uses hierarchically scaled parametric curves in over-lapping 2D planes to create synthetic river valleys. The approach is able to simulate 3D stream geometry from paired 2D descriptions and can allow experimental insight into form-process relationships in addition to visualizing past measurements of channel form that are limited to two dimension descriptions. Results are presented that illustrate the models ability to simulate fluvial topography representative of real world rivers as well as how channel geometric elements can be adjusted. The testing of synthetic river valleys would open up a wealth of knowledge as to why some 3D attributes of river channels are more prevalent than others as well as bridging the gap between the 2D descriptions that have dominated fluvial geomorphology the past century and modern, more complete, 3D

  3. Antimicrobial activity of a new synthetic peptide loaded in polylactic acid or poly(lactic-co-glycolic) acid nanoparticles against Pseudomonas aeruginosa, Escherichia coli O157:H7 and methicillin resistant Staphylococcus aureus (MRSA)

    Science.gov (United States)

    Cruz, J.; Flórez, J.; Torres, R.; Urquiza, M.; Gutiérrez, J. A.; Guzmán, F.; Ortiz, C. C.

    2017-03-01

    Nanocarrier systems are currently being developed for peptide, protein and gene delivery to protect them in the blood circulation and in the gastrointestinal tract. Polylactic acid (PLA) and poly(lactic-co-glycolic) acid (PLGA) nanoparticles loaded with a new antimicrobial GIBIM-P5S9K peptide were obtained by the double emulsion solvent extraction/evaporation method. PLA- and PLGA-NPs were spherical with sizes between 300 and 400 nm for PLA and 200 and 300 nm for PLGA and 20 mV. The peptide-loading efficiency of PLA-NP and PLGA-NPs was 75% and 55%, respectively. PLA- and PLGA-NPs released around 50% of this peptide over 8 h. In 10% human sera the size of peptide loaded PLA- and PLGA-NPs increased between 25.2% and 39.3%, the PDI changed from 3.2 to 5.1 and the surface charge from ‑7.15 to 14.6 mV. Both peptide loaded PLA- and PLGA-NPs at 0.5 μM peptide concentration inhibited the growth of Escherichia coli O157:H7 (E. coli O157:H7), methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas. aeruginosa (P. aeruginosa). In contrast, free peptide inhibited at 10 μM but did not inhibit at 0.5 and 1 μM. These PLA- and PLGA-NPs presented <10% hemolysis indicating that they are hemocompatible and promising for delivery and protection system of GIBIM-P5S9K peptide.

  4. Peptides as new smart bionanomaterials: molecular-recognition and self-assembly capabilities.

    Science.gov (United States)

    Sawada, Toshiki; Mihara, Hisakazu; Serizawa, Takeshi

    2013-04-01

    Biomolecules express exquisite properties that are required for molecular recognition and self-assembly on the nanoscale. These smart capabilities have developed through evolution and such biomolecules operate based on smart functions in natural systems. Recently, these remarkable smart capabilities have been utilized in not only biologically related fields, but also in materials science and engineering. A peptide-screening technology that uses phage-display systems has been developed based on this natural smart evolution for the generation of new functional peptide bionanomaterials. We focused on peptides that specifically bound to synthetic polymers. These polymer-binding peptides were screened by using a phage-display peptide library to recognize nanostructures that were derived from polymeric structural features and were utilized for possible applications as new bionanomaterials. We also focused on self-assembling peptides with β-sheet structures that formed nanoscale, fibrous structures for applications in new bottom-up nanomaterials. Moreover, nanofiber-binding peptides were also screened to introduce the desired functionalities into nanofibers without the need for additional molecular design. Our approach to construct new bionanomaterials that employ peptides will open up excellent opportunities for the next generation of materials science and technology. Copyright © 2013 The Chemical Society of Japan and Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. The crystal structures of the calcium-bound con-G and con-T[K7gamma] dimeric peptides demonstrate a metal-dependent helix-forming motif.

    Science.gov (United States)

    Cnudde, Sara E; Prorok, Mary; Dai, Qiuyun; Castellino, Francis J; Geiger, James H

    2007-02-14

    Short peptides that have the ability to form stable alpha-helices in solution are rare, and a number of strategies have been used to produce them, including the use of metal chelation to stabilize folding of the backbone. However, no example exists of a structurally well-defined helix stabilized exclusively through metal ion chelation. Conantokins (con)-G and -T are short peptides that are potent antagonists of N-methyl-D-aspartate receptor channels. While con-G exhibits no helicity alone, it undergoes a structural transition to a helical conformation in the presence of a variety of multivalent cations, especially Mg2+ and Ca2+. This complexation also results in antiparallel dimerization of two peptide helices in the presence of Ca2+, but not Mg2+. A con-T variant, con-T[K7gamma], displays very similar behavior. We have solved the crystal structures of both Ca2+/con-G and Ca2+/con-T [K7gamma] at atomic resolution. These structures clearly show the nature of the metal-dependent dimerization and helix formation and surprisingly also show that the con-G dimer interface is completely different from the con-T[K7gamma] interface, even though the metal chelation is similar in the two peptides. This represents a new paradigm in helix stabilization completely independent of the hydrophobic effect, which we define as the "metallo-zipper."

  6. Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells

    Directory of Open Access Journals (Sweden)

    Ringhieri P

    2017-01-01

    Full Text Available Paola Ringhieri,1 Silvia Mannucci,2 Giamaica Conti,2 Elena Nicolato,2 Giulio Fracasso,3 Pasquina Marzola,4 Giancarlo Morelli,1 Antonella Accardo1 1Department of Pharmacy and Interuniversity Research Centre on Bioactive Peptides (CIRPeB, University of Naples “Federico II”, Napoli, 2Department of Neurological Biomedical and Movement Sciences, 3Section of Immunology, Department of Medicine, 4Department of Informatics, University of Verona, Verona, Italy Abstract: Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd] have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2 receptors. Derivatization of liposomal surface with targeting peptides is achieved using the postmodification method: the alkyne-peptide derivative Pra-KCCYSL reacts, through click chemistry procedures, with a synthetic surfactant modified with 1, 2, or 4 azido moieties previously inserted in liposome formulation. Preliminary in vitro data on MDA-MB-231 and BT-474 cells indicated that liposomes functionalized with P6.1 peptide in its tetrameric form had better binding to and uptake into BT-474 cells compared to liposomes decorated with monomeric or dimeric versions of the P6.1 peptide. BT-474 cells treated with liposomes functionalized with the tetrameric form of P6.1 showed high degree of liposome uptake, which was comparable with the uptake of anti-HER-2 antibodies such as Herceptin. Moreover, magnetic MRI experiments have demonstrated the potential of liposomes to act as MRI contrast agents. Keywords: anti-HER2 liposomes, target peptide, KCCYSL peptide, breast cancer, click chemistry, branched peptides 

  7. Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes.

    Science.gov (United States)

    Hirose, Yukihiro; Nagahori, Hirohisa; Yamada, Tomoya; Deguchi, Yoshihito; Tomigahara, Yoshitaka; Nishioka, Kazuhiko; Uwagawa, Satoshi; Kawamura, Satoshi; Isobe, Naohiko; Lake, Brian G; Okuno, Yasuyoshi

    2009-04-05

    High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 microM MTF and 50 microM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2'-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10-1000 microM MTF and 100-500 microM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5-50 ng/ml epidermal growth factor or 5-100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.

  8. Synthetic Cannabinoids.

    Science.gov (United States)

    Mills, Brooke; Yepes, Andres; Nugent, Kenneth

    2015-07-01

    Synthetic cannabinoids (SCBs), also known under the brand names of "Spice," "K2," "herbal incense," "Cloud 9," "Mojo" and many others, are becoming a large public health concern due not only to their increasing use but also to their unpredictable toxicity and abuse potential. There are many types of SCBs, each having a unique binding affinity for cannabinoid receptors. Although both Δ-tetrahydrocannabinol (THC) and SCBs stimulate the same receptors, cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), studies have shown that SCBs are associated with higher rates of toxicity and hospital admissions than is natural cannabis. This is likely due to SCBs being direct agonists of the cannabinoid receptors, whereas THC is a partial agonist. Furthermore, the different chemical structures of SCBs found in Spice or K2 may interact in unpredictable ways to elicit previously unknown, and the commercial products may have unknown contaminants. The largest group of users is men in their 20s who participate in polydrug use. The most common reported toxicities with SCB use based on studies using Texas Poison Control records are tachycardia, agitation and irritability, drowsiness, hallucinations, delusions, hypertension, nausea, confusion, dizziness, vertigo and chest pain. Acute kidney injury has also been strongly associated with SCB use. Treatment mostly involves symptom management and supportive care. More research is needed to identify which contaminants are typically found in synthetic marijuana and to understand the interactions between different SBCs to better predict adverse health outcomes.

  9. Toxin Inhibition - Deconvolution Strategies and Assay Screening of Combinatorial Peptide Libraries

    Science.gov (United States)

    2007-08-01

    to micellar electrokinetic chromatography (MEKC) [34, 35, 36]. At 50 mM concentration SDS forms micelles; the negatively charged polar sulfate head...synthetic derivatives of peptide hormones by capillary zone electrophoresis and micellar electrokinetic chromatography with ultraviolet-absorption and...BoNT MI Mean inhibition MEKC Micellar electrokinetic chromatography SNAP-25 Synapotsomal associated Protein 25 kDa S, P1, P2 Substrate and product I

  10. Natural - synthetic - artificial!

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    The terms "natural," "synthetic" and "artificial" are discussed in relation to synthetic and artificial chromosomes and genomes, synthetic and artificial cells and artificial life.......The terms "natural," "synthetic" and "artificial" are discussed in relation to synthetic and artificial chromosomes and genomes, synthetic and artificial cells and artificial life....

  11. Pore forming polyalkylpyridinium salts from marine sponges versus synthetic lipofection systems: distinct tools for intracellular delivery of cDNA and siRNA

    Directory of Open Access Journals (Sweden)

    Blagbrough Ian S

    2006-01-01

    Full Text Available Abstract Background Haplosclerid marine sponges produce pore forming polyalkylpyridinium salts (poly-APS, which can be used to deliver macromolecules into cells. The aim of this study was to investigate the delivery of DNA, siRNA and lucifer yellow into cells mediated by poly-APS and its potential mechanisms as compared with other lipofection systems (lipofectamine and N4,N9-dioleoylspermine (LipoGen. DNA condensation was evaluated and HEK 293 and HtTA HeLa cells were used to investigate pore formation and intracellular delivery of cDNA, siRNA and lucifer yellow. Results Poly-APS and LipoGen were both found to be highly efficient DNA condensing agents. Fura-2 calcium imaging was used to measure calcium transients indicative of cell membrane pore forming activity. Calcium transients were evoked by poly-APS but not LipoGen and lipofectamine. The increases in intracellular calcium produced by poly-APS showed temperature sensitivity with greater responses being observed at 12°C compared to 21°C. Similarly, delivery of lucifer yellow into cells with poly-APS was enhanced at lower temperatures. Transfection with cDNA encoding for the expression enhanced green fluorescent protein was also evaluated at 12°C with poly-APS, lipofectamine and LipoGen. Intracellular delivery of siRNA was achieved with knockdown in beta-actin expression when lipofectamine and LipoGen were used as transfection reagents. However, intracellular delivery of siRNA was not achieved with poly-APS. Conclusion Poly-APS mediated pore formation is critical to its activity as a transfection reagent, but lipofection systems utilise distinct mechanisms to enable delivery of DNA and siRNA into cells.

  12. The tachykinin peptide neurokinin B binds copper forming an unusual [CuII(NKB)2] complex and inhibits copper uptake into 1321N1 astrocytoma cells.

    Science.gov (United States)

    Russino, Debora; McDonald, Elle; Hejazi, Leila; Hanson, Graeme R; Jones, Christopher E

    2013-10-16

    Neurokinin B (NKB) is a member of the tachykinin family of neuropeptides that have neuroinflammatory, neuroimmunological, and neuroprotective functions. In a neuroprotective role, tachykinins can help protect cells against the neurotoxic processes observed in Alzheimer's disease. A change in copper homeostasis is a clear feature of Alzheimer's disease, and the dysregulation may be a contributory factor in toxicity. Copper has recently been shown to interact with neurokinin A and neuropeptide γ and can lead to generation of reactive oxygen species and peptide degradation, which suggests that copper may have a place in tachykinin function and potentially misfunction. To explore this, we have utilized a range of spectroscopic techniques to show that NKB, but not substance P, can bind Cu(II) in an unusual [Cu(II)(NKB)2] neutral complex that utilizes two N-terminal amine and two imidazole nitrogen ligands (from each molecule of NKB) and the binding substantially alters the structure of the peptide. Using 1321N1 astrocytoma cells, we show that copper can enter the cells and subsequently open plasma membrane calcium channels but when bound to neurokinin B copper ion uptake is inhibited. This data suggests a novel role for neurokinin B in protecting cells against copper-induced calcium changes and implicates the peptide in synaptic copper homeostasis.

  13. Synthetic Brainbows

    KAUST Repository

    Wan, Y.

    2013-06-01

    Brainbow is a genetic engineering technique that randomly colorizes cells. Biological samples processed with this technique and imaged with confocal microscopy have distinctive colors for individual cells. Complex cellular structures can then be easily visualized. However, the complexity of the Brainbow technique limits its applications. In practice, most confocal microscopy scans use different florescence staining with typically at most three distinct cellular structures. These structures are often packed and obscure each other in rendered images making analysis difficult. In this paper, we leverage a process known as GPU framebuffer feedback loops to synthesize Brainbow-like images. In addition, we incorporate ID shuffing and Monte-Carlo sampling into our technique, so that it can be applied to single-channel confocal microscopy data. The synthesized Brainbow images are presented to domain experts with positive feedback. A user survey demonstrates that our synthetic Brainbow technique improves visualizations of volume data with complex structures for biologists.

  14. A novel alkaloid antioxidant, Boldine and synthetic antioxidant, reduced form of RU486, inhibit the oxidation of LDL in-vitro and atherosclerosis in vivo in LDLR(-/-) mice.

    Science.gov (United States)

    Santanam, N; Penumetcha, M; Speisky, H; Parthasarathy, S

    2004-04-01

    A corollary to the oxidation hypothesis of atherosclerosis is that the consumption of antioxidants is beneficial. However, the literature is divided in support of this conclusion. In this study, Boldine, an alkaloid of Peumus boldus and reduced form of RU486, was tested for their antioxidant potency both in, in vitro oxidation system and in mouse models. Boldine decreased the ex-vivo oxidation of low-density lipoprotein (LDL). Two different in vivo studies were performed to study the effect of these compounds on the atherosclerotic lesion formation in LDLR(-/-) mice. In study I, three groups of LDLR(-/-) mice (N = 12 each) were fed an atherogenic diet. Group 1 was given vehicle and group 2 and 3 were given 1mg of Boldine or Red RU per day for 12 weeks. In study II, two groups of LDLR(-/-) mice N = 10 each) were fed an atherogenic diet. Group 1 was given vehicle and group 2 was given 5mg of Boldine per day. The results indicated that there was a decrease in lesion formation reaching a 40% reduction due to Boldine and 45% reduction by Red RU compared to controls. The in vivo tolerance of Boldine in humans (has been used as an herbal medicine in other diseases) should make it an attractive alternative to Vitamin E.

  15. Chlorination of bromide-containing waters: enhanced bromate formation in the presence of synthetic metal oxides and deposits formed in drinking water distribution systems.

    Science.gov (United States)

    Liu, Chao; von Gunten, Urs; Croué, Jean-Philippe

    2013-09-15

    Bromate formation from the reaction between chlorine and bromide in homogeneous solution is a slow process. The present study investigated metal oxides enhanced bromate formation during chlorination of bromide-containing waters. Selected metal oxides enhanced the decay of hypobromous acid (HOBr), a requisite intermediate during the oxidation of bromide to bromate, via (i) disproportionation to bromate in the presence of nickel oxide (NiO) and cupric oxide (CuO), (ii) oxidation of a metal to a higher valence state in the presence of cuprous oxide (Cu2O) and (iii) oxygen formation by NiO and CuO. Goethite (α-FeOOH) did not enhance either of these pathways. Non-charged species of metal oxides seem to be responsible for the catalytic disproportionation which shows its highest rate in the pH range near the pKa of HOBr. Due to the ability to catalyze HOBr disproportionation, bromate was formed during chlorination of bromide-containing waters in the presence of CuO and NiO, whereas no bromate was detected in the presence of Cu2O and α-FeOOH for analogous conditions. The inhibition ability of coexisting anions on bromate formation at pH 8.6 follows the sequence of phosphate > sulfate > bicarbonate/carbonate. A black deposit in a water pipe harvested from a drinking water distribution system exerted significant residual oxidant decay and bromate formation during chlorination of bromide-containing waters. Energy dispersive spectroscopy (EDS) analyses showed that the black deposit contained copper (14%, atomic percentage) and nickel (1.8%, atomic percentage). Cupric oxide was further confirmed by X-ray diffraction (XRD). These results indicate that bromate formation may be of concern during chlorination of bromide-containing waters in distribution systems containing CuO and/or NiO. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells

    Science.gov (United States)

    Ringhieri, Paola; Mannucci, Silvia; Conti, Giamaica; Nicolato, Elena; Fracasso, Giulio; Marzola, Pasquina; Morelli, Giancarlo; Accardo, Antonella

    2017-01-01

    Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd]) have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide) sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2) receptors. Derivatization of liposomal surface with targeting peptides is achieved using the postmodification method: the alkyne-peptide derivative Pra-KCCYSL reacts, through click chemistry procedures, with a synthetic surfactant modified with 1, 2, or 4 azido moieties previously inserted in liposome formulation. Preliminary in vitro data on MDA-MB-231 and BT-474 cells indicated that liposomes functionalized with P6.1 peptide in its tetrameric form had better binding to and uptake into BT-474 cells compared to liposomes decorated with monomeric or dimeric versions of the P6.1 peptide. BT-474 cells treated with liposomes functionalized with the tetrameric form of P6.1 showed high degree of liposome uptake, which was comparable with the uptake of anti-HER-2 antibodies such as Herceptin. Moreover, magnetic MRI experiments have demonstrated the potential of liposomes to act as MRI contrast agents. PMID:28144135

  17. An Electrospray Ionization Mass Spectrometry Study on the "In Vacuo" Hetero-Oligomers Formed by the Antimicrobial Peptides, Surfactin and Gramicidin S

    Science.gov (United States)

    Rautenbach, Marina; Vlok, N. Maré; Eyéghé-Bickong, Hans A.; van der Merwe, Marthinus J.; Stander, Marietjie A.

    2017-08-01

    It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS Srf↔GS+Srf correlated to apparent K d values of 6-9 μM in gas-phase and 1 μM in aqueous solution. The apparent K d values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low μM K d app values of GS Srf↔GS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na+ nor Ca2+ had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca2+ and Na+ from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation that depend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment.

  18. Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form.

    Science.gov (United States)

    Qian, Wen-Jian; Lai, Christopher C; Kelley, James A; Burke, Terrence R

    2014-05-01

    The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  19. Synthetic Astrobiology

    Science.gov (United States)

    Rothschild, Lynn J.

    2017-01-01

    "Are we alone?" is one of the primary questions of astrobiology, and whose answer defines our significance in the universe. Unfortunately, this quest is hindered by the fact that we have only one confirmed example of life, that of earth. While this is enormously helpful in helping to define the minimum envelope for life, it strains credulity to imagine that life, if it arose multiple times, has not taken other routes. To help fill this gap, our lab has begun using synthetic biology - the design and construction of new biological parts and systems and the redesign of existing ones for useful purposes - as an enabling technology. One theme, the "Hell Cell" project, focuses on creating artificial extremophiles in order to push the limits for Earth life, and to understand how difficult it is for life to evolve into extreme niches. In another project, we are re-evolving biotic functions using only the most thermodynamically stable amino acids in order to understand potential capabilities of an early organism with a limited repertoire of amino acids.

  20. Liposome entrapment and immunogenic studies of a synthetic lipophilic multiple antigenic peptide bearing VP1 and VP3 domains of the hepatitis A virus: a robust method for vaccine design.

    Science.gov (United States)

    Haro, Isabel; Pérez, Silvia; García, Mónica; Chan, Weng C; Ercilla, Guadalupe

    2003-04-10

    Multiple antigen peptides (MAP) have been demonstrated to be efficient immunological reagents for the induction of immune responses to a variety of infectious agents. Several peptide domains of the hepatitis A virus (HAV) capsid proteins, mainly VP1 and VP3, are the immunodominant targets for a protective antibody response. In the present study we analyse the immunogenic properties of a tetrameric heterogeneous palmitoyl-derivatised MAP containing two defined HAV peptide sequences, VP1(11-25) and VP3(102-121), in rabbits immunised with either Freund's adjuvant or multilamellar liposomes. The immune response was evaluated with a specific enzyme immunoassay using MAP[VP1+VP3], VP1 and VP3 as targets. The avidity of the immune response was measured by a non-competitive enzyme-linked immunosorbent assay and by the surface plasmon resonance technology. Antisera raised against the lipo-MAP peptide entrapped in liposomes demonstrated high avidity of binding with affinity rate constants approximately one order of magnitude greater than those obtained with the Freund's protocol.

  1. Treatment and prevention of Staphylococcus epidermidis experimental biomaterial-associated infection by bactericidal peptide 2

    NARCIS (Netherlands)

    P.H.S. Kwakman; A.A.T. Velde; C.M.J.E. Vandenbroucke-Grauls; S.J.H. van Deventer; S.A.J. Zaat

    2006-01-01

    Biomaterial-associated infections (BAI) are the major cause of failure of indwelling medical devices and are predominantly caused by staphylococci, especially Staphylococcus epidermidis. We investigated the in vitro microbicidal activity of the synthetic antimicrobial peptide bactericidal peptide 2

  2. Pro-opiomelanocortin-derived peptides in the pig pituitary: alpha- and gamma 1-melanocyte-stimulating hormones and their glycine-extended forms

    DEFF Research Database (Denmark)

    Fenger, M

    1988-01-01

    )-related material consisted of the glycine-extended intermediate ACTH-(1-14) and analogs. In contrast only one fourth to one third of the N-terminal part of POMC (N-POMC) was processed to amidated gamma-MSH and its C-terminal glycine-extended precursor. The relative amount of amidated gamma-MSH was the same...... as alpha-MSH and analogs (94%). However, more than 95% of these peptides were of high molecular weight. In the anterior lobe 2.3% of N-POMC was processed and 94% was amidated gamma-MSH of only high molecular weight. These results show that gamma-MSH and alpha-MSH are amidated to the same extent...... and that gamma 1-MSH and gamma 2-MSH immunoreactivity are present in both the anterior lobe and the neurointermediate lobe. The results suggest that the production of amidated peptides is not regulated by the amidation process itself but at an earlier step (e.g. at the proteolytic cleavage)....

  3. A linker peptide with high affinity towards silica-containing materials.

    Science.gov (United States)

    Sunna, Anwar; Chi, Fei; Bergquist, Peter L

    2013-06-25

    A peptide sequence with affinity to silica-containing materials was fused to a truncated form of Streptococcus strain G148 Protein G. The resulting recombinant Linker-Protein G (LPG) was produced in Escherichia coli and purified to apparent homogeneity. It displayed high affinity towards two natural clinoptilolite zeolites. The LPG also displayed high binding affinity towards commercial-grade synthetic zeolite, silica and silica-containing materials. A commercial sample of the truncated Protein G and a basic protein, both without the linker, did not bind to natural or synthetic zeolites or silica. We conclude that the zeolite-binding affinity is mediated by the linker peptide sequence. As a consequence, these data may imply that the binding affinity is directed to the SiO2 component rather than to the atomic orientation on the zeolite crystal surface as previously assumed.

  4. Modulation of autoimmunity with artificial peptides

    Science.gov (United States)

    La Cava, Antonio

    2010-01-01

    The loss of immune tolerance to self antigens leads to the development of autoimmune responses. Since self antigens are often multiple and/or their sequences may not be known, one approach to restore immune tolerance uses synthetic artificial peptides that interfere or compete with self peptides in the networks of cellular interactions that drive the autoimmune process. This review describes the rationale behind the use of artificial peptides in autoimmunity and their mechanisms of action. Examples of use of artificial peptides in preclinical studies and in the management of human autoimmune diseases are provided. PMID:20807590

  5. Therapeutical Potential of Venom Peptides

    Directory of Open Access Journals (Sweden)

    İlker Kelle

    2006-01-01

    Full Text Available The term of pharmazooticals is known as a few amount of drugs derived from natural sources such as plants, venomous species of snakes, spiders, scorpions, frogs, lizards and cone snails. Peptide components of venoms are directed against wide variety of pharmacological targets such as ion channels and receptors. At the beginning, a number of these peptides have been used in experimental studies for defining the physiological, biochemical and immunological activities of organisms like mammalians. In recent studies, it has been shown that venom peptides can be valuable in treatment of acute and chronic pain, autoimmune and cardiovascular diseases, neurological disorders and chronic inflammatory and tumoral processes. Therefore particularly in clinical approaches, these peptide molecules or their synthetic analogues are considered as alternative agents that can be used instead of classical drugs for many clinical disorders due to their potent activity besides very few side effects.

  6. DNA recognition by synthetic constructs.

    Science.gov (United States)

    Pazos, Elena; Mosquera, Jesús; Vázquez, M Eugenio; Mascareñas, José L

    2011-09-05

    The interaction of transcription factors with specific DNA sites is key for the regulation of gene expression. Despite the availability of a lar