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Sample records for acidic amphipathic peptides

  1. Interaction of 18-residue peptides derived from amphipathic helical ...

    Indian Academy of Sciences (India)

    Madhsudhan

    interaction of proteins and peptides with membranes (Segrest ... favour surface activity have been described by Eisenberg et ... amphipathicity and propensity for α-helical conformation ..... Membrane destabilisation occurs due to electrostatic.

  2. Peptides, polypeptides and peptide-polymer hybrids as nucleic acid carriers.

    Science.gov (United States)

    Ahmed, Marya

    2017-10-24

    Cell penetrating peptides (CPPs), and protein transduction domains (PTDs) of viruses and other natural proteins serve as a template for the development of efficient peptide based gene delivery vectors. PTDs are sequences of acidic or basic amphipathic amino acids, with superior membrane trespassing efficacies. Gene delivery vectors derived from these natural, cationic and cationic amphipathic peptides, however, offer little flexibility in tailoring the physicochemical properties of single chain peptide based systems. Owing to significant advances in the field of peptide chemistry, synthetic mimics of natural peptides are often prepared and have been evaluated for their gene expression, as a function of amino acid functionalities, architecture and net cationic content of peptide chains. Moreover, chimeric single polypeptide chains are prepared by a combination of multiple small natural or synthetic peptides, which imparts distinct physiological properties to peptide based gene delivery therapeutics. In order to obtain multivalency and improve the gene delivery efficacies of low molecular weight cationic peptides, bioactive peptides are often incorporated into a polymeric architecture to obtain novel 'polymer-peptide hybrids' with improved gene delivery efficacies. Peptide modified polymers prepared by physical or chemical modifications exhibit enhanced endosomal escape, stimuli responsive degradation and targeting efficacies, as a function of physicochemical and biological activities of peptides attached onto a polymeric scaffold. The focus of this review is to provide comprehensive and step-wise progress in major natural and synthetic peptides, chimeric polypeptides, and peptide-polymer hybrids for nucleic acid delivery applications.

  3. Structure-activity relationship study of Aib-containing amphipathic helical peptide-cyclic RGD conjugates as carriers for siRNA delivery.

    Science.gov (United States)

    Wada, Shun-Ichi; Takesada, Anna; Nagamura, Yurie; Sogabe, Eri; Ohki, Rieko; Hayashi, Junsuke; Urata, Hidehito

    2017-12-15

    The conjugation of Aib-containing amphipathic helical peptide with cyclo(-Arg-Gly-Asp-d-Phe-Cys-) (cRGDfC) at the C-terminus of the helix peptide (PI) has been reported to be useful for constructing a carrier for targeted siRNA delivery into cells. In order to explore structure-activity relationships for the development of potential carriers for siRNA delivery, we synthesized conjugates of Aib-containing amphipathic helical peptide with cRGDfC at the N-terminus (PII) and both the N- and C-termini (PIII) of the helical peptide. Furthermore, to examine the influence of PI helical chain length on siRNA delivery, truncated peptides containing 16 (PIV), 12 (PV), and 8 (PVI) amino acid residues at the N-terminus of the helical chain were synthesized. PII and PIII, as well as PI, could deliver anti-luciferase siRNA into cells to induce the knockdown of luciferase stably expressed in cells. In contrast, all of the truncated peptides were unlikely to transport siRNA into cells. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2018-02-01

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

  5. Comparative mode of action of novel hybrid peptide CS-1a and its rearranged amphipathic analogue CS-2a.

    Science.gov (United States)

    Joshi, Seema; Bisht, Gopal S; Rawat, Diwan S; Maiti, Souvik; Pasha, Santosh

    2012-10-01

    Cell selective, naturally occurring, host defence cationic peptides present a good template for the design of novel peptides with the aim of achieving a short length with improved antimicrobial potency and selectivity. A novel, short peptide CS-1a (14 residues) was derived using a sequence hybridization approach on sarcotoxin I (39 residues) and cecropin B (35 residues). The sequence of CS-1a was rearranged to enhance amphipathicity with the help of a Schiffer-Edmundson diagram to obtain CS-2a. Both peptides showed good antibacterial activity in the concentration range 4-16 μg·mL(-1) against susceptible as well as drug-resistant bacterial strains, including the clinically relevant pathogens Acenatobacter sp. and methicillin-resistant Staphylococcus aureus. The major thrust of these peptides is their nonhaemolytic activity against human red blood cells up to a high concentration of 512 μg·mL(-1). Compared to CS-1a, amphipathic peptide CS-2a showed a more pronounced α-helical conformation, along with a better membrane insertion depth in bacterial mimic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) small unilamellar vesicles. With equivalent lipid-binding affinity, the two peptides assumed different pathways of membrane disruption, as demonstrated by calcein leakage and the results of transmission electron microscopy on model bacterial mimic large unilamellar vesicles. Extending the work from model membranes to intact Escherichia coli cells, differences in membrane perturbation were visible in microscopic images of peptide-treated E. coli. The present study describes two novel short peptides with potent activity, cell selectivity and divergent modes of action that will aid in the future design of peptides with better therapeutic potential. © 2012 The Authors Journal compilation © 2012 FEBS.

  6. In Vivo Imaging of the Stability and Sustained Cargo Release of an Injectable Amphipathic Peptide-Based Hydrogel.

    Science.gov (United States)

    Oyen, Edith; Martin, Charlotte; Caveliers, Vicky; Madder, Annemieke; Van Mele, Bruno; Hoogenboom, Richard; Hernot, Sophie; Ballet, Steven

    2017-03-13

    Hydrogels are promising materials for biomedical applications such as tissue engineering and controlled drug release. In the past two decades, the peptide hydrogel subclass has attracted an increasing level of interest from the scientific community because of its numerous advantages, such as biocompatibility, biodegradability, and, most importantly, injectability. Here, we report on a hydrogel consisting of the amphipathic hexapeptide H-FEFQFK-NH 2 , which has previously shown promising in vivo properties in terms of releasing morphine. In this study, the release of a small molecule, a peptide, and a protein cargo as representatives of the three major drug classes is directly visualized by in vivo fluorescence and nuclear imaging. In addition, the in vivo stability of the peptide hydrogel system is investigated through the use of a radiolabeled hydrogelator sequence. Although it is shown that the hydrogel remains present for several days, the largest decrease in volume takes place within the first 12 h of subcutaneous injection, which is also the time frame wherein the cargos are released. Compared to the situation in which the cargos are injected in solution, a prolonged release profile is observed up to 12 h, showing the potential of our hydrogel system as a scaffold for controlled drug delivery. Importantly, this study elucidates the release mechanism of the peptide hydrogel system that seems to be based on erosion of the hydrogel providing a generally applicable controlled release platform for small molecule, peptide, and protein drugs.

  7. Regulator of differentiation 1 (ROD1) binds to the amphipathic C-terminal peptide of thrombospondin-4 and is involved in its mitogenic activity.

    Science.gov (United States)

    Sadvakassova, Gulzhakhan; Dobocan, Monica C; Difalco, Marcos R; Congote, Luis F

    2009-09-01

    The matrix protein thrombospondin-4 has an acidic amphipathic C-terminal peptide (C21) which stimulates erythroid cell proliferation. Here we show that C21 stimulates red cell formation in anemic mice in vivo. In vitro experiments indicated that the peptide-mediated increase of erythroid colony formation in cultures of human CD34+ hematopoietic progenitor cells was possible only under continuous presence of erythropoietin. In the absence of this cytokine, C21 stimulated exclusively myeloid colony formation. Therefore, the peptide is not a specific erythroid differentiation factor. In fact, it is mitogenic in non-erythroid cells, such as skin fibroblasts and kidney epithelial cells. In erythroleukemic TF-1 cells, it actually decreased the production of the erythroid differentiation marker glycophorin A. C21-affinity chromatography revealed regulator of differentiation 1 (ROD1) as a major C21-binding protein. ROD1 is the hematopoietic cell paralog of polypyrimidine tract binding proteins (PTBs), RNA splice regulators which regulate differentiation by repressing tissue-specific exons. ROD1 binding to C21 was strongly inhibited by synthetic RNAs in the order poly A > poly U > poly G = poly C and was weakly inhibited by a synthetic phosphorylated peptide mimicking the C-terminal domain of RNA polymerase II. Cellular overexpression or knockdown experiments of ROD1 suggest a role for this protein in the mitogenic activity of C21. Since the nuclear proteins ROD1 and PTBs regulate differentiation at a posttranscriptional level and there is a fast nuclear uptake of C21, we put forward the idea that the peptide is internalized, goes to the nucleus and maintains cells in a proliferative state by supporting ROD1-mediated inhibition of differentiation.

  8. A non-covalent peptide-based carrier for in vivo delivery of DNA mimics

    OpenAIRE

    Morris, May C.; Gros, Edwige; Aldrian-Herrada, Gudrun; Choob, Michael; Archdeacon, John; Heitz, Frederic; Divita, Gilles

    2007-01-01

    The dramatic acceleration in identification of new nucleic-acid-based therapeutic molecules has provided new perspectives in pharmaceutical research. However, their development is limited by their poor cellular uptake and inefficient trafficking. Here we describe a short amphipathic peptide, Pep-3, that combines a tryptophan/phenylalanine domain with a lysine/arginine-rich hydrophilic motif. Pep-3 forms stable nano-size complexes with peptide-nucleic acid analogues and promotes their efficien...

  9. Anti-plasmodial action of de novo-designed, cationic, lysine-branched, amphipathic, helical peptides

    Directory of Open Access Journals (Sweden)

    Kaushik Naveen K

    2012-08-01

    Full Text Available Abstract Background A lack of vaccine and rampant drug resistance demands new anti-malarials. Methods In vitro blood stage anti-plasmodial properties of several de novo-designed, chemically synthesized, cationic, amphipathic, helical, antibiotic peptides were examined against Plasmodium falciparum using SYBR Green assay. Mechanistic details of anti-plasmodial action were examined by optical/fluorescence microscopy and FACS analysis. Results Unlike the monomeric decapeptides {(Ac-GXRKXHKXWA-NH2 (X = F,ΔF (Fm, ΔFm IC50 >100 μM}, the lysine-branched,dimeric versions showed far greater potency {IC50 (μM Fd 1.5 , ΔFd 1.39}. The more helical and proteolytically stable ΔFd was studied for mechanistic details. ΔFq, a K-K2 dendrimer of ΔFm and (ΔFm2 a linear dimer of ΔFm showed IC50 (μM of 0.25 and 2.4 respectively. The healthy/infected red cell selectivity indices were >35 (ΔFd, >20 (ΔFm2 and 10 (ΔFq. FITC-ΔFd showed rapid and selective accumulation in parasitized red cells. Overlaying DAPI and FITC florescence suggested that ΔFd binds DNA. Trophozoites and schizonts incubated with ΔFd (2.5 μM egressed anomalously and Band-3 immunostaining revealed them not to be associated with RBC membrane. Prematurely egressed merozoites from peptide-treated cultures were found to be invasion incompetent. Conclusion Good selectivity (>35, good resistance index (1.1 and low cytotoxicity indicate the promise of ΔFd against malaria.

  10. Neutron diffraction studies of amphipathic helices in phospholipid bilayers

    Energy Technology Data Exchange (ETDEWEB)

    Bradshaw, J.P.; Gilchrist, P.J. [Univ. of Edinburgh (United Kingdom); Duff, K.C. [Univ. of Edinburgh Medical School (United Kingdom); Saxena, A.M. [Brookhaven National Laboratory, Upton, NY (United States)

    1994-12-31

    The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt to investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein.

  11. Neutron diffraction studies of amphipathic helices in phospholipid bilayers

    International Nuclear Information System (INIS)

    Bradshaw, J.P.; Gilchrist, P.J.; Duff, K.C.; Saxena, A.M.

    1994-01-01

    The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt to investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein

  12. Recurrent Neural Network Model for Constructive Peptide Design.

    Science.gov (United States)

    Müller, Alex T; Hiss, Jan A; Schneider, Gisbert

    2018-02-26

    We present a generative long short-term memory (LSTM) recurrent neural network (RNN) for combinatorial de novo peptide design. RNN models capture patterns in sequential data and generate new data instances from the learned context. Amino acid sequences represent a suitable input for these machine-learning models. Generative models trained on peptide sequences could therefore facilitate the design of bespoke peptide libraries. We trained RNNs with LSTM units on pattern recognition of helical antimicrobial peptides and used the resulting model for de novo sequence generation. Of these sequences, 82% were predicted to be active antimicrobial peptides compared to 65% of randomly sampled sequences with the same amino acid distribution as the training set. The generated sequences also lie closer to the training data than manually designed amphipathic helices. The results of this study showcase the ability of LSTM RNNs to construct new amino acid sequences within the applicability domain of the model and motivate their prospective application to peptide and protein design without the need for the exhaustive enumeration of sequence libraries.

  13. Cell-penetrating recombinant peptides for potential use in agricultural pest control applications.

    Science.gov (United States)

    Hughes, Stephen R; Dowd, Patrick F; Johnson, Eric T

    2012-09-28

    Several important areas of interest intersect in a class of peptides characterized by their highly cationic and partly hydrophobic structure. These molecules have been called cell-penetrating peptides (CPPs) because they possess the ability to translocate across cell membranes. This ability makes these peptides attractive candidates for delivery of therapeutic compounds, especially to the interior of cells. Compounds with characteristics similar to CPPs and that, in addition, have antimicrobial properties are being investigated as antibiotics with a reduced risk of causing resistance. These CPP-like membrane-acting antimicrobial peptides (MAMPs) are α-helical amphipathic peptides that interact with and perturb cell membranes to produce their antimicrobial effects. One source of MAMPs is spider venom. Because these compounds are toxic to insects, they also show promise for development as biological agents for control of insecticide-resistant agricultural pests. Spider venom is a potential source of novel insect-specific peptide toxins. One example is the small amphipathic α-helical peptide lycotoxin-1 (Lyt-1 or LCTX) from the wolf spider (Lycosa carolinensis). One side of the α-helix has mostly hydrophilic and the other mainly hydrophobic amino acid residues. The positive charge of the hydrophilic side interacts with negatively charged prokaryotic membranes and the hydrophobic side associates with the membrane lipid bilayer to permeabilize it. Because the surface of the exoskeleton, or cuticle, of an insect is highly hydrophobic, to repel water and dirt, it would be expected that amphipathic compounds could permeabilize it. Mutagenized lycotoxin 1 peptides were produced and expressed in yeast cultures that were fed to fall armyworm (Spodoptera frugiperda) larvae to identify the most lethal mutants. Transgenic expression of spider venom toxins such as lycotoxin-1 in plants could provide durable insect resistance.

  14. Cysteine-containing peptides having antioxidant properties

    Science.gov (United States)

    Bielicki, John K [Castro Valley, CA

    2008-10-21

    Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I.sub.Milano (R173C) and apoA-I.sub.Paris, (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

  15. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  16. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  17. Helical 1:1 α/Sulfono-γ-AA Heterogeneous Peptides with Antibacterial Activity

    Energy Technology Data Exchange (ETDEWEB)

    She, Fengyu; Nimmagadda, Alekhya; Teng, Peng; Su, Ma; Zuo, Xiaobing; Cai, Jianfeng

    2016-05-09

    As one of the greatest threats facing in 21st century, antibiotic resistance is now a major public health concern. Host-defense peptides (HDPs) offer an alternative approach to combat emerging multidrug-resistant bacteria. It is known that helical HDPs such as magainin 2 and its analogs adopt cationic amphipathic conformations upon interaction with bacterial membranes, leading to membrane disruption and subsequent bacterial cell death. We have previously shown that amphipathic sulfono-γ-AApeptides could mimic magainin 2 and exhibit bactericidal activity. In this article, we demonstrate for the first time that amphipathic helical 1:1 α/sulfono-γ-AA heterogeneous peptides, in which regular amino acids and sulfono-γ-AApeptide building blocks are alternatively present in a 1:1 pattern, display potent antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens. Small Angle X-ray Scattering (SAXS) suggests that the lead sequences adopt defined helical structures. The subsequent studies including 2 fluorescence microscopy and time-kill experiments indicate that these hybrid peptides exert antimicrobial activity by mimicking the mechanism of HDPs. Our findings may lead to the development of HDP-mimicking antimicrobial peptidomimetics that combat drug-resistant bacterial pathogens. In addition, our results also demonstrate the effective design of a new class of helical foldamer, which could be employed to interrogate other important biological targets such as protein-protein interactions in the future.

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

    Science.gov (United States)

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

    2004-04-15

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

  19. Roles of d-Amino Acids on the Bioactivity of Host Defense Peptides

    Directory of Open Access Journals (Sweden)

    Hao Li

    2016-06-01

    Full Text Available Host defense peptides (HDPs are positively-charged and amphipathic components of the innate immune system that have demonstrated great potential to become the next generation of broad spectrum therapeutic agents effective against a vast array of pathogens and tumor. As such, many approaches have been taken to improve the therapeutic efficacy of HDPs. Amongst these methods, the incorporation of d-amino acids (d-AA is an approach that has demonstrated consistent success in improving HDPs. Although, virtually all HDP review articles briefly mentioned about the role of d-AA, however it is rather surprising that no systematic review specifically dedicated to this topic exists. Given the impact that d-AA incorporation has on HDPs, this review aims to fill that void with a systematic discussion of the impact of d-AA on HDPs.

  20. Double-Stranded Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2001-01-01

    A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker.......A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  1. Coexistence of a two-states organization for a cell-penetrating peptide in lipid bilayer.

    Science.gov (United States)

    Plénat, Thomas; Boichot, Sylvie; Dosset, Patrice; Milhiet, Pierre-Emmanuel; Le Grimellec, Christian

    2005-12-01

    Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P(alpha), a primary amphipathic cell-penetrating peptide which remains alpha-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P(alpha) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P(alpha) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P(alpha). The simultaneous two-states organization of P(alpha), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.

  2. Designing anticancer peptides by constructive machine learning.

    Science.gov (United States)

    Grisoni, Francesca; Neuhaus, Claudia; Gabernet, Gisela; Müller, Alex; Hiss, Jan; Schneider, Gisbert

    2018-04-21

    Constructive machine learning enables the automated generation of novel chemical structures without the need for explicit molecular design rules. This study presents the experimental application of such a generative model to design membranolytic anticancer peptides (ACPs) de novo. A recurrent neural network with long short-term memory cells was trained on alpha-helical cationic amphipathic peptide sequences and then fine-tuned with 26 known ACPs. This optimized model was used to generate unique and novel amino acid sequences. Twelve of the peptides were synthesized and tested for their activity on MCF7 human breast adenocarcinoma cells and selectivity against human erythrocytes. Ten of these peptides were active against cancer cells. Six of the active peptides killed MCF7 cancer cells without affecting human erythrocytes with at least threefold selectivity. These results advocate constructive machine learning for the automated design of peptides with desired biological activities. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

  4. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  5. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  6. The signal peptide anchors apolipoprotein M in plasma lipoproteins and prevents rapid clearance of apolipoprotein M from plasma

    DEFF Research Database (Denmark)

    Christoffersen, Christina; Ahnström, Josefin; Axler, Olof

    2008-01-01

    Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic alpha-helixes and beta-sheets. Plasma apolipoprotein (apo) M lacks an external amphipathic motif but, nevertheless, is exclusively associated with lipoproteins...... (mainly high density lipoprotein). Uniquely, however, apoM is secreted to plasma without cleavage of its hydrophobic NH(2)-terminal signal peptide. To test whether the signal peptide serves as a lipoprotein anchor for apoM in plasma, we generated mice expressing a mutated apoM(Q22A) cDNA in the liver (apoM......(Q22A)-Tg mice (transgenic mice)) and compared them with mice expressing wild-type human apoM (apoM-Tg mice). The substitution of the amino acid glutamine 22 with alanine in apoM(Q22A) results in secretion of human apoM without a signal peptide. The human apoM mRNA level in liver and the amount...

  7. IFITM3 requires an amphipathic helix for antiviral activity.

    Science.gov (United States)

    Chesarino, Nicholas M; Compton, Alex A; McMichael, Temet M; Kenney, Adam D; Zhang, Lizhi; Soewarna, Victoria; Davis, Matthew; Schwartz, Olivier; Yount, Jacob S

    2017-10-01

    Interferon-induced transmembrane protein 3 (IFITM3) is a cellular factor that blocks virus fusion with cell membranes. IFITM3 has been suggested to alter membrane curvature and fluidity, though its exact mechanism of action is unclear. Using a bioinformatic approach, we predict IFITM3 secondary structures and identify a highly conserved, short amphipathic helix within a hydrophobic region of IFITM3 previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for the IFITM3-dependent inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections. The homologous amphipathic helix within IFITM1 is also required for the inhibition of infection, indicating that IFITM proteins possess a conserved mechanism of antiviral action. We further demonstrate that the amphipathic helix of IFITM3 is required to block influenza virus hemagglutinin-mediated membrane fusion. Overall, our results provide evidence that IFITM proteins utilize an amphipathic helix for inhibiting virus fusion. © 2017 The Authors.

  8. A proline-hinge alters the characteristics of the amphipathic α-helical AMPs.

    Science.gov (United States)

    Lee, Jong Kook; Gopal, Ramamourthy; Park, Seong-Cheol; Ko, Hyun Sook; Kim, Yangmee; Hahm, Kyung-Soo; Park, Yoonkyung

    2013-01-01

    HP (2-20) is a 19-aa, amphipathic, α-helical peptide with antimicrobial properties that was derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously showed that increasing the net hydrophobicity of HP (2-20) by substituting Trp for Gln(17) and Asp(19) (Anal 3) increased the peptide's antimicrobial activity. In hydrophobic medium, Anal 3 forms an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and an extended helical region (residues 6-20). To investigate the structure-activity relationship of Anal 3, we substituted Pro for Glu(9) (Anal 3-Pro) and then examined the new peptide's three-dimensional structure, antimicrobial activity and mechanism of action. Anal 3-Pro had an α-helical structure in the presence of trifluoroethanol (TFE) and sodium dodecyl sulfate (SDS). NMR spectroscopic analysis of Anal 3-Pro's tertiary structure in SDS micelles confirmed that the kink potential introduced by Pro(10) was responsible for the helix distortion. We also found that Anal 3-Pro exhibited about 4 times greater antimicrobial activity than Anal 3. Fluorescence activated flow cytometry and confocal fluorescence microscopy showed that incorporating a Pro-hinge into Anal 3 markedly reduced its membrane permeability so that it accumulated in the cytoplasm without remaining in the cell membrane. To investigate the translocation mechanism, we assessed its ability to release of FITC-dextran. The result showed Anal 3-Pro created a pore Candida albicans revealed that Anal 3-Pro and buforin II exert similar effects on cell membranes, whereas magainin 2 exerts a different, more damaging, effect. In addition, Anal 3-Pro assumed a helix-hinge-helix structure in the presence of biological membranes and formed micropores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly bound to DNA. These results indicate that the bending region of Anal 3- Pro peptide is prerequisite for effective

  9. Antimicrobial peptides design by evolutionary multiobjective optimization.

    Directory of Open Access Journals (Sweden)

    Giuseppe Maccari

    Full Text Available Antimicrobial peptides (AMPs are an abundant and wide class of molecules produced by many tissues and cell types in a variety of mammals, plant and animal species. Linear alpha-helical antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement in innate defense. Recently, AMPs have received increasing attention as potential therapeutic agents, owing to their broad activity spectrum and their reduced tendency to induce resistance. The introduction of non-natural amino acids will be a key requisite in order to contrast host resistance and increase compound's life. In this work, the possibility to design novel AMP sequences with non-natural amino acids was achieved through a flexible computational approach, based on chemophysical profiles of peptide sequences. Quantitative structure-activity relationship (QSAR descriptors were employed to code each peptide and train two statistical models in order to account for structural and functional properties of alpha-helical amphipathic AMPs. These models were then used as fitness functions for a multi-objective evolutional algorithm, together with a set of constraints for the design of a series of candidate AMPs. Two ab-initio natural peptides were synthesized and experimentally validated for antimicrobial activity, together with a series of control peptides. Furthermore, a well-known Cecropin-Mellitin alpha helical antimicrobial hybrid (CM18 was optimized by shortening its amino acid sequence while maintaining its activity and a peptide with non-natural amino acids was designed and tested, demonstrating the higher activity achievable with artificial residues.

  10. Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics.

    Science.gov (United States)

    Charpentier, S; Amiche, M; Mester, J; Vouille, V; Le Caer, J P; Nicolas, P; Delfour, A

    1998-06-12

    Analysis of antimicrobial activities that are present in the skin secretions of the South American frog Phyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic alpha-helical peptides, 24-33 residues long, termed dermaseptins B1 to B6, respectively. Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first 3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of precursors for D-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species. The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations, biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved "secretory cassette" exon.

  11. Structure, synthesis, and activity of dermaseptin b, a novel vertebrate defensive peptide from frog skin: relationship with adenoregulin.

    Science.gov (United States)

    Mor, A; Amiche, M; Nicolas, P

    1994-05-31

    A novel antimicrobial peptide, designated dermaseptin b, was isolated from the skin of the arboreal frog Phyllomedusa bicolor. This 27-residue peptide amide is basic, containing 3 lysine residues that punctuate an alternating hydrophobic and hydrophilic sequence. In helix-inducing solvent, dermaseptin b adopts an amphipathic alpha-helical conformation that most closely resembles class L amphipathic helixes, with all lysine residues on the polar face of the helix. The peptide exhibits growth inhibition activity in vitro against a broad spectrum of pathogenic microorganisms including yeast and bacteria as well as various filamentous fungi that are responsible for severe opportunistic infections accompanying acquired immunodeficiency syndrome and the use of immunosuppressive agents. Maximized pairwise sequence alignment of dermaseptin b and dermaseptin s, a 34-residue antimicrobial peptide previously isolated from Phyllomedusa sauvagii, reveals 81% amino acid identity. No other significant similarity was found between dermaseptin b and any prokaryotic or eukaryotic protein, but similarity was found with adenoregulin (38% amino acid postional identity), a 33-residue peptide that enhances binding of agonists to the A1 adenosine receptor. The synthetic replicates of dermaseptin b and adenoregulin displayed similar but nonidentical spectra of antimicrobial activity, and both peptides were devoid of lytic effect on mammalian cells. Accordingly, the observation that adenoregulin enhances binding of agonists to the adenosine receptor may in fact be a consequence of its ability to alter the structure of biological membranes and to produce signal transduction via interactions with the lipid bilayer, bypassing cell surface receptor interactions.

  12. Peptide stabilized amphotericin B nanodisks

    Science.gov (United States)

    Tufteland, Megan; Pesavento, Joseph B.; Bermingham, Rachelle L.; Hoeprich, Paul D.; Ryan, Robert O.

    2007-01-01

    Nanometer scale apolipoprotein A-I stabilized phospholipid disk complexes (nanodisks; ND) have been formulated with the polyene antibiotic amphotericin B (AMB). The present studies were designed to evaluate if a peptide can substitute for the function of the apolipoprotein component of ND with respect to particle formation and stability. An 18-residue synthetic amphipathic α-helical peptide, termed 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2), solubilized vesicles comprised of egg phosphatidylcholine (egg PC), dipentadecanoyl PC or dimyristoylphosphatidylcholine (DMPC) at rates greater than or equal to solubilization rates observed with human apolipoprotein A-I (apoA-I; 243 amino acids). Characterization studies revealed that interaction with DMPC induced a near doubling of 4F tryptophan fluorescence emission quantum yield (excitation 280 nm) and a ~7 nm blue shift in emission wavelength maximum. Inclusion of AMB in the vesicle substrate resulted in formation of 4F AMB-ND. Spectra of AMB containing particles revealed the antibiotic is a highly effective quencher of 4F tryptophan fluorescence emission, giving rise to a Ksv = 7.7 × 104. Negative stain electron microscopy revealed that AMB-ND prepared with 4F possessed a disk shaped morphology similar to ND prepared without AMB or prepared with apoA-I. In yeast and pathogenic fungi growth inhibition assays, 4F AMB-ND was as effective as apoA-I AMB-ND. The data indicate that AMB-ND generated using an amphipathic peptide in lieu of apoA-I form a discrete population of particles that possess potent biological activity. Given their intrinsic versatility, peptides may be preferred for scale up and clinical application of AMB-ND. PMID:17293004

  13. Jumping Hurdles: Peptides Able To Overcome Biological Barriers.

    Science.gov (United States)

    Sánchez-Navarro, Macarena; Teixidó, Meritxell; Giralt, Ernest

    2017-08-15

    The cell membrane, the gastrointestinal tract, and the blood-brain barrier (BBB) are good examples of biological barriers that define and protect cells and organs. They impose different levels of restriction, but they also share common features. For instance, they all display a high lipophilic character. For this reason, hydrophilic compounds, like peptides, proteins, or nucleic acids have long been considered as unable to bypass them. However, the discovery of cell-penetrating peptides (CPPs) opened a vast field of research. Nowadays, CPPs, homing peptides, and blood-brain barrier peptide shuttles (BBB-shuttles) are good examples of peptides able to target and to cross various biological barriers. CPPs are a group of peptides able to interact with the plasma membrane and enter the cell. They display some common characteristics like positively charged residues, mainly arginines, and amphipathicity. In this field, our group has been focused on the development of proline rich CPPs and in the analysis of the importance of secondary amphipathicity in the internalization process. Proline has a privileged structure being the only amino acid with a secondary amine and a cyclic side chain. These features constrain its structure and hamper the formation of H-bonds. Taking advantage of this privileged structure, three different families of proline-rich peptides have been developed, namely, a proline-rich dendrimer, the sweet arrow peptide (SAP), and a group of foldamers based on γ-peptides. The structure and the mechanism of internalization of all of them has been evaluated and analyzed. BBB-shuttles are peptides able to cross the BBB and to carry with them compounds that cannot reach the brain parenchyma unaided. These peptides take advantage of the natural transport mechanisms present at the BBB, which are divided in active and passive transport mechanisms. On the one hand, we have developed BBB-shuttles that cross the BBB by a passive transport mechanism, like

  14. Panurgines, novel antimicrobial peptides from the venom of communal bee Panurgus calcaratus (Hymenoptera: Andrenidae).

    Science.gov (United States)

    Čujová, Sabína; Slaninová, Jiřina; Monincová, Lenka; Fučík, Vladimír; Bednárová, Lucie; Štokrová, Jitka; Hovorka, Oldřich; Voburka, Zdeněk; Straka, Jakub; Čeřovský, Václav

    2013-07-01

    Three novel antimicrobial peptides (AMPs), named panurgines (PNGs), were isolated from the venom of the wild bee Panurgus calcaratus. The dodecapeptide of the sequence LNWGAILKHIIK-NH₂ (PNG-1) belongs to the category of α-helical amphipathic AMPs. The other two cyclic peptides containing 25 amino acid residues and two intramolecular disulfide bridges of the pattern Cys8-Cys23 and Cys11-Cys19 have almost identical sequence established as LDVKKIICVACKIXPNPACKKICPK-OH (X=K, PNG-K and X=R, PNG-R). All three peptides exhibited antimicrobial activity against Gram-positive bacteria and Gram-negative bacteria, antifungal activity, and low hemolytic activity against human erythrocytes. We prepared a series of PNG-1 analogs to study the effects of cationicity, amphipathicity, and hydrophobicity on the biological activity. Several of them exhibited improved antimicrobial potency, particularly those with increased net positive charge. The linear analogs of PNG-K and PNG-R having all Cys residues substituted by α-amino butyric acid were inactive, thus indicating the importance of disulfide bridges for the antimicrobial activity. However, the linear PNG-K with all four cysteine residues unpaired, exhibited antimicrobial activity. PNG-1 and its analogs induced a significant leakage of fluorescent dye entrapped in bacterial membrane-mimicking large unilamellar vesicles as well as in vesicles mimicking eukaryotic cell membrane. On the other hand, PNG-K and PNG-R exhibited dye-leakage activity only from vesicles mimicking bacterial cell membrane.

  15. Histidine-Containing Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2000-01-01

    Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics.......Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics....

  16. A proline-hinge alters the characteristics of the amphipathic α-helical AMPs.

    Directory of Open Access Journals (Sweden)

    Jong Kook Lee

    Full Text Available HP (2-20 is a 19-aa, amphipathic, α-helical peptide with antimicrobial properties that was derived from the N-terminus of Helicobacter pylori ribosomal protein L1. We previously showed that increasing the net hydrophobicity of HP (2-20 by substituting Trp for Gln(17 and Asp(19 (Anal 3 increased the peptide's antimicrobial activity. In hydrophobic medium, Anal 3 forms an amphipathic structure consisting of an N-terminal random coil region (residues 2-5 and an extended helical region (residues 6-20. To investigate the structure-activity relationship of Anal 3, we substituted Pro for Glu(9 (Anal 3-Pro and then examined the new peptide's three-dimensional structure, antimicrobial activity and mechanism of action. Anal 3-Pro had an α-helical structure in the presence of trifluoroethanol (TFE and sodium dodecyl sulfate (SDS. NMR spectroscopic analysis of Anal 3-Pro's tertiary structure in SDS micelles confirmed that the kink potential introduced by Pro(10 was responsible for the helix distortion. We also found that Anal 3-Pro exhibited about 4 times greater antimicrobial activity than Anal 3. Fluorescence activated flow cytometry and confocal fluorescence microscopy showed that incorporating a Pro-hinge into Anal 3 markedly reduced its membrane permeability so that it accumulated in the cytoplasm without remaining in the cell membrane. To investigate the translocation mechanism, we assessed its ability to release of FITC-dextran. The result showed Anal 3-Pro created a pore <1.8 nm in diameter, which is similar to buforin II. Notably, scanning electron microscopic observation of Candida albicans revealed that Anal 3-Pro and buforin II exert similar effects on cell membranes, whereas magainin 2 exerts a different, more damaging, effect. In addition, Anal 3-Pro assumed a helix-hinge-helix structure in the presence of biological membranes and formed micropores in both bacterial and fungal membranes, through which it entered the cytoplasm and tightly

  17. Adsorption of amphipathic dendrons on polystyrene nanoparticles.

    Science.gov (United States)

    Sakthivel, T; Florence, A T

    2003-03-18

    Adsorption of dendrons onto nanoparticles may provide new model structures which may be useful in drug and gene delivery. Tritiated amphipathic dendrons having three lipidic (C(14)) chains coupled to branched (dendritic) lysine head groups with 8, 16 or 32 free terminal amino groups have been synthesised by solid phase peptide techniques. The interaction between these tritiated dendrons and 200 nm polystyrene latex nanoparticles was investigated in phosphate buffered saline. The amount of dendron adsorbed increased with increasing concentration of dendrons and then decreased. Maximum adsorption of dendrons per gram of nanoparticles was found to be between 8.2 and 84 x 10(-6)M, the amounts adsorbed being inversely proportional to the number of amino groups present in the molecule. The number of dendron molecules adsorbed per nanoparticle was found to be between 430 and 4421. The degree of adsorption was found to be slightly altered by the temperature. Copyright 2002 Elsevier Science B.V.

  18. Peptide Nucleic Acids Having Amino Acid Side Chains

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than the corresponding DNA or RNA strands, and exhibit increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a group consisting...

  19. Synthetic Procedures for Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  20. A non-covalent peptide-based carrier for in vivo delivery of DNA mimics.

    Science.gov (United States)

    Morris, May C; Gros, Edwige; Aldrian-Herrada, Gudrun; Choob, Michael; Archdeacon, John; Heitz, Frederic; Divita, Gilles

    2007-01-01

    The dramatic acceleration in identification of new nucleic-acid-based therapeutic molecules has provided new perspectives in pharmaceutical research. However, their development is limited by their poor cellular uptake and inefficient trafficking. Here we describe a short amphipathic peptide, Pep-3, that combines a tryptophan/phenylalanine domain with a lysine/arginine-rich hydrophilic motif. Pep-3 forms stable nano-size complexes with peptide-nucleic acid analogues and promotes their efficient delivery into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. We demonstrate that Pep-3-mediated delivery of antisense-cyclin B1-charged-PNA blocks tumour growth in vivo upon intratumoral and intravenous injection. Moreover, we show that PEGylation of Pep-3 significantly improves complex stability in vivo and consequently the efficiency of antisense cyclin B1 administered intravenously. Given the biological characteristics of these vectors, we believe that peptide-based delivery technologies hold a true promise for therapeutic applications of DNA mimics.

  1. Cleaving Double-Stranded DNA with Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1997-01-01

    Peptide nucleic acids and analogues of peptide nucleic acids are used to form duplex, triplex, and other structures with nucleic acids and to modify nucleic acids. The peptide nucleic acids and analogues thereof also are used to modulate protein activity through, for example, transcription arrest......, transcription initiation, and site specific cleavage of nucleic acids....

  2. Enhanced mitochondrial degradation of yeast cytochrome c with amphipathic structures.

    Science.gov (United States)

    Chen, Xi; Moerschell, Richard P; Pearce, David A; Ramanan, Durga D; Sherman, Fred

    2005-02-01

    The dispensable N-terminus of iso-1-cytochrome c (iso-1) in the yeast Saccharomyces cerevisiae was replaced by 11 different amphipathic structures. Rapid degradation of the corresponding iso-1 occurred, with the degree of degradation increasing with the amphipathic moments; and this amphipathic-dependent degradation was designated ADD. ADD occurred with the holo-forms in the mitochondria but not as the apo-forms in the cytosol. The extreme mutant type degraded with a half-life of approximately 12 min, whereas the normal iso-1 was stable over hours. ADD was influenced by the rho+/rho- state and by numerous chromosomal genes. Most importantly, ADD appeared to be specifically suppressed to various extents by deletions of any of the YME1, AFG3, or RCA1 genes encoding membrane-associated mitochondrial proteases, probably because the amphipathic structures caused a stronger association with the mitochondrial inner membrane and its associated proteases. The use of ADD assisted in the differentiation of substrates of different mitochondrial degradation pathways.

  3. Modeling of the Ebola Virus Delta Peptide Reveals a Potential Lytic Sequence Motif

    Directory of Open Access Journals (Sweden)

    William R. Gallaher

    2015-01-01

    Full Text Available Filoviruses, such as Ebola and Marburg viruses, cause severe outbreaks of human infection, including the extensive epidemic of Ebola virus disease (EVD in West Africa in 2014. In the course of examining mutations in the glycoprotein gene associated with 2014 Ebola virus (EBOV sequences, a differential level of conservation was noted between the soluble form of glycoprotein (sGP and the full length glycoprotein (GP, which are both encoded by the GP gene via RNA editing. In the region of the proteins encoded after the RNA editing site sGP was more conserved than the overlapping region of GP when compared to a distant outlier species, Tai Forest ebolavirus. Half of the amino acids comprising the “delta peptide”, a 40 amino acid carboxy-terminal fragment of sGP, were identical between otherwise widely divergent species. A lysine-rich amphipathic peptide motif was noted at the carboxyl terminus of delta peptide with high structural relatedness to the cytolytic peptide of the non-structural protein 4 (NSP4 of rotavirus. EBOV delta peptide is a candidate viroporin, a cationic pore-forming peptide, and may contribute to EBOV pathogenesis.

  4. BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

    DEFF Research Database (Denmark)

    Madsen, Kenneth Lindegaard; Bhatia, V K; Gether, U

    2010-01-01

    /ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can...

  5. MDR1 P-glycoprotein transports endogenous opioid peptides

    NARCIS (Netherlands)

    Oude Elferink, R. P.; Zadina, J.

    2001-01-01

    MDR1 P-glycoprotein is generally regarded as an efflux pump for amphipathic toxic compounds. The question remains, however, whether certain endogenous compounds are also substrates for this transporter. Certain peptides have been shown to interact with MDR1 Pgp as well and we have therefore

  6. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    Science.gov (United States)

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  7. The Spider Venom Peptide Lycosin-II Has Potent Antimicrobial Activity against Clinically Isolated Bacteria

    Directory of Open Access Journals (Sweden)

    Yongjun Wang

    2016-04-01

    Full Text Available Antimicrobial peptides have been accepted as excellent candidates for developing novel antibiotics against drug-resistant bacteria. Recent studies indicate that spider venoms are the source for the identification of novel antimicrobial peptides. In the present study, we isolated and characterized an antibacterial peptide named lycosin-II from the venom of the spider Lycosa singoriensis. It contains 21 amino acid residue lacking cysteine residues and forms a typical linear amphipathic and cationic α-helical conformation. Lycosin-II displays potent bacteriostatic effect on the tested drug-resistant bacterial strains isolated from hospital patients, including multidrug-resistant A. baumannii, which has presented a huge challenge for the infection therapy. The inhibitory ability of lycosin-II might derive from its binding to cell membrane, because Mg2+ could compete with the binding sites to reduce the bacteriostatic potency of lycosin-II. Our data suggest that lycosin-II might be a lead in the development of novel antibiotics for curing drug-resistant bacterial infections.

  8. Peptide Nucleic Acids Having 2,6-Diaminopurine Nucleobases

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. The peptide nucleic acids of the invention comprise ligands selected from a group...

  9. Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silk worm, Bombyx mori.

    Science.gov (United States)

    Li, Bao-Cun; Zhang, Shuang-Quan; Dan, Wen-Bing; Chen, Yu-Qing; Cao, Peng

    2007-07-01

    The antibacterial peptide CM4 (ABP-CM4), isolated from Chinese Bombys mori, is a 35-residue cationic, amphipathic alpha-helical peptide that exhibits a broad range of antimicrobial activity. To explore a new approach for the expression of ABP-CM4 in E. coli, the gene ABP-CM4, obtained by recursive PCR (rPCR), was cloned into the vector pET32a to construct a fusion expression plasmid. The fusion protein Trx-CM4 was expressed in soluble form, purified by Ni(2+)-chelating chromatography, and cleaved by formic acid to release recombinant CM4. Purification of rCM4 was achieved by affinity chromatography and reverse-phase HPLC. The purified of recombinant peptide showed antimicrobial activities against E. coli K(12)D(31), Penicillium chrysogenum, Aspergillus niger and Gibberella saubinetii. According to the antimicrobial peptide database (http://aps.unmc.edu/AP/main.html), 116 peptides contain a Met residue, but only 5 peptides contain the AspPro site, indicating a broader application of formic acid than CNBr in cleaving fusion protein. The successful application to the expression of the ABP-CM4 indicates that the system is a low-cost, efficient way of producting milligram quantities of ABP-CM4 that is biologically active.

  10. Effect of hydrocarbon stapling on the properties of alpha-helical antimicrobial peptides isolated from the venom of hymenoptera

    Czech Academy of Sciences Publication Activity Database

    Chapuis, Hubert Jean; Slaninová, Jiřina; Bednárová, Lucie; Monincová, Lenka; Buděšínský, Miloš; Čeřovský, Václav

    2012-01-01

    Roč. 43, č. 5 (2012), s. 2047-2058 ISSN 0939-4451 R&D Projects: GA ČR GA203/08/0536 Institutional research plan: CEZ:AV0Z40550506 Keywords : antimicrobial peptides * stapled peptides * amphipathic helix * CD spectroscopy Subject RIV: CC - Organic Chemistry Impact factor: 3.914, year: 2012

  11. Evaluation of Novel Antimicrobial Peptides as Topical Anti-Infectives with Broad-Spectrum Activity against Combat-Related Bacterial and Fungal Wound Infections

    Science.gov (United States)

    2017-10-01

    that the dHDPs kill by disrupting membrane function . This is consistent with the amphipathic properties of the dHDPs and is a mechanism to which...recalcitrant biofilm are major obstacles in treating wounds. Antimicrobial peptides ( AMPs ), also known as host defense peptides, are evolutionarily highly...Designed antimicrobial peptides (dAMPs) are synthesized peptides that have been rationally designed based on sequences found in naturally occurring AMPs

  12. Formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations - A review.

    Science.gov (United States)

    Zhao, Cindy J; Schieber, Andreas; Gänzle, Michael G

    2016-11-01

    Fermented foods are valued for their rich and complex odour and taste. The metabolic activity of food-fermenting microorganisms determines food quality and generates odour and taste compounds. This communication reviews the formation of taste-active amino acids, amino acid derivatives and peptides in food fermentations. Pathways of the generation of taste compounds are presented for soy sauce, cheese, fermented meats, and bread. Proteolysis or autolysis during food fermentations generates taste-active amino acids and peptides; peptides derived from proteolysis particularly impart umami taste (e.g. α-glutamyl peptides) or bitter taste (e.g. hydrophobic peptides containing proline). Taste active peptide derivatives include pyroglutamyl peptides, γ-glutamyl peptides, and succinyl- or lactoyl amino acids. The influence of fermentation microbiota on proteolysis, and peptide hydrolysis, and the metabolism of glutamate and arginine is well understood, however, the understanding of microbial metabolic activities related to the formation of taste-active peptide derivatives is incomplete. Improved knowledge of the interactions between taste-active compounds will enable the development of novel fermentation strategies to develop tastier, less bitter, and low-salt food products, and may provide novel and "clean label" ingredients to improve the taste of other food products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Histidine-lysine peptides as carriers of nucleic acids.

    Science.gov (United States)

    Leng, Qixin; Goldgeier, Lisa; Zhu, Jingsong; Cambell, Patricia; Ambulos, Nicholas; Mixson, A James

    2007-03-01

    With their biodegradability and diversity of permutations, peptides have significant potential as carriers of nucleic acids. This review will focus on the sequence and branching patterns of peptide carriers composed primarily of histidines and lysines. While lysines within peptides are important for binding to the negatively charged phosphates, histidines are critical for endosomal lysis enabling nucleic acids to reach the cytosol. Histidine-lysine (HK) polymers by either covalent or ionic bonds with liposomes augment transfection compared to liposome carriers alone. More recently, we have examined peptides as sole carriers of nucleic acids because of their intrinsic advantages compared to the bipartite HK/liposome carriers. With a protocol change and addition of a histidine-rich tail, HK peptides as sole carriers were more effective than liposomes alone in several cell lines. While four-branched polymers with a primary repeating sequence pattern of -HHK- were more effective as carriers of plasmids, eight-branched polymers with a sequence pattern of -HHHK- were more effective as carriers of siRNA. Compared to polyethylenimine, HK carriers of siRNA and plasmids had reduced toxicity. When injected intravenously, HK polymers in complex with plasmids encoding antiangiogenic proteins significantly decreased tumor growth. Furthermore, modification of HK polymers with polyethylene glycol and vascular-specific ligands increased specificity of the polyplex to the tumor by more than 40-fold. Together with further development and insight on the structure of HK polyplexes, HK peptides may prove to be useful as carriers of different forms of nucleic acids both in vitro and in vivo.

  14. Analysis of Peptides and Conjugates by Amino Acid Analysis

    DEFF Research Database (Denmark)

    Højrup, Peter

    2015-01-01

    Amino acid analysis is a highly accurate method for characterization of the composition of synthetic peptides. Together with mass spectrometry, it gives a reliable control of peptide quality and quantity before conjugation and immunization.Peptides are hydrolyzed, preferably in gas phase, with 6 M...... HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by ion-exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and cysteine also, unless derivatized, and the amides, glutamine and asparagine, are deamidated...... to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5 % can usually be obtained....

  15. Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir

    2008-01-01

    Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway...... for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases...... with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates...

  16. Amino Acid and Peptide Immobilization on Oxidized Nanocellulose: Spectroscopic Characterization

    Science.gov (United States)

    Barazzouk, Saïd; Daneault, Claude

    2012-01-01

    In this work, oxidized nanocellulose (ONC) was synthesized and chemically coupled with amino acids and peptides using a two step coupling method at room temperature. First, ONC was activated by N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride, forming a stable active ester in the presence of N-hydroxysuccinimide. Second, the active ester was reacted with the amino group of the amino acid or peptide, forming an amide bond between ONC and the grafted molecule. Using this method, the intermolecular interaction of amino acids and peptides was avoided and uniform coupling of these molecules on ONC was achieved. The coupling reaction was very fast in mild conditions and without alteration of the polysaccharide. The coupling products (ONC-amino acids and ONC-peptides) were characterized by transmission electron microscopy and by the absorption, emission, Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) spectroscopic techniques. PMID:28348303

  17. Amino Acid and Peptide Immobilization on Oxidized Nanocellulose: Spectroscopic Characterization

    Directory of Open Access Journals (Sweden)

    Claude Daneault

    2012-06-01

    Full Text Available In this work, oxidized nanocellulose (ONC was synthesized and chemically coupled with amino acids and peptides using a two step coupling method at room temperature. First, ONC was activated by N-ethyl-N’-(3-dimethylaminopropyl carbodiimide hydrochloride, forming a stable active ester in the presence of N-hydroxysuccinimide. Second, the active ester was reacted with the amino group of the amino acid or peptide, forming an amide bond between ONC and the grafted molecule. Using this method, the intermolecular interaction of amino acids and peptides was avoided and uniform coupling of these molecules on ONC was achieved. The coupling reaction was very fast in mild conditions and without alteration of the polysaccharide. The coupling products (ONC-amino acids and ONC-peptides were characterized by transmission electron microscopy and by the absorption, emission, Fourier transform infrared spectroscopy (FTIR and X-ray photoelectron spectroscopy (XPS spectroscopic techniques.

  18. Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein Erns.

    Science.gov (United States)

    Aberle, Daniel; Oetter, Kay-Marcus; Meyers, Gregor

    2015-01-01

    Pestiviruses express a peculiar protein named Erns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of Erns from the infected cell. Retention/secretion of Erns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of Erns binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of Erns had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for Erns proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained Erns is not determined by the lipid affinity of the membrane anchor.

  19. Lipid Binding of the Amphipathic Helix Serving as Membrane Anchor of Pestivirus Glycoprotein Erns.

    Directory of Open Access Journals (Sweden)

    Daniel Aberle

    Full Text Available Pestiviruses express a peculiar protein named Erns representing envelope glycoprotein and RNase, which is important for control of the innate immune response and persistent infection. The latter functions are connected with secretion of a certain amount of Erns from the infected cell. Retention/secretion of Erns is most likely controlled by its unusual membrane anchor, a long amphipathic helix attached in plane to the membrane. Here we present results of experiments conducted with a lipid vesicle sedimentation assay able to separate lipid-bound from unbound protein dissolved in the water phase. Using this technique we show that a protein composed of tag sequences and the carboxyterminal 65 residues of Erns binds specifically to membrane vesicles with a clear preference for compositions containing negatively charged lipids. Mutations disturbing the helical folding and/or amphipathic character of the anchor as well as diverse truncations and exchange of amino acids important for intracellular retention of Erns had no or only small effects on the proteins membrane binding. This result contrasts the dramatically increased secretion rates observed for Erns proteins with equivalent mutations within cells. Accordingly, the ratio of secreted versus cell retained Erns is not determined by the lipid affinity of the membrane anchor.

  20. Transgenic tobacco expressing a modified spider peptide inhibits the growth of plant pathogens and insect larvae

    Science.gov (United States)

    The gene encoding lycotoxin I, an amphipathic pore-forming peptide, was modified to increase oral toxicity to insects. One of the most active modified genes was then constitutively expressed in tobacco (Nicotiana tabacum) and transformants were evaluated for insect and disease resistance. Pathogenic...

  1. Reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5

    OpenAIRE

    Veerman, Enno C. I.; Nazmi, Kamran; van '​t HOF, Wim; Bolscher, Jan G. M.; den Hertog, Alice L.; Nieuw Amerongen, Arie V.

    2004-01-01

    The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an...

  2. Structural similarity between β(3)-peptides synthesized from β(3)-homo-amino acids and aspartic acid monomers.

    Science.gov (United States)

    Ahmed, Sahar; Sprules, Tara; Kaur, Kamaljit

    2014-07-01

    Formation of stable secondary structures by oligomers that mimic natural peptides is a key asset for enhanced biological response. Here we show that oligomeric β(3)-hexapeptides synthesized from L-aspartic acid monomers (β(3)-peptides 1, 5a, and 6) or homologated β(3)-amino acids (β(3)-peptide 2), fold into similar stable 14-helical secondary structures in solution, except that the former form right-handed 14-helix and the later form left-handed 14-helix. β(3)-Peptides from L-Asp monomers contain an additional amide bond in the side chains that provides opportunities for more hydrogen bonding. However, based on the NMR solution structures, we found that β(3)-peptide from L-Asp monomers (1) and from homologated amino acids (2) form similar structures with no additional side-chain interactions. These results suggest that the β(3)-peptides derived from L-Asp are promising peptide-mimetics that can be readily synthesized using L-Asp monomers as well as the right-handed 14-helical conformation of these β(3)-peptides (such as 1 and 6) may prove beneficial in the design of mimics for right-handed α-helix of α-peptides. © 2014 Wiley Periodicals, Inc.

  3. Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling.

    Directory of Open Access Journals (Sweden)

    Zhongqiu Ni

    Full Text Available A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl phenyl-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis.

  4. Human Antimicrobial Peptides and Proteins

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2014-05-01

    Full Text Available As the key components of innate immunity, human host defense antimicrobial peptides and proteins (AMPs play a critical role in warding off invading microbial pathogens. In addition, AMPs can possess other biological functions such as apoptosis, wound healing, and immune modulation. This article provides an overview on the identification, activity, 3D structure, and mechanism of action of human AMPs selected from the antimicrobial peptide database. Over 100 such peptides have been identified from a variety of tissues and epithelial surfaces, including skin, eyes, ears, mouths, gut, immune, nervous and urinary systems. These peptides vary from 10 to 150 amino acids with a net charge between −3 and +20 and a hydrophobic content below 60%. The sequence diversity enables human AMPs to adopt various 3D structures and to attack pathogens by different mechanisms. While α-defensin HD-6 can self-assemble on the bacterial surface into nanonets to entangle bacteria, both HNP-1 and β-defensin hBD-3 are able to block cell wall biosynthesis by binding to lipid II. Lysozyme is well-characterized to cleave bacterial cell wall polysaccharides but can also kill bacteria by a non-catalytic mechanism. The two hydrophobic domains in the long amphipathic α-helix of human cathelicidin LL-37 lays the basis for binding and disrupting the curved anionic bacterial membrane surfaces by forming pores or via the carpet model. Furthermore, dermcidin may serve as ion channel by forming a long helix-bundle structure. In addition, the C-type lectin RegIIIα can initially recognize bacterial peptidoglycans followed by pore formation in the membrane. Finally, histatin 5 and GAPDH(2-32 can enter microbial cells to exert their effects. It appears that granulysin enters cells and kills intracellular pathogens with the aid of pore-forming perforin. This arsenal of human defense proteins not only keeps us healthy but also inspires the development of a new generation of personalized

  5. Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication

    DEFF Research Database (Denmark)

    Ndeboko, Benedicte; Ramamurthy, Narayan; Lemamy, Guy Joseph

    2017-01-01

    Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA...

  6. Stereoselective determination of amino acids in beta-amyloid peptides and senile plaques.

    Science.gov (United States)

    Thorsén, G; Bergquist, J; Westlind-Danielsson, A; Josefsson, B

    2001-06-01

    A novel method for the determination of the enantiomeric composition of peptides is presented. In this paper, the focus has been on beta-amyloid peptides from deceased Alzheimer's disease patients. The peptides are hydrolyzed using mineral acid. The free amino acids are derivatized with the chiral reagent (+)- or (-)-1-(9-anthryl)-2-propyl chloroformate and subsequently separated using micellar electrokinetic chromatography (MEKC) and detected using laser-induced fluorescence (LIF) detection. The high separation efficiency of the MEKC-LIF system, yielding approximately 1 million theoretical plates/m for most amino acids, facilitates the simultaneous chiral determination of nine amino acids. The samples that have been analyzed were standard 1-40 beta-amyloid peptides, in vitro precipitated beta-amyloid fibrils, and human senile plaque samples.

  7. Structure-activity study of macropin, a novel antimicrobial peptide from the venom of solitary bee Macropis fulvipes (Hymenoptera: Melittidae).

    Science.gov (United States)

    Monincová, Lenka; Veverka, Václav; Slaninová, Jiřina; Buděšínský, Miloš; Fučík, Vladimír; Bednárová, Lucie; Straka, Jakub; Ceřovský, Václav

    2014-06-01

    A novel antimicrobial peptide, designated macropin (MAC-1) with sequence Gly-Phe-Gly-Met-Ala-Leu-Lys-Leu-Leu-Lys-Lys-Val-Leu-NH2 , was isolated from the venom of the solitary bee Macropis fulvipes. MAC-1 exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria, antifungal activity, and moderate hemolytic activity against human red blood cells. A series of macropin analogs were prepared to further evaluate the effect of structural alterations on antimicrobial and hemolytic activities and stability in human serum. The antimicrobial activities of several analogs against pathogenic Pseudomonas aeruginosa were significantly increased while their toxicity against human red blood cells was decreased. The activity enhancement is related to the introduction of either l- or d-lysine in selected positions. Furthermore, all-d analog and analogs with d-amino acid residues introduced at the N-terminal part of the peptide chain exhibited better serum stability than did natural macropin. Data obtained by CD spectroscopy suggest a propensity of the peptide to adopt an amphipathic α-helical secondary structure in the presence of trifluoroethanol or membrane-mimicking sodium dodecyl sulfate. In addition, the study elucidates the structure-activity relationship for the effect of d-amino acid substitutions in MAC-1 using NMR spectroscopy. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  8. Melectin: A novel antimicrobial peptide from the venom of the cleptoparasitic bee Melecta albifrons

    Czech Academy of Sciences Publication Activity Database

    Čeřovský, Václav; Hovorka, Oldřich; Cvačka, Josef; Voburka, Zdeněk; Bednárová, Lucie; Borovičková, Lenka; Slaninová, Jiřina; Fučík, Vladimír

    2008-01-01

    Roč. 9, č. 17 (2008), s. 2815-2821 ISSN 1439-4227 R&D Projects: GA ČR(CZ) GA203/08/0536 Institutional research plan: CEZ:AV0Z40550506 Keywords : amphipathicity * antimicrobial activity * helical structures * peptides * solitary bee venom Subject RIV: CC - Organic Chemistry Impact factor: 3.322, year: 2008

  9. The remarkable stability of chimeric, sialic acid-derived alpha/delta-peptides in human blood plasma.

    Science.gov (United States)

    Saludes, Jonel P; Natarajan, Arutselvan; DeNardo, Sally J; Gervay-Hague, Jacquelyn

    2010-05-01

    Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-alpha-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric alpha/delta-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en alpha/delta-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with (111)In, incubated in human blood plasma at 37 degrees C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural alpha-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.

  10. A Diverse Family of Host-Defense Peptides (Piscidins Exhibit Specialized Anti-Bacterial and Anti-Protozoal Activities in Fishes.

    Directory of Open Access Journals (Sweden)

    Scott A Salger

    Full Text Available Conventional antibiotics and other chemical-based drugs are currently one of the most common methods used to control disease-related mortality in animal agriculture. Use of the innate immune system to decrease disease related mortalities is a novel alternative to conventional drugs. One component of the innate immune system is the host-defense peptides, also known as antimicrobial peptides. Host-defense peptides are typically small, amphipathic, α-helical peptides with a broad-spectrum of action against viral, bacterial, fungal, and/or protozoal pathogens. Piscidins are host-defense peptides first discovered in the hybrid striped bass (white bass, Morone chrysops, x striped bass, M. saxatilis. In this paper we identify four new piscidin isoforms in the hybrid striped bass and describe their tissue distributions. We also determine the progenitor species of origin of each piscidin (orthology and propose a revised nomenclature for this newly described piscidin family based on a three class system. The Class I piscidins (22 amino acids in length; striped bass and white bass piscidin 1 and piscidin 3 show broad-spectrum activity against bacteria and ciliated protozoans, while the Class III piscidins (55 amino acids in length; striped bass and white bass piscidin 6 and striped bass piscidin 7 primarily show anti-protozoal activity. The Class II piscidins (44-46 amino acids in length; striped bass and white bass piscidin 4 and white bass piscidin 5 have a level of activity against bacteria and protozoans intermediate to Classes I and III. Knowledge of piscidin function and activity may help in the future development of disease-resistant lines of striped bass and white bass that could be used to produce superior hybrids for aquaculture.

  11. A Diverse Family of Host-Defense Peptides (Piscidins) Exhibit Specialized Anti-Bacterial and Anti-Protozoal Activities in Fishes.

    Science.gov (United States)

    Salger, Scott A; Cassady, Katherine R; Reading, Benjamin J; Noga, Edward J

    2016-01-01

    Conventional antibiotics and other chemical-based drugs are currently one of the most common methods used to control disease-related mortality in animal agriculture. Use of the innate immune system to decrease disease related mortalities is a novel alternative to conventional drugs. One component of the innate immune system is the host-defense peptides, also known as antimicrobial peptides. Host-defense peptides are typically small, amphipathic, α-helical peptides with a broad-spectrum of action against viral, bacterial, fungal, and/or protozoal pathogens. Piscidins are host-defense peptides first discovered in the hybrid striped bass (white bass, Morone chrysops, x striped bass, M. saxatilis). In this paper we identify four new piscidin isoforms in the hybrid striped bass and describe their tissue distributions. We also determine the progenitor species of origin of each piscidin (orthology) and propose a revised nomenclature for this newly described piscidin family based on a three class system. The Class I piscidins (22 amino acids in length; striped bass and white bass piscidin 1 and piscidin 3) show broad-spectrum activity against bacteria and ciliated protozoans, while the Class III piscidins (55 amino acids in length; striped bass and white bass piscidin 6 and striped bass piscidin 7) primarily show anti-protozoal activity. The Class II piscidins (44-46 amino acids in length; striped bass and white bass piscidin 4 and white bass piscidin 5) have a level of activity against bacteria and protozoans intermediate to Classes I and III. Knowledge of piscidin function and activity may help in the future development of disease-resistant lines of striped bass and white bass that could be used to produce superior hybrids for aquaculture.

  12. Amphipathic motifs in BAR domains are essential for membrane curvature sensing

    DEFF Research Database (Denmark)

    Bhatia, Vikram K; Madsen, Kenneth L; Bolinger, Pierre-Yves

    2009-01-01

    BAR (Bin/Amphiphysin/Rvs) domains and amphipathic alpha-helices (AHs) are believed to be sensors of membrane curvature thus facilitating the assembly of protein complexes on curved membranes. Here, we used quantitative fluorescence microscopy to compare the binding of both motifs on single...... nanosized liposomes of different diameters and therefore membrane curvature. Characterization of members of the three BAR domain families showed surprisingly that the crescent-shaped BAR dimer with its positively charged concave face is not able to sense membrane curvature. Mutagenesis on BAR domains showed...... that membrane curvature sensing critically depends on the N-terminal AH and furthermore that BAR domains sense membrane curvature through hydrophobic insertion in lipid packing defects and not through electrostatics. Consequently, amphipathic motifs, such as AHs, that are often associated with BAR domains...

  13. Detection of cancer cells using a peptide nanotube–folic acid modified graphene electrode

    DEFF Research Database (Denmark)

    Castillo, John J.; Svendsen, Winnie Edith; Rozlosnik, Noemi

    2013-01-01

    This article describes the preparation of a graphene electrode modified with a new conjugate of peptide nanotubes and folic acid for the selective detection of human cervical cancer cells over-expressing folate receptors. The functionalization of peptide nanotubes with folic acid was confirmed...... by fluorescence microscopy and atomic force microscopy. The peptide nanotube–folic acid modified graphene electrode was characterized by scanning electron microscopy and cyclic voltammetry. The modification of the graphene electrode with peptide nanotube–folic acid led to an increase in the current signal....... The human cervical cancer cells were bound to the modified electrode through the folic acid–folate receptor interaction. Cyclic voltammograms in the presence of [Fe(CN)6]3/4 as a redox species demonstrated that the binding of the folate receptor from human cervical cancer cells to the peptide nanotube...

  14. Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications

    Science.gov (United States)

    Powell, Thomas; Bowra, Steve; Cooper, Helen J.

    2017-09-01

    Previously we have shown that subcritical water may be used as an alternative to enzymatic digestion in the proteolysis of proteins for bottom-up proteomics. Subcritical water hydrolysis of proteins was shown to result in protein sequence coverages greater than or equal to that obtained following digestion with trypsin; however, the percentage of peptide spectral matches for the samples treated with trypsin were consistently greater than for those treated with subcritical water. This observation suggests that in addition to cleavage of the peptide bond, subcritical water treatment results in other hydrolysis products, possibly due to modifications of amino acid side chains. Here, a model peptide comprising all common amino acid residues (VQSIKCADFLHYMENPTWGR) and two further model peptides (VCFQYMDRGDR and VQSIKADFLHYENPTWGR) were treated with subcritical water with the aim of probing any induced amino acid side-chain modifications. The hydrolysis products were analyzed by direct infusion electrospray tandem mass spectrometry, either collision-induced dissociation or electron transfer dissociation, and liquid chromatography collision-induced dissociation tandem mass spectrometry. The results show preferential oxidation of cysteine to sulfinic and sulfonic acid, and oxidation of methionine. In the absence of cysteine and methionine, oxidation of tryptophan was observed. In addition, water loss from aspartic acid and C-terminal amidation were observed in harsher subcritical water conditions. [Figure not available: see fulltext.

  15. Systems chemistry of α-amino acids and peptides

    Directory of Open Access Journals (Sweden)

    Danger Grégoire

    2014-02-01

    Full Text Available Pathways have been disclosed in the past decade, which support the possibility that α-amino acids could have contributed to self-organization processes leading to the emergence of life. It is proposed that the systems chemistry of these simple building blocks may have led to features of self-organization through the realization of protometabolisms based on unidirectional loops involving both peptide formation and breakdown and additional feedback processes. Potential peptide activating agents have been identified. Scenarios of peptide elongation are proposed to account for peptide elongation both at the N-terminus and the C-terminus and new indications that these processes could be involved in symmetry breaking have been provided.

  16. Antimicrobial peptides: the role of hydrophobicity in the alpha helical structure

    Directory of Open Access Journals (Sweden)

    Pandurangan Perumal

    2013-12-01

    Full Text Available The antimicrobial peptides (AMPs are a class of molecule obtained from plants, insects, animals, and humans. These peptides have been classified into five categories: 1. Anionic peptide, 2. Linear alpha helical cationic peptide, 3. Cationic peptide, 4. Anionic and cationic peptides with disulphide bonds, and 5. Anionic and cationic peptide fragments of larger proteins. Factors affecting AMPs are sequence, size, charge, hydrophobicity, amphipathicity, structure and conformation. Synthesis of these peptides is convenient by using solid phase peptide synthesis by using FMOC chemistry protocol. The secondary structures of three synthetic peptides were determined by circular dichroism. Also, it was compared the stability of the α-helical structure and confirmed the percentage of helix of these peptides by using circular dichroism. Some of these AMPs show therapeutic properties like antimicrobial, antiviral, contraceptive, and anticancer. The formulations of some peptides have been entered into the phase I, II, or III of clinical trials. This article to review briefly the sources, classification, factors affecting AMPs activity, synthesis, characterization, mechanism of action and therapeutic concern of AMPs and mainly focussed on percentage of α-helical structure in various medium.

  17. Septal membrane localization by C-terminal amphipathic α-helices of MinD in Bacillus subtilis mutant cells lacking MinJ or DivIVA.

    Science.gov (United States)

    Ishikawa, Kazuki; Matsuoka, Satoshi; Hara, Hiroshi; Matsumoto, Kouji

    2017-10-18

    The Min system, which inhibits assembly of the cytokinetic protein FtsZ, is largely responsible for positioning the division site in rod-shaped bacteria. It has been reported that MinJ, which bridges DivIVA and MinD, is targeted to the cell poles by an interaction with DivIVA, and that MinJ in turn recruits MinCD to the cell poles. MinC, however, is located primarily at active division sites at mid-cell when expressed from its native promoter. Surprisingly, we found that Bacillus subtilis MinD is located at nascent septal membranes and at an asymmetric site on lateral membranes between nascent septal membranes in filamentous cells lacking MinJ or DivIVA. Bacillus subtilis MinD has two amphipathic α-helices rich in basic amino acid residues at its C-terminus; one of these, named MTS1 here, is the counterpart of the membrane targeting sequence (MTS) in Escherichia coli MinD while the other, named MTS-like sequence (MTSL), is the nearest helix to MTS1. These amphipathic helices were located independently at nascent septal membranes in cells lacking MinJ or DivIVA, whereas elimination of the helices from the wild type protein reduced its localization considerably. MinD variants with altered MTS1 and MTSL, in which basic amino acid residues were replaced with proline or acidic residues, were not located at nascent septal membranes, indicating that the binding to the nascent septal membranes requires basic residues and a helical structure. The septal localization of MTSL, but not of MTS1, was dependent on host cell MinD. These results suggest that MinD is targeted to nascent septal membranes via its C-terminal amphipathic α-helices in B. subtilis cells lacking MinJ or DivIVA. Moreover, the diffuse distribution of MinD lacking both MTSs suggests that only a small fraction of MinD depends on MinJ for its localization to nascent septal membranes.

  18. Oxidative diversification of amino acids and peptides by small-molecule iron catalysis.

    Science.gov (United States)

    Osberger, Thomas J; Rogness, Donald C; Kohrt, Jeffrey T; Stepan, Antonia F; White, M Christina

    2016-09-08

    Secondary metabolites synthesized by non-ribosomal peptide synthetases display diverse and complex topologies and possess a range of biological activities. Much of this diversity derives from a synthetic strategy that entails pre- and post-assembly oxidation of both the chiral amino acid building blocks and the assembled peptide scaffolds. The vancomycin biosynthetic pathway is an excellent example of the range of oxidative transformations that can be performed by the iron-containing enzymes involved in its biosynthesis. However, because of the challenges associated with using such oxidative enzymes to carry out chemical transformations in vitro, chemical syntheses guided by these principles have not been fully realized in the laboratory. Here we report that two small-molecule iron catalysts are capable of facilitating the targeted C-H oxidative modification of amino acids and peptides with preservation of α-centre chirality. Oxidation of proline to 5-hydroxyproline furnishes a versatile intermediate that can be transformed to rigid arylated derivatives or flexible linear carboxylic acids, alcohols, olefins and amines in both monomer and peptide settings. The value of this C-H oxidation strategy is demonstrated in its capacity for generating diversity: four 'chiral pool' amino acids are transformed to twenty-one chiral unnatural amino acids representing seven distinct functional group arrays; late-stage C-H functionalizations of a single proline-containing tripeptide furnish eight tripeptides, each having different unnatural amino acids. Additionally, a macrocyclic peptide containing a proline turn element is transformed via late-stage C-H oxidation to one containing a linear unnatural amino acid.

  19. Recent Developments in Peptide-Based Nucleic Acid Delivery

    Directory of Open Access Journals (Sweden)

    Tobias Restle

    2008-07-01

    Full Text Available Despite the fact that non-viral nucleic acid delivery systems are generally considered to be less efficient than viral vectors, they have gained much interest in recent years due to their superior safety profile compared to their viral counterpart. Among these synthetic vectors are cationic polymers, branched dendrimers, cationic liposomes and cellpenetrating peptides (CPPs. The latter represent an assortment of fairly unrelated sequences essentially characterised by a high content of basic amino acids and a length of 10-30 residues. CPPs are capable of mediating the cellular uptake of hydrophilic macromolecules like peptides and nucleic acids (e.g. siRNAs, aptamers and antisenseoligonucleotides, which are internalised by cells at a very low rate when applied alone. Up to now, numerous sequences have been reported to show cell-penetrating properties and many of them have been used to successfully transport a variety of different cargos into mammalian cells. In recent years, it has become apparent that endocytosis is a major route of internalisation even though the mechanisms underlying the cellular translocation of CPPs are poorly understood and still subject to controversial discussions. In this review, we will summarise the latest developments in peptide-based cellular delivery of nucleic acid cargos. We will discuss different mechanisms of entry, the intracellular fate of the cargo, correlation studies of uptake versus biological activity of the cargo as well as technical problems and pitfalls.

  20. Negative Ion In-Source Decay Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry for Sequencing Acidic Peptides

    Science.gov (United States)

    McMillen, Chelsea L.; Wright, Patience M.; Cassady, Carolyn J.

    2016-05-01

    Matrix-assisted laser desorption/ionization (MALDI) in-source decay was studied in the negative ion mode on deprotonated peptides to determine its usefulness for obtaining extensive sequence information for acidic peptides. Eight biological acidic peptides, ranging in size from 11 to 33 residues, were studied by negative ion mode ISD (nISD). The matrices 2,5-dihydroxybenzoic acid, 2-aminobenzoic acid, 2-aminobenzamide, 1,5-diaminonaphthalene, 5-amino-1-naphthol, 3-aminoquinoline, and 9-aminoacridine were used with each peptide. Optimal fragmentation was produced with 1,5-diaminonphthalene (DAN), and extensive sequence informative fragmentation was observed for every peptide except hirudin(54-65). Cleavage at the N-Cα bond of the peptide backbone, producing c' and z' ions, was dominant for all peptides. Cleavage of the N-Cα bond N-terminal to proline residues was not observed. The formation of c and z ions is also found in electron transfer dissociation (ETD), electron capture dissociation (ECD), and positive ion mode ISD, which are considered to be radical-driven techniques. Oxidized insulin chain A, which has four highly acidic oxidized cysteine residues, had less extensive fragmentation. This peptide also exhibited the only charged localized fragmentation, with more pronounced product ion formation adjacent to the highly acidic residues. In addition, spectra were obtained by positive ion mode ISD for each protonated peptide; more sequence informative fragmentation was observed via nISD for all peptides. Three of the peptides studied had no product ion formation in ISD, but extensive sequence informative fragmentation was found in their nISD spectra. The results of this study indicate that nISD can be used to readily obtain sequence information for acidic peptides.

  1. A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.

    Science.gov (United States)

    Chua, Ngee Kiat; Howe, Vicky; Jatana, Nidhi; Thukral, Lipi; Brown, Andrew J

    2017-12-08

    Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Rational development of a cytotoxic peptide to trigger cell death.

    Science.gov (United States)

    Boohaker, Rebecca J; Zhang, Ge; Lee, Michael W; Nemec, Kathleen N; Santra, Santimukul; Perez, J Manuel; Khaled, Annette R

    2012-07-02

    Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.

  3. Improving surface functional properties of tofu whey-derived peptides by chemical modification with fatty acids.

    Science.gov (United States)

    Matemu, Athanasia Oswald; Katayama, Shigeru; Kayahara, Hisataka; Murasawa, Hisashi; Nakamura, Soichiro

    2012-04-01

    Effect of acylation with saturated fatty acids on surface functional properties of tofu whey-derived peptides was investigated. Tofu whey (TW) and soy proteins (7S, 11S, and acid-precipitated soy protein [APP]) were hydrolyzed by Protease M 'Amano' G, and resulting peptide mixtures were acylated with esterified fatty acids of different chain length (6C to 18C) to form a covalent linkage between the carboxyl group of fatty acid and the free amino groups of peptide. Acylation significantly (P properties of 7S, 11S, and APP peptides independent of fatty acid chain length. Acylation decreased water binding capacity although oil binding capacity of acylated tofu whey ultra filtered fraction (UFTW acids had shown significant higher surface hydrophobicity as in contrast with acylated UFTW acids can further affect functional properties of soy proteins. © 2012 Institute of Food Technologists®

  4. Antibiotic and synergistic effect of Leu-Lys rich peptide against antibiotic resistant microorganisms isolated from patients with cholelithiasis.

    Science.gov (United States)

    Jeong, Nari; Kim, Jin-Young; Park, Seong-Cheol; Lee, Jong-Kook; Gopal, Ramamourthy; Yoo, Suyeon; Son, Byoung Kwan; Hahm, Joon Soo; Park, Yoonkyung; Hahm, Kyung-Soo

    2010-09-03

    Pseudomonas aeruginosa has eventually developed resistance against flomoxef sodium, isepamicin and cefpiramide. Therefore, in this study, the antibacterial activity and synergistic effects of the amphipathic-derived P5-18mer antimicrobial peptide were tested against pathogens associated with cholelithiasis that have developed resistance against commonly used antibiotics. The results were then compared with the activities of the amphipathic-derived peptide, P5-18mer, melittin and common antibiotics. Growth inhibition of planktonic bacteria was tested using the National Committee for Clinical Laboratory Standards (NCCLS). The bactericidal activity of the antimicrobial peptides was measured using time-kill curves. Synergistic effects were evaluated by testing the effects of P5-18mer alone and in combination with flomoxef sodium, isepamicin or cefpiramide at 0.5xMIC. P5-18mer peptide displayed strong activity against pathogens and flomoxef sodium, isepamicin and cefpiramide-resistant bacteria cell lines obtained from a patient with gallstones; however, it did not exert cytotoxicity against the human keratinocyte HaCat cell line. In addition, the results of time-kill curves indicated that P5-18mer peptide exerted bactericidal activity against four strains of P. aeruginosa. Finally, the use of P5-18mer and antibiotics exerted synergistic effects against cell lines that were resistant to commonly used antibiotics. These results indicate that this class of peptides has a rapid microbicidal effect on flomoxef sodium, isepamicin and cefpiramide-resistant strains of P. aeruginosa. Therefore, these peptides may be used as a lead drug for the treatment of acquired pathogens from patients with cholelithiasis who are affected with antibiotic-resistant bacteria. Copyright 2010 Elsevier Inc. All rights reserved.

  5. Role of amphipathic helix of a herpesviral protein in membrane deformation and T cell receptor downregulation.

    Directory of Open Access Journals (Sweden)

    Chan-Ki Min

    2008-11-01

    Full Text Available Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip of T lymphotropic Herpesvirus saimiri (HVS is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip's transmembrane (TM domain mediates lipid raft localization and membrane deformation. In turn, this motif directs Tip's lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation.

  6. Far UV irradiation of DNA in the presence of proteins, amino acids or peptides

    International Nuclear Information System (INIS)

    Larcom, L.L.; Rains, C.A.

    1985-01-01

    The DNA of bacteriophage SPO2c12 was subjected to 254 nm irradiation in solutions containing lysozyme or histone. The sensitivity of phage DNA to biological inactivation by UV increased as the amount of lysozyme bound per DNA strand increased. Although binding constants could not be measured for the DNA-histone interaction, this protein had a protective effect which was greater under conditions which cause enhanced binding. No crosslinking of either protein could be detected. Irradiation was also performed in the presence of various amino acids and short peptides. These were chosen to include amino acids which: (1) are positively charged, (2) absorb UV of this wavelength or (3) form UV-induced crosslinks to DNA. None of the amino acids tested affected sensitivity of the DNA to biological inactivation. Peptides containing a UV-absorbing amino acid and a positively charged amino acid enhanced sensitivity. For each of these peptides, a mixture of the constituent amino acids had the same effect as the peptide itself. Under the conditions used, no evidence for formation of DNA-amino acid crosslinks was found. The results indicate that proteins and peptides can sensitize DNA to UV inactivation by mechanisms other than covalent crosslink formation. (author)

  7. NMR Determination of Protein Partitioning into Membrane Domains with Different Curvatures and Application to the Influenza M2 Peptide

    Science.gov (United States)

    Wang, Tuo; Cady, Sarah D.; Hong, Mei

    2012-01-01

    The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use 31P and 13C solid-state NMR to examine M2-induced membrane curvature. M2(22–46), which includes only the transmembrane (TM) helix, and M2(21–61), which contains an additional amphipathic helix, are studied. 31P chemical shift lineshapes indicate that M2(21–61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22–46) has minimal effect. The lamellar and isotropic domains have distinct 31P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. 31P- and 13C-detected 1H T2 relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21–61) preferentially binds to the high-curvature domain. 31P linewidths indicate that the isotropic vesicles induced by M2(21–61) are 10–35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides. PMID:22385849

  8. Construction of tunable peptide nucleic acid junctions.

    Science.gov (United States)

    Duan, Tanghui; He, Liu; Tokura, Yu; Liu, Xin; Wu, Yuzhou; Shi, Zhengshuang

    2018-03-15

    We report here the construction of 3-way and 4-way peptide nucleic acid (PNA) junctions as basic structural units for PNA nanostructuring. The incorporation of amino acid residues into PNA chains makes PNA nanostructures with more structural complexity and architectural flexibility possible, as exemplified by building 3-way PNA junctions with tunable nanopores. Given that PNA nanostructures have good thermal and enzymatic stabilities, they are expected to have broad potential applications in biosensing, drug delivery and bioengineering.

  9. Prediction of anticancer peptides against MCF-7 breast cancer cells from the peptidomes of Achatina fulica mucus fractions.

    Science.gov (United States)

    E-Kobon, Teerasak; Thongararm, Pennapa; Roytrakul, Sittiruk; Meesuk, Ladda; Chumnanpuen, Pramote

    2016-01-01

    Several reports have shown antimicrobial and anticancer activities of mucous glycoproteins extracted from the giant African snail Achatina fulica. Anticancer properties of the snail mucous peptides remain incompletely revealed. The aim of this study was to predict anticancer peptides from A. fulica mucus. Two of HPLC-separated mucous fractions (F2 and F5) showed in vitro cytotoxicity against the breast cancer cell line (MCF-7) and normal epithelium cell line (Vero). According to the mass spectrometric analysis, 404 and 424 peptides from the F2 and F5 fractions were identified. Our comprehensive bioinformatics workflow predicted 16 putative cationic and amphipathic anticancer peptides with diverse structures from these two peptidome data. These peptides would be promising molecules for new anti-breast cancer drug development.

  10. Human acid β-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site

    International Nuclear Information System (INIS)

    Dinur, T.; Osiecki, K.M.; Legler, G.; Gatt, S.; Desnick, R.J.; Grabowski, G.A.

    1986-01-01

    Human acid β-glucosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) cleaves the glucosidic bonds of glucosylceramide and synthetic β-glucosides. The deficient activity of this hydrolase is the enzymatic defect in the subtypes and variants of Gaucher disease, the most prevalent lysosomal storage disease. To isolate and characterize the catalytic site of the normal enzyme, brominated 3 H-labeled conduritol B epoxide ( 3 H-Br-CBE), which inhibits the enzyme by binding covalently to this site, was used as an affinity label. Under optimal conditions 1 mol of 3 H-Br-CBE bound to 1 mol of pure enzyme protein, indicating the presence of a single catalytic site per enzyme subunit. After V 8 protease digestion of the 3 H-Br-CBE-labeled homogeneous enzyme, three radiolabeled peptides, designated peptide A, B, or C, were resolved by reverse-phase HPLC. The partial amino acid sequence (37 residues) of peptide A (M/sub r/, 5000) was determined. The sequence of this peptide, which contained the catalytic site, had exact homology to the sequence near the carboxyl terminus of the protein, as predicted from the nucleotide sequence of the full-length cDNA encoding acid β-glucosidase

  11. Peptide nucleic acids and their potential applications in biotechnology

    DEFF Research Database (Denmark)

    Buchardt, O.; Egholm, M.; Berg, R.H.

    1993-01-01

    Peptide nucleic acids (PNAs) are novel DNA mimics in which the sugar-phosphate backbone has been replaced with a backbone based on amino acids1-3. PNAs exhibit sequence-specific binding to DNA and RNA with higher affinities and specificities than unmodified DNA. They,are resistant to nuclease...

  12. Biogenic and Synthetic Peptides with Oppositely Charged Amino Acids as Binding Sites for Mineralization.

    Science.gov (United States)

    Lemloh, Marie-Louise; Altintoprak, Klara; Wege, Christina; Weiss, Ingrid M; Rothenstein, Dirk

    2017-01-28

    Proteins regulate diverse biological processes by the specific interaction with, e.g., nucleic acids, proteins and inorganic molecules. The generation of inorganic hybrid materials, such as shell formation in mollusks, is a protein-controlled mineralization process. Moreover, inorganic-binding peptides are attractive for the bioinspired mineralization of non-natural inorganic functional materials for technical applications. However, it is still challenging to identify mineral-binding peptide motifs from biological systems as well as for technical systems. Here, three complementary approaches were combined to analyze protein motifs consisting of alternating positively and negatively charged amino acids: (i) the screening of natural biomineralization proteins; (ii) the selection of inorganic-binding peptides derived from phage display; and (iii) the mineralization of tobacco mosaic virus (TMV)-based templates. A respective peptide motif displayed on the TMV surface had a major impact on the SiO₂ mineralization. In addition, similar motifs were found in zinc oxide- and zirconia-binding peptides indicating a general binding feature. The comparative analysis presented here raises new questions regarding whether or not there is a common design principle based on acidic and basic amino acids for peptides interacting with minerals.

  13. Cathelicidins from the bullfrog Rana catesbeiana provides novel template for peptide antibiotic design.

    Directory of Open Access Journals (Sweden)

    Guiying Ling

    Full Text Available Cathelicidins, a class of gene-encoded effector molecules of vertebrate innate immunity, provide a first line of defense against microbial invasions. Although cathelicidins from mammals, birds, reptiles and fishes have been extensively studied, little is known about cathelicidins from amphibians. Here we report the identification and characterization of two cathelicidins (cathelicidin-RC1 and cathelicidin-RC2 from the bullfrog Rana catesbeiana. The cDNA sequences (677 and 700 bp, respectively encoding the two peptides were successfully cloned from the constructed lung cDNA library of R. catesbeiana. And the deduced mature peptides are composed of 28 and 33 residues, respectively. Structural analysis indicated that cathelicidin-RC1 mainly assumes an amphipathic alpha-helical conformation, while cathelicidin-RC2 could not form stable amphipathic structure. Antimicrobial and bacterial killing kinetic analysis indicated that the synthetic cathelicidin-RC1 possesses potent, broad-spectrum and rapid antimicrobial potency, while cathelicidin-RC2 exhibited very weak antimicrobial activity. Besides, the antimicrobial activity of cathelicidin-RC1 is salt-independent and highly stable. Scanning electron microscopy (SEM analysis indicated that cathelicidin-RC1 kills microorganisms through the disruption of microbial membrane. Moreover, cathelicidin-RC1 exhibited low cytotoxic activity against mammalian normal or tumor cell lines, and low hemolytic activity against human erythrocytes. The potent, broad-spectrum and rapid antimicrobial activity combined with the salt-independence, high stability, low cytotoxic and hemolytic activities make cathelicidin-RC1 an ideal template for the development of novel peptide antibiotics.

  14. A Novel Apolipoprotein C-II Mimetic Peptide That Activates Lipoprotein Lipase and Decreases Serum Triglycerides in Apolipoprotein E–Knockout Mice

    Science.gov (United States)

    Sakurai, Toshihiro; Sakurai-Ikuta, Akiko; Sviridov, Denis; Freeman, Lita; Ahsan, Lusana; Remaley, Alan T.

    2015-01-01

    Apolipoprotein A-I (apoA-I) mimetic peptides are currently being developed as possible new agents for the treatment of cardiovascular disease based on their ability to promote cholesterol efflux and their other beneficial antiatherogenic properties. Many of these peptides, however, have been reported to cause transient hypertriglyceridemia due to inhibition of lipolysis by lipoprotein lipase (LPL). We describe a novel bihelical amphipathic peptide (C-II-a) that contains an amphipathic helix (18A) for binding to lipoproteins and stimulating cholesterol efflux as well as a motif based on the last helix of apolipoprotein C-II (apoC-II) that activates lipolysis by LPL. The C-II-a peptide promoted cholesterol efflux from ATP-binding cassette transporter ABCA1-transfected BHK cells similar to apoA-I mimetic peptides. Furthermore, it was shown in vitro to be comparable to the full-length apoC-II protein in activating lipolysis by LPL. When added to serum from a patient with apoC-II deficiency, it restored normal levels of LPL-induced lipolysis and also enhanced lipolysis in serum from patients with type IV and V hypertriglyceridemia. Intravenous injection of C-II-a (30 mg/kg) in apolipoprotein E–knockout mice resulted in a significant reduction of plasma cholesterol and triglycerides of 38 ± 6% and 85 ± 7%, respectively, at 4 hours. When coinjected with the 5A peptide (60 mg/kg), the C-II-a (30 mg/kg) peptide was found to completely block the hypertriglyceridemic effect of the 5A peptide in C57Bl/6 mice. In summary, C-II-a is a novel peptide based on apoC-II, which promotes cholesterol efflux and lipolysis and may therefore be useful for the treatment of apoC-II deficiency and other forms of hypertriglyceridemia. PMID:25395590

  15. Investigation of solid-phase hydrogenation of amino acids and peptides

    International Nuclear Information System (INIS)

    Zolotarev, Yu.A.; Myasoedov, N.F.; Zajtsev, D.A.; Lubnin, M.Yu.; Tatur, V.Yu.; Kozik, V.S.; Dorokhova, E.M.; Rozenberg, S.N.

    1990-01-01

    The possibility of synthesizing amino acids and peptides multiply labelled with tritium or deuterium by the method of solid-phase isotopic exchange with gaseous hydrogen isotopes was verified. Establishment of the isotopic hydrogen equilibrium between the gaseous phase and the solid phase formed by the amino acid molecules was found experimentally. The activation energy of the isotopic exchange is 13 kcal/mol. A mathematical model was set up for the isotopic exchange with a probable substitution of hydrogen atoms. Uniformly labelled amino acids were obtained in a high optical purity and with 80 to 90% hydrogen substitution by deuterium and tritium. Tritiated peptides were prepared in high yields at molar activities of 1.5 to 3.7 TBq/mmol. (author). 4 tabs

  16. Effect of chenodeoxycholic acid and the bile acid sequestrant colesevelam on glucagon-like peptide-1 secretion

    DEFF Research Database (Denmark)

    Hansen, Morten; Scheltema, Matthijs J; Sonne, David P

    2016-01-01

    AIMS: In patients with type 2 diabetes, rectal administration of bile acids increases glucagon-like peptide-1 (GLP-1) secretion and reduces plasma glucose. In addition, oral bile acid sequestrants (BASs) reduce blood glucose by an unknown mechanism. In this study we evaluated the effects...... of the primary human bile acid, chenodeoxycholic acid (CDCA), and the BAS, colesevelam, instilled into the stomach, on plasma levels of GLP-1, glucose-dependent insulinotropic polypeptide, glucose, insulin, C-peptide, glucagon, cholecystokinin and gastrin as well as gastric emptying, gallbladder volume, appetite......, and delayed gastric emptying. We speculate that bile acid-induced activation of TGR5 on L cells increases GLP-1 secretion, which in turn may result in amplification of glucose-stimulated insulin secretion. Furthermore our data suggest that colesevelam does not have an acute effect on GLP-1 secretion in humans....

  17. Interaction of MreB-derived antimicrobial peptides with membranes.

    Science.gov (United States)

    Saikia, Karabi; Chaudhary, Nitin

    2018-03-25

    Antimicrobial peptides are critical components of defense systems in living forms. The activity is conferred largely by the selective membrane-permeabilizing ability. In our earlier work, we derived potent antimicrobial peptides from the 9-residue long, N-terminal amphipathic helix of E. coli MreB protein. The peptides display broad-spectrum activity, killing not only Gram-positive and Gram-negative bacteria but opportunistic fungus, Candida albicans as well. These results proved that membrane-binding stretches of bacterial proteins could turn out to be self-harming when applied from outside. Here, we studied the membrane-binding and membrane-perturbing potential of these peptides. Steady-state tryptophan fluorescence studies with tryptophan extended peptides, WMreB 1-9 and its N-terminal acetylated analog, Ac-WMreB 1-9 show preferential binding to negatively-charged liposomes. Both the peptides cause permeabilization of E. coli inner and outer-membranes. Tryptophan-lacking peptides, though permeabilize the outer-membrane efficiently, little permeabilization of the inner-membrane is observed. These data attest membrane-destabilization as the mechanism of rapid bacterial killing. This study is expected to motivate the research in identifying microbes' self-sequences to combat them. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Phospholyl(borane) Amino Acids and Peptides: Stereoselective Synthesis and Fluorescent Properties with Large Stokes Shift.

    Science.gov (United States)

    Arribat, Mathieu; Rémond, Emmanuelle; Clément, Sébastien; Lee, Arie Van Der; Cavelier, Florine

    2018-01-24

    The synthesis of phospholyl(borane) amino acids was stereoselectively achieved by reaction of phospholide anion with iodo α-amino ester derived from l-aspartic acid or l-serine, followed by in situ complexation with borane. Phospholyl(borane) amino acids are easy to store and can be subjected to direct transformation into the corresponding free phospholyl, gold complex, oxide or sulfur derivatives as well as phospholinium salts, thus offering a variety of side chains. After selective deprotection of carboxylic function or amine, C- or N- peptide coupling with an alanine moiety proved the possible incorporation into peptides. Such phospholyl amino acid and peptide derivatives exhibit fluorescent properties with a large Stokes shift (160 nm) and fluorescence up to 535 nm, depending on the phosphole aromaticity and the chemical environment. These phospholyl(borane) amino acids constitute a new class of unnatural amino acids useful for structure-activities relationship studies and appear to be promising fluorophores for the development of labeled peptides.

  19. Femtomolar Ln(III) affinity in peptide-based ligands containing unnatural chelating amino acids.

    Science.gov (United States)

    Niedźwiecka, Agnieszka; Cisnetti, Federico; Lebrun, Colette; Delangle, Pascale

    2012-05-07

    The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P(22), which incorporates two unnatural amino acids Ada(2) with aminodiacetate chelating arms, showed picomolar affinity for Tb(3+). To design peptides with higher denticity, expected to show higher affinity for Ln(3+), we synthesized the novel unnatural amino acid Ed3a(2) which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a(2)(tBu)(3)-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P(HD2) (Ac-Trp-Ed3a(2)-Pro-Gly-Ada(2)-Gly-NH(2)) and P(HD5) (Ac-Trp-Ada(2)-Pro-Gly-Ed3a(2)-Gly-NH(2)) led to octadentate Tb(3+) complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a(2) in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P(HD5) promotes the formation of polymetallic species in excess of Ln(3+), P(HD2) forms exclusively the mononuclear complex. The octadentate coordination of Tb(3+) by both P(HD) leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P(HD2) forms a unique octadentate Tb(3+) complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP(22) complex by a factor of 4.

  20. Acidic-Basic Properties of Three Alanine-Based Peptides Containing Acidic and Basic Side Chains: Comparison Between Theory and Experiment

    OpenAIRE

    Makowska, Joanna; Bagińska, Katarzyna; Liwo, Adam; Chmurzyński, Lech; Scheraga, Harold A.

    2008-01-01

    The purpose of this work was to evaluate the effect of the nature of the ionizable end groups, and the solvent, on their acid-base properties in alanine-based peptides. Hence, the acid-base properties of three alanine-based peptides: Ac-KK-(A)7-KK-NH2 (KAK), Ac-OO-(A)7-DD-NH2 (OAD), Ac-KK-(A)7-EE-NH2 (KAE), where A, D, E, K, and O denote alanine, aspartic acid, glutamic acid, lysine, and ornithine, respectively, were determined in water and in methanol by potentiometry. With the availability ...

  1. Prediction of anticancer peptides against MCF-7 breast cancer cells from the peptidomes of Achatina fulica mucus fractions

    Directory of Open Access Journals (Sweden)

    Teerasak E-kobon

    2016-01-01

    Full Text Available Several reports have shown antimicrobial and anticancer activities of mucous glycoproteins extracted from the giant African snail Achatina fulica. Anticancer properties of the snail mucous peptides remain incompletely revealed. The aim of this study was to predict anticancer peptides from A. fulica mucus. Two of HPLC-separated mucous fractions (F2 and F5 showed in vitro cytotoxicity against the breast cancer cell line (MCF-7 and normal epithelium cell line (Vero. According to the mass spectrometric analysis, 404 and 424 peptides from the F2 and F5 fractions were identified. Our comprehensive bioinformatics workflow predicted 16 putative cationic and amphipathic anticancer peptides with diverse structures from these two peptidome data. These peptides would be promising molecules for new anti-breast cancer drug development.

  2. The synthesis of amphipathic prodrugs of 1,2-diol drugs with saccharide conjugates by high regioselective enzymatic protocol.

    Science.gov (United States)

    Quan, Jing; Chen, Zhichun; Han, Chengyou; Lin, Xianfu

    2007-02-15

    A facile, high regioselective enzymatic synthesis approach for the preparation of amphipathic prodrugs with saccharides of mephenesin and chlorphenesin was developed. Firstly, transesterification of two drugs with divinyl dicarboxylates with different carbon chain length was performed under the catalysis of Candida antarctica lipase acrylic resin and Lipozyme in anhydrous acetone at 50 degrees C, respectively. A series of lipophilic derivatives with vinyl groups of mephenesin and chlorphenesin were prepared. The influences of different organic solvents, enzyme sources, reaction time, and the acylation reagents on the synthesis of vinyl esters were investigated. And then, protease-catalyzed high regioselective acylation of D-glucose and D-mannose with vinyl esters of mephenesin and chlorphenesin gave drug-saccharide derivatives in good yields. The studies of lipophilicity and hydrolysis in vitro of prodrugs verified that drug-saccharide derivatives had amphipathic properties, and both lipophilic and amphipathic drug derivatives had obvious controlled release characteristics.

  3. Gene Targeting and Expression Modulation by Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Peptide nucleic acids (PNA) are artificial structural mimics of nucleic acids capable of sequence specific hybridization to both RNA and DNA. Thus they have obvious potential as gene targeting agents for drug discovery approaches. An overview with emphasis on recent progress on RNA "interference...

  4. Amino acids and peptides. XXXII: A bifunctional poly(ethylene glycol) hybrid of fibronectin-related peptides.

    Science.gov (United States)

    Maeda, M; Izuno, Y; Kawasaki, K; Kaneda, Y; Mu, Y; Tsutsumi, Y; Lem, K W; Mayumi, T

    1997-12-18

    An amino acid type poly(ethylene glycol) (aaPPEG) was prepared and its application to a drug carrier was examined. The peptides, Arg-Gly-Asp (RGD) and Glu-Ile-Leu-Asp-Val (EILDV) which were reported as active fragments of Fibronectin (a cell adhesion protein), were conjugated with aaPEG (molecular weight, 10,000). The hybrid, RGD-aaPEG-EILDV, was prepared by a combination of the solid-phase method and the solution method. Antiadhesive activity of the peptides was not lost by its hybrid formation with the large aaPEG molecule. A mixture of RGD (0.43 mmol) and EILDV (0.43 mmol) did not demonstrate an antiadhesive effect, but the hybrid containing 0.43 mmol of each peptide did exhibit this effect.

  5. Structural analysis and taste evaluation of γ-glutamyl peptides comprising sulfur-containing amino acids.

    Science.gov (United States)

    Amino, Yusuke; Wakabayashi, Hidehiko; Akashi, Satoko; Ishiwatari, Yutaka

    2018-03-01

    The structures, flavor-modifying effects, and CaSR activities of γ-glutamyl peptides comprising sulfur-containing amino acids were investigated. The chemical structures, including the linkage mode of the N-terminal glutamic acid, of γ-L-glutamyl-S-(2-propenyl)-L-cysteine (γ-L-glutamyl-S-allyl-L-cysteine) and its sulfoxide isolated from garlic were established by comparing their NMR spectra with those of authentic peptides prepared using chemical methods. Mass spectrometric analysis also enabled determination of the linkage modes in the glutamyl dipeptides by their characteristic fragmentation. In sensory evaluation, these peptides exhibited flavor-modifying effects (continuity) in umami solutions less pronounced but similar to that of glutathione. Furthermore, the peptides exhibited intrinsic flavor due to the sulfur-containing structure, which may be partially responsible for their flavor-modifying effects. In CaSR assays, γ-L-glutamyl-S-methyl-L-cysteinylglycine was most active, which indicates that the presence of a medium-sized aliphatic substituent at the second amino acid residue in γ-glutamyl peptides enhances CaSR activity.

  6. Thermodynamics of the interactions of some amino acids and peptides with dodecyltrimethylammonium bromide and tetradecyltrimethylammonium bromide

    International Nuclear Information System (INIS)

    Talele, Paurnima; Kishore, Nand

    2014-01-01

    Highlights: • Interactions of amino acids and peptides were studied with two cationic surfactants. • Partial molar properties and hydration numbers did not change significantly. • Measured properties indicate balance of polar and non-polar interactions. • Peptide bonds did not strengthen the extent of polar interactions with surfactant. • Results provide quantitative fine details of cationic surfactant–amino acids/peptides interactions. -- Abstract: The values of apparent molar volume V 2,ϕ and apparent molar adiabatic compressibility K S,2,ϕ of amino acids glycine, L-alanine, DL-α-amino-n-butyric acid, L-valine, L-leucine and peptides glycyl-glycine, glycyl-glycyl-glycine and glycyl-leucine have been determined in aqueous solutions of cationic surfactants dodecyltrimethylammonium bromide (DTAB) and tetradecyltrimethylammonium bromide (TTAB) by means of density and sound velocity measurements. The heat evolved or absorbed (q) during the course of interactions of amino acids and peptides with the aqueous solutions of surfactants were determined by isothermal titration calorimetry at T = 298.15 K. The values of standard partial molar volume V 2,m 0 and standard partial molar adiabatic compressibility K s,2,m 0 at infinite dilution were calculated from the values of V 2,ϕ and K S,2,ϕ . Similarly the values of limiting enthalpies of dilution (Δ dil H 0 ) of the amino acids/peptides were calculated from heat evolved or absorbed during calorimetric experiments. The standard partial molar quantities of transfer from water to aqueous surfactant solutions have been used to identify the interactions of amino acids and peptides with surfactants in terms of ionic–ionic, ionic–hydrophobic and hydrophobic–hydrophobic group interactions

  7. Conformational study of melectin and antapin antimicrobial peptides in model membrane environments

    Science.gov (United States)

    Kocourková, Lucie; Novotná, Pavlína; Čujová, Sabína; Čeřovský, Václav; Urbanová, Marie; Setnička, Vladimír

    2017-01-01

    Antimicrobial peptides have long been considered as promising compounds against drug-resistant pathogens. In this work, we studied the secondary structure of antimicrobial peptides melectin and antapin using electronic (ECD) and vibrational circular dichroism (VCD) spectroscopies that are sensitive to peptide secondary structures. The results from quantitative ECD spectral evaluation by Dichroweb and CDNN program and from the qualitative evaluation of the VCD spectra were compared. The antimicrobial activity of the selected peptides depends on their ability to adopt an amphipathic α-helical conformation on the surface of the bacterial membrane. Hence, solutions of different zwitterionic and negatively charged liposomes and micelles were used to mimic the eukaryotic and bacterial biological membranes. The results show a significant content of α-helical conformation in the solutions of negatively charged liposomes mimicking the bacterial membrane, thus correlating with the antimicrobial activity of the studied peptides. On the other hand in the solutions of zwitterionic liposomes used as models of the eukaryotic membranes, the fraction of α-helical conformation was lower, which corresponds with their moderate hemolytic activity.

  8. Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

    Science.gov (United States)

    Periat, Aurélie; Krull, Ira S; Guillarme, Davy

    2015-02-01

    This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity. Hydrophilic interaction chromatography is also recognized as a powerful technique for peptide analysis, and there are a lot of papers showing its applicability for proteomic applications (peptide mapping). It is expected that its use for peptide mapping will continue to grow in the future, particularly because this analytical strategy can be combined with reversed-phase liquid chromatography, in a two-dimensional setup, to reach very high resolving power. Finally, the interest in hydrophilic interaction chromatography for intact proteins analysis is less evident due to possible solubility issues and a lack of suitable hydrophilic interaction chromatography stationary phases. To date, it has been successfully employed only for the characterization of membrane proteins, histones, and the separation of glycosylated isoforms of an intact glycoprotein. From our point of view, the number of hydrophilic interaction chromatography columns compatible with intact proteins (higher upper temperature limit, large pore size, etc.) is still too limited. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 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.

  10. G-CSF receptor-binding cyclic peptides designed with artificial amino-acid linkers

    International Nuclear Information System (INIS)

    Shibata, Kenji; Maruyama-Takahashi, Kumiko; Yamasaki, Motoo; Hirayama, Noriaki

    2006-01-01

    Designing small molecules that mimic the receptor-binding local surface structure of large proteins such as cytokines or growth factors is fascinating and challenging. In this study, we designed cyclic peptides that reproduce the receptor-binding loop structures of G-CSF. We found it is important to select a suitable linker to join two or more discontinuous sequences and both termini of the peptide corresponding to the receptor-binding loop. Structural simulations based on the crystallographic structure of KW-2228, a stable and potent analog of human G-CSF, led us to choose 4-aminobenzoic acid (Abz) as a part of the linker. A combination of 4-Abz with β-alanine or glycine, and disulfide bridges between cysteins or homocysteins, gave a structure suitable for receptor binding. In this structure, the side-chains of several amino acids important for the interactions with the receptor are protruding from one side of the peptide ring. This artificial peptide showed G-CSF antagonistic activity in a cell proliferation assay

  11. Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Pankratova, Stanislava; Nielsen, Peter E

    2005-01-01

    Cell-penetrating peptides have been widely used to improve cellular delivery of a variety of proteins and antisense agents. However, recent studies indicate that such cationic peptides are predominantly entering cells via an endosomal pathway. We now show that the nuclear antisense effect in He......La cells of a variety of peptide nucleic acid (PNA) peptide conjugates is significantly enhanced by addition of 6 mM Ca(2+) (as well as by the lysosomotrophic agent chloroquine). In particular, the antisense activities of Tat(48-60) and heptaarginine-conjugated PNAs were increased 44-fold and 8.5-fold......, respectively. Evidence is presented that the mechanism involves endosomal release. The present results show that Ca(2+) can be used as an effective enhancer for in vitro cellular delivery of cationic peptide-conjugated PNA oligomers, and also emphasize the significance of the endosomal escape route...

  12. Anti-Inflammatory Action of an Antimicrobial Model Peptide That Suppresses the TRIF-Dependent Signaling Pathway via Inhibition of Toll-Like Receptor 4 Endocytosis in Lipopolysaccharide-Stimulated Macrophages.

    Directory of Open Access Journals (Sweden)

    Do-Wan Shim

    Full Text Available Antimicrobial peptides (AMPs, also called host defense peptides, particularly those with amphipathic helical structures, are emerging as target molecules for therapeutic development due to their immunomodulatory properties. Although the antimicrobial activity of AMPs is known to be exerted primarily by permeation of the bacterial membrane, the mechanism underlying its anti-inflammatory activity remains to be elucidated. We report potent anti-inflammatory activity of WALK11.3, an antimicrobial model peptide with an amphipathic helical conformation, in lipopolysaccharide (LPS-stimulated RAW264.7 cells. This peptide inhibited the expression of inflammatory mediators, including nitric oxide, COX-2, IL-1β, IL-6, INF-β, and TNF-α. Although WALK11.3 did not exert a major effect on all downstream signaling in the MyD88-dependent pathway, toll-like receptor 4 (TLR4- mediated pro-inflammatory signals were markedly attenuated in the TRIF-dependent pathway due to inhibition of the phosphorylation of STAT1 by attenuation of IRF3 phosphorylation. WALK11.3 specifically inhibited the endocytosis of TLR4, which is essential for triggering TRIF-mediated signaling in macrophage cells. Hence, we suggest that specific interference with TLR4 endocytosis could be one of the major modes of the anti-inflammatory action of AMPs. Our designed WALK11 peptides, which possess both antimicrobial and anti-inflammatory activities, may be promising molecules for the development of therapies for infectious inflammation.

  13. Iminodiacetic acid as bifunctional linker for dimerization of cyclic RGD peptides

    International Nuclear Information System (INIS)

    Xu, Dong; Zhao, Zuo-Quan; Chen, Shu-Ting; Yang, Yong; Fang, Wei; Liu, Shuang

    2017-01-01

    Introduction: In this study, I2P-RGD 2 was used as the example to illustrate a novel approach for dimerization of cyclic RGD peptides. The main objective of this study was to explore the impact of bifunctional linkers (glutamic acid vs. iminodiacetic acid) on tumor-targeting capability and excretion kinetics of the 99m Tc-labeled dimeric cyclic RGD peptides. Methods: HYNIC-I2P-RGD 2 was prepared by reacting I2P-RGD 2 with HYNIC-OSu in the presence of diisopropylethylamine, and was evaluated for its α v β 3 binding affinity against 125 I-echistatin bound to U87MG glioma cells. 99m Tc-I2P-RGD 2 was prepared with high specific activity (~185 GBq/μmol). The athymic nude mice bearing U87MG glioma xenografts were used to evaluate its biodistribution properties and image quality in comparison with those of 99m Tc-3P-RGD 2 . Results: The IC 50 value for HYNIC-I2P-RGD 2 was determined to be 39 ± 6 nM, which was very close to that (IC 50 = 33 ± 5 nM) of HYNIC-3P-RGD 2 . Replacing glutamic acid with iminodiacetic acid had little impact on α v β 3 binding affinity of cyclic RGD peptides. 99m Tc-I2P-RGD 2 and 99m Tc-3P-RGD 2 shared similar tumor uptake values over the 2 h period, and its α v β 3 -specificity was demonstrated by a blocking experiment. The uptake of 99m Tc-I2P-RGD 2 was significantly lower than 99m Tc-3P-RGD 2 in the liver and kidneys. The U87MG glioma tumors were visualized by SPECT with excellent contrast using both 99m Tc-I2P-RGD 2 and 99m Tc-3P-RGD 2 . Conclusion: Iminodiacetic acid is an excellent bifunctional linker for dimerization of cyclic RGD peptides. Bifunctional linkers have significant impact on the excretion kinetics of 99m Tc radiotracers. Because of its lower liver uptake and better tumor/liver ratios, 99m Tc-I2P-RGD 2 may have advantages over 99m Tc-3P-RGD 2 for diagnosis of tumors in chest region. -- Graphical abstract: This report presents novel approach for dimerization of cyclic RGD peptides using iminodiacetic acid as a

  14. Molecular basis for endotoxin neutralization by amphipathic peptides derived from the alpha-helical cationic core-region of NK-lysin.

    Science.gov (United States)

    Brandenburg, Klaus; Garidel, Patrick; Fukuoka, Satoshi; Howe, Jörg; Koch, Michel H J; Gutsmann, Thomas; Andrä, Jörg

    2010-08-01

    An analysis of the interaction of the NK-lysin derived peptide NK-2 and of analogs thereof with bacterial lipopolysaccharide (LPS, endotoxin) was performed to determine the most important biophysical parameters for an effective LPS neutralization. We used microcalorimetry, FTIR spectroscopy, Zeta potential measurements, and small-angle X-ray scattering to analyze the peptide:LPS binding enthalpy, the accessible LPS surface charge, the fluidity of the LPS hydrocarbon chains, their phase transition enthalpy change, the aggregate structure of LPS, and how these parameters are modulated by the peptides. We conclude that (i) a high peptide:LPS binding affinity, which is facilitated by electrostatic and hydrophobic interactions and which leads to a positive Zeta potential, (ii) the formation of peptide-enriched domains, which destabilize the lipid packing, demonstrated by a drastic decrease of phase transition enthalpy change of LPS, and (iii) the multilamellarization of the LPS aggregate structure are crucial for an effective endotoxin neutralization by cationic peptides.

  15. Interaction of a novel antimicrobial peptide isolated from the venom of solitary bee Colletes daviesanus with phospholipid vesicles and Escherichia coli cells.

    Science.gov (United States)

    Čujová, Sabína; Bednárová, Lucie; Slaninová, Jiřina; Straka, Jakub; Čeřovský, Václav

    2014-11-01

    The peptide named codesane (COD), consisting of 18 amino acid residues and isolated from the venom of wild bee Colletes daviesanus (Hymenoptera : Colletidae), falls into the category of cationic α-helical amphipathic antimicrobial peptides. In our investigations, synthetic COD exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria and Candida albicans but also noticeable hemolytic activity. COD and its analogs (collectively referred to as CODs) were studied for the mechanism of their action. The interaction of CODs with liposomes led to significant leakage of calcein entrapped in bacterial membrane-mimicking large unilamellar vesicles made preferentially from anionic phospholipids while no calcein leakage was observed from zwitterionic liposomes mimicking membranes of erythrocytes. The preference of CODs for anionic phospholipids was also established by the blue shift in the tryptophan emission spectra maxima when the interactions of tryptophan-containing COD analogs with liposomes were examined. Those results were in agreement with the antimicrobial and hemolytic activities of CODs. Moreover, we found that the studied peptides permeated both the outer and inner cytoplasmic membranes of Escherichia coli. This was determined by measuring changes in the fluorescence of probe N-phenyl-1-naphthylamine and detecting cytoplasmic β-galactosidase released during the interaction of peptides with E. coli cells. Transmission electron microscopy revealed that treatment of E. coli with one of the COD analogs caused leakage of bacterial content mainly from the septal areas of the cells. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  16. Distinguishing Aspartic and Isoaspartic Acids in Peptides by Several Mass Spectrometric Fragmentation Methods

    Science.gov (United States)

    DeGraan-Weber, Nick; Zhang, Jun; Reilly, James P.

    2016-12-01

    Six ion fragmentation techniques that can distinguish aspartic acid from its isomer, isoaspartic acid, were compared. MALDI post-source decay (PSD), MALDI 157 nm photodissociation, tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) charge tagging in PSD and photodissociation, ESI collision-induced dissociation (CID), electron transfer dissociation (ETD), and free-radical initiated peptide sequencing (FRIPS) with CID were applied to peptides containing either aspartic or isoaspartic acid. Diagnostic ions, such as the y-46 and b+H2O, are present in PSD, photodissociation, and charge tagging. c•+57 and z-57 ions are observed in ETD and FRIPS experiments. For some molecules, aspartic and isoaspartic acid yield ion fragments with significantly different intensities. ETD and charge tagging appear to be most effective at distinguishing these residues.

  17. Dendropsophin 1, a novel antimicrobial peptide from the skin secretion of the endemic Colombian frog Dendropsophus columbianus.

    Science.gov (United States)

    Triana-Vidal, Luz Elena; Castro, Mariana Souza; Pires Júnior, Osmindo Rodrigues; Álvares, Alice Cunha Morales; de Freitas, Sonia Maria; Fontes, Wagner; Vargas, Jimmy Alexander Guerrero; Zúñiga-Baos, Jorge Alberto; Correia Batista, Isabel de Fátima; Grellier, Philippe; Charneau, Sébastien

    2018-06-01

    In efforts to find new antimicrobial peptides (AMPs), we studied the skin secretion of the endemic Colombian frog Dendropsophus columbianus belonging to a genus that has not been investigated previously. From HPLC-fractionated secretion, we identified one peptide with slightly antibacterial activity. Its peptide sequence showed no sequence similarity to current annotated peptides. We named this novel peptide dendropsophin 1 (Dc1). Afterward, two analogues were designed (Dc1.1 and Dc1.2) to improve the cationic and amphipathic features. Then, their antiproliferative and cytotoxic properties were evaluated against several pathogens including bacteria, fungi, protozoa and also mammalian cells. Dc1 and its two analogues exhibited moderate antibacterial activities and no hemolytic and cytotoxic effects on mammalian cells. Analogue Dc1.2 showed slightly improved antibacterial properties. Their secondary structures were characterised using CD spectroscopy and Dc1.2 displayed a higher α-helix content and thermal stability compared to Dc1 and Dc1.1 in hydrophobic experimental conditions.

  18. Nanocarriers Conjugated with Cell Penetrating Peptides: New Trojan Horses by Modern Ulysses.

    Science.gov (United States)

    Zappavigna, Silvia; Misso, Gabriella; Falanga, Annarita; Perillo, Emiliana; Novellino, Ettore; Galdiero, Massimiliano; Grieco, Paolo; Caraglia, Michele; Galdiero, Stefania

    Nanomedicine has opened the way to the design of more efficient diagnostics and therapeutics. Moreover, recent literature has illustrated the use of short cationic and/or amphipathic peptides, known as cell-penetrating peptides (CPPs), for mediating advanced drug delivery. CPPs exploit their ability to enter cells and enhance the uptake of many cargoes ranging from small molecules to proteins. The distinctive properties of nanocarriers (NC) based systems provide unforeseen benefits over pure drugs for biomedical applications and constitute a challenging research field particularly focused on imaging and delivery; nonetheless, several problems have to be overcome to make them a viable option in clinic. The use of CPPs improves significantly their delivery to specific intracellular targets and thus readily contributes to their use both for effective tumor therapy and gene therapy. A key issue is related to their mechanism of uptake, because although classical CPPs enhance NCs' uptake, the entry mechanism involves the endocytic pathway, which means that the delivered material is sequestered within vesicles and only a small amount will escape from this environment and reach the desired target. In this review, we will summarize recent advances in the use of CPP for enhanced delivery of nanocarriers, nucleic acids, and drugs, we will discuss their uptake mechanisms and we will describe novel approaches to improve endosomal escape of internalized nanosystems.

  19. Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

    Science.gov (United States)

    Kato, Ryuji; Kaga, Chiaki; Kunimatsu, Mitoshi; Kobayashi, Takeshi; Honda, Hiroyuki

    2006-06-01

    Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have

  20. Isolation of dermatoxin from frog skin, an antibacterial peptide encoded by a novel member of the dermaseptin genes family.

    Science.gov (United States)

    Amiche, M; Seon, A A; Wroblewski, H; Nicolas, P

    2000-07-01

    A 32-residue peptide, named dermatoxin, has been extracted from the skin of a single specimen of the tree frog Phyllomedusa bicolor, and purified to homogeneity using a four-step protocol. Mass spectral analysis and sequencing of the purified peptide, as well as chemical synthesis and cDNA analysis were consistent with the structure: SLGSFLKGVGTTLASVGKVVSDQF GKLLQAGQ. This peptide proved to be bactericidal towards mollicutes (wall-less eubacteria) and Gram-positive eubacteria, and also, though to a lesser extent, towards Gram-negative eubacteria. Measurement of the bacterial membrane potential revealed that the plasma membrane is the primary target of dermatoxin. Observation of bacterial cells using reflected light fluorescence microscopy after DNA-staining was consistent with a mechanism of cell killing based upon the alteration of membrane permeability rather than membrane solubilization, very likely by forming ion-conducting channels through the plasma membrane. CD spectroscopy and secondary structure predictions indicated that dermatoxin assumes an amphipathic alpha-helical conformation in low polarity media which mimic the lipophilicity of the membrane of target microorganisms. PCR analysis coupled with cDNA cloning and sequencing revealed that dermatoxin is expressed in the skin, the intestine and the brain. Preprodermatoxin from the brain and the intestine have the same sequence as the skin preproform except for two amino-acid substitutions in the preproregion of the brain precursor. The dermatoxin precursor displayed the characteristic features of preprodermaseptins, a family of peptide precursors found in the skin of Phyllomedusa ssp. Precursors of this family have a common N-terminal preproregion followed by markedly different C-terminal domains that give rise to 19-34-residue peptide antibiotics named dermaseptins B and phylloxin, and to the D-amino-acid-containing opioid heptapeptides dermorphins and deltorphins. Because the structures and cidal

  1. Influence of specific amino acid side-chains on the antimicrobial activity and structure of bovine lactoferrampin.

    Science.gov (United States)

    Haney, Evan F; Nazmi, Kamran; Bolscher, Jan G M; Vogel, Hans J

    2012-06-01

    Lactoferrin is an 80 kDa iron binding protein found in the secretory fluids of mammals and it plays a major role in host defence. An antimicrobial peptide, lactoferrampin, was identified through sequence analysis of bovine lactoferrin and its antimicrobial activity against a wide range of bacteria and yeast species is well documented. In the present work, the contribution of specific amino acid residues of lactoferrampin was examined to evaluate the role that they play in membrane binding and bilayer disruption. The structures of all the bovine lactoferrampin derivatives were examined with circular dichroism and nuclear magnetic resonance spectroscopy, and their interactions with phospholipids were evaluated with differential scanning calorimetry and isothermal titration calorimetry techniques. From our results it is apparent that the amphipathic N-terminal helix anchors the peptide to membranes with Trp 268 and Phe 278 playing important roles in determining the strength of the interaction and for inducing peptide folding. In addition, the N-terminal helix capping residues (DLI) increase the affinity for negatively charged vesicles and they mediate the depth of membrane insertion. Finally, the unique flexibility in the cationic C-terminal region of bovine lactoferrampin does not appear to be essential for the antimicrobial activity of the peptide.

  2. Characterisation of neuroprotective efficacy of modified poly-arginine-9 (R9) peptides using a neuronal glutamic acid excitotoxicity model.

    Science.gov (United States)

    Edwards, Adam B; Anderton, Ryan S; Knuckey, Neville W; Meloni, Bruno P

    2017-02-01

    In a recent study, we highlighted the importance of cationic charge and arginine residues for the neuroprotective properties of poly-arginine and arginine-rich peptides. In this study, using cortical neuronal cultures and an in vitro glutamic acid excitotoxicity model, we examined the neuroprotective efficacy of different modifications to the poly-arginine-9 peptide (R9). We compared an unmodified R9 peptide with R9 peptides containing the following modifications: (i) C-terminal amidation (R9-NH2); (ii) N-terminal acetylation (Ac-R9); (iii) C-terminal amidation with N-terminal acetylation (Ac-R9-NH2); and (iv) C-terminal amidation with D-amino acids (R9D-NH2). The three C-terminal amidated peptides (R9-NH2, Ac-R9-NH2, and R9D-NH2) displayed neuroprotective effects greater than the unmodified R9 peptide, while the N-terminal acetylated peptide (Ac-R9) had reduced efficacy. Using the R9-NH2 peptide, neuroprotection could be induced with a 10 min peptide pre-treatment, 1-6 h before glutamic acid insult, or when added to neuronal cultures up to 45 min post-insult. In addition, all peptides were capable of reducing glutamic acid-mediated neuronal intracellular calcium influx, in a manner that reflected their neuroprotective efficacy. This study further highlights the neuroprotective properties of poly-arginine peptides and provides insight into peptide modifications that affect efficacy.

  3. Peptides with Dual Antimicrobial and Anticancer Activities

    Science.gov (United States)

    Felício, Mário R.; Silva, Osmar N.; Gonçalves, Sônia; Santos, Nuno C.; Franco, Octávio L.

    2017-02-01

    In recent years, the number of people suffering from cancer and multi-resistant infections has increased, such that both diseases are already seen as current and future major causes of death. Moreover, chronic infections are one of the main causes of cancer, due to the instability in the immune system that allows cancer cells to proliferate. Likewise, the physical debility associated with cancer or with anticancer therapy itself often paves the way for opportunistic infections. It is urgent to develop new therapeutic methods, with higher efficiency and lower side effects. Antimicrobial peptides (AMPs) are found in the innate immune system of a wide range of organisms. Identified as the most promising alternative to conventional molecules used nowadays against infections, some of them have been shown to have dual activity, both as antimicrobial and anticancer peptides (ACPs). Highly cationic and amphipathic, they have demonstrated efficacy against both conditions, with the number of nature-driven or synthetically designed peptides increasing year by year. With similar properties, AMPs that can also act as ACPs are viewed as future chemotherapeutic drugs, with the advantage of low propensity to resistance, which started this paradigm in the pharmaceutical market. These peptides have already been described as molecules presenting killing mechanisms at the membrane level, but also acting towards intracellular targets, which increases their success comparatively to specific one-target drugs. This review will approach the desirable characteristics of small peptides that demonstrated dual activity against microbial infections and cancer, as well as the peptides engaged in clinical trials.

  4. Observation of the side chain O-methylation of glutamic acid or aspartic acid containing model peptides by electrospray ionization-mass spectrometry.

    Science.gov (United States)

    Atik, A Emin; Guray, Melda Z; Yalcin, Talat

    2017-03-15

    O-methylation of the side chains of glutamic acid (E) and aspartic acid (D) residues is generally observed modification when an acidified methanol/water (MeOH/dH 2 O) mixture is used as a solvent system during sample preparation for proteomic research. This chemical modification may result misidentification with endogenous protein methylation; therefore, a special care should be taken during sample handling prior to mass spectrometric analysis. In the current study, we systematically examined the extent of E/D methylation and C-terminus carboxyl group of synthetic model peptides in terms of different incubation temperatures, storage times, and added acid types as well as its percentages. To monitor these effects, C-terminus amidated and free acid forms of synthetic model peptides comprised of E or D residue(s) have been analyzed by electrospray ionization-mass spectrometry (ESI-MS). Additionally, LC-MS/MS experiments were performed to confirm the formation of methylated peptide product. The results showed that the rate of methylation was increased as the temperature increases along with prolong incubation times. Moreover, the extent of methylation was remarkably high when formic acid (FA) used as a protonation agent instead of acetic acid (AA). In addition, it was found that the degree of methylation was significantly decreased by lowering acid percentages in ESI solution. More than one acidic residue containing model peptides have been also used to explore the extent of multiple methylation reaction. Lastly, the ethanol (EtOH) and isopropanol (iPrOH) have been substituted separately with MeOH in sample preparation step to investigate the extent of esterification reaction under the same experimental conditions. However, in the positive perspective of view, this method can be used as a simple, rapid and cheap method for methylation of acidic residues under normal laboratory conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Creating diversity by site-selective peptide modification: a customizable unit affords amino acids with high optical purity.

    Science.gov (United States)

    Romero-Estudillo, Ivan; Boto, Alicia

    2013-11-15

    The development of peptide libraries by site-selective modification of a few parent peptides would save valuable time and materials in discovery processes, but still is a difficult synthetic challenge. Herein natural hydroxyproline is introduced as a "convertible" unit for the production of a variety of optically pure amino acids, including expensive N-alkyl amino acids, and to achieve the mild, efficient, and site-selective modification of peptides.

  6. Influences of conformations of peptides on stereoinversions and/or isomerizations of aspartic acid residues.

    Science.gov (United States)

    Oda, Akifumi; Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Kurimoto, Eiji; Takahashi, Ohgi

    2018-07-01

    Recently, non-enzymatic stereoinversions of aspartic acid (Asp) residues in proteins and peptides have been reported. Here, we performed replica exchange molecular dynamics (REMD) simulations of model peptides (exon 6, 26A-1, and 26A-2) extracted from elastin to investigate their structural features, thereby revealing the factor that influences stereoinversions. For REMD trajectories, we calculated distances between carboxyl carbon in Asp and amide nitrogen in the (n + 1) residue (CN distances). Because bond formation between carbon and nitrogen is indispensable to the formation of a succinimide intermediate the distance between them seems to play an important role in stereoinversion. Moreover, we calculated polar surface areas (PSAs) for the trajectories, finding that CN distances and PSA were different for each peptide, with the longest CN distance and smallest PSA observed for exon 6 peptide, where stereoinversion of Asp is the slowest. Although the average CN distance was shorter for exon 26A-1 peptide than for exon 26A-2 peptide, the number of conformations with CN distances acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Antimicrobial peptides with selective antitumor mechanisms: prospect for anticancer applications.

    Science.gov (United States)

    Deslouches, Berthony; Di, Y Peter

    2017-07-11

    In the last several decades, there have been significant advances in anticancer therapy. However, the development of resistance to cancer drugs and the lack of specificity related to actively dividing cells leading to toxic side effects have undermined these achievements. As a result, there is considerable interest in alternative drugs with novel antitumor mechanisms. In addition to the recent approach using immunotherapy, an effective but much cheaper therapeutic option of pharmaceutical drugs would still provide the best choice for cancer patients as the first line treatment. Ribosomally synthesized cationic antimicrobial peptides (AMPs) or host defense peptides (HDP) display broad-spectrum activity against bacteria based on electrostatic interactions with negatively charged lipids on the bacterial surface. Because of increased proportions of phosphatidylserine (negatively charged) on the surface of cancer cells compared to normal cells, cationic amphipathic peptides could be an effective source of anticancer agents that are both selective and refractory to current resistance mechanisms. We reviewed herein the prospect for AMP application to cancer treatment, with a focus on modes of action of cationic AMPs.

  8. Ampulexins: A New Family of Peptides in Venom of the Emerald Jewel Wasp, Ampulex compressa.

    Science.gov (United States)

    Moore, Eugene L; Arvidson, Ryan; Banks, Christopher; Urenda, Jean Paul; Duong, Elizabeth; Mohammed, Haroun; Adams, Michael E

    2018-03-27

    The parasitoid wasp Ampulex compressa injects venom directly into the brain and subesophageal ganglion of the cockroach Periplaneta americana, inducing a 7 to 10 day lethargy termed hypokinesia. Hypokinesia presents as a significant reduction in both escape response and spontaneous walking. We examined aminergic and peptidergic components of milked venom with HPLC and MALDI-TOF mass spectrometry. HPLC coupled with electrochemical detection confirmed the presence of dopamine in milked venom, while mass spectrometry revealed that the venom gland and venom sac have distinct peptide profiles, with milked venom predominantly composed of venom sac peptides. We isolated and characterized novel α-helical, amphipathic venom sac peptides that constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin 1, into the subesophageal ganglion of cockroaches resulted in a short-term increase in escape threshold. Neither milked venom nor venom peptides interfered with growth of Escherichia coli or Bacillus thuringiensis on agar plates, and exposure to ampulexins or milked venom did not induce cell death in Chinese hamster ovary cells (CHO-K1) or Hi5 cells ( Trichoplusia ni).

  9. Glucagon-like peptide-1 7-36 amide and peptide YY from the L-cell of the ileal mucosa are potent inhibitors of vagally induced gastric acid secretion in man

    DEFF Research Database (Denmark)

    Wettergren, A; Petersen, H; Orskov, C

    1994-01-01

    BACKGROUND: Glucagon-like peptide (GLP-1) 7-36 amide and peptide YY (PYY) from the L-cell of the ileal mucosa are potent inhibitors of gastric acid secretion in man. It is not clear, however, by which mechanism(s) they inhibit acid secretion. In dogs the inhibitory effect of PYY on acid secretion...

  10. Synthesis of mutual azo prodrugs of anti-inflammatory agents and peptides facilitated by α-aminoisobutyric acid.

    Science.gov (United States)

    Kennedy, David A; Vembu, Nagarajan; Fronczek, Frank R; Devocelle, Marc

    2011-12-02

    Reported is the synthesis of azo mutual prodrugs of the nonsteroidal anti-inflammatory agents (NSAIDs) 4-aminophenylacetic acid (4-APAA) or 5-aminosalicylic acid (5-ASA) with peptides, including an antibiotic peptide temporin analogue modified at the amino terminal by an α-aminoisobutyric acid (Aib) residue. These prodrugs are designed for colonic delivery of two agents to treat infection and inflammation by the bacterial pathogen Clostridium difficile . © 2011 American Chemical Society

  11. Quantification of amino acids and peptides in an ionic liquid based aqueous two-phase system by LC-MS analysis.

    Science.gov (United States)

    Oppermann, Sebastian; Oppermann, Christina; Böhm, Miriam; Kühl, Toni; Imhof, Diana; Kragl, Udo

    2018-04-25

    Aqueous two-phase systems (ATPS) occur by the mixture of two polymers or a polymer and an inorganic salt in water. It was shown that not only polymers but also ionic liquids in combination with inorganic cosmotrophic salts are able to build ATPS. Suitable for the formation of ionic liquid-based ATPS systems are hydrophilic water miscible ionic liquids. To understand the driving force for amino acid and peptide distribution in IL-ATPS at different pH values, the ionic liquid Ammoeng 110™ and K 2 HPO 4 have been chosen as a test system. To quantify the concentration of amino acids and peptides in the different phases, liquid chromatography and mass spectrometry (LC-MS) technologies were used. Therefore the peptides and amino acids have been processed with EZ:faast™-Kit from Phenomenex for an easy and reliable quantification method even in complex sample matrices. Partitioning is a surface-dependent phenomenon, investigations were focused on surface-related amino acid respectively peptide properties such as charge and hydrophobicity. Only a very low dependence between the amino acids or peptides hydrophobicity and the partition coefficient was found. Nevertheless, the presented results show that electrostatic respectively ionic interactions between the ionic liquid and the amino acids or peptides have a strong impact on their partitioning behavior.

  12. Improving cell penetration of helical peptides stabilized by N-terminal crosslinked aspartic acids.

    Science.gov (United States)

    Zhao, Hui; Jiang, Yanhong; Tian, Yuan; Yang, Dan; Qin, Xuan; Li, Zigang

    2017-01-04

    Cell penetration and nucleus translocation efficiency are important for the cellular activities of peptide therapeutics. For helical peptides stabilized by N-terminal crosslinked aspartic acid, correlations between their penetration efficiency/nucleus translocation and physicochemical properties were studied. An increase in hydrophobicity and isoelectric point will promote cellular uptake and nucleus translocation of stabilized helices.

  13. Peptide chemistry toolbox - Transforming natural peptides into peptide therapeutics.

    Science.gov (United States)

    Erak, Miloš; Bellmann-Sickert, Kathrin; Els-Heindl, Sylvia; Beck-Sickinger, Annette G

    2018-06-01

    The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Rigid amphipathic fusion inhibitors demonstrate antiviral activity against African swine fever virus.

    Science.gov (United States)

    Hakobyan, Astghik; Galindo, Inmaculada; Nañez, Almudena; Arabyan, Erik; Karalyan, Zaven; Chistov, Alexey A; Streshnev, Philipp P; Korshun, Vladimir A; Alonso, Covadonga; Zakaryan, Hovakim

    2018-01-01

    Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments.

  15. The diversity and evolution of anuran skin peptides.

    Science.gov (United States)

    König, Enrico; Bininda-Emonds, Olaf R P; Shaw, Chris

    2015-01-01

    Amphibians exhibit various, characteristic adaptations related to their "incomplete" shift from the aquatic to the terrestrial habitat. In particular, the integument was subject to a number of specialized modifications during the evolution of these animals. In this review, we place special emphasis on endogenous host-defence skin peptides from the cuteanous granular glands anuran amphibians (frogs and toads). The overview on the two broad groups of neuroactive and antimicrobial peptides (AMPs) goes beyond a simple itemization in that we provide a new perspective into the evolution and function of anuran AMPs. Briefly, these cationic, amphipathic and α-helical peptides are traditionally viewed as being part of the innate immune system, protecting the moist skin against invading microorganisms through their cytolytic action. However, the complete record of anuran species investigated to date suggests that AMPs are distributed sporadically (i.e., non-universally) across Anura. Together with the intriguing observation that virtually all anurans known to produce neuropeptides in their granular glands also co-secrete cytolytic peptides, we call the traditional role for AMPs as being purely antimicrobial into question and present an alternative scenario. We hypothesize AMPs to assist neuroactive peptides in their antipredator role through their cytolytic action increasing the delivery of the latter to the endocrine and nervous system of the predator. Thus, AMPs are more accurately viewed as cytolysins and their contribution to the immune system is better regarded as an accessory benefit. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.

    Science.gov (United States)

    Runge, Steffen; Thøgersen, Henning; Madsen, Kjeld; Lau, Jesper; Rudolph, Rainer

    2008-04-25

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.

  17. Identification of 11-amino acid peptides that disrupt Notch-mediated processes in Drosophila

    Directory of Open Access Journals (Sweden)

    Yeh Hsiao-Fong

    2011-06-01

    Full Text Available Abstract Background The conserved Notch signaling pathway regulates cell fate decisions and maintains stem cells in multicellular organisms. Up-regulation of Notch signaling is observed in several types of cancer and is causally involved in proliferation and survival of cancer cells. Thus, it is of great interest to look for anti-Notch reagents for therapeutic purposes. In model animal Drosophila, Notch signaling restricts selection of sensory organ precursors (SOPs during external sensory (ES organ development. To look for novel genes that can suppress Notch signaling, we performed a gain-of-function modifier screen to look for genes that enhance the phenotype of ectopic ES organs induced by overexpression of phyllopod, a gene required for SOP specification. Results From the gain-of-function screen, we discovered that overexpression of polished rice/tarsal-less (pri/tal increases the numbers of ES organs as well as SOPs. pri/tal is a polycistronic gene that contains four short open reading frames encoding three 11-amino acid and one 32-amino acid peptides. Ectopic expression of the 11 amino-acid peptides recapitulates the pri/tal misexpression phenotype in ectopic ES organ formation. In situ hybridization experiment reveals that pri/tal mRNA is expressed in the SOPs of the chemosensory organs and the stretch-sensing chordotonal organs. In Drosophila wing development, the Notch signaling pathway mediates the formation of the dorsal-ventral (DV compartmental boundary and the restriction of the vein width from the primordial veins, the proveins. We also found that pri/tal mRNA is expressed in the DV boundary and the longitudinal proveins, and overexpression of Pri/Tal peptides disrupts the DV boundary formation and helps to expand the width of the wing vein. Genetic analyses further show that a Notch loss-of-function allele strongly enhances these two phenotypes. Cut and E(splmβ are target genes of the Notch pathway in DV boundary formation and

  18. D-amino acid substitution enhances the stability of antimicrobial peptide polybia-CP.

    Science.gov (United States)

    Jia, Fengjing; Wang, Jiayi; Peng, Jinxiu; Zhao, Ping; Kong, Ziqing; Wang, Kairong; Yan, Wenjin; Wang, Rui

    2017-10-01

    With the increasing emergence of resistant microbes toward conventional antimicrobial agents, there is an urgent need for the development of antimicrobial agents with novel action mode. Antimicrobial peptides (AMPs) are believed to be one kind of ideal alternatives. However, AMPs can be easily degraded by protease, which limited their therapeutic use. In the present study, D-amino acid substitution strategy was employed to enhance the stability of polybia-CP. We investigated the stability of peptides against the degradation of trypsin and chymotrypsin by determining the antimicrobial activity or determining the HPLC profile of peptides after incubation with proteases. Our results showed that both the all D-amino acid derivative (D-CP) and partial D-lysine substitution derivative (D-lys-CP) have an improved stability against trypsin and chymotrypsin. Although D-CP takes left-hand α-helical conformation and D-lys-CP loses some α-helical content, both of the D-amino acid-substituted derivatives maintain their parental peptides' membrane active action mode. In addition, D-lys-CP showed a slight weaker antimicrobial activity than polybia-CP, but the hemolytic activity decreased greatly. These results suggest that D-CP and D-lys-CP can offer strategy to improve the property of AMPs and may be leading compounds for the development of novel antimicrobial agents. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Synthetic histatin analogues with broad-spectrum antimicrobial activity.

    OpenAIRE

    Helmerhorst, E J; Van't Hof, W; Veerman, E C; Simoons-Smit, I; Nieuw Amerongen, A V

    1997-01-01

    Histatins are salivary histidine-rich cationic peptides, ranging from 7 to 38 amino acid residues in length, that exert a potent killing effect in vitro on Candida albicans. Starting from the C-terminal fungicidal domain of histatin 5 (residues 11-24, called dh-5) a number of substitution analogues were chemically synthesized to study the effect of amphipathicity of the peptide in helix conformation on candidacidal activity. Single substitutions in dh-5 at several positions did not have any e...

  20. Cyclic Sulfamidate Enabled Syntheses of Amino Acids, Peptides, Carbohydrates, and Natural Products

    Science.gov (United States)

    This article reviews the emergence of cyclic sulfamidates as versatile intermediatesfor the synthesis of unnatural amino acids, chalcogen peptides, modified sugars, drugs and drug candidates, and important natural products.

  1. Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody

    International Nuclear Information System (INIS)

    Smith, J.W.; Dey, B.; Knauer, D.J.

    1990-01-01

    Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. The authors have previously proposed that the heparin binding site of ATIII residues within a region extending from amino acid residues 114-156. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation

  2. Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Hyun Suk; Ko, Yeong Mu; Shim, Jae Won [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, Gwangju (Korea, Republic of); Lim, Yun Kyong; Kook, Joong-Ki [Department of Oral Biochemistry, School of Dentistry, Chosun University, Gwangju (Korea, Republic of); Cho, Dong-Lyun [School of Applied Chemical Engineering and Center for Functional Nano Fine Chemicals, Chonnam National University, Gwangju (Korea, Republic of); Kim, Byung Hoon, E-mail: kim5055@chosun.ac.kr [Department of Dental Materials, School of Dentistry, MRC Center, Chosun University, Gwangju (Korea, Republic of)

    2010-11-01

    Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH{sub 2} of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.

  3. 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.

  4. Antioxidant activity of yoghurt peptides: Part 2 – Characterisationof peptide fractions

    DEFF Research Database (Denmark)

    Farvin, Sabeena; Baron, Caroline; Nielsen, Nina Skall

    2010-01-01

    the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained...

  5. C9/12 Ribbon-Like Structures in Hybrid Peptides Alternating α- and Thiazole-Based γ-Amino Acids.

    Science.gov (United States)

    Bonnel, Clément; Legrand, Baptiste; Simon, Matthieu; Martinez, Jean; Bantignies, Jean-Louis; Kang, Young Kee; Wenger, Emmanuel; Hoh, Francois; Masurier, Nicolas; Maillard, Ludovic T

    2017-12-11

    According to their restricted conformational freedom, heterocyclic γ-amino acids are usually considered to be related to Z-vinylogous γ-amino acids. In this context, oligomers alternating α-amino acids and thiazole-based γ-amino acids (ATCs) were expected to fold into a canonical 12-helical shape as described for α/γ-hybrid peptides composed of cis-α/β-unsaturated γ-amino acids. However, through a combination of X-ray crystallography, NMR spectroscopy, FTIR experiments, and DFT calculations, it was determined that the folding behavior of ATC-containing hybrid peptides is much more complex. The homochiral α/(S)-ATC sequences were unable to adopt a stable conformation, whereas the heterochiral α/(R)-ATC peptides displayed novel ribbon structures stabilized by unusual C 9/12 -bifurcated hydrogen bonds. These ribbon structures could be considered as a succession of pre-organized γ/α dipeptides and may provide the basis for designing original α-helix mimics. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  7. 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.

  8. Biosynthesis of 2-aminooctanoic acid and its use to terminally modify a lactoferricin B peptide derivative for improved antimicrobial activity.

    Science.gov (United States)

    Almahboub, Sarah A; Narancic, Tanja; Devocelle, Marc; Kenny, Shane T; Palmer-Brown, William; Murphy, Cormac; Nikodinovic-Runic, Jasmina; O'Connor, Kevin E

    2018-01-01

    Terminal modification of peptides is frequently used to improve their hydrophobicity. While N-terminal modification with fatty acids (lipidation) has been reported previously, C-terminal lipidation is limited as it requires the use of linkers. Here we report the use of a biocatalyst for the production of an unnatural fatty amino acid, (S)-2-aminooctanoic acid (2-AOA) with enantiomeric excess > 98% ee and the subsequent use of 2-AOA to modify and improve the activity of an antimicrobial peptide. A transaminase originating from Chromobacterium violaceum was employed with a conversion efficiency 52-80% depending on the ratio of amino group donor to acceptor. 2-AOA is a fatty acid with amino functionality, which allowed direct C- and N-terminal conjugation respectively to an antimicrobial peptide (AMP) derived from lactoferricin B. The antibacterial activity of the modified peptides was improved by up to 16-fold. Furthermore, minimal inhibitory concentrations (MIC) of C-terminally modified peptide were always lower than N-terminally conjugated peptides. The C-terminally modified peptide exhibited MIC values of 25 μg/ml for Escherichia coli, 50 μg/ml for Bacillus subtilis, 100 μg/ml for Salmonella typhimurium, 200 μg/ml for Pseudomonas aeruginosa and 400 μg/ml for Staphylococcus aureus. The C-terminally modified peptide was the only peptide tested that showed complete inhibition of growth of S. aureus.

  9. CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

    Science.gov (United States)

    Duffy, Fergal J; Verniere, Mélanie; Devocelle, Marc; Bernard, Elise; Shields, Denis C; Chubb, Anthony J

    2011-04-25

    We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .

  10. Tritium labeling of amino acids and peptides with liquid and solid tritium

    International Nuclear Information System (INIS)

    Peng, C.T.; Hua, R.L.; Souers, P.C.; Coronado, P.R.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21 K and 9 K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenyl-alanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritium are potentially useful agents for labeling peptides and proteins. 11 refs., 1 fig., 3 tabs

  11. Tritium labeling of amino acids and peptides with liquid and solid tritium

    International Nuclear Information System (INIS)

    Souers, P.C.; Coronado, P.R.; Peng, C.T.; Hua, R.L.

    1988-01-01

    Amino acids and peptides were labeled with liquid and solid tritium at 21/degree/K and 9/degree/K. At these low temperatures radiation degradation is minimal, and tritium incorporation increases with tritium concentration and exposure time. Ring saturation in L-phenylalanine does not occur. Peptide linkage in oligopeptides is stable toward tritium. Deiodination in 3-iodotyrosine and 3,5-diiodotyrosine occurs readily and proceeds in steps by losing one iodine atom at a time. Nickel and noble metal supported catalysts when used as supports for dispersion of the substrate promote tritium labeling at 21 K. Our study shows that both liquid and solid tritiums are potentially useful agents for labeling peptides and proteins

  12. Acidity-Triggered Tumor Retention/Internalization of Chimeric Peptide for Enhanced Photodynamic Therapy and Real-Time Monitoring of Therapeutic Effects.

    Science.gov (United States)

    Han, Kai; Zhang, Wei-Yun; Ma, Zhao-Yu; Wang, Shi-Bo; Xu, Lu-Ming; Liu, Jia; Zhang, Xian-Zheng; Han, He-You

    2017-05-17

    Photodynamic therapy (PDT) holds great promise in tumor treatment. Nevertheless, it remains highly desirable to develop easy-to-fabricated PDT systems with improved tumor accumulation/internalization and timely therapeutic feedback. Here, we report a tumor-acidity-responsive chimeric peptide for enhanced PDT and noninvasive real-time apoptosis imaging. Both in vitro and in vivo studies revealed that a tumor mildly acidic microenvironment could trigger rapid protonation of carboxylate anions in chimeric peptide, which led to increased ζ potential, improved hydrophobicity, controlled size enlargement, and precise morphology switching from sphere to spherocylinder shape of the chimeric peptide. All of these factors realized superfast accumulation and prolonged retention in the tumor region, selective cellular internalization, and enhanced PDT against the tumor. Meanwhile, this chimeric peptide could further generate reactive oxygen species and initiate cell apoptosis during PDT. The subsequent formation of caspase-3 enzyme hydrolyzed the chimeric peptide, achieving a high signal/noise ratio and timely fluorescence feedback. Importantly, direct utilization of the acidity responsiveness of a biofunctional Asp-Glu-Val-Asp-Gly (DEVDG, caspase-3 enzyme substrate) peptide sequence dramatically simplified the preparation and increased the performance of the chimeric peptide furthest.

  13. The role of citric acid in oral peptide and protein formulations

    DEFF Research Database (Denmark)

    Welling, Søren H; Hubálek, František; Jacobsen, Jette

    2014-01-01

    not occur significantly at the acidic pH values where it effectively inhibits proteolysis, which is its dominant action in oral peptide formulations. On account of insulin's low basal permeability, inclusion of alternative permeation enhancers is likely to be necessary to achieve sufficient oral...

  14. Reverse engineering truncations of an antimicrobial peptide dimer to identify the origins of potency and broad spectrum of action.

    Science.gov (United States)

    Anantharaman, Aparna; Sahal, Dinkar

    2010-08-26

    Antimicrobial peptides hold promise against antibiotic resistant pathogens. Here, to find the physicochemical origins of potency and broad spectrum antimicrobial action, we report the structure-activity relationships of synthetic intermediates (peptides A-D) of a potent lysine branched dimeric antibacterial peptide DeltaFd. Our studies show that a tetracationic character in a weak helical fold (peptide C) elicits potent but narrow spectrum antimicrobial activity [Minimum inhibitory concentrations (MICs) E. coli 10 microM, S. aureus>100 microM]. In contrast, a hexacationic character in a strong, amphipathic helix (DeltaFd) confers potent and broad spectrum action [MICs E. coli 2.5 microM, S. aureus 5 microM]. While DeltaFd caused rapid and potent permeabilization of the E. coli membranes, the less helical intermediates (peptides A-D) showed slow and weak to no responses. Two seminal findings that may aid future drug design are (a) at identical helicity, increasing charge enhanced outer membrane permeabilization, and (b) at identical charge, increasing helicity stimulated rate of outer membrane permeabilization and kill kinetics besides enhancing potency leading to broad spectrum action.

  15. Acidic-basic properties of three alanine-based peptides containing acidic and basic side chains: comparison between theory and experiment.

    Science.gov (United States)

    Makowska, Joanna; Bagińska, Katarzyna; Liwo, Adam; Chmurzyński, Lech; Scheraga, Harold A

    2008-01-01

    The purpose of this work was to evaluate the effect of the nature of the ionizable end groups, and the solvent, on their acid-base properties in alanine-based peptides. Hence, the acid-base properties of three alanine-based peptides: Ac-KK-(A)(7)-KK-NH(2) (KAK), Ac-OO-(A)(7)-DD-NH(2) (OAD), Ac-KK-(A)(7)-EE-NH(2) (KAE), where A, D, E, K, and O denote alanine, aspartic acid, glutamic acid, lysine, and ornithine, respectively, were determined in water and in methanol by potentiometry. With the availability of these data, the ability of two theoretical methods to simulate pH-metric titration of those peptides was assessed: (i) the electrostatically driven Monte Carlo method with the ECEPP/3 force field and the Poisson-Boltzmann approach to compute solvation energy (EDMC/PB/pH), and (ii) the molecular dynamics method with the AMBER force field and the Generalized Born model (MD/GB/pH). For OAD and KAE, pK(a1) and pK(a2) correspond to the acidic side chains. For all three compounds in both solvents, the pK(a1) value is remarkably lower than the pK(a) of a compound modeling the respective isolated side chain, which can be explained by the influence of the electrostatic field from positively charged ornithine or lysine side chains. The experimental titration curves are reproduced well by the MD/GB/pH approach, the agreement being better if restraints derived from NMR measurements are incorporated in the conformational search. Poorer agreement is achieved by the EDMC/PB/pH method.

  16. AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion, Androctonus aeneas: Structural Characterisation, Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities

    Directory of Open Access Journals (Sweden)

    Qiang Du

    2015-01-01

    Full Text Available The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3'- and 5'-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation.

  17. AaeAP1 and AaeAP2: novel antimicrobial peptides from the venom of the scorpion, Androctonus aeneas: structural characterisation, molecular cloning of biosynthetic precursor-encoding cDNAs and engineering of analogues with enhanced antimicrobial and anticancer activities.

    Science.gov (United States)

    Du, Qiang; Hou, Xiaojuan; Wang, Lei; Zhang, Yingqi; Xi, Xinping; Wang, Hui; Zhou, Mei; Duan, Jinao; Wei, Minjie; Chen, Tianbao; Shaw, Chris

    2015-01-23

    The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3'- and 5'-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation.

  18. Acid-base titration of melanocortin peptides: evidence of Trp rotational conformers interconversion.

    Science.gov (United States)

    Fernandez, Roberto M; Vieira, Renata F F; Nakaie, Clóvis R; Lamy, M Teresa; Ito, Amando S

    2005-01-01

    Tryptophantime-resolved fluorescence was used to monitor acid-base titration properties of alpha-melanocyte stimulating hormone (alpha-MSH) and the biologically more potent analog [Nle4, D-Phe7]alpha -MSH (NDP-MSH), labeled or not with the paramagnetic amino acid probe 2,2,6,6-tetramthylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Global analysis of fluorescence decay profiles measured in the pH range between 2.0 and 11.0 showed that, for each peptide, the data could be well fitted to three lifetimes whose values remained constant. The less populated short lifetime component changed little with pH and was ascribed to Trp g+ chi1 rotamer, in which electron transfer deactivation predominates over fluorescence. The long and intermediate lifetime preexponential factors interconverted along that pH interval and the result was interpreted as due to interconversion between Trp g- and trans chi1 rotamers, driven by conformational changes promoted by modifications in the ionization state of side-chain residues. The differences in the extent of interconversion in alpha-MSH and NDP-MSH are indicative of structural differences between the peptides, while titration curves suggest structural similarities between each peptide and its Toac-labeled species, in aqueous solution. Though less sensitive than fluorescence, the Toac electron spin resonance (ESR) isotropic hyperfine splitting parameter can also monitor the titration of side-chain residues located relatively far from the probe. Copyright (c) 2005 Wiley Periodicals, Inc.

  19. Multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer: Design, synthesis, and dissolving thrombus.

    Science.gov (United States)

    Zhang, Shao-Fei; Lü, Shaoyu; Gao, Chunmei; Yang, Jiandong; Yan, Xiang; Li, Tao; Wen, Na; Huang, Mengjie; Liu, Mingzhu

    2018-06-01

    Thrombotic events affect many individuals in a number of ways, all of which can cause significant morbidity and mortality. Nattokinase (NK), as a novel thrombolytic drug, has been used for thrombolytic therapy. It not only possesses plasminogen activator activity, but also directly digests fibrin through limited proteolysis. However, it may undergo inactivation and denaturation in the harsh external environment. In this study, a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer was fabricated and used as a carrier for NK protection and delivery. Different arm numbers of polyethylene glycol-polyglutamic acid peptide dendrimers (x-PEG(G 3 ) x , x = 2, 4, 6, 8) were designed, prepared, and characterized by 1 H NMR and FTIR. Then, x-PEG(G 3 ) x were loaded with NK to form nanocomposites. Their size and morphology were determined by dynamic light scattering and transmission electron microscopy. Enzyme activity was evaluated via UV-Vis absorbance spectra, fluorescence spectra, circular dichroism spectra, and zeta potential measurements. The study reveals that the obtained x-PEG(G 3 ) x /NK nanocomposites possess high enzyme activity. In addition, the nanocomposites show increased viability of rat macrophage cells, and excellent thrombolysis ability in vitro and in vivo. This work establishes a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer with potential application in NK carrier and thrombolytic therapy. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1687-1696, 2018. © 2018 Wiley Periodicals, Inc.

  20. Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva.

    Science.gov (United States)

    Schlesinger, D H; Hay, D I

    1977-03-10

    The complete amino acid sequence of human salivary statherin, a peptide which strongly inhibits precipitation from supersaturated calcium phosphate solutions, and therefore stabilizes supersaturated saliva, has been determined. The NH2-terminal half of this Mr=5380 (43 amino acids) polypeptide was determined by automated Edman degradations (liquid phase) on native statherin. The peptide was digested separately with trypsin, chymotrypsin, and Staphylococcus aureus protease, and the resulting peptides were purified by gel filtration. Manual Edman degradations on purified peptide fragments yielded peptides that completed the amino acid sequence through the penultimate COOH-terminal residue. These analyses, together with carboxypeptidase digestion of native statherin and of peptide fragments of statherin, established the complete sequence of the molecule. The 2 serine residues (positions 2 and 3) in statherin were identified as phosphoserine. The amino acid sequence of human salivary statherin is striking in a number of ways. The NH2-terminal one-third is highly polar and includes three polar dipeptides: H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds of the molecule is hydrophobic, containing several repeating dipeptides: four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-, and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage sites in the statherin sequence obtained with chymotrypsin and S. aureus protease were also noted.

  1. Highly potent antimicrobial peptides from N-terminal membrane-binding region of E. coli MreB.

    Science.gov (United States)

    Saikia, Karabi; Sravani, Yalavarthi Durga; Ramakrishnan, Vibin; Chaudhary, Nitin

    2017-02-23

    Microbial pathogenesis is a serious health concern. The threat escalates as the existing conventional antimicrobials are losing their efficacy against the evolving pathogens. Peptides hold promise to be developed into next-generation antibiotics. Antimicrobial peptides adopt amphipathic structures that could selectively bind to and disrupt the microbial membranes. Interaction of proteins with membranes is central to all living systems and we reasoned that the membrane-binding domains in microbial proteins could be developed into efficient antimicrobials. This is an interesting approach as self-like sequences could elude the microbial strategies of degrading the antimicrobial peptides, one of the mechanisms of showing resistance to antimicrobials. We selected the 9-residue-long membrane-binding region of E. coli MreB protein. The 9-residue peptide (C-terminal amide) and its N-terminal acetylated analog displayed broad-spectrum activity, killing Gram-negative bacteria, Gram-positive bacteria, and fungi. Extension with a tryptophan residue at the N-terminus drastically improved the activity of the peptides with lethal concentrations ≤10 μM against all the organisms tested. The tryptophan-extended peptides caused complete killing of C. albicans as well as gentamicin and methicillin resistant S. aureus at 5 μM concentration. Lipid-binding studies and electron microscopic analyses of the peptide-treated microbes suggest membrane disruption as the mechanism of killing.

  2. The neuroprotective efficacy of cell-penetrating peptides TAT, penetratin, Arg-9, and Pep-1 in glutamic acid, kainic acid, and in vitro ischemia injury models using primary cortical neuronal cultures.

    Science.gov (United States)

    Meloni, Bruno P; Craig, Amanda J; Milech, Nadia; Hopkins, Richard M; Watt, Paul M; Knuckey, Neville W

    2014-03-01

    Cell-penetrating peptides (CPPs) are small peptides (typically 5-25 amino acids), which are used to facilitate the delivery of normally non-permeable cargos such as other peptides, proteins, nucleic acids, or drugs into cells. However, several recent studies have demonstrated that the TAT CPP has neuroprotective properties. Therefore, in this study, we assessed the TAT and three other CPPs (penetratin, Arg-9, Pep-1) for their neuroprotective properties in cortical neuronal cultures following exposure to glutamic acid, kainic acid, or in vitro ischemia (oxygen-glucose deprivation). Arg-9, penetratin, and TAT-D displayed consistent and high level neuroprotective activity in both the glutamic acid (IC50: 0.78, 3.4, 13.9 μM) and kainic acid (IC50: 0.81, 2.0, 6.2 μM) injury models, while Pep-1 was ineffective. The TAT-D isoform displayed similar efficacy to the TAT-L isoform in the glutamic acid model. Interestingly, Arg-9 was the only CPP that displayed efficacy when washed-out prior to glutamic acid exposure. Neuroprotection following in vitro ischemia was more variable with all peptides providing some level of neuroprotection (IC50; Arg-9: 6.0 μM, TAT-D: 7.1 μM, penetratin/Pep-1: >10 μM). The positive control peptides JNKI-1D-TAT (JNK inhibitory peptide) and/or PYC36L-TAT (AP-1 inhibitory peptide) were neuroprotective in all models. Finally, in a post-glutamic acid treatment experiment, Arg-9 was highly effective when added immediately after, and mildly effective when added 15 min post-insult, while the JNKI-1D-TAT control peptide was ineffective when added post-insult. These findings demonstrate that different CPPs have the ability to inhibit neurodamaging events/pathways associated with excitotoxic and ischemic injuries. More importantly, they highlight the need to interpret neuroprotection studies when using CPPs as delivery agents with caution. On a positive note, the cytoprotective properties of CPPs suggests they are ideal carrier molecules to

  3. Glucagon-like peptide 2 stimulates glucagon secretion, enhances lipid absorption, and inhibits gastric acid secretion in humans

    DEFF Research Database (Denmark)

    Meier, Juris J; Nauck, Michael A; Pott, Andrea

    2006-01-01

    or placebo during the ingestion of a solid test meal. Gastric emptying was determined using a 13C-sodium-octanote breath test. Plasma concentrations of glucose, insulin, C-peptide, glucagon, GLP-2, free fatty acids, free glycerol, and triglycerides were determined. RESULTS: GLP-2 administration led...... (P = .07). GLP-2 administration caused an approximately 15% reduction in pentagastrin-stimulated gastric acid and chloride secretion (P gastric emptying was not affected (P = .99). CONCLUSIONS: GLP-2 reduces gastric acid secretion but does not seem to have an influence on gastric......BACKGROUND & AIMS: The gut-derived peptide glucagon-like peptide 2 (GLP-2) has been suggested as a potential drug candidate for the treatment of various intestinal diseases. However, the acute effects of GLP-2 on gastric functions as well as on glucose and lipid homeostasis in humans are less well...

  4. N-terminal amphipathic helix as a trigger of hemolytic activity in antimicrobial peptides: a case study in latarcins.

    Science.gov (United States)

    Polyansky, Anton A; Vassilevski, Alexander A; Volynsky, Pavel E; Vorontsova, Olga V; Samsonova, Olga V; Egorova, Natalya S; Krylov, Nicolay A; Feofanov, Alexei V; Arseniev, Alexander S; Grishin, Eugene V; Efremov, Roman G

    2009-07-21

    In silico structural analyses of sets of alpha-helical antimicrobial peptides (AMPs) are performed. Differences between hemolytic and non-hemolytic AMPs are revealed in organization of their N-terminal region. A parameter related to hydrophobicity of the N-terminal part is proposed as a measure of the peptide propensity to exhibit hemolytic and other unwanted cytotoxic activities. Based on the information acquired, a rational approach for selective removal of these properties in AMPs is suggested. A proof of concept is gained through engineering specific mutations that resulted in elimination of the hemolytic activity of AMPs (latarcins) while leaving the beneficial antimicrobial effect intact.

  5. Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles

    Science.gov (United States)

    Nakamura, Shota; Ono, Chikako; Shiokawa, Mai; Yamamoto, Satomi; Motomura, Takashi; Okamoto, Toru; Okuzaki, Daisuke; Yamamoto, Masahiro; Saito, Izumu; Wakita, Takaji; Koike, Kazuhiko; Matsuura, Yoshiharu

    2014-01-01

    Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly. PMID:25502789

  6. ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

    DEFF Research Database (Denmark)

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo

    2017-01-01

    and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation....... The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots...

  7. Distinguishing d- and l-aspartic and isoaspartic acids in amyloid β peptides with ultrahigh resolution ion mobility spectrometry.

    Science.gov (United States)

    Zheng, Xueyun; Deng, Liulin; Baker, Erin S; Ibrahim, Yehia M; Petyuk, Vladislav A; Smith, Richard D

    2017-07-11

    While α-linked amino acids in the l-form are exclusively utilized in mammalian protein building, β-linked and d-form amino acids also have important biological roles. Unfortunately, the structural elucidation and separation of these different amino acid types in peptides has been analytically challenging to date due to the numerous isomers present, limiting our knowledge about their existence and biological roles. Here, we utilized an ultrahigh resolution ion mobility spectrometry platform coupled with mass spectrometry (IMS-MS) to separate amyloid β (Aβ) peptides containing l-aspartic acid, d-aspartic acid, l-isoaspartic acid, and d-isoaspartic acid residues which span α- and β-linked amino acids in both d- and l-forms. The results illustrate how IMS-MS could be used to better understand age-related diseases or protein folding disorders resulting from amino acid modifications.

  8. Efficacy of peptide nucleic acid and selected conjugates against specific cellular pathologies of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Browne, Elisse C; Parakh, Sonam; Duncan, Luke F; Langford, Steven J; Atkin, Julie D; Abbott, Belinda M

    2016-04-01

    Cellular studies have been undertaken on a nonamer peptide nucleic acid (PNA) sequence, which binds to mRNA encoding superoxide dismutase 1, and a series of peptide nucleic acids conjugated to synthetic lipophilic vitamin analogs including a recently prepared menadione (vitamin K) analog. Reduction of both mutant superoxide dismutase 1 inclusion formation and endoplasmic reticulum stress, two of the key cellular pathological hallmarks in amyotrophic lateral sclerosis, by two of the prepared PNA oligomers is reported for the first time. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  9. Bile acids induce glucagon-like peptide 2 secretion with limited effects on intestinal adaptation in early weaned pigs

    DEFF Research Database (Denmark)

    Ipharraguerre, Ignacio R; Tedó, Gemma; Menoyo, David

    2013-01-01

    Early weaning is a stressful event characterized by a transient period of intestinal atrophy that may be mediated by reduced secretion of glucagon-like peptide (GLP) 2. We tested whether enterally fed bile acids or plant sterols could increase nutrient-dependent GLP-2 secretion and improve.......05) but did not affect plasma GLP-1 and feed intake. The intestinal expression of glucagon-like peptide 2 receptor, sodium-dependent bile acid transporter, farnesoid X receptor, and guanosine protein-coupled bile acid receptor genes were not affected by CDC treatment. The intragastric administration of CDC...

  10. Analysis of in vitro toxicity of five cell-penetrating peptides by metabolic profiling

    International Nuclear Information System (INIS)

    Kilk, Kalle; Mahlapuu, Riina; Soomets, Ursel; Langel, Ulo

    2009-01-01

    Cell-penetrating peptides (CPPs) are promising candidates for safe and efficient delivery vectors for a wide range of cargoes. However, any compound that is internalized into cells may affect the cell homeostasis. The plethora of possible biological responses makes large scale 'omics' studies appealing approaches for hunting any unsuspected side-effects and evaluate the toxicity of drug candidates. Here we have compared the alterations in cytosolic metabolome of CHO cells caused by five representatives of the most common CPPs: transportan (TP), penetratin (pAntp), HIV Tat derived peptide (pTat), nonaarginine (R 9 ) and model amphipathic peptide (MAP). Analysis was done by liquid chromatography-mass spectrometry techniques, principal component analysis and heatmap displays. Results showed that the intracellular metabolome was the most affected by TP followed by pTat and MAP. Only minor changes could be associated with pAntp or R 9 treatment. The cells could recover from a treatment with 5 μM TP, but no recovery was observed at higher concentration. Both metabolomic and control experiments showed that TP affected cellular redox potential, depleted energy and the pools of purines and pyrimidines. In conclusion, we have performed a metabolomic analysis comparing the safety of cell-penetrating peptides and demonstrate the toxicity of one of them.

  11. Selective rhodium-catalyzed reduction of tertiary amides in amino acid esters and peptides.

    Science.gov (United States)

    Das, Shoubhik; Li, Yuehui; Bornschein, Christoph; Pisiewicz, Sabine; Kiersch, Konstanze; Michalik, Dirk; Gallou, Fabrice; Junge, Kathrin; Beller, Matthias

    2015-10-12

    Efficient reduction of the tertiary amide bond in amino acid derivatives and peptides is described. Functional group selectivity has been achieved by applying a commercially available rhodium precursor and bis(diphenylphosphino)propane (dppp) ligand together with phenyl silane as a reductant. This methodology allows for specific reductive derivatization of biologically interesting peptides and offers straightforward access to a variety of novel peptide derivatives for chemical biology studies and potential pharmaceutical applications. The catalytic system tolerates a variety of functional groups including secondary amides, ester, nitrile, thiomethyl, and hydroxy groups. This convenient hydrosilylation reaction proceeds at ambient conditions and is operationally safe because no air-sensitive reagents or highly reactive metal hydrides are needed. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Antimicrobial activity and self-assembly behavior of antimicrobial peptide chensinin-1b with lipophilic alkyl tails.

    Science.gov (United States)

    Dong, Weibing; Liu, Ziang; Sun, Liying; Wang, Cui; Guan, Yue; Mao, Xiaoman; Shang, Dejing

    2018-04-25

    The threshold hydrophobicity and amphipathic structure of the peptidic chain are important for the biological function of antimicrobial peptides. Chensinin-1b exhibits broad-spectrum bactericidal activity with no hemolytic activity but has almost no anticancer ability against the selected cancer cell lines. In this study, the conjugation of aliphatic acid was designed with different lengths of N-terminal of chensinin-1b, the antimicrobial activity of the resulting lipo-chensinin-1b was examined, in which OA-C1b showed much stronger activity than those of cheninin-1b and the other two lipopeptides. The membrane interaction between the lipo-chensinin-1b and real/mimetic bacterial cell membrane was investigated. Electrostatic interactions between the lipo-chensinin-1b and lipopolysaccharides were detected by isothermal titration calorimetry and the binding affinities were 10.83 μM, 8.77 μM and 7.35 μM for OA-C1b, LA-C1b and PA-C1b, respectively. The antimicrobial activity and membrane interaction ability of the lipo-chensinin-1b followed this order: OA-C1b > chensinin-1b > LA-C1b > PA-C1b. In addition, the lipo-chensinin-1b also exhibited lytic activity against various cancer cells and demonstrated the ability to inhibit LPS-stimulated cytokine release from human U937 cells. The CD spectra indicated that the helical or β-strands contents were existed as the main components in TFE or LPS solution, respectively. The self-assembly behavior was trigged by the solution pH and affected by the length of carbon chain, in which chensinin-1b, OA-C1b, LA-C1b and PA-C1b formed micelles at neutral pH and the micelle size increased for chensinin-1b, OA-C1b and LA-C1b. PA-C1b formed nanofibers in an acidic environment indicated by TEM experiments, and the peptides formed aggregates in an acidic environment and re-dissociated when the pH was adjusted to neutral. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  13. Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

    Science.gov (United States)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  14. Conformational Plasticity of the Cell-Penetrating Peptide SAP As Revealed by Solid-State 19F-NMR and Circular Dichroism Spectroscopies.

    Science.gov (United States)

    Afonin, Sergii; Kubyshkin, Vladimir; Mykhailiuk, Pavel K; Komarov, Igor V; Ulrich, Anne S

    2017-07-13

    The cell-penetrating peptide SAP, which was designed as an amphipathic poly-l-proline helix II (PPII), was suggested to self-assemble into regular fibrils that are relevant for its internalization. Herein we have analyzed the structure of SAP in the membrane-bound state by solid-state 19 F-NMR, which revealed other structural states, in addition to the expected surface-aligned PPII. Trifluoromethyl-bicyclopentyl-glycine (CF 3 -Bpg) and two rigid isomers of trifluoromethyl-4,5-methanoprolines (CF 3 -MePro) were used as labels for 19 F-NMR analysis. The equilibria between different conformations of SAP were studied and were found to be shifted by the substituents at Pro-11. Synchrotron-CD results suggested that substituting Pro-11 by CF 3 -MePro governed the coil-to-PPII equilibrium in solution and in the presence of a lipid bilayer. Using CD and 19 F-NMR, we examined the slow kinetics of the association of SAP with membranes and the dependence of the SAP conformational dynamics on the lipid composition. The peptide did not bind to lipids in the solid ordered phase and aggregated only in the liquid ordered "raft"-like bilayers. Self-association could not be detected in solution or in the presence of liquid disordered membranes. Surface-bound amphipathic SAP in a nonaggregated state was structured as a mixture of nonideal extended conformations reflecting the equilibrium already present in solution, i.e., before binding to the membrane.

  15. Partition Coefficients of Amino Acids, Peptides, and Enzymes in Dextran + Poly(Ethylene Glycol) + Water Aqueous Two-Phase Systems

    Energy Technology Data Exchange (ETDEWEB)

    Kakisaka, Keijiro.; Shindo, Takashi.; Iwai, Yoshio.; Arai, Yasuhiko. [Kyushu University, Fukuoka (Japan). Department of Chemical Systems and Engineering

    1998-12-01

    Partition coefficients are measured for five amino acids(aspartic acid, asparagine, methionine, cysteine and histidine) and tow peptides(glycyl-glycine and hexa-glycine) in dextran + poly(ethylene glycol) + water aqueous two-phase system. The partition coefficients of the amino acids and peptides are aorrelated using the osmotic virial equation. The interaction coefficients contained in the equation can be calculated by hydrophilic group parameters. The partition coefficients of {alpha}-amylase calculated by the osmotic virial equation with the hydrophilic group parameters are in fairly good agreement with the experimental data, though a relatively large discrepancy is shown for {beta}-amylase. (author)

  16. Partition Coefficients of Amino Acids, Peptides, and Enzymes in Dextran + Poly(Ethylene Glycol) + Water Aqueous Two-Phase Systems

    Energy Technology Data Exchange (ETDEWEB)

    Kakisaka, Keijiro.; Shindo, Takashi.; Iwai, Yoshio.; Arai, Yasuhiko. (Kyushu University, Fukuoka (Japan). Department of Chemical Systems and Engineering)

    1998-12-01

    Partition coefficients are measured for five amino acids(aspartic acid, asparagine, methionine, cysteine and histidine) and tow peptides(glycyl-glycine and hexa-glycine) in dextran + poly(ethylene glycol) + water aqueous two-phase system. The partition coefficients of the amino acids and peptides are aorrelated using the osmotic virial equation. The interaction coefficients contained in the equation can be calculated by hydrophilic group parameters. The partition coefficients of [alpha]-amylase calculated by the osmotic virial equation with the hydrophilic group parameters are in fairly good agreement with the experimental data, though a relatively large discrepancy is shown for [beta]-amylase. (author)

  17. The C-Terminal O-S Acyl Shift Pathway under Acidic Condition to Propose Peptide-Thioesters

    Directory of Open Access Journals (Sweden)

    Bo Mi Kim

    2016-11-01

    Full Text Available Peptide-thioester is a pivotal intermediate for peptide ligation and N-, C-terminal cyclization. In this study, desired pathway and the side products of two C-terminal handles, hydroxyethylthiol (HET and hydroxypropylthiol (HPT are described in different conditions as well as kinetic studies. In addition, a new mechanism of C-terminal residue racemization is proposed on the basis of differentiation of products derived from the two C-terminal handles in preparing peptide thioesters through an acid-catalyzed tandem thiol switch, first by an intramolecular O-S acyl shift, and then by an intermolecular S-S exchange.

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

    Science.gov (United States)

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

    2015-10-01

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

  19. Reaction of hypochlorite with amino acids and peptides : EPR evidence for rapid rearrangement and fragmentation of nitrogen-centred radicals

    International Nuclear Information System (INIS)

    Hawkins, C.L.; Davies, M.J.

    1998-01-01

    Various amino acid side chains have been shown to be particularly susceptible to attack and modification by hypochlorite (HOCl). It is known that tyrosine is readily chlorinated by HOCl to give 3-chlorotyrosine and this product has been employed as a marker of HOCl-mediated damage to proteins. Cysteine and methionine react rapidly with HOCl to give oxy acids and cystine (from cysteine) and sulphoxides (from methionine). Lysine and amino acids which lack the above functional groups also react with HOCl via the free amino group which results in the generation of unstable chloramine intermediates; subsequent decomposition of these species gives NH 3 , CO 2 and aldehydes. While the products of reaction of HOCl with amino acids and peptides are reasonably well characterised, the mechanism(s) by which these products arise is less well understood. Electron paramagnetic resonance (EPR) spectroscopy with spin trapping and UV/visible spectroscopy has been employed to examine the reaction of HOCl with amino acids and some small peptides. Reaction of HOCl with N-acetyl amino acids or small peptides gives radicals predominantly at α-carbon sites via reaction at N-terminal free amino groups or amide (peptide) bonds. It is proposed that these carbon-centred radicals are produced as a result of the rearrangement of initial nitrogen-centred radicals formed on cleavage of the N-CI bond of the chloramine/chloramide species by a 1,2-shift reaction

  20. Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis.

    Science.gov (United States)

    Shin, Byung-Sik; Katoh, Takayuki; Gutierrez, Erik; Kim, Joo-Ran; Suga, Hiroaki; Dever, Thomas E

    2017-08-21

    Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

  1. Purification of Peptide Components including Melittin from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis

    Directory of Open Access Journals (Sweden)

    Young Chon Choi

    2006-06-01

    Full Text Available Objectives : This study was conducted to carry out Purification of Melittin and other peptide components from Bee Venom using gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis Methods : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. Results : Melittin and other peptide components were separated from bee venom by using gel filtration chromatography on Sephadex G-50 column in 0.05M ammonium acetate buffer. The fractions obtained from gel filtration chromatography was analyzed by using SDS-PAGE and propionic acid/urea polyacrylamide gel electrophoresis. The melittin obtained from the gel filtration contained residual amount of phospholipase A2 and a protein with molecular weight of 6,000. The contaminating proteins were removed by the second gel filtration chromatography. Conclusion : Gel filtration chromatography and propionic acid/urea polyacrylamide gel electrophoresis are useful to separate peptide components including melittin from bee venom.

  2. Characterizing hydrophobicity of amino acid side chains in a protein environment via measuring contact angle of a water nanodroplet on planar peptide network.

    Science.gov (United States)

    Zhu, Chongqin; Gao, Yurui; Li, Hui; Meng, Sheng; Li, Lei; Francisco, Joseph S; Zeng, Xiao Cheng

    2016-11-15

    Hydrophobicity of macroscopic planar surface is conventionally characterized by the contact angle of water droplets. However, this engineering measurement cannot be directly extended to surfaces of proteins, due to the nanometer scale of amino acids and inherent nonplanar structures. To measure the hydrophobicity of side chains of proteins quantitatively, numerous parameters were developed to characterize behavior of hydrophobic solvation. However, consistency among these parameters is not always apparent. Herein, we demonstrate an alternative way of characterizing hydrophobicity of amino acid side chains in a protein environment by constructing a monolayer of amino acids (i.e., artificial planar peptide network) according to the primary and the β-sheet secondary structures of protein so that the conventional engineering measurement of the contact angle of a water droplet can be brought to bear. Using molecular dynamics simulations, contact angles θ of a water nanodroplet on the planar peptide network, together with excess chemical potentials of purely repulsive methane-sized Weeks-Chandler-Andersen solute, are computed. All of the 20 types of amino acids and the corresponding planar peptide networks are studied. Expectedly, all of the planar peptide networks with nonpolar amino acids are hydrophobic due to θ [Formula: see text] 90°, whereas all of the planar peptide networks of the polar and charged amino acids are hydrophilic due to θ [Formula: see text] 90°. Planar peptide networks of the charged amino acids exhibit complete-wetting behavior due to θ [Formula: see text] 0°. This computational approach for characterization of hydrophobicity can be extended to artificial planar networks of other soft matter.

  3. Alanine scan of the peptide antibiotic feglymycin: assessment of amino acid side chains contributing to antimicrobial activity.

    Science.gov (United States)

    Hänchen, Anne; Rausch, Saskia; Landmann, Benjamin; Toti, Luigi; Nusser, Antje; Süssmuth, Roderich D

    2013-03-18

    The antibiotic feglymycin is a linear 13-mer peptide synthesized by the bacterium Streptomyces sp. DSM 11171. It mainly consists of the nonproteinogenic amino acids 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine. An alanine scan of feglymycin was performed by solution-phase peptide synthesis in order to assess the significance of individual amino acid side chains for biological activity. Hence, 13 peptides were synthesized from di- and tripeptide building blocks, and subsequently tested for antibacterial activity against Staphylococcus aureus strains. Furthermore we tested the inhibition of peptidoglycan biosynthesis enzymes MurA and MurC, which are inhibited by feglymycin. Whereas the antibacterial activity is significantly based on the three amino acids D-Hpg1, L-Hpg5, and L-Phe12, the inhibitory activity against MurA and MurC depends mainly on L-Asp13. The difference in the position dependence for antibacterial activity and enzyme inhibition suggests multiple molecular targets in the modes of action of feglymycin. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Microbial degradation rates of small peptides and amino acids in the oxygen minimum zone of Chilean coastal waters

    Science.gov (United States)

    Pantoja, Silvio; Rossel, Pamela; Castro, Rodrigo; Cuevas, L. Antonio; Daneri, Giovanni; Córdova, Candy

    2009-07-01

    We found similar microbial degradation rates of labile dissolved organic matter in oxic and suboxic waters off northern Chile. Rates of peptide hydrolysis and amino acid uptake in unconcentrated water samples were not low in the water column where oxygen concentration was depleted. Hydrolysis rates ranged from 65 to 160 nmol peptide L -1 h -1 in the top 20 m, 8-28 nmol peptide L -1 h -1 between 100 and 300 m (O 2-depleted zone), and 14-19 nmol peptide L -1 h -1 between 600 and 800 m. Dissolved free amino acid uptake rates were 9-26, 3-17, and 6 nmol L -1 h -1 at similar depth intervals. Since these findings are consistent with a model of comparable potential activity of microbes in degrading labile substrates of planktonic origin, we suggest, as do other authors, that differences in decomposition rates with high and low oxygen concentrations may be a matter of substrate lability. The comparison between hydrolysis and uptake rates indicates that microbial peptide hydrolysis occurs at similar or faster rates than amino acid uptake in the water column, and that the hydrolysis of peptides is not a rate-limiting step for the complete remineralization of labile macromolecules. Low O 2 waters process about 10 tons of peptide carbon per h, double the amount processed in surface-oxygenated water. In the oxygen minimum zone, we suggest that the C balance may be affected by the low lability of the dissolved organic matter when this is upwelled to the surface. An important fraction of dissolved organic matter is processed in the oxygen minimum layer, a prominent feature of the coastal ocean in the highly productive Humboldt Current System.

  5. d-Amino acid mutation of PMI as potent dual peptide inhibitors of p53-MDM2/MDMX interactions.

    Science.gov (United States)

    Li, Xiang; Liu, Chao; Chen, Si; Hu, Honggang; Su, Jiacan; Zou, Yan

    2017-10-15

    According to the previously reported potent dual l-peptide PMI of p53-MDM2/MDMX interactions, a series of d-amino acid mutational PMI analogues, PMI-1-4, with enhanced proteolytic resistence and in vitro tumor cell inhibitory activities were reported, of which Liposome-PMI-1 showed a stronger inhibitory activity against the U87 cell lines than Nutlin-3. This d-amino acid mutation strategy may give a hand for enhancing the potential of peptide drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Effect of micelle interface on the binding of anticoccidial PW2 peptide

    International Nuclear Information System (INIS)

    Tinoco, Luzineide W.; Gomes-Neto, Francisco; Valente, Ana Paula; Almeida, Fabio C. L.

    2007-01-01

    PW2 is an anticoccidial peptide active against Eimeria acervulina and Eimeria tenella. We determined the structure of PW2 in dodecylphosphocholine micelles. The structure showed two distinct regions: an amphipathic N-terminal 3 10 helix and an aromatic region containing WWR interface-binding motif. The aromatic region acted as a scaffold of the protein in the interface and shared the same structure in both DPC and SDS micelles. N-terminal helix interacted with DPC but not with SDS interface. Chemical shift change was slow when SDS was added to PW2 in DPC and fast when DPC was added to PW2 in SDS, indicating that interaction with DPC micelles was kinetically more stable than with SDS micelles. Also, DPC interface was able to accommodate PW2, but it maintained the conformational arrangement in the aromatic region observed for SDS micelles. This behavior, which is different from that observed for other antimicrobial peptides with WWR motif, may be associated with the absence of PW2 antibacterial activity and its selectivity for Eimeria parasites

  7. Effect of micelle interface on the binding of anticoccidial PW2 peptide

    Energy Technology Data Exchange (ETDEWEB)

    Tinoco, Luzineide W. [Universidade Federal do Rio de Janeiro, Nucleo de Pesquisas de Produtos Naturais (Brazil); Gomes-Neto, Francisco; Valente, Ana Paula; Almeida, Fabio C. L. [Universidade Federal do Rio de Janeiro, Centro Nacional de Ressonancia Magnetica Nuclear Jiri Jonas, Instituto de Bioquimica Medica, Programa de Biologia Estrutural (Brazil)], E-mail: falmeida@cnrmn.bioqmed.ufrj.br

    2007-12-15

    PW2 is an anticoccidial peptide active against Eimeria acervulina and Eimeria tenella. We determined the structure of PW2 in dodecylphosphocholine micelles. The structure showed two distinct regions: an amphipathic N-terminal 3{sub 10} helix and an aromatic region containing WWR interface-binding motif. The aromatic region acted as a scaffold of the protein in the interface and shared the same structure in both DPC and SDS micelles. N-terminal helix interacted with DPC but not with SDS interface. Chemical shift change was slow when SDS was added to PW2 in DPC and fast when DPC was added to PW2 in SDS, indicating that interaction with DPC micelles was kinetically more stable than with SDS micelles. Also, DPC interface was able to accommodate PW2, but it maintained the conformational arrangement in the aromatic region observed for SDS micelles. This behavior, which is different from that observed for other antimicrobial peptides with WWR motif, may be associated with the absence of PW2 antibacterial activity and its selectivity for Eimeria parasites.

  8. Promotion of double-duplex invasion of peptide nucleic acids through conjugation with nuclear localization signal peptide.

    Science.gov (United States)

    Aiba, Yuichiro; Honda, Yuta; Komiyama, Makoto

    2015-03-02

    Pseudo-complementary peptide nucleic acid (pcPNA), as one of the most widely used synthetic DNA analogues, invades double-stranded DNA according to Watson-Crick rules to form invasion complexes. This unique mode of DNA recognition induces structural changes at the invasion site and can be used for a range of applications. In this paper, pcPNA is conjugated with a nuclear localization signal (NLS) peptide, and its invading activity is notably promoted both thermodynamically and kinetically. Thus, the double-duplex invasion complex is formed promptly at low pcPNA concentrations under high salt conditions, where the invasion otherwise never occurs. Furthermore, NLS-modified pcPNA is successfully employed for site-selective DNA scission, and the targeted DNA is selectively cleaved under conditions that are not conducive for DNA cutters using unmodified pcPNAs. This strategy of pcPNA modification is expected to be advantageous and promising for a range of in vitro and in vivo applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Expression of the cationic antimicrobial peptide lactoferricin fused with the anionic peptide in Escherichia coli.

    Science.gov (United States)

    Kim, Ha-Kun; Chun, Dae-Sik; Kim, Joon-Sik; Yun, Cheol-Ho; Lee, Ju-Hoon; Hong, Soon-Kwang; Kang, Dae-Kyung

    2006-09-01

    Direct expression of lactoferricin, an antimicrobial peptide, is lethal to Escherichia coli. For the efficient production of lactoferricin in E. coli, we developed an expression system in which the gene for the lysine- and arginine-rich cationic lactoferricin was fused to an anionic peptide gene to neutralize the basic property of lactoferricin, and successfully overexpressed the concatemeric fusion gene in E. coli. The lactoferricin gene was linked to a modified magainin intervening sequence gene by a recombinational polymerase chain reaction, thus producing an acidic peptide-lactoferricin fusion gene. The monomeric acidic peptide-lactoferricin fusion gene was multimerized and expressed in E. coli BL21(DE3) upon induction with isopropyl-beta-D-thiogalactopyranoside. The expression levels of the fusion peptide reached the maximum at the tetramer, while further increases in the copy number of the fusion gene substantially reduced the peptide expression level. The fusion peptides were isolated and cleaved to generate the separate lactoferricin and acidic peptide. About 60 mg of pure recombinant lactoferricin was obtained from 1 L of E. coli culture. The purified recombinant lactoferricin was found to have a molecular weight similar to that of chemically synthesized lactoferricin. The recombinant lactoferricin showed antimicrobial activity and disrupted bacterial membrane permeability, as the native lactoferricin peptide does.

  10. Interaction of antimicrobial peptide Plantaricin149a and four analogs with lipid bilayers and bacterial membranes

    Directory of Open Access Journals (Sweden)

    José Luiz de Souza Lopes

    2013-12-01

    Full Text Available The amidated analog of Plantaricin149, an antimicrobial peptide from Lactobacillus plantarum NRIC 149, directly interacts with negatively charged liposomes and bacterial membranes, leading to their lysis. In this study, four Pln149-analogs were synthesized with different hydrophobic groups at their N-terminus with the goal of evaluating the effect of the modifications at this region in the peptide's antimicrobial properties. The interaction of these peptides with membrane models, surface activity, their hemolytic effect on red blood cells, and antibacterial activity against microorganisms were evaluated. The analogs presented similar action of Plantaricin149a; three of them with no hemolytic effect (< 5% until 0.5 mM, in addition to the induction of a helical element when binding to negative liposomes. The N-terminus difference between the analogs and Plantaricin149a retained the antibacterial effect on S. aureus and P. aeruginosa for all peptides (MIC50 of 19 µM and 155 µM to Plantaricin149a, respectively but resulted in a different mechanism of action against the microorganisms, that was bactericidal for Plantaricin149a and bacteriostatic for the analogs. This difference was confirmed by a reduction in leakage action for the analogs. The lytic activity of Plantaricin149a is suggested to be a result of the peptide-lipid interactions from the amphipathic helix and the hydrophobic residues at the N-terminus of the antimicrobial peptide.

  11. Global analysis of myocardial peptides containing cysteines with irreversible sulfinic and sulfonic Acid post-translational modifications

    DEFF Research Database (Denmark)

    Paulech, Jana; Liddy, Kiersten A; Engholm-Keller, Kasper

    2015-01-01

    ) and others (Cys sulfinic [Cys-SO2H] and sulfonic [Cys-SO3H] acids) that are considered "irreversible." We developed an enrichment method to isolate Cys-SO2H/SO3H-containing peptides from complex tissue lysates that is compatible with tandem mass spectrometry (MS/MS). The acidity of these post...

  12. Interaction study of amino acids and the peptide aspartame with lanthanide (III) ions

    International Nuclear Information System (INIS)

    Carubelli, C.R.

    1990-01-01

    The interactions between the Nd(III) ion with the amino acids L-aspartic acid, L-glutamic acid and L-histidine and the peptide aspartame in aqueous solution were studied. The study was conducted by means of electronic spectroscopy with the Judd-Ofelt formalism for transition intensity parameters calculations. Several coordination compounds involving Nd(III), Eu(III), and Tb(III) and the ligands L-histidine and aspartame were synthesized and characterized in the solid state. Mixed compounds involving Eu(III) and Tb(III) with the same ligands were synthesized and characterized also. The characterization were achieved by chemical analysis, melting points, vibrational spectroscopy (IR) and powder X-ray diffractometry. (author)

  13. Oostatic peptides containing d-amino acids: synthesis, oostatic activity, degradation, accumulation in ovaries and NMR study

    Czech Academy of Sciences Publication Activity Database

    Hlaváček, Jan; Tykva, Richard; Holík, Josef; Bennettová, Blanka; Buděšínský, Miloš; Vlasáková, Věra; Černý, Bohuslav; Slaninová, Jiřina

    2012-01-01

    Roč. 42, č. 5 (2012), s. 1715-1725 ISSN 0939-4451 R&D Projects: GA ČR GA203/06/1272 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50380511; CEZ:AV0Z50070508 Keywords : D-amino acids * oostatic peptide synthesis * H-3 labeling * oostatic activity in Neobellieria bullata * H-3 incorporation * Peptide degradation * NMR study Subject RIV: CC - Organic Chemistry Impact factor: 3.914, year: 2012

  14. Peptides containing internal residues of pyroglutamic acid: proton NMR characteristics

    International Nuclear Information System (INIS)

    Khan, S.A.

    1986-01-01

    The proton NMR characteristics of internal pyroglutamic acid (Glp; 5-oxoproline) residues in seven tripeptides of the general structure Boc-Xxx-Glp-Yyy-NH 2 were studied. In general, the chemical shifts of several diagnostic protons moved downfield on going from the Glu-containing peptides (Boc-Xxx-Glu-Yyy-NH 2 ) to the corresponding Glp-containing peptides. The C-2 proton of the Xxx residue was shifted by about 1.1 ppm. The N-2 proton of the Yyy residue was shifted by about 0.5 ppm. The C-2 proton of the Glx residue itself was shifted by about 0.5 ppm. One of the Glx C-3 protons was also shifted by about 0.5 ppm, but the other remained essentially unchanged. Finally, the Glx C-4 protons were shifted by about 0.3 ppm. Internal Glu residues are readily converted chemically into internal Glp residues. This conversion also occurs as a side reaction during HP cleavage of the protecting group from Glu(OBzl) residues. The spontaneous fragmentation of serum proteins C3, C4 and λ 2 -macroglobulin under denaturing conditions is probably due to regioselective hydrolysis of an internal Glp residue formed in each of these proteins upon denaturation. These proton NMR characteristics may be useful in establishing the presence of internal Glp residues in synthetic and natural peptides

  15. Collision-Induced Dissociation of Deprotonated Peptides. Relative Abundance of Side-Chain Neutral Losses, Residue-Specific Product Ions, and Comparison with Protonated Peptides.

    Science.gov (United States)

    Liang, Yuxue; Neta, Pedatsur; Yang, Xiaoyu; Stein, Stephen E

    2018-03-01

    High-accuracy MS/MS spectra of deprotonated ions of 390 dipeptides and 137 peptides with three to six residues are studied. Many amino acid residues undergo neutral losses from their side chains. The most abundant is the loss of acetaldehyde from threonine. The abundance of losses from the side chains of other amino acids is estimated relative to that of threonine. While some amino acids lose the whole side chain, others lose only part of it, and some exhibit two or more different losses. Side-chain neutral losses are less abundant in the spectra of protonated peptides, being significant mainly for methionine and arginine. In addition to the neutral losses, many amino acid residues in deprotonated peptides produce specific negative ions after peptide bond cleavage. An expanded list of fragment ions from protonated peptides is also presented and compared with those of deprotonated peptides. Fragment ions are mostly different for these two cases. These lists of fragments are used to annotate peptide mass spectral libraries and to aid in the confirmation of specific amino acids in peptides. Graphical Abstract ᅟ.

  16. Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson-Crick base pairing.

    Science.gov (United States)

    Berger, Or; Adler-Abramovich, Lihi; Levy-Sakin, Michal; Grunwald, Assaf; Liebes-Peer, Yael; Bachar, Mor; Buzhansky, Ludmila; Mossou, Estelle; Forsyth, V Trevor; Schwartz, Tal; Ebenstein, Yuval; Frolow, Felix; Shimon, Linda J W; Patolsky, Fernando; Gazit, Ehud

    2015-04-01

    The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs-CG, GC and GG-could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

  17. Metal-Free, Site-Selective Peptide Modification by Conversion of "Customizable" Units into β-Substituted Dehydroamino Acids.

    Science.gov (United States)

    Saavedra, Carlos J; Hernández, Dácil; Boto, Alicia

    2018-01-12

    Our site-selective modification of serine or threonine units in peptides allows the generation of β-substituted dehydroamino acids, which increase peptide resistance to hydrolysis and may improve their biological properties. Both the terminal and internal positions can be modified, and different customizable units can be activated separately. Remarkably, high Z selectivity is achieved, even at internal positions. The conversion involves a one-pot oxidative radical scission/phosphorylation process by using the low-toxicity (diacetoxyiodo)benzene/iodine system as the scission reagent. The resulting α-amino phosphonates undergo a Horner-Wadsworth-Emmons reaction to produce the dehydroamino acid derivatives (in a Z/E ratio of usually >98:2) under mild and metal-free conditions. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. 2-Aminobenzamide and 2-Aminobenzoic Acid as New MALDI Matrices Inducing Radical Mediated In-Source Decay of Peptides and Proteins

    Science.gov (United States)

    Smargiasso, Nicolas; Quinton, Loic; de Pauw, Edwin

    2012-03-01

    One of the mechanisms leading to MALDI in-source decay (MALDI ISD) is the transfer of hydrogen radicals to analytes upon laser irradiation. Analytes such as peptides or proteins may undergo ISD and this method can therefore be exploited for top-down sequencing. When performed on peptides, radical-induced ISD results in production of c- and z-ions, as also found in ETD and ECD activation. Here, we describe two new compounds which, when used as MALDI matrices, are able to efficiently induce ISD of peptides and proteins: 2-aminobenzamide and 2-aminobenzoic acid. In-source reduction of the disulfide bridge containing peptide Calcitonin further confirmed the radicalar mechanism of the ISD process. ISD of peptides led, in addition to c- and z-ions, to the generation of a-, x-, and y-ions both in positive and in negative ion modes. Finally, good sequence coverage was obtained for the sequencing of myoglobin (17 kDa protein), confirming the effectiveness of both 2-aminobenzamide and 2-aminobenzoic acid as MALDI ISD matrices.

  19. OK, thanks! A new mutualism between Chlamydomonas and methylobacteria facilitates growth on amino acids and peptides.

    Science.gov (United States)

    Calatrava, Victoria; Hom, Erik F Y; Llamas, Ángel; Fernández, Emilio; Galván, Aurora

    2018-04-01

    Nitrogen is a key nutrient for land plants and phytoplankton in terrestrial and aquatic ecosystems. The model alga Chlamydomonas reinhardtii can grow efficiently on several inorganic nitrogen sources (e.g. ammonium, nitrate, nitrite) as well as many amino acids. In this study, we show that Chlamydomonas is unable to use proline, hydroxyproline and peptides that contain these amino acids. However, we discovered that algal growth on these substrates is supported in association with Methylobacterium spp., and that a mutualistic carbon-nitrogen metabolic exchange between Chlamydomonas and Methylobacterium spp. is established. Specifically, the mineralization of these amino acids and peptides by Methylobacterium spp. produces ammonium that can be assimilated by Chlamydomonas, and CO2 photosynthetically fixed by Chlamydomonas yields glycerol that can be assimilated by Methylobacterium. As Chlamydomonas is an algal ancestor to land plants and Methylobacterium is a plant growth-promoting bacterium, this new model of mutualism may facilitate insights into the ecology and evolution of plant-bacterial interactions and design principles of synthetic ecology.

  20. Hydroxamic acid derivatives as potent peptide deformylase inhibitors and antibacterial agents.

    Science.gov (United States)

    Apfel, C; Banner, D W; Bur, D; Dietz, M; Hirata, T; Hubschwerlen, C; Locher, H; Page, M G; Pirson, W; Rossé, G; Specklin, J L

    2000-06-15

    Low-molecular-weight beta-sulfonyl- and beta-sulfinylhydroxamic acid derivatives have been synthesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most of the compounds synthesized and tested displayed antibacterial activities that cover several pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these compounds as antibacterial agents is discussed with respect to selectivity, intracellular concentrations in bacteria, and potential for resistance development.

  1. Capping biological quantum dots with the peptide CLPFFD to increase stability and to reduce effects on cell viability

    Energy Technology Data Exchange (ETDEWEB)

    Riveros, A. L., E-mail: ariveros@postqyf.uchile.cl [Universidad de Chile, Facultad de Ciencias Químicas y Farmacéuticas (Chile); Astudillo, J., E-mail: jason.astudillo@usach.cl; Vásquez, C. C., E-mail: claudio.vasquez@usach.cl [Universidad de Santiago de Chile, Facultad de Química y Biología (Chile); Jara, Danilo H., E-mail: Danilo.H.JaraQuinteros.1@nd.edu [University of Notre Dame, Radiation Laboratory, Department of Chemistry and Biochemistry (United States); Guerrero, Ariel R. [Universidad de Chile, Facultad de Ciencias Químicas y Farmacéuticas (Chile); Guzman, F., E-mail: fanny.guzman@ucv.cl [Pontificia Universidad Católica de Valparaíso, Núcleo de Biotecnología Curauma (Chile); Osorio-Roman, I. O., E-mail: igor.orlando@gmail.com [University of Windsor, Department of Chemistry and Biochemistry (Canada); Kogan, M. J., E-mail: mkogan@ciq.uchile.cl [Universidad de Chile, Facultad de Ciencias Químicas y Farmacéuticas (Chile)

    2016-08-15

    Highly fluorescent nanoparticles, or quantum dots, have multiple applications in biology and biomedicine; however, in most cases, it is necessary to functionalize them to enhance their biocompatibility and selectivity. Generally, functionalization is performed after nanoparticle synthesis and involves the use of molecules or macromolecules having two important traits: specific biological activity and functional groups that facilitate nanoparticle capping (i.e. atom–atom interaction). For this reason, we carried out a simple protocol for the chemical synthesis of cadmium telluride quantum dots capped with glutathione, and we then functionalized these nanoparticles with the amphipathic peptide CLPFFD. This peptide attaches selectively to β-Amyloid fibres, which are involved in Alzheimer’s disease. Our results show that the optical properties of the quantum dots are not affected by functionalization with this peptide. Infrared spectra showed that cadmium telluride quantum dots were functionalized with the peptide CLPFFD. In addition, no significant differences were observed between the surface charge of the quantum dots with or without CLPFFD and the nanocrystal size calculated for HR-TEM was 4.2 nm. Finally, our results show that quantum dots with CLPFFD are stable and that they resulted in a significantly reduced cytotoxicity with respect to that induced by quantum dots not conjugated with the peptide. Moreover, the results show that the CLPFFD-functionalized nanoparticles bind to β-Amyloid fibres.

  2. Enhanced peptide nucleic acid binding to supercoiled DNA: possible implications for DNA "breathing" dynamics

    DEFF Research Database (Denmark)

    Bentin, T; Nielsen, Peter E.

    1996-01-01

    The influence of DNA topology on peptide nucleic acid (PNA) binding was studied. Formation of sequence-specific PNA2/dsDNA (double-stranded DNA) complexes was monitored by a potassium permanganate probing/primer extension assay. At low ionic strengths, the binding of PNA was 2-3 times more...

  3. Overcoming the Refractory Expression of Secreted Recombinant Proteins in Mammalian Cells through Modification of the Signal Peptide and Adjacent Amino Acids.

    Science.gov (United States)

    Güler-Gane, Gülin; Kidd, Sara; Sridharan, Sudharsan; Vaughan, Tristan J; Wilkinson, Trevor C I; Tigue, Natalie J

    2016-01-01

    The expression and subsequent purification of mammalian recombinant proteins is of critical importance to many areas of biological science. To maintain the appropriate tertiary structure and post-translational modifications of such proteins, transient mammalian expression systems are often adopted. The successful utilisation of these systems is, however, not always forthcoming and some recombinant proteins prove refractory to expression in mammalian hosts. In this study we focussed on the role of different N-terminal signal peptides and residues immediately downstream, in influencing the level of secreted recombinant protein obtained from suspension HEK293 cells. Using secreted alkaline phosphatase (SEAP) as a model protein, we identified that the +1/+2 downstream residues flanking a heterologous signal peptide significantly affect secreted levels. By incorporating these findings we conducted a comparison of different signal peptide sequences and identified the most productive as secrecon, a computationally-designed sequence. Importantly, in the context of the secrecon signal peptide and SEAP, we also demonstrated a clear preference for specific amino acid residues at the +1 position (e.g. alanine), and a detrimental effect of others (cysteine, proline, tyrosine and glutamine). When proteins that naturally contain these "undesirable" residues at the +1 position were expressed with their native signal peptide, the heterologous secrecon signal peptide, or secrecon with an additional alanine at the +1 or +1 and +2 position, the level of expression differed significantly and in an unpredictable manner. For each protein, however, at least one of the panel of signal peptide/adjacent amino acid combinations enabled successful recombinant expression. In this study, we highlight the important interplay between a signal peptide and its adjacent amino acids in enabling protein expression, and we describe a strategy that could enable recombinant proteins that have so far

  4. Development of novel ligands for peptide GPCRs.

    Science.gov (United States)

    Moran, Brian M; McKillop, Aine M; O'Harte, Finbarr Pm

    2016-12-01

    Incretin based glucagon-like peptide-1 receptor (GLP-1R) agonists which target a G-protein coupled receptor (GPCR) are currently used in the treatment of type 2 diabetes. This review focuses on GPCRs from pancreatic β-cells, including GLP-1, glucose-dependent insulinotropic polypeptide (GIP), glucagon, somatostatin, pancreatic polypeptide (PP), cholecystokinin (CCK), peptide YY (PYY), oxyntomodulin (OXM) and ghrelin receptors. In addition, fatty acids GPCRs are thought to have an increasing role in regulating peptide secretions namely short fatty acids GPCR (GPR41, GPR43), medium chain fatty acid GPCR (GPR84), long chain fatty acid GPCR (GPR40, GPR120) and cannabinoid-like GPCR (GPR55, GPR119). Several pre-clinical and clinical trials are currently ongoing in peptide GPCR based therapies, including dual and triple agonist peptides which activate two or more GPCRs simultaneously. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. PH dependent adhesive peptides

    Science.gov (United States)

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  6. 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

    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...... 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...

  7. Radiation-induced reductive modifications of sulfur-containing amino acids within peptides and proteins.

    Science.gov (United States)

    Chatgilialoglu, Chryssostomos; Ferreri, Carla; Torreggiani, Armida; Salzano, Anna Maria; Renzone, Giovanni; Scaloni, Andrea

    2011-10-19

    The complex scenario of radical stress reactions affecting peptides/proteins can be better elucidated through the design of biomimetic studies simulating the consequences of the different free radicals attacking amino acids. In this context, ionizing radiations allowed to examine the specific damages caused by H-atoms and electrons coupled with protons, thus establishing the molecular basis of reductive radical stress. This is an innovative concept that complements the well-known oxidative stress also in view of a complete understanding of the global consequences of radical species reactivities on living systems. This review summarizes the knowledge of the chemical changes present in sulfur-containing amino acids occurring in polypeptides under reductive radical conditions, in particular the transformation of Met and Cys residues into α-amino butyric acid and alanine, respectively. Reductive radical stress causing a desulfurization process, is therefore coupled with the formation of S-centered radicals, which in turn can diffuse apart and become responsible of the damage transfer from proteins to lipids. These reductive modifications assayed in different peptide/protein sequences constitute an integration of the molecular inventories that up to now take into account only oxidative transformations. They can be useful to achieve an integrated vision of the free radical reactivities in a multifunctional system and, overall, for wider applications in the redox proteomics field. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Estimation of acidity constants, ionic mobilities and charges of antimicrobial peptides by capillary electrophoresis

    Czech Academy of Sciences Publication Activity Database

    Tůmová, Tereza; Monincová, Lenka; Čeřovský, Václav; Kašička, Václav

    2016-01-01

    Roč. 37, 23/24 (2016), s. 3186-3195 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GA15-01948S Institutional support: RVO:61388963 Keywords : acid dissociation constant * antimicrobial peptides * capillary electrophoresis * charge * mobility * Pka Subject RIV: CB - Analytical Chemistry , Separation Impact factor: 2.744, year: 2016

  9. Small surfactant-like peptides can drive soluble proteins into active aggregates

    Directory of Open Access Journals (Sweden)

    Zhou Bihong

    2012-01-01

    Full Text Available Abstract Background Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF and a short beta structure peptide ELK16 (LELELKLKLELELKLK have a similar property. Results In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6 were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used, Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same, Bacillus pumilus xylosidase (XynB, and green fluorescent protein (GFP, and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core. Conclusions This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in

  10. Determination of acidity constants and ionic mobilities of polyprotic peptide hormones by CZE

    Czech Academy of Sciences Publication Activity Database

    Šolínová, Veronika; Kašička, Václav

    2013-01-01

    Roč. 34, č. 18 (2013), s. 2655-2665 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR(CZ) GA13-17224S Institutional support: RVO:61388963 Keywords : acid dissociation constant * gonadotropin-releasing hormones * ionization constant * peptides * pK(a) Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.161, year: 2013

  11. Amphipathic alpha-helices and putative cholesterol binding domains of the influenza virus matrix M1 protein are crucial for virion structure organisation.

    Science.gov (United States)

    Tsfasman, Tatyana; Kost, Vladimir; Markushin, Stanislav; Lotte, Vera; Koptiaeva, Irina; Bogacheva, Elena; Baratova, Ludmila; Radyukhin, Victor

    2015-12-02

    The influenza virus matrix M1 protein is an amphitropic membrane-associated protein, forming the matrix layer immediately beneath the virus raft membrane, thereby ensuring the proper structure of the influenza virion. The objective of this study was to elucidate M1 fine structural characteristics, which determine amphitropic properties and raft membrane activities of the protein, via 3D in silico modelling with subsequent mutational analysis. Computer simulations suggest the amphipathic nature of the M1 α-helices and the existence of putative cholesterol binding (CRAC) motifs on six amphipathic α-helices. Our finding explains for the first time many features of this protein, particularly the amphitropic properties and raft/cholesterol binding potential. To verify these results, we generated mutants of the A/WSN/33 strain via reverse genetics. The M1 mutations included F32Y in the CRAC of α-helix 2, W45Y and W45F in the CRAC of α-helix 3, Y100S in the CRAC of α-helix 6, M128A and M128S in the CRAC of α-helix 8 and a double L103I/L130I mutation in both a putative cholesterol consensus motif and the nuclear localisation signal. All mutations resulted in viruses with unusual filamentous morphology. Previous experimental data regarding the morphology of M1-gene mutant influenza viruses can now be explained in structural terms and are consistent with the pivotal role of the CRAC-domains and amphipathic α-helices in M1-lipid interactions. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Effect of amino acid sequence and pH on nanofiber formation of self-assembling peptides EAK16-II and EAK16-IV.

    Science.gov (United States)

    Hong, Yooseong; Legge, Raymond L; Zhang, S; Chen, P

    2003-01-01

    Atomic force microscopy (AFM) and axisymmetric drop shape analysis-profile (ASDA-P) were used to investigate the mechanism of self-assembly of peptides. The peptides chosen consisted of 16 alternating hydrophobic and hydrophilic amino acids, where the hydrophilic residues possess alternating negative and positive charges. Two types of peptides, AEAEAKAKAEAEAKAK (EAK16-II) and AEAEAEAEAKAKAKAK (EAK16-IV), were investigated in terms of nanostructure formation through self-assembly. The experimental results, which focused on the effects of the amino acid sequence and pH, show that the nanostructures formed by the peptides are dependent on the amino acid sequence and the pH of the solution. For pH conditions around neutrality, one of the peptides used in this study, EAK16-IV, forms globular assemblies and has lower surface tension at air-water interfaces than another peptide, EAK16-II, which forms fibrillar assemblies at the same pH. When the pH is lowered below 6.5 or raised above 7.5, there is a transition from globular to fibrillar structures for EAK16-IV, but EAK16-II does not show any structural transition. Surface tension measurements using ADSA-P showed different surface activities of peptides at air-water interfaces. EAK16-II does not show a significant difference in surface tension for the pH range between 4 and 9. However, EAK16-IV shows a noticeable decrease in surface tension at pH around neutrality, indicating that the formation of globular assemblies is related to the molecular hydrophobicity.

  13. Effect of BMAP-28 antimicrobial peptides on Leishmania major promastigote and amastigote growth: role of leishmanolysin in parasite survival.

    Directory of Open Access Journals (Sweden)

    Miriam A Lynn

    Full Text Available Protozoan parasites, such as Leishmania, still pose an enormous public health problem in many countries throughout the world. Current measures are outdated and have some associated drug resistance, prompting the search into novel therapies. Several innovative approaches are under investigation, including the utilization of host defence peptides (HDPs as emerging anti-parasitic therapies. HDPs are characterised by their small size, amphipathic nature and cationicity, which induce permeabilization of cell membranes, whilst modulating the immune response of the host. Recently, members of the cathelicidin family of HDPs have demonstrated significant antimicrobial activities against various parasites including Leishmania. The cathelicidin bovine myeloid antimicrobial peptide 28 (BMAP-28 has broad antimicrobial activities and confers protection in animal models of bacterial infection or sepsis. We tested the effectiveness of the use of BMAP-28 and two of its isomers the D-amino acid form (D-BMAP-28 and the retro-inverso form (RI-BMAP-28, as anti-leishmanial agents against the promastigote and amastigote intracellular Leishmania major lifecycle stages.An MTS viability assay was utilized to show the potent antiparasitic activity of BMAP-28 and its protease resistant isomers against L. major promastigotes in vitro. Cell membrane permeability assays, caspase 3/7, Tunel assays and morphologic studies suggested that this was a late stage apoptotic cell death with early osmotic cell lysis caused by the antimicrobial peptides. Furthermore, BMAP-28 and its isomers demonstrated anti-leishmanial activities against intracellular amastigotes within a macrophage infection model.Interestingly, D-BMAP-28 appears to be the most potent antiparasitic of the three isomers against wild type L. major promastigotes and amastigotes. These exciting results suggest that BMAP-28 and its protease resistant isomers have significant therapeutic potential as novel anti-leishmanials.

  14. Purification of the bacteriocin bavaricin MN and characterization of its mode of action against Listeria monocytogenes Scott A cells and lipid vesicles.

    OpenAIRE

    Kaiser, A L; Montville, T J

    1996-01-01

    Bavaricin MN was purified from Lactobacillus sake culture supernatant 135-fold with a final yield of 11%. Sequence analysis revealed bavaricin MN to be a 42-amino-acid peptide having a molecular weight of 4,769 and a calculated pI of 10.0. Computer analysis indicated that the C-terminal region may form an alpha-helical structure with an amphipathic nature deemed important in the interaction of bacteriocins with biological membranes. Bavaricin MN rapidly depleted the membrane potential (delta ...

  15. Development of a method for environmentally friendly chemical peptide synthesis in water using water-dispersible amino acid nanoparticles

    Directory of Open Access Journals (Sweden)

    Fukumori Yoshinobu

    2011-08-01

    Full Text Available Abstract Due to the vast importance of peptides in biological processes, there is an escalating need for synthetic peptides to be used in a wide variety of applications. However, the consumption of organic solvent is extremely large in chemical peptide syntheses because of the multiple condensation steps in organic solvents. That is, the current synthesis method is not environmentally friendly. From the viewpoint of green sustainable chemistry, we focused on developing an organic solvent-free synthetic method using water, an environmentally friendly solvent. Here we described in-water synthesis technology using water-dispersible protected amino acids.

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

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  17. [Plant signaling peptides. Cysteine-rich peptides].

    Science.gov (United States)

    Ostrowski, Maciej; Kowalczyk, Stanisław

    2015-01-01

    Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation.

  18. Membrane-Active Epithelial Keratin 6A Fragments (KAMPs) Are Unique Human Antimicrobial Peptides with a Non-αβ Structure

    Science.gov (United States)

    Lee, Judy T. Y.; Wang, Guangshun; Tam, Yu Tong; Tam, Connie

    2016-01-01

    Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs). Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS) micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αβ structure for human antimicrobial peptides. Direct observation of Staphylococcus aureus and Pseudomonas aeruginosa by scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αβ peptide scaffolds for the design of novel peptide-based antibiotics. PMID:27891122

  19. Membrane-Active Epithelial Keratin 6A Fragments (KAMPs Are Unique Human Antimicrobial Peptides with a Non-αβ Structure

    Directory of Open Access Journals (Sweden)

    Judy Tsz Ying Lee

    2016-11-01

    Full Text Available Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs. Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αβ structure for human antimicrobial peptides. Direct observation of Staphylococcus aureus and Pseudomonas aeruginosa by scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αβ peptide scaffolds for the design of novel peptide-based antibiotics.

  20. Statistically significant dependence of the Xaa-Pro peptide bond conformation on secondary structure and amino acid sequence

    Directory of Open Access Journals (Sweden)

    Leitner Dietmar

    2005-04-01

    Full Text Available Abstract Background A reliable prediction of the Xaa-Pro peptide bond conformation would be a useful tool for many protein structure calculation methods. We have analyzed the Protein Data Bank and show that the combined use of sequential and structural information has a predictive value for the assessment of the cis versus trans peptide bond conformation of Xaa-Pro within proteins. For the analysis of the data sets different statistical methods such as the calculation of the Chou-Fasman parameters and occurrence matrices were used. Furthermore we analyzed the relationship between the relative solvent accessibility and the relative occurrence of prolines in the cis and in the trans conformation. Results One of the main results of the statistical investigations is the ranking of the secondary structure and sequence information with respect to the prediction of the Xaa-Pro peptide bond conformation. We observed a significant impact of secondary structure information on the occurrence of the Xaa-Pro peptide bond conformation, while the sequence information of amino acids neighboring proline is of little predictive value for the conformation of this bond. Conclusion In this work, we present an extensive analysis of the occurrence of the cis and trans proline conformation in proteins. Based on the data set, we derived patterns and rules for a possible prediction of the proline conformation. Upon adoption of the Chou-Fasman parameters, we are able to derive statistically relevant correlations between the secondary structure of amino acid fragments and the Xaa-Pro peptide bond conformation.

  1. pH dependence of MHC class I-restricted peptide presentation

    DEFF Research Database (Denmark)

    Stryhn, A; Pedersen, L O; Romme, T

    1996-01-01

    The function of MHC class I molecules is to bind and present antigenic peptides to cytotoxic T cells. Here, we report that class I-restricted peptide presentation is strongly pH dependent. The presentation of some peptides was enhanced at acidic pH, whereas the presentation of others was inhibited....... Biochemical peptide-MHC class I binding assays demonstrated that peptide-MHC class I complexes are more stable at neutral pH than at acidic pH. We suggest that acid-dependent peptide dissociation can generate empty class I molecules and that the resulting binding potential can be exploited by a subset...

  2. Chenodeoxycholic acid stimulates glucagon-like peptide-1 secretion in patients after Roux-en-Y gastric bypass

    DEFF Research Database (Denmark)

    Nielsen, Signe; Svane, Maria S; Kuhre, Rune E

    2017-01-01

    (BMI 29.1 ± 1.2, age 37.0 ± 3.2 years, time from RYGB 32.3 ± 1.1 months, weight loss after RYGB 37.0 ± 3.1 kg) were studied in a placebo-controlled, crossover-study. On three different days, participants ingested (1) placebo (water), (2) UDCA 750 mg, (3) CDCA 1250 mg (highest recommended doses). Oral...... intake of CDCA increased plasma concentrations of GLP-1, C-peptide, glucagon, peptide YY, neurotensin, total bile acids, and fibroblast growth factor 19 significantly compared with placebo (all P

  3. Peptide design using alpha,beta-dehydro amino acids: from beta-turns to helical hairpins.

    Science.gov (United States)

    Mathur, Puniti; Ramakumar, S; Chauhan, V S

    2004-01-01

    Incorporation of alpha,beta-dehydrophenylalanine (DeltaPhe) residue in peptides induces folded conformations: beta-turns in short peptides and 3(10)-helices in larger ones. A few exceptions-namely, alpha-helix or flat beta-bend ribbon structures-have also been reported in a few cases. The most favorable conformation of DeltaPhe residues are (phi,psi) approximately (-60 degrees, -30 degrees ), (-60 degrees, 150 degrees ), (80 degrees, 0 degrees ) or their enantiomers. DeltaPhe is an achiral and planar residue. These features have been exploited in designing DeltaPhe zippers and helix-turn-helix motifs. DeltaPhe can be incorporated in both right and left-handed helices. In fact, consecutive occurrence of three or more DeltaPhe amino acids induce left-handed screw sense in peptides containing L-amino acids. Weak interactions involving the DeltaPhe residue play an important role in molecular association. The C--H.O==C hydrogen bond between the DeltaPhe side-chain and backbone carboxyl moiety, pi-pi stacking interactions between DeltaPhe side chains belonging to enantiomeric helices have shown to stabilize folding. The unusual capability of a DeltaPhe ring to form the hub of multicentered interactions namely, a donor in aromatic C--H.pi and C--H.O==C and an acceptor in a CH(3).pi interaction suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures. Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004

  4. Osmotic Pressure Simulations of Amino Acids and Peptides Highlight Potential Routes to Protein Force Field Parameterization

    Science.gov (United States)

    Miller, Mark S.; Lay, Wesley K.

    2016-01-01

    Recent molecular dynamics (MD) simulations of proteins have suggested that common force fields overestimate the strength of amino acid interactions in aqueous solution. In an attempt to determine the causes of these effects, we have measured the osmotic coefficients of a number of amino acids using the AMBER ff99SB-ILDN force field with two popular water models, and compared the results with available experimental data. With TIP4P-Ew water, interactions between aliphatic residues agree well with experiment, but interactions of the polar residues serine and threonine are found to be excessively attractive. For all tested amino acids, the osmotic coefficients are lower when the TIP3P water model is used. Additional simulations performed on charged amino acids indicate that the osmotic coefficients are strongly dependent on the parameters assigned to the salt ions, with a reparameterization of the sodium:carboxylate interaction reported by the Aksimentiev group significantly improving description of the osmotic coefficient for glutamate. For five neutral amino acids, we also demonstrate a decrease in solute-solute attractions using the recently reported TIP4P-D water model and using the KBFF force field. Finally, we show that for four two-residue peptides improved agreement with experiment can be achieved by re-deriving the partial charges for each peptide. PMID:27052117

  5. Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

    Science.gov (United States)

    Marin, Milenen Hernández; Rodríguez-Tanty, Chryslaine; Higginson-Clarke, David; Bocalandro, Yadaris Márquez; Peña, Lilliam Pozo

    2005-10-28

    Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

  6. Toward Peptide Nucleic Acid (PNA) Directed Peptide Translation Using Ester Based Aminoacyl Transfer

    DEFF Research Database (Denmark)

    Singhal, Abhishek; Bagnacani, Valentina; Corradini, Roberto

    2014-01-01

    Peptide synthesis is a fundamental feature of life. However, it still remains unclear how the contemporary translation apparatus evolved from primitive prebiotic systems and at which stage of the evolution peptide synthesis emerged. Using simple molecular architectures, in which aminoacyl transfe...

  7. The 4-pyridylmethyl ester as a protecting group for glutamic and aspartic acids: 'flipping' peptide charge states for characterization by positive ion mode ESI-MS.

    Science.gov (United States)

    Garapati, Sriramya; Burns, Colin S

    2014-03-01

    Use of the 4-pyridylmethyl ester group for side-chain protection of glutamic acid residues in solid-phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI-MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4-pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

  8. A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo

    Science.gov (United States)

    Lehto, Taavi; Simonson, Oscar E; Mäger, Imre; Ezzat, Kariem; Sork, Helena; Copolovici, Dana-Maria; Viola, Joana R; Zaghloul, Eman M; Lundin, Per; Moreno, Pedro MD; Mäe, Maarja; Oskolkov, Nikita; Suhorutšenko, Julia; Smith, CI Edvard; Andaloussi, Samir EL

    2011-01-01

    Finding suitable nonviral delivery vehicles for nucleic acid–based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice. PMID:21343913

  9. Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs.

    Directory of Open Access Journals (Sweden)

    Mohamed F Mohamed

    Full Text Available Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan with minimum inhibitory concentration50 (MIC50 of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide and IK8 "D isoform" demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF3K (two cell penetrating peptides were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin

  10. Flanking signal and mature peptide residues influence signal peptide cleavage

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2008-12-01

    Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

  11. Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids

    DEFF Research Database (Denmark)

    Lonkar, Pallavi; Kim, Ki-Hyun; Kuan, Jean Y

    2009-01-01

    Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures...

  12. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    International Nuclear Information System (INIS)

    Garrison, W.M.

    1981-12-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the α,α'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The α,α'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the α,α'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized

  13. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    International Nuclear Information System (INIS)

    Garrison, W.M.

    1979-03-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the α,α'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The α,α'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and pathogenic toxins. All of the available data strongly suggest that the α,α'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized

  14. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods

    Energy Technology Data Exchange (ETDEWEB)

    Garrison, W. M.

    1981-12-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides, and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and bacterial products. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

  15. Effects of the substitution of amino acid residues, through chemical synthesis, on the conformation and activity of antimicrobial peptides

    Directory of Open Access Journals (Sweden)

    Regina C. Adão

    2012-06-01

    Full Text Available Antimicrobial peptides make up an assorted group of molecules which contain from 12 to 50 amino acid residues and which may be produced by microorganisms, plants and animals. From the discovery that these biomolecules are lethal to bacteria, inhibiting the pathogenic organism’s growth, and are also related to innate and adapted defense mechanisms, the investigation of such molecules came to be an emergent research field, in which more than 1800 antimicrobial peptides have so far been discovered throughout the last three decades. These molecules are potential representatives of a new generation of antibiotic agents and the main motivation for such use is their activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria as well as fungi and viruses. An important class of comprising some of these peptides may be found in anurans, from which it has been isolated, a considerable number of antimicrobial peptides with diverse sequences and structures, including linear and dimeric ones. In this work monomeric chains (CH1 e CH2 of the heterodimeric antimicrobial peptide distinctin (isolated in 1999 from Phyllomedusa distincta anurans, as well as its mutated monomers (CH1-S and CH2-S and the heterodimer itself were synthesized. The distinctin is the peptide with two chains of different sequences (Table 1 bound each other by disulfide bond from the cystein residues constituting the heterodimer. To investigate the effects on the biological activity by amino acids substitution at normal distinctin CH1 and CH2 chains, both were synthesized as well as their similar chains (CH1-S and CH2-S in which the cystein (Fig.1 a residues of each chain were changed by serin residues (Fig. 1 b. The new chains were named mutants. The synthesis was carried out in solid phase, using Fmoc strategy. The heterodimer distinctin was obtained from CH1 and CH2 chains coupling through cystein residues air oxidation. The results from HPLC

  16. 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.

  17. Exploring Alternative Radiolabeling Strategies for Sialic Acid-Binding Immunoglobulin-Like Lectin 9 Peptide: [68Ga]Ga- and [18F]AlF-NOTA-Siglec-9

    Directory of Open Access Journals (Sweden)

    Olli Moisio

    2018-01-01

    Full Text Available Amino acid residues 283–297 from sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9 form a cyclic peptide ligand targeting vascular adhesion protein-1 (VAP-1. VAP-1 is associated with the transfer of leukocytes from blood to tissues upon inflammation. Therefore, analogs of Siglec-9 peptide are good candidates for visualizing inflammation non-invasively using positron emission tomography (PET. Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA-conjugated Siglec-9 has been evaluated extensively for this purpose. Here, we explored two alternative strategies for radiolabeling Siglec-9 peptide using a 1,4,7-triazacyclononane-triacetic acid (NOTA-chelator to bind [68Ga]Ga or [18F]AlF. The radioligands were evaluated by in vivo PET imaging and ex vivo γ-counting of turpentine-induced sterile skin/muscle inflammation in Sprague-Dawley rats. Both tracers showed clear accumulation in the inflamed tissues. The whole-body biodistribution patterns of the tracers were similar.

  18. Evidence connecting old, new and neglected glucose-lowering drugs to bile acid-induced GLP-1 secretion

    DEFF Research Database (Denmark)

    Kårhus, Martin L; Brønden, Andreas; Sonne, David P

    2017-01-01

    Bile acids are amphipathic water-soluble steroid-based molecules best known for their important lipid-solubilizing role in the assimilation of fat. Recently, bile acids have emerged as metabolic integrators with glucose-lowering potential. Among a variety of gluco-metabolic effects, bile acids have...... current evidence connecting established glucose-lowering drugs to bile acid-induced GLP-1 secretion and discusses whether bile acid-induced GLP-1 secretion may constitute a new basis for understanding how metformin, inhibitors of the apical sodium-dependent bile acids transporter, and bile acid...... sequestrants - old, new and neglected glucose-lowering drugs - improve glucose metabolism....

  19. Photochemical addition of amino acids and peptides to homopolyribonucleotides of the major DNA bases

    International Nuclear Information System (INIS)

    Shetlar, M.D.; Hom, K.; Carbone, J.; Moy, D.; Steady, E.; Watanabe, M.

    1984-01-01

    The photochemical quantum yields for addition of glycine and the L-amino acids commonly occurring in proteins to polyadenylic acid, polycytidylic acid, polyguanylic acid and polyribothymidylic acid have been determined in deoxygenated phosphate buffer using a fluorescamine assay technique. Polyadenylic acid was reactive with eleven of the twenty amino acids tested, with phenylalanine, tyrosine, glutamine, lysine and asparagine having the highest quantum yields. Polyguanylic acid reacted with sixteen amino acids; phenylalanine, arginine, cysteine, tyrosine, and lysine displayed the largest quantum yields. Polycytidylic acid showed reactivity with fifteen amino acids with lysine, phenylalanine, cysteine, tyrosine and arginine having the greatest quantum yields. Polyribothymidylic acid, reactive with fifteen of nineteen amino acids surveyed, showed the highest quantum yields for cysteine, phenylalanine, tyrosine, lysine and asparagine. None of the polynucleotides were reactive with aspartic acid or glutamic acid. The quantum yields for photoaddition of eighteen dipeptides of the form gycyl X (X being an amino acid), and of L-alanyl-L-tryptophan, L-seryl-L-seryl-L-serine, L-threonyl-L-threonyl-L-threonine, L-cystine-bis-glycine, and Nsup(α)-acetyllysine to polyadenylic acid, polycytidylic acid and polyguanylic acid were measured. All were found to add photochemically to each of these polymers. Polyribothymidylic acid, tested with eleven peptides and with Nsup(α)-acetyllysine, was found to be reactive with all. (author)

  20. Killing of Mycobacterium avium by lactoferricin peptides: improved activity of arginine- and D-amino-acid-containing molecules.

    Science.gov (United States)

    Silva, Tânia; Magalhães, Bárbara; Maia, Sílvia; Gomes, Paula; Nazmi, Kamran; Bolscher, Jan G M; Rodrigues, Pedro N; Bastos, Margarida; Gomes, Maria Salomé

    2014-06-01

    Mycobacterium avium causes respiratory disease in susceptible individuals, as well as disseminated infections in immunocompromised hosts, being an important cause of morbidity and mortality among these populations. Current therapies consist of a combination of antibiotics taken for at least 6 months, with no more than 60% overall clinical success. Furthermore, mycobacterial antibiotic resistance is increasing worldwide, urging the need to develop novel classes of antimicrobial drugs. One potential and interesting alternative strategy is the use of antimicrobial peptides (AMP). These are present in almost all living organisms as part of their immune system, acting as a first barrier against invading pathogens. In this context, we investigated the effect of several lactoferrin-derived AMP against M. avium. Short peptide sequences from both human and bovine lactoferricins, namely, hLFcin1-11 and LFcin17-30, as well as variants obtained by specific amino acid substitutions, were evaluated. All tested peptides significantly inhibited the axenic growth of M. avium, the bovine peptides being more active than the human. Arginine residues were found to be crucial for the display of antimycobacterial activity, whereas the all-d-amino-acid analogue of the bovine sequence displayed the highest mycobactericidal activity. These findings reveal the promising potential of lactoferricins against mycobacteria, thus opening the way for further research on their development and use as a new weapon against mycobacterial infections. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  1. Enzyme active site mimics based on TriAzaCyclophane (TAC)-scaffolded peptides and amino acid residues

    NARCIS (Netherlands)

    Albada, H.B.

    2009-01-01

    This thesis describes the scope and limitations of the application of TriAzaCyclophane (TAC)-scaffolded peptides or amino acid residues as enzyme active site mimics, as ligands in asymmetric catalysis and as hydrolysis catalysts attached to vancomycin. For the mimicry of functional group enzymes, of

  2. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from –1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical ...

  3. Three-dimensional solution structure of lactoferricin B, an antimicrobial peptide derived from bovine lactoferrin.

    Science.gov (United States)

    Hwang, P M; Zhou, N; Shan, X; Arrowsmith, C H; Vogel, H J

    1998-03-24

    The solution structure of bovine lactoferricin (LfcinB) has been determined using 2D 1H NMR spectroscopy. LfcinB is a 25-residue antimicrobial peptide released by pepsin cleavage of lactoferrin, an 80 kDa iron-binding glycoprotein with many immunologically important functions. The NMR structure of LfcinB reveals a somewhat distorted antiparallel beta-sheet. This contrasts with the X-ray structure of bovine lactoferrin, in which residues 1-13 (of LfcinB) form an alpha-helix. Hence, this region of lactoferricin B appears able to adopt a helical or sheetlike conformation, similar to what has been proposed for the amyloidogenic prion proteins and Alzheimer's beta-peptides. LfcinB has an extended hydrophobic surface comprised of residues Phe1, Cys3, Trp6, Trp8, Pro16, Ile18, and Cys20. The side chains of these residues are well-defined in the NMR structure. Many hydrophilic and positively charged residues surround the hydrophobic surface, giving LfcinB an amphipathic character. LfcinB bears numerous similarities to a vast number of cationic peptides which exert their antimicrobial activities through membrane disruption. The structures of many of these peptides have been well characterized, and models of their membrane-permeabilizing mechanisms have been proposed. The NMR solution structure of LfcinB may be more relevant to membrane interaction than that suggested by the X-ray structure of intact lactoferrin. Based on the solution structure, it is now possible to propose potential mechanisms for the antimicrobial action of LfcinB.

  4. Bioactive dietary peptides and amino acids in inflammatory bowel disease.

    Science.gov (United States)

    Zhang, Hua; Hu, Chien-An A; Kovacs-Nolan, Jennifer; Mine, Yoshinori

    2015-10-01

    Inflammatory bowel disease (IBD), most commonly ulcerative colitis (UC) and Crohn's disease (CD), is a chronic inflammation of the gastrointestinal tract. Patients affected with IBD experience symptoms including abdominal pain, persistent diarrhea, rectal bleeding, and weight loss. There is no cure for IBD; thus treatments typically focus on preventing complications, inducing and maintaining remission, and improving quality of life. During IBD, dysregulation of the intestinal immune system leads to increased production of pro-inflammatory cytokines, such as TNF-α and IL-6, and recruitment of activated immune cells to the intestine, causing tissue damage and perpetuating the inflammatory response. Recent biological therapies targeting specific inflammatory cytokines or pathways, in particular TNF-α, have shown promise, but not all patients respond to treatment, and some individuals become intolerant to treatment over time. Dietary peptides and amino acids (AAs) have been shown to modulate intestinal immune functions and influence inflammatory responses, and may be useful as alternative or ancillary treatments in IBD. This review focuses on dietary interventions for IBD treatment, in particular the role of dietary peptides and AAs in reducing inflammation, oxidative stress, and apoptosis in the gut, as well as recent advances in the cellular mechanisms responsible for their anti-inflammatory activity.

  5. Formation of active inclusion bodies induced by hydrophobic self-assembling peptide GFIL8.

    Science.gov (United States)

    Wang, Xu; Zhou, Bihong; Hu, Weike; Zhao, Qing; Lin, Zhanglin

    2015-06-16

    In the last few decades, several groups have observed that proteins expressed as inclusion bodies (IBs) in bacteria could still be biologically active when terminally fused to an appropriate aggregation-prone partner such as pyruvate oxidase from Paenibacillus polymyxa (PoxB). More recently, we have demonstrated that three amphipathic self-assembling peptides, an alpha helical peptide 18A, a beta-strand peptide ELK16, and a surfactant-like peptide L6KD, have properties that induce target proteins into active IBs. We have developed an efficient protein expression and purification approach for these active IBs by introducing a self-cleavable intein molecule. In this study, the self-assembling peptide GFIL8 (GFILGFIL) with only hydrophobic residues was analyzed, and this peptide effectively induced the formation of cytoplasmic IBs in Escherichia coli when terminally attached to lipase A and amadoriase II. The protein aggregates in cells were confirmed by transmission electron microscopy analysis and retained ~50% of their specific activities relative to the native counterparts. We constructed an expression and separation coupled tag (ESCT) by incorporating an intein molecule, the Mxe GyrA intein. Soluble target proteins were successfully released from active IBs upon cleavage of the intein between the GFIL8 tag and the target protein, which was mediated by dithiothreitol. A variant of GFIL8, GFIL16 (GFILGFILGFILGFIL), improved the ESCT scheme by efficiently eliminating interference from the soluble intein-GFIL8 molecule. The yields of target proteins at the laboratory scale were 3.0-7.5 μg/mg wet cell pellet, which is comparable to the yields from similar ESCT constructs using 18A, ELK16, or the elastin-like peptide tag scheme. The all-hydrophobic self-assembling peptide GFIL8 induced the formation of active IBs in E. coli when terminally attached to target proteins. GFIL8 and its variant GFIL16 can act as a "pull-down" tag to produce purified soluble proteins with

  6. Implementation of antimicrobial peptides for sample preparation prior to nucleic acid amplification in point-of-care settings.

    Science.gov (United States)

    Krõlov, Katrin; Uusna, Julia; Grellier, Tiia; Andresen, Liis; Jevtuševskaja, Jekaterina; Tulp, Indrek; Langel, Ülo

    2017-12-01

    A variety of sample preparation techniques are used prior to nucleic acid amplification. However, their efficiency is not always sufficient and nucleic acid purification remains the preferred method for template preparation. Purification is difficult and costly to apply in point-of-care (POC) settings and there is a strong need for more robust, rapid, and efficient biological sample preparation techniques in molecular diagnostics. Here, the authors applied antimicrobial peptides (AMPs) for urine sample preparation prior to isothermal loop-mediated amplification (LAMP). AMPs bind to many microorganisms such as bacteria, fungi, protozoa and viruses causing disruption of their membrane integrity and facilitate nucleic acid release. The authors show that incubation of E. coli with antimicrobial peptide cecropin P1 for 5 min had a significant effect on the availability of template DNA compared with untreated or even heat treated samples resulting in up to six times increase of the amplification efficiency. These results show that AMPs treatment is a very efficient sample preparation technique that is suitable for application prior to nucleic acid amplification directly within biological samples. Furthermore, the entire process of AMPs treatment was performed at room temperature for 5 min thereby making it a good candidate for use in POC applications.

  7. Acyclic peptides incorporating the d-Phe-2-Abz turn motif: Investigations on antimicrobial activity and propensity to adopt β-hairpin conformations.

    Science.gov (United States)

    Cameron, Alan J; Varnava, Kyriakos G; Edwards, Patrick J B; Harjes, Elena; Sarojini, Vijayalekshmi

    2018-06-14

    Three linear peptides incorporating d-Phe-2-Abz as the turn motif are reported. Peptide 1, a hydrophobic β-hairpin, served as a proof of principle for the design strategy with both NMR and CD spectra strongly suggesting a β-hairpin conformation. Peptides 2 and 3, designed as amphipathic antimicrobials, exhibited broad spectrum antimicrobial activity, with potency in the nanomolar range against Staphylococcus aureus. Both compounds possess a high degree of selectivity, proving non-haemolytic at concentrations 500 to 800 times higher than their respective minimal inhibitory concentrations (MICs) against S. aureus. Peptide 2 induced cell membrane and cell wall disintegration in both S. aureus and Pseudomonas aeruginosa as observed by transmission electron microscopy. Peptide 2 also demonstrated moderate antifungal activity against Candida albicans with an MIC of 50 μM. Synergism was observed with sub-MIC levels of amphotericin B (AmB), leading to nanomolar MICs against C. albicans for peptide 2. Based on circular dichroism spectra, both peptides 2 and 3 appear to exist as a mixture of conformers with the β-hairpin as a minor conformer in aqueous solution, and a slight increase in hairpin population in 50% trifluoroethanol, which was more pronounced for peptide 3. NMR spectra of peptide 2 in a 1:1 CD 3 CN/H 2 O mixture and 30 mM deuterated sodium dodecyl sulfate showed evidence of an extended backbone conformation of the β-strand residues. However, inter-strand rotating frame Overhauser effects (ROE) could not be detected and a loosely defined divergent hairpin structure resulted from ROE structure calculation in CD 3 CN/H 2 O. The loosely defined hairpin conformation is most likely a result of the electrostatic repulsions between cationic strand residues which also probably contribute towards maintaining low haemolytic activity. Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.

  8. Multiple native-like conformations trapped via self-association-induced hydrophobic collapse of the 33-residue beta-sheet domain from platelet factor 4.

    OpenAIRE

    Ilyina, E; Mayo, K H

    1995-01-01

    Native platelet factor 4 (PF4) (70 residues) has a hydrophobic three-stranded anti-parallel beta-sheet domain on to which is folded an amphipathic C-terminal alpha-helix and an aperiodic N-terminal domain. The 33-amino acid beta-sheet domain from PF4 (residues 23-55) has been synthesized and studied by c.d. and n.m.r. At 10 degrees C and low concentration, peptide 23-55 appears to exist in aqueous solution in a random-coil distribution of highly flexible conformational states. Some preferred ...

  9. Poly aspartic acid peptide-linked PLGA based nanoscale particles: potential for bone-targeting drug delivery applications.

    Science.gov (United States)

    Jiang, Tao; Yu, Xiaohua; Carbone, Erica J; Nelson, Clarke; Kan, Ho Man; Lo, Kevin W-H

    2014-11-20

    Delivering drugs specifically to bone tissue is very challenging due to the architecture and structure of bone tissue. Poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs) hold great promise for the delivery of therapeutics to bone tissue. The goal of the present research was to formulate a PLGA-based NP drug delivery system for bone tissue exclusively. Since poly-aspartic acids (poly-Asp) peptide sequence has been shown to bind to hydroxyapatite (HA), and has been suggested as a molecular tool for bone-targeting applications, we fabricated PLGA-based NPs linked with poly-Asp peptide sequence. Nanoparticles made of methoxy - poly(ethylene glycol) (PEG)-PLGA and maleimide-PEG-PLGA were prepared using a water-in-oil-in-water double emulsion and solvent evaporation method. Fluorescein isothiocyanate (FITC)-tagged poly-Asp peptide was conjugated to the surface of the nanoparticles via the alkylation reaction between the sulfhydryl groups at the N-terminal of the peptide and the CC double bond of maleimide at one end of the polymer chain to form thioether bonds. The conjugation of FITC-tagged poly-Asp peptide to PLGA NPs was confirmed by NMR analysis and fluorescent microscopy. The developed nanoparticle system is highly aqueous dispersible with an average particle size of ∼80 nm. In vitro binding analyses demonstrated that FITC-poly-Asp NPs were able to bind to HA gel as well as to mineralized matrices produced by human mesenchymal stem cells and mouse bone marrow stromal cells. Using a confocal microscopy technique, an ex vivo binding study of mouse major organ ground sections revealed that the FITC-poly-Asp NPs were able to bind specifically to the bone tissue. In addition, proliferation studies indicated that our FITC-poly-Asp NPs did not induce cytotoxicity to human osteoblast-like MG63 cell lines. Altogether, these promising results indicated that this nanoscale targeting system was able to bind to bone tissue specifically and might have a great

  10. Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation

    NARCIS (Netherlands)

    Herberts, Carla A.; Neijssen, Joost J.; de Haan, Jolanda; Janssen, Lennert; Drijfhout, Jan Wouter; Reits, Eric A.; Neefjes, Jacques J.

    2006-01-01

    Ag presentation by MHC class I is a highly inefficient process because cytosolic peptidases destroy most peptides after proteasomal generation. Various mechanisms shape the MHC class I peptidome. We define a new one: intracellular peptide stability. Peptides with two N-terminal basic amino acids are

  11. Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

    Science.gov (United States)

    Sahin, Deniz; Taflan, Sevket Onur; Yartas, Gizem; Ashktorab, Hassan; Smoot, Duane T

    2018-04-25

    Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency of traditional techniques both in diagnosis and therapy of the disease makes the development of alternative and novel techniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be used to increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of this study is screening and identification of individual peptides specifically targeted to human gastric cancer cells using a phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptide sequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cells were used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide library were applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were established by enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clones after five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequencies of each amino acid in best binding clones to determine positively overexpressed amino acids for designing novel peptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific binding on MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acid frequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45 cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, but more importantly, data extracted from eluted phage clones may be used to design theoretical peptides with better binding properties than even experimentally selected ones

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

    Science.gov (United States)

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

    2005-06-07

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

  13. Antibacterial Peptide Nucleic Acid-Antimicrobial Peptide (PNA-AMP) Conjugates

    DEFF Research Database (Denmark)

    Hansen, Anna Mette; Bonke, Gitte; Larsen, Camilla Josephine

    2016-01-01

    . In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential acpP gene. The results demonstrate that buforin 2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59...

  14. [Distiller Yeasts Producing Antibacterial Peptides].

    Science.gov (United States)

    Klyachko, E V; Morozkina, E V; Zaitchik, B Ts; Benevolensky, S V

    2015-01-01

    A new method of controlling lactic acid bacteria contamination was developed with the use of recombinant Saccharomyces cerevisiae strains producing antibacterial peptides. Genes encoding the antibacterial peptides pediocin and plantaricin with codons preferable for S. cerevisiae were synthesized, and a system was constructed for their secretory expression. Recombinant S. cerevisiae strains producing antibacterial peptides effectively inhibit the growth of Lactobacillus sakei, Pediacoccus pentasaceus, Pediacoccus acidilactici, etc. The application of distiller yeasts producing antibacterial peptides enhances the ethanol yield in cases of bacterial contamination. Recombinant yeasts producing the antibacterial peptides pediocin and plantaricin can successfully substitute the available industrial yeast strains upon ethanol production.

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

    Czech Academy of Sciences Publication Activity Database

    Paleček, Emil; Trefulka, Mojmír; Fojta, Miroslav

    2009-01-01

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

  16. Hydroxyapatite-binding peptides for bone growth and inhibition

    Science.gov (United States)

    Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

    2011-09-20

    Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

  17. Biosynthesis of cardiac natriuretic peptides

    DEFF Research Database (Denmark)

    Goetze, Jens Peter

    2010-01-01

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

  18. Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry.

    OpenAIRE

    Jensen, O N; Kulkarni, S; Aldrich, J V; Barofsky, D F

    1996-01-01

    Two peptide-oligothymidylic acids, prepared by joining an 11 residue synthetic peptide containing one internal carboxyl group (Asp side chain) to amino-linker-5'pdT6 and amino-linker-5'pdT10 oligonucleotides, were analyzed by matrix-assisted laser desorption/ionization (MALDI) on a linear time-of-flight mass spectrometer and by electrospray ionization (ESI) on a triple-quadrupole system. These synthetic compounds model peptide-nucleic acid heteroconjugates encountered in antisense research an...

  19. Escherichia coli Peptide Binding Protein OppA Has a Preference for Positively Charged Peptides

    NARCIS (Netherlands)

    Klepsch, M. M.; Kovermann, M.; Löw, C.; Balbach, J.; Permentier, H. P.; Fusetti, F.; de Gier, J. W.; Gier, Jan-Willem de; Slotboom, D. J.; Berntsson, R. P. -A.

    2011-01-01

    The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity.

  20. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    International Nuclear Information System (INIS)

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-01-01

    E2, along with E rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, 818 CPIGWTGVIEC 828 , containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP 818 CPIGWTGVIEC 828 indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion

  1. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Sainz, I.J. [Plum Island Animal Disease Center, ARS, USDA (United States); Largo, E. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Gladue, D.P.; Fletcher, P. [Plum Island Animal Disease Center, ARS, USDA (United States); O’Donnell, V. [Plum Island Animal Disease Center, ARS, USDA (United States); Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Holinka, L.G. [Plum Island Animal Disease Center, ARS, USDA (United States); Carey, L.B. [Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), E-08003 Barcelona (Spain); Lu, X. [Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Nieva, J.L. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Borca, M.V., E-mail: manuel.borca@ars.usda.gov [Plum Island Animal Disease Center, ARS, USDA (United States)

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  2. Chlorination of tyrosyl residues in peptides and proteins by hypochlorous acid

    International Nuclear Information System (INIS)

    Kettle, A.J.; Chapman, A.L.P.; Senthilmohan, R.; Vile, G.F.; Chai, L.L.

    1998-01-01

    Full text: Hypochlorous acid (HOCI) is the major strong oxidant produced by neutrophils. These granulocytic cells use HOCI to kill bacteria and it is also proposed to promote inflammation. Previously, it was shown that HOCI converts tyrosyl residues in proteins to 3-chlorotyrosine. This chlorinated molecule is an ideal biomarker for determining the precise roles HOCI plays in bacterial killing and inflammatory tissue damage. We have investigated the reaction of HOCI with tyrosyl residues in peptides and proteins to establish whether or not chlorinated products in addition to 3-chlorotyrosine are formed. When 200μM HOCI was added to 500μg/ml of bovine serum albumin both 3-chlorotyrosine and 3,5-dichlorotyrosine were formed. The monochlorinated amino acid was the predominant product and its formation was complete by 20 minutes whereas levels of 3,5-dichlorotyrosine continued to increase for up to an hour. Amounts of both chlorinated products increased with increasing concentrations of HOCI until a plateau was reached at about 800μM. At all concentrations of HOCI a substantial amount of the tyrosine that had reacted was unaccounted for as either 3-chlorotyrosine or 3,5-dichlorotyrosine. Similar results were obtained with small peptides containing tyrosine. Sub-stoichiometric concentrations of HOCI converted tyrosyl residues in GGYR to 3-chlorotyrosine. At higher concentrations of HOCI, chlorination was rapid and both 3-chlorotyrosine and 3,5-dichlorotyrosine were produced but they accounted for less than 50% of the products. To identify the additional products of the reaction, we reacted HOCI with tyrosine analogues including N-acetyltyrosine, phydroxyphenylacetic acid, and 4-propylphenol. Separation of the reaction mixture by HPLC revealed that numerous products were formed besides mono and dichlorinated derivatives of the parent compounds. Analysis of the products by gas chromatography/mass spectrometry strongly indicated that mono and dichlorinated

  3. Highly sensitive detection of influenza virus by boron-doped diamond electrode terminated with sialic acid-mimic peptide.

    Science.gov (United States)

    Matsubara, Teruhiko; Ujie, Michiko; Yamamoto, Takashi; Akahori, Miku; Einaga, Yasuaki; Sato, Toshinori

    2016-08-09

    The progression of influenza varies according to age and the presence of an underlying disease; appropriate treatment is therefore required to prevent severe disease. Anti-influenza therapy, such as with neuraminidase inhibitors, is effective, but diagnosis at an early phase of infection before viral propagation is critical. Here, we show that several dozen plaque-forming units (pfu) of influenza virus (IFV) can be detected using a boron-doped diamond (BDD) electrode terminated with a sialic acid-mimic peptide. The peptide was used instead of the sialyloligosaccharide receptor, which is the common receptor of influenza A and B viruses required during the early phase of infection, to capture IFV particles. The peptide, which was previously identified by phage-display technology, was immobilized by click chemistry on the BDD electrode, which has excellent electrochemical characteristics such as low background current and weak adsorption of biomolecules. Electrochemical impedance spectroscopy revealed that H1N1 and H3N2 IFVs were detectable in the range of 20-500 pfu by using the peptide-terminated BDD electrode. Our results demonstrate that the BDD device integrated with the receptor-mimic peptide has high sensitivity for detection of a low number of virus particles in the early phase of infection.

  4. Amphipathic lignin derivatives to accelerate simultaneous saccharification and fermentation of unbleached softwood pulp for bioethanol production.

    Science.gov (United States)

    Cheng, Ningning; Yamamoto, Yoko; Koda, Keiichi; Tamai, Yutaka; Uraki, Yasumitsu

    2014-12-01

    Amphipathic lignin derivatives (A-LDs) were already demonstrated to improve enzymatic saccharification of lignocellulose. Based on this knowledge, two kinds of A-LDs prepared from black liquor of soda pulping of Japanese cedar were applied to a fed-batch simultaneous saccharification and fermentation (SSF) process for unbleached soda pulp of Japanese cedar to produce bioethanol. Both lignin derivatives slightly accelerated yeast fermentation of glucose but not inhibited it. In addition, ethanol yields based on the theoretical maximum ethanol production in the fed-batch SSF process was increased from 49% without A-LDs to 64% in the presence of A-LDs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Software-aided approach to investigate peptide structure and metabolic susceptibility of amide bonds in peptide drugs based on high resolution mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Tatiana Radchenko

    Full Text Available Interest in using peptide molecules as therapeutic agents due to high selectivity and efficacy is increasing within the pharmaceutical industry. However, most peptide-derived drugs cannot be administered orally because of low bioavailability and instability in the gastrointestinal tract due to protease activity. Therefore, structural modifications peptides are required to improve their stability. For this purpose, several in-silico software tools have been developed such as PeptideCutter or PoPS, which aim to predict peptide cleavage sites for different proteases. Moreover, several databases exist where this information is collected and stored from public sources such as MEROPS and ExPASy ENZYME databases. These tools can help design a peptide drug with increased stability against proteolysis, though they are limited to natural amino acids or cannot process cyclic peptides, for example. We worked to develop a new methodology to analyze peptide structure and amide bond metabolic stability based on the peptide structure (linear/cyclic, natural/unnatural amino acids. This approach used liquid chromatography / high resolution, mass spectrometry to obtain the analytical data from in vitro incubations. We collected experimental data for a set (linear/cyclic, natural/unnatural amino acids of fourteen peptide drugs and four substrate peptides incubated with different proteolytic media: trypsin, chymotrypsin, pepsin, pancreatic elastase, dipeptidyl peptidase-4 and neprilysin. Mass spectrometry data was analyzed to find metabolites and determine their structures, then all the results were stored in a chemically aware manner, which allows us to compute the peptide bond susceptibility by using a frequency analysis of the metabolic-liable bonds. In total 132 metabolites were found from the various in vitro conditions tested resulting in 77 distinct cleavage sites. The most frequent observed cleavage sites agreed with those reported in the literature. The

  6. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

    of novel peptide-based protease inhibitors, efforts were made towards improved methods for peptide synthesis. The coupling of Fmoc-amino acids onto N-methylated peptidyl resins was investigated. These couplings can be low yielding and the effect of the use of microwave heating combined with the coupling...

  7. Poly(amino acid)-based fibrous scaffolds modified with surface-pendant peptides for cartilage tissue engineering

    Czech Academy of Sciences Publication Activity Database

    Svobodová, Jana; Proks, Vladimír; Karabiyik, Ö.; Calikoglu Koyuncu, A. C.; Köse, G. T.; Rypáček, František; Studenovská, Hana

    2017-01-01

    Roč. 11, č. 3 (2017), s. 831-842 ISSN 1932-6254 R&D Projects: GA ČR GAP108/12/1629; GA ČR GAP108/12/1538 Grant - others:AV ČR, TUBITAK(CZ) 111M031 Institutional support: RVO:61389013 Keywords : poly(amino acid) * fibrous scaffolds * adhesion peptide Subject RIV: CD - Macromolecular Chemistry OBOR OECD: Polymer science Impact factor: 3.989, year: 2016

  8. New potentially antihypertensive peptides liberated in milk during fermentation with selected lactic acid bacteria and kombucha cultures.

    Science.gov (United States)

    Elkhtab, Ebrahim; El-Alfy, Mohamed; Shenana, Mohamed; Mohamed, Abdelaty; Yousef, Ahmed E

    2017-12-01

    Compounds with the ability to inhibit angiotensin-converting enzyme (ACE) are used medically to treat human hypertension. The presence of such compounds naturally in food is potentially useful for treating the disease state. The goal of this study was to screen lactic acid bacteria, including species commonly used as dairy starter cultures, for the ability to produce new potent ACE-inhibiting peptides during milk fermentation. Strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus, Lactobacillus paracasei, Lactococcus lactis, Leuconostoc mesenteroides, and Pediococcus acidilactici were tested in this study. Additionally, a symbiotic consortium of yeast and bacteria, used commercially to produce kombucha tea, was tested. Commercially sterile milk was inoculated with lactic acid bacteria strains and kombucha culture and incubated at 37°C for up to 72 h, and the liberation of ACE-inhibiting compounds during fermentation was monitored. Fermented milk was centrifuged and the supernatant (crude extract) was subjected to ultrafiltration using 3- and 10-kDa cut-off filters. Crude and ultrafiltered extracts were tested for ACE-inhibitory activity. The 10-kDa filtrate resulting from L. casei ATCC 7469 and kombucha culture fermentations (72 h) showed the highest ACE-inhibitory activity. Two-step purification of these filtrates was done using HPLC equipped with a reverse-phase column. Analysis of HPLC-purified fractions by liquid chromatography-mass spectrometry/mass spectrometry identified several new peptides with potent ACE-inhibitory activities. Some of these peptides were synthesized, and their ACE-inhibitory activities were confirmed. Use of organisms producing these unique peptides in food fermentations could contribute positively to human health. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. Helleborus purpurascens—Amino Acid and Peptide Analysis Linked to the Chemical and Antiproliferative Properties of the Extracted Compounds

    Directory of Open Access Journals (Sweden)

    Adina-Elena Segneanu

    2015-12-01

    Full Text Available There is a strong drive worldwide to discover and exploit the therapeutic potential of a large variety of plants. In this work, an alcoholic extract of Helleborus purpurascens (family Ranunculaceae was investigated for the identification of amino acids and peptides with putative antiproliferative effects. In our work, a separation strategy was developed using solvents of different polarity in order to obtain active compounds. Biochemical components were characterized through spectroscopic (mass spectroscopy and chromatographic techniques (RP-HPLC and GC-MS. The biological activity of the obtained fractions was investigated in terms of their antiproliferative effects on HeLa cells. Through this study, we report an efficient separation of bioactive compounds (amino acids and peptides from a plant extract dependent on solvent polarity, affording fractions with unaffected antiproliferative activities. Moreover, the two biologically tested fractions exerted a major antiproliferative effect, thereby suggesting potential anticancer therapeutic activity.

  10. Helleborus purpurascens-Amino Acid and Peptide Analysis Linked to the Chemical and Antiproliferative Properties of the Extracted Compounds.

    Science.gov (United States)

    Segneanu, Adina-Elena; Grozescu, Ioan; Cziple, Florentina; Berki, Daniel; Damian, Daniel; Niculite, Cristina Mariana; Florea, Alexandru; Leabu, Mircea

    2015-12-11

    There is a strong drive worldwide to discover and exploit the therapeutic potential of a large variety of plants. In this work, an alcoholic extract of Helleborus purpurascens (family Ranunculaceae) was investigated for the identification of amino acids and peptides with putative antiproliferative effects. In our work, a separation strategy was developed using solvents of different polarity in order to obtain active compounds. Biochemical components were characterized through spectroscopic (mass spectroscopy) and chromatographic techniques (RP-HPLC and GC-MS). The biological activity of the obtained fractions was investigated in terms of their antiproliferative effects on HeLa cells. Through this study, we report an efficient separation of bioactive compounds (amino acids and peptides) from a plant extract dependent on solvent polarity, affording fractions with unaffected antiproliferative activities. Moreover, the two biologically tested fractions exerted a major antiproliferative effect, thereby suggesting potential anticancer therapeutic activity.

  11. Novel angiotensin-converting enzyme (ACE) inhibitory peptides derived from boneless chicken leg meat.

    Science.gov (United States)

    Terashima, Masaaki; Baba, Takako; Ikemoto, Narumi; Katayama, Midori; Morimoto, Tomoko; Matsumura, Saki

    2010-06-23

    Four peptides that inhibit angiotensin-converting enzyme (ACE) were separated from the hydorlysate of boneless chicken leg meat digested with artificial gastric juice (pepsin). Two peptides were identified as the peptides encrypted in myosin heavy chain. The peptide P1 (MNVKHWPWMK) corresponds to the amino acid sequence from amino acids 825 to 834 of myosin heavy chain, and the peptide P4 (VTVNPYKWLP) corresponds to the amino acid sequence from amino acids 125 to 135 of myosin heavy chain. They are novel ACE inhibitory peptides derived from chicken, and IC(50) values of P1 and P4 were determined as 228 and 5.5 microM, respectively. Although these values were much larger than 0.022 microM for captopril, a typical synthetic ACE inhibitor, they are comparable to IC(50) values reported for various ACE inhibitory peptides derived from foods. Because the peptide P4 has a relatively low IC(50) value, it is a good starting substance for designing food supplements for hypertensive patients.

  12. Peptidomic approach identifies cruzioseptins, a new family of potent antimicrobial peptides in the splendid leaf frog, Cruziohyla calcarifer.

    Science.gov (United States)

    Proaño-Bolaños, Carolina; Zhou, Mei; Wang, Lei; Coloma, Luis A; Chen, Tianbao; Shaw, Chris

    2016-09-02

    Phyllomedusine frogs are an extraordinary source of biologically active peptides. At least 8 families of antimicrobial peptides have been reported in this frog clade, the dermaseptins being the most diverse. By a peptidomic approach, integrating molecular cloning, Edman degradation sequencing and tandem mass spectrometry, a new family of antimicrobial peptides has been identified in Cruziohyla calcarifer. These 15 novel antimicrobial peptides of 20-32 residues in length are named cruzioseptins. They are characterized by having a unique shared N-terminal sequence GFLD- and the sequence motifs -VALGAVSK- or -GKAAL(N/G/S) (V/A)V- in the middle of the peptide. Cruzioseptins have a broad spectrum of antimicrobial activity and low haemolytic effect. The most potent cruzioseptin was CZS-1 that had a MIC of 3.77μM against the Gram positive bacterium, Staphylococcus aureus and the yeast Candida albicans. In contrast, CZS-1 was 3-fold less potent against the Gram negative bacterium, Escherichia coli (MIC 15.11μM). CZS-1 reached 100% haemolysis at 120.87μM. Skin secretions from unexplored species such as C. calcarifer continue to demonstrate the enormous molecular diversity hidden in the amphibian skin. Some of these novel peptides may provide lead structures for the development of a new class of antibiotics and antifungals of therapeutic use. Through the combination of molecular cloning, Edman degradation sequencing, tandem mass spectrometry and MALDI-TOF MS we have identified a new family of 15 antimicrobial peptides in the skin secretion of Cruziohyla calcarifer. The novel family is named "Cruzioseptins" and contains cationic amphipathic peptides of 20-32 residues. They have a broad range of antimicrobial activity that also includes effective antifungals with low haemolytic activity. Therefore, C. calcarifer has proven to be a rich source of novel peptides, which could become leading structures for the development of novel antibiotics and antifungals of clinical

  13. Synthesis of peptide .alpha.-thioesters

    Science.gov (United States)

    Camarero, Julio A [Livermore, CA; Mitchell, Alexander R [Livermore, CA; De Yoreo, James J [Clayton, CA

    2008-08-19

    Disclosed herein is a new method for the solid phase peptide synthesis (SPPS) of C-terminal peptide .alpha. thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. The oxidation step converts the acyl-hydrazine group into a highly reactive acyl-diazene intermediate which reacts with an .alpha.-amino acid alkylthioester (H-AA-SR) to yield the corresponding peptide .alpha.-thioester in good yield. A variety of peptide thioesters, cyclic peptides and a fully functional Src homology 3 (SH3) protein domain have been successfully prepared.

  14. Enzymatic characterization of peptidic materials isolated from aqueous solutions of ammonium cyanide (pH 9) and hydrocyanic acid (pH 6) exposed to ionizing radiation.

    Science.gov (United States)

    Niketic, V; Draganić, Z; Nesković, S; Draganić, I

    1982-01-01

    The enzymatic digestion of some radiolytically produced peptidic materials was examined. The substrates were compounds isolated from 0.1 molar solutions of NH4CN (pH 9) and HCN (pH 6), after their exposure to gamma rays from a 60Co source (15-20 Mrad doses). Commercial proteolytic enzymes pronase and aminopeptidase M were used. The examined materials were of composite nature and proteolytic action was systematically observed after their subsequent purification. In some fractions the effect was found to be positive with up to 30% of peptide bonds cleaved with respect to the amino acid content. These findings support our previous conclusions on the free radical induced formation of peptidic backbones without the intervention of amino acids. Some side effects were also noted which might be of interest in observations on enzymatic cleavage of other composite peptidic materials of abiotic origin.

  15. Peptide nucleic acid (PNA) antisense effects in Escherichia coli

    DEFF Research Database (Denmark)

    Good, L; Nielsen, P E

    1999-01-01

    Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene...... and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible...... to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake...

  16. Expression of an engineered heterologous antimicrobial peptide in potato alters plant development and mitigates normal abiotic and biotic responses.

    Directory of Open Access Journals (Sweden)

    Ravinder K Goyal

    Full Text Available Antimicrobial cationic peptides (AMPs are ubiquitous small proteins used by living cells to defend against a wide spectrum of pathogens. Their amphipathic property helps their interaction with negatively charged cellular membrane of the pathogen causing cell lysis and death. AMPs also modulate signaling pathway(s and cellular processes in animal models; however, little is known of cellular processes other than the pathogen-lysis phenomenon modulated by AMPs in plants. An engineered heterologous AMP, msrA3, expressed in potato was previously shown to cause resistance of the transgenic plants against selected fungal and bacterial pathogens. These lines together with the wild type were studied for growth habits, and for inducible defense responses during challenge with biotic (necrotroph Fusarium solani and abiotic stressors (dark-induced senescence, wounding and temperature stress. msrA3-expression not only conferred protection against F. solani but also delayed development of floral buds and prolonged vegetative phase. Analysis of select gene transcript profiles showed that the transgenic potato plants were suppressed in the hypersensitive (HR and reactive oxygen species (ROS responses to both biotic and abiotic stressors. Also, the transgenic leaves accumulated lesser amounts of the defense hormone jasmonic acid upon wounding with only a slight change in salicylic acid as compared to the wild type. Thus, normal host defense responses to the pathogen and abiotic stressors were mitigated by msrA3 expression suggesting MSRA3 regulates a common step(s of these response pathways. The stemming of the pathogen growth and mitigating stress response pathways likely contributes to resource reallocation for higher tuber yield.

  17. Thalassospiramide G, a New γ-Amino-Acid-Bearing Peptide from the Marine Bacterium Thalassospira sp.

    Directory of Open Access Journals (Sweden)

    Sang Kook Lee

    2013-02-01

    Full Text Available In the chemical investigation of marine unicellular bacteria, a new peptide, thalassospiramide G (1, along with thalassospiramides A and D (2–3, was discovered from a large culture of Thalassospira sp. The structure of thalassospiramide G, bearing γ-amino acids, such as 4-amino-5-hydroxy-penta-2-enoic acid (AHPEA, 4-amino-3,5-dihydroxy-pentanoic acid (ADPA, and unique 2-amino-1-(1H-indol-3-yl ethanone (AIEN, was determined via extensive spectroscopic analysis. The absolute configuration of thalassospiramide D (3, including 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA, was rigorously determined by 1H–1H coupling constant analysis and chemical derivatization. Thalassospiramides A and D (2–3 inhibited nitric oxide (NO production in lipopolysaccharide (LPS-stimulated mouse macrophage RAW 264.7 cells, with IC50 values of 16.4 and 4.8 μM, respectively.

  18. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte

    2013-01-01

    properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

  19. Peptoid-Peptide hybrid backbone architectures

    DEFF Research Database (Denmark)

    Olsen, Christian Adam

    2010-01-01

    Peptidomimetic oligomers and foldamers have received considerable attention for over a decade, with beta-peptides and the so-called peptoids (N-alkylglycine oligomers) representing prominent examples of such architectures. Lately, hybrid or mixed backbones consisting of both alpha- and beta......-amino acids (alpha/beta-peptides) have been investigated in some detail as well. The present Minireview is a survey of the literature concerning hybrid structures of alpha-amino acids and peptoids, including beta-peptoids (N-alkyl-beta-alanine oligomers), and is intended to give an overview of this area...

  20. Albumin-derived peptides efficiently reduce renal uptake of radiolabelled peptides

    International Nuclear Information System (INIS)

    Vegt, Erik; Eek, Annemarie; Oyen, Wim J.G.; Gotthardt, Martin; Boerman, Otto C.; Jong, Marion de

    2010-01-01

    In peptide-receptor radionuclide therapy (PRRT), the maximum activity dose that can safely be administered is limited by high renal uptake and retention of radiolabelled peptides. The kidney radiation dose can be reduced by coinfusion of agents that competitively inhibit the reabsorption of radiolabelled peptides, such as positively charged amino acids, Gelofusine, or trypsinised albumin. The aim of this study was to identify more specific and potent inhibitors of the kidney reabsorption of radiolabelled peptides, based on albumin. Albumin was fragmented using cyanogen bromide and six albumin-derived peptides with different numbers of electric charges were selected and synthesised. The effect of albumin fragments (FRALB-C) and selected albumin-derived peptides on the internalisation of 111 In-albumin, 111 In-minigastrin, 111 In-exendin and 111 In-octreotide by megalin-expressing cells was assessed. In rats, the effect of Gelofusine and albumin-derived peptides on the renal uptake and biodistribution of 111 In-minigastrin, 111 In-exendin and 111 In-octreotide was determined. FRALB-C significantly reduced the uptake of all radiolabelled peptides in vitro. The albumin-derived peptides showed different potencies in reducing the uptake of 111 In-albumin, 111 In-exendin and 111 In-minigastrin in vitro. The most efficient albumin-derived peptide (peptide 6), was selected for in vivo testing. In rats, 5 mg of peptide 6 very efficiently inhibited the renal uptake of 111 In-minigastrin, by 88%. Uptake of 111 In-exendin and 111 In-octreotide was reduced by 26 and 33%, respectively. The albumin-derived peptide 6 efficiently inhibited the renal reabsorption of 111 In-minigastrin, 111 In-exendin and 111 In-octreotide and is a promising candidate for kidney protection in PRRT. (orig.)

  1. Method for predicting peptide detection in mass spectrometry

    Science.gov (United States)

    Kangas, Lars [West Richland, WA; Smith, Richard D [Richland, WA; Petritis, Konstantinos [Richland, WA

    2010-07-13

    A method of predicting whether a peptide present in a biological sample will be detected by analysis with a mass spectrometer. The method uses at least one mass spectrometer to perform repeated analysis of a sample containing peptides from proteins with known amino acids. The method then generates a data set of peptides identified as contained within the sample by the repeated analysis. The method then calculates the probability that a specific peptide in the data set was detected in the repeated analysis. The method then creates a plurality of vectors, where each vector has a plurality of dimensions, and each dimension represents a property of one or more of the amino acids present in each peptide and adjacent peptides in the data set. Using these vectors, the method then generates an algorithm from the plurality of vectors and the calculated probabilities that specific peptides in the data set were detected in the repeated analysis. The algorithm is thus capable of calculating the probability that a hypothetical peptide represented as a vector will be detected by a mass spectrometry based proteomic platform, given that the peptide is present in a sample introduced into a mass spectrometer.

  2. Antimicrobial Peptides in 2014

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2015-03-01

    Full Text Available This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

  3. Ultrasmall Peptides Self-Assemble into Diverse Nanostructures: Morphological Evaluation and Potential Implications

    Directory of Open Access Journals (Sweden)

    Charlotte A.E. Hauser

    2011-09-01

    Full Text Available In this study, we perform a morphological evaluation of the diverse nanostructures formed by varying concentration and amino acid sequence of a unique class of ultrasmall self-assembling peptides. We modified these peptides by replacing the aliphatic amino acid at the C-aliphatic terminus with different aromatic amino acids. We tracked the effect of introducing aromatic residues on self-assembly and morphology of resulting nanostructures. Whereas aliphatic peptides formed long, helical fibers that entangle into meshes and entrap >99.9% water, the modified peptides contrastingly formed short, straight fibers with a flat morphology. No helical fibers were observed for the modified peptides. For the aliphatic peptides at low concentrations, different supramolecular assemblies such as hollow nanospheres and membrane blebs were found. Since the ultrasmall peptides are made of simple, aliphatic amino acids, considered to have existed in the primordial soup, study of these supramolecular assemblies could be relevant to understanding chemical evolution leading to the origin of life on Earth. In particular, we propose a variety of potential applications in bioengineering and nanotechnology for the diverse self-assembled nanostructures.

  4. Two functional motifs define the interaction, internalization and toxicity of the cell-penetrating antifungal peptide PAF26 on fungal cells.

    Directory of Open Access Journals (Sweden)

    Alberto Muñoz

    Full Text Available The synthetic, cell penetrating hexapeptide PAF26 (RKKWFW is antifungal at low micromolar concentrations and has been proposed as a model for cationic, cell-penetrating antifungal peptides. Its short amino acid sequence facilitates the analysis of its structure-activity relationships using the fungal models Neurospora crassa and Saccharomyces cerevisiae, and human and plant pathogens Aspergillus fumigatus and Penicillium digitatum, respectively. Previously, PAF26 at low fungicidal concentrations was shown to be endocytically internalized, accumulated in vacuoles and then actively transported into the cytoplasm where it exerts its antifungal activity. In the present study, two PAF26 derivatives, PAF95 (AAAWFW and PAF96 (RKKAAA, were designed to characterize the roles of the N-terminal cationic and the C-terminal hydrophobic motifs in PAF26's mode-of-action. PAF95 and PAF96 exhibited substantially reduced antifungal activity against all the fungi analyzed. PAF96 localized to fungal cell envelopes and was not internalized by the fungi. In contrast, PAF95 was taken up into vacuoles of N. crassa, wherein it accumulated and was trapped without toxic effects. Also, the PAF26 resistant Δarg1 strain of S. cerevisiae exhibited increased PAF26 accumulation in vacuoles. Live-cell imaging of GFP-labelled nuclei in A. fumigatus showed that transport of PAF26 from the vacuole to the cytoplasm was followed by nuclear breakdown and dissolution. This work demonstrates that the amphipathic PAF26 possesses two distinct motifs that allow three stages in its antifungal action to be defined: (i its interaction with the cell envelope; (ii its internalization and transport to vacuoles mediated by the aromatic hydrophobic domain; and (iii its transport from vacuoles to the cytoplasm. Significantly, cationic residues in PAF26 are important not only for the electrostatic attraction and interaction with the fungal cell but also for transport from the vacuole to the

  5. New peptides players in metabolic disorders

    Directory of Open Access Journals (Sweden)

    Agata Mierzwicka

    2016-08-01

    Full Text Available Among new peptides responsible for the pathogenesis of metabolic disorders and carbohydrate metabolism, adipokines are of great importance. Adipokines are substances of hormonal character, secreted by adipose tissue. Apart from the well-known adipokines, adropin and preptin are relatively newly discovered, hence their function is not fully understood. They are peptides not secreted by adipose tissue but their role in the metabolic regulations seems to be significant. Preptin is a 34-amino acid peptide, a derivative of proinsulin growth factor II (pro-IGF-II, secreted by pancreatic β cells, considered to be a physiological enhancer of insulin secretion. Additionally, preptin has a stimulating effect on osteoblasts, inducing their proliferation, differentiation and survival. Adropin is a 76-amino acid peptide, encoded by the energy homeostasis associated gene (Enho, mainly in liver and brain, and its expression is dependent on a diet. Adropin is believed to play an important role in metabolic homeostasis, fatty acids metabolism control, insulin resistance prevention, dyslipidemia, and impaired glucose tolerance. The results of studies conducted so far show that the diseases resulting from metabolic syndrome, such as obesity, type 2 diabetes mellitus, polycystic ovary syndrome, non-alcoholic fatty liver disease, or cardiovascular disease are accompanied by significant changes in the concentration of these peptides. It is also important to note that preptin has an anabolic effect on bone tissue, which might be preventive in osteoporosis.

  6. Peptides, proteins and peptide/protein-polymer conjugates as drug delivery system.

    Science.gov (United States)

    Mukherjee, Biswajit; Karmakar, Swapna D; Hossain, Chowdhury M; Bhattacharya, Sanchari

    2014-01-01

    In the last few decades, novel drug delivery strategies have been a big priority to the formulation scientists. Peptides and proteins have drawn a special attention for their wide scope in the area. Serum albumin, transferrin, recom- binant proteins, virus capsids etc. are used as carrier for drug and biomolecules. Conjugates of polymers with proteins have also shown strong potency in the field of drug delivery. Polyethylene glycol is one of the most successful polymers that has been used extensively to develop protein conjugated formulations. Besides, polyvinyl pyrrolidone, polylactic-co- glycolic acid, N-(2-hydroxypropyl) methacrylamide copolymer, polyglutamic acid have also been investigated. In this re- view, we will highlight on the most recent overview of various advantages, limitations and marketed products of proteins, peptides and protein/peptide-polymer conjugates as drug carriers, such products in clinical trials and their various uses in the field of modern drug delivery. Understanding the key features of these materials and the vigorous research in this field will develop new drug formulations that will combat various types of life-threatening diseases.

  7. Dermaseptins and Magainins: Antimicrobial Peptides from Frogs' Skin—New Sources for a Promising Spermicides Microbicides—A Mini Review

    Directory of Open Access Journals (Sweden)

    Amira Zairi

    2009-01-01

    Full Text Available Sexually transmitted infections (STIs and human immunodeficiency virus (HIV, the causative agents of acquired immunodeficiency syndrome (AIDS, are two great concerns in the reproductive health of women. Thus, the challenge is to find products with a double activity, on the one hand having antimicrobial/antiviral properties with a role in the reduction of STI, and on the other hand having spermicidal action to be used as a contraceptive. In the absence of an effective microbicide along with the disadvantages of the most commonly used spermicidal contraceptive worldwide, nonoxynol-9, new emphasis has been focused on the development of more potential intravaginal microbicidal agents. Topical microbicides spermicides would ideally provide a female-controlled method of self-protection against HIV as well as preventing pregnancies. Nonoxynol-9, the only recommended microbicide spermicide, damages cervicovaginal epithelium because of its membrane-disruptive properties. Clearly, there is an urgent need to identify new compounds with dual potential microbicidal properties; antimicrobial peptides should be candidates for such investigations. Dermaseptins and magainins are two classes of cationic, amphipathic α-helical peptides that have been identified in the skin extracts of frogs Phyllomedusa sauvagei and Xenopus laevis. Regarding their contraceptive activities and their effect against various STI-causing pathogens, we believe that these two peptides are appropriate candidates in the evaluation of newer and safer microbicides spermicides in the future.

  8. Ammonia production by human faecal bacteria, and the enumeration, isolation and characterization of bacteria capable of growth on peptides and amino acids

    Directory of Open Access Journals (Sweden)

    Richardson Anthony J

    2013-01-01

    Full Text Available Abstract Background The products of protein breakdown in the human colon are considered to be detrimental to gut health. Amino acid catabolism leads to the formation of sulfides, phenolic compounds and amines, which are inflammatory and/or precursors to the formation of carcinogens, including N-nitroso compounds. The aim of this study was to investigate the kinetics of protein breakdown and the bacterial species involved. Results Casein, pancreatic casein hydrolysate (mainly short-chain peptides or amino acids were incubated in vitro with suspensions of faecal bacteria from 3 omnivorous and 3 vegetarian human donors. Results from the two donor groups were similar. Ammonia production was highest from peptides, followed by casein and amino acids, which were similar. The amino acids metabolized most extensively were Asp, Ser, Lys and Glu. Monensin inhibited the rate of ammonia production from amino acids by 60% (P = 0.001, indicating the involvement of Gram-positive bacteria. Enrichment cultures were carried out to investigate if, by analogy with the rumen, there was a significant population of asaccharolytic, obligately amino acid-fermenting bacteria (‘hyper-ammonia-producing’ bacteria; HAP in the colon. Numbers of bacteria capable of growth on peptides or amino acids alone averaged 3.5% of the total viable count, somewhat higher than the rumen. None of these were HAP, however. The species enriched included Clostridium spp., one of which was C. perfringens, Enterococcus, Shigella and Escherichia coli. Conclusions Protein fermentation by human faecal bacteria in the absence of sugars not only leads to the formation of hazardous metabolic products, but also to the possible proliferation of harmful bacteria. The kinetics of protein metabolism were similar to the rumen, but HAP bacteria were not found.

  9. How Does Amino Acid Ligand Modulate Au Core Structure and Characteristics in Peptide Coated Au Nanocluster?

    Science.gov (United States)

    Li, Nan; Li, Xu; Zhao, Hongkang; Zhao, Lina

    2018-03-01

    The atomic structures and the corresponding physicochemical properties of peptide coated Au nanoclusters determine their distinctive biological targeting applications. To learn the modulation of amino acid ligand on the atomic structure and electronic characteristics of coated Au core is the fundamental knowledge for peptide coated Au nanocluster design and construction. Based on our recent coated Au nanocluster configuration study (Nanoscale, 2016, 8, 11454), we built the typically simplified Au13(Cys-Au-Cys) system to more clearly learn the basic modulation information of amino acid ligand on Au core by the density functional theory (DFT) calculations. There are two isomers as ligand adjacent bonding (Iso1) and diagonal bonding (Iso2) to Au13 cores. The geometry optimizations indicate the adjacent bonding Iso1 is more stable than Iso2. More important, the Au13 core of Iso1 distorts much more significantly than that of Iso2 by Cys-Au-Cys bonding through the root-mean-square deviation (RMSD) analysis, which modulate their electronic characteristics in different ways. In addition, the frontier molecular orbital results of Au13(Cys-Au-Cys) isomers confirm that the Au cores mainly determine the blue shifts of Au13(Cys-Au-Cys) systems versus the original Au13 core in their UV-visible absorption spectrum studies. The configuration of Au13 core performs deformation under Cys-Au-Cys ligand modulation to reach new stability with distinct atomic structure and electronic properties, which could be the theory basis for peptide coated AuNCs design and construction.

  10. Radiation chemistry of amino acids, peptides and proteins in relation to the radiation sterilization of high-protein foods. [106 references

    Energy Technology Data Exchange (ETDEWEB)

    Garrison, W.M.

    1979-03-01

    An important source of information on the question of whether or not toxic or other deleterious substances are formed in the radiation sterilization of foods is the chemical study of reaction products and reaction mechanisms in the radiolysis of individual food components. The present evaluation of the radiation chemistry of amino acids, peptides and proteins outlines the various radiation-induced processes which lead to amino acid degradation and to the synthesis of amino acid derivatives of higher molecular weight. Among the latter are the ..cap alpha..,..cap alpha..'-diamino dicarboxylic acids which are formed as major products in the radiolysis of peptides both in aqueous solution and in the solid state. The ..cap alpha..,..cap alpha..'-diamino acids are of particular interest as irradiation products because they represent a class of compounds not normally encountered in plant and animal protein sources. Such compounds have, however, been isolated from certain types of bacteria and pathogenic toxins. All of the available data strongly suggest that the ..cap alpha..,..cap alpha..'-diamino acids are produced in significant yield in the radiation sterilization of high protein foods. The importance of initiating extensive chemical and biological studies of initiating extensive chemical and biological studies of these and of other high molecular weight products in irradiated food is emphasized.

  11. Peptide (Lys-Leu) and amino acids (Lys and Leu) supplementations improve physiological activity and fermentation performance of brewer's yeast during very high-gravity (VHG) wort fermentation.

    Science.gov (United States)

    Yang, Huirong; Zong, Xuyan; Cui, Chun; Mu, Lixia; Zhao, Haifeng

    2017-12-22

    Lys and Leu were generally considered as the key amino acids for brewer's yeast during beer brewing. In the present study, peptide Lys-Leu and a free amino acid (FAA) mixture of Lys and Leu (Lys + Leu) were supplemented in 24 °P wort to examine their effects on physiological activity and fermentation performance of brewer's yeast during very high-gravity (VHG) wort fermentation. Results showed that although both peptide Lys-Leu and their FAA mixture supplementations could increase the growth and viability, intracellular trehalose and glycerol content, wort fermentability, and ethanol content for brewer's yeast during VHG wort fermentation, and peptide was better than their FAA mixture at promoting growth and fermentation for brewer's yeast when the same dose was kept. Moreover, peptide Lys-Leu supplementation significantly increased the assimilation of Asp, but decreased the assimilation of Gly, Ala, Val, (Cys)2, Ile, Leu, Tyr, Phe, Lys, Arg, and Pro. However, the FAA mixture supplementation only promoted the assimilation of Lys and Leu, while reduced the absorption of total amino acids to a greater extent. Thus, the peptide Lys-Leu was more effective than their FAA mixture on the improvement of physiological activity, fermentation performance, and nitrogen metabolism of brewer's yeast during VHG wort fermentation. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  12. D-Amino acids incorporation in the frog skin-derived peptide esculentin-1a(1-21)NH2 is beneficial for its multiple functions.

    Science.gov (United States)

    Di Grazia, Antonio; Cappiello, Floriana; Cohen, Hadar; Casciaro, Bruno; Luca, Vincenzo; Pini, Alessandro; Di, Y Peter; Shai, Yechiel; Mangoni, Maria Luisa

    2015-12-01

    Naturally occurring antimicrobial peptides (AMPs) represent promising future antibiotics. We have previously isolated esculentin-1a(1-21)NH2, a short peptide derived from the frog skin AMP esculentin-1a, with a potent anti-Pseudomonal activity. Here, we investigated additional functions of the peptide and properties responsible for these activities. For that purpose, we synthesized the peptide, as well as its structurally altered analog containing two D-amino acids. The peptides were then biophysically and biologically investigated for their cytotoxicity and immunomodulating activities. The data revealed that compared to the wild-type, the diastereomer: (1) is significantly less toxic towards mammalian cells, in agreement with its lower α-helical structure, as determined by circular dichroism spectroscopy; (2) is more effective against the biofilm form of Pseudomonas aeruginosa (responsible for lung infections in cystic fibrosis sufferers), while maintaining a high activity against the free-living form of this important pathogen; (3) is more stable in serum; (4) has a higher activity in promoting migration of lung epithelial cells, and presumably in healing damaged lung tissue, and (5) disaggregates and detoxifies the bacterial lipopolysaccharide (LPS), albeit less than the wild-type. Light scattering studies revealed a correlation between anti-LPS activity and the ability to disaggregate the LPS. Besides shedding light on the multifunction properties of esculentin-1a(1-21)NH2, the D-amino acid containing isomer may serve as an attractive template for the development of new anti-Pseudomonal compounds with additional beneficial properties. Furthermore, together with other studies, incorporation of D-amino acids may serve as a general approach to optimize the future design of new AMPs.

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

    NARCIS (Netherlands)

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

    2007-01-01

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

  14. Dinosaur peptides suggest mechanisms of protein survival.

    Science.gov (United States)

    San Antonio, James D; Schweitzer, Mary H; Jensen, Shane T; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P R O

    2011-01-01

    Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.

  15. Dinosaur Peptides Suggest Mechanisms of Protein Survival

    Energy Technology Data Exchange (ETDEWEB)

    San Antonio, James D.; Schweitzer, Mary H.; Jensen, Shane T.; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P.R.O. (Harvard-Med); (IIT); (NCSU); (UPENN); (Manchester); (Orthovita)

    2011-09-16

    Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.

  16. Radiopharmaceutical development of radiolabelled peptides

    Energy Technology Data Exchange (ETDEWEB)

    Fani, Melpomeni; Maecke, Helmut R. [University Hospital Freiburg, Department of Nuclear Medicine, Freiburg (Germany)

    2012-02-15

    Receptor targeting with radiolabelled peptides has become very important in nuclear medicine and oncology in the past few years. The overexpression of many peptide receptors in numerous cancers, compared to their relatively low density in physiological organs, represents the molecular basis for in vivo imaging and targeted radionuclide therapy with radiolabelled peptide-based probes. The prototypes are analogs of somatostatin which are routinely used in the clinic. More recent developments include somatostatin analogs with a broader receptor subtype profile or with antagonistic properties. Many other peptide families such as bombesin, cholecystokinin/gastrin, glucagon-like peptide-1 (GLP-1)/exendin, arginine-glycine-aspartic acid (RGD) etc. have been explored during the last few years and quite a number of potential radiolabelled probes have been derived from them. On the other hand, a variety of strategies and optimized protocols for efficient labelling of peptides with clinically relevant radionuclides such as {sup 99m}Tc, M{sup 3+} radiometals ({sup 111}In, {sup 86/90}Y, {sup 177}Lu, {sup 67/68}Ga), {sup 64/67}Cu, {sup 18}F or radioisotopes of iodine have been developed. The labelling approaches include direct labelling, the use of bifunctional chelators or prosthetic groups. The choice of the labelling approach is driven by the nature and the chemical properties of the radionuclide. Additionally, chemical strategies, including modification of the amino acid sequence and introduction of linkers/spacers with different characteristics, have been explored for the improvement of the overall performance of the radiopeptides, e.g. metabolic stability and pharmacokinetics. Herein, we discuss the development of peptides as radiopharmaceuticals starting from the choice of the labelling method and the conditions to the design and optimization of the peptide probe, as well as some recent developments, focusing on a selected list of peptide families, including somatostatin

  17. Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters.

    Science.gov (United States)

    Ollivier, Nathalie; Raibaut, Laurent; Blanpain, Annick; Desmet, Rémi; Dheur, Julien; Mhidia, Reda; Boll, Emmanuelle; Drobecq, Hervé; Pira, Silvain L; Melnyk, Oleg

    2014-02-01

    Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

  18. PEPTIDE SOLUBILITY, STRUCTURE AND CHARGE POSITION EFFECT ON ADSORPTION BY ALUMINIUM HYDROXIDE

    Directory of Open Access Journals (Sweden)

    Mary Trujillo

    2008-04-01

    Full Text Available Solubility, structure and position of charges in a peptide antigen sequence can be mentioned as being amongst the basic features of adsorption. In order to study their effect on adsorption, seven analogue series were synthesized from a MSP-1 peptide sequence by systematically replacing each one of the positions in the peptide sequence by aspartic acid, glutamic acid, serine, alanine, asparagine, glutamine or lysine. Such modifications in analogue peptide sequences showed a non-regular tendency regarding solubility and adsorption data. Aspartic acid and Glutamic acid analogue series showed great improvements in adsorption, especially in peptides where Lysine in position 6 and Arginine in position 13 were replaced. Solubility of position 5 analogue was greater than the position 6 analogue in Aspartic acid series; however, the position 6 analogue showed best adsorption results whilst the Aspartic acid in position 5 analogue showed no adsorption in the same conditions. Nuclear Magnetic Resonance structural analysis revealed differences in the -helical structureextension between these analogues. The Aspartic acid in position 6, located in the polar side of the helix, may allow this analogueto fit better onto the adsorption regions suggesting that the local electrostatic charge is responsible for this behavior.

  19. Characterizing Peptide Neutral Losses Induced by Negative Electron-Transfer Dissociation (NETD)

    Science.gov (United States)

    Rumachik, Neil G.; McAlister, Graeme C.; Russell, Jason D.; Bailey, Derek J.; Wenger, Craig D.; Coon, Joshua J.

    2012-01-01

    We implemented negative electron-transfer dissociation (NETD) on a hybrid ion trap/Orbitrap mass spectrometer to conduct ion/ion reactions using peptide anions and radical reagent cations. In addition to sequence-informative ladders of a•- and x-type fragment ions, NETD generated intense neutral loss peaks corresponding to the entire or partial side-chain cleavage from amino acids constituting a given peptide. Thus, a critical step towards the characterization of this recently introduced fragmentation technique is a systematic study of synthetic peptides to identify common neutral losses and preferential fragmentation pathways. Examining 46 synthetic peptides with high mass accuracy and high resolution analysis permitted facile determination of the chemical composition of each neutral loss. We identified 19 unique neutral losses from 14 amino acids and three modified amino acids, and assessed the specificity and sensitivity of each neutral loss using a database of 1542 confidently identified peptides generated from NETD shotgun experiments employing high-pH separations and negative electrospray ionization. As residue-specific neutral losses indicate the presence of certain amino acids, we determined that many neutral losses have potential diagnostic utility. We envision this catalogue of neutral losses being incorporated into database search algorithms to improve peptide identification specificity and to further advance characterization of the acidic proteome. PMID:22290482

  20. Effect of surface modification of nanofibres with glutamic acid peptide on calcium phosphate nucleation and osteogenic differentiation of marrow stromal cells.

    Science.gov (United States)

    Karaman, Ozan; Kumar, Ankur; Moeinzadeh, Seyedsina; He, Xuezhong; Cui, Tong; Jabbari, Esmaiel

    2016-02-01

    Biomineralization is mediated by extracellular matrix (ECM) proteins with amino acid sequences rich in glutamic acid. The objective of this study was to investigate the effect of calcium phosphate deposition on aligned nanofibres surface-modified with a glutamic acid peptide on osteogenic differentiation of rat marrow stromal cells. Blend of EEGGC peptide (GLU) conjugated low molecular weight polylactide (PLA) and high molecular weight poly(lactide-co-glycolide) (PLGA) was electrospun to form aligned nanofibres (GLU-NF). The GLU-NF microsheets were incubated in a modified simulated body fluid for nucleation of calcium phosphate crystals on the fibre surface. To achieve a high calcium phosphate to fibre ratio, a layer-by-layer approach was used to improve diffusion of calcium and phosphate ions inside the microsheets. Based on dissipative particle dynamics simulation of PLGA/PLA-GLU fibres, > 80% of GLU peptide was localized to the fibre surface. Calcium phosphate to fibre ratios as high as 200%, between those of cancellous (160%) and cortical (310%) bone, was obtained with the layer-by-layer approach. The extent of osteogenic differentiation and mineralization of marrow stromal cells seeded on GLU-NF microsheets was directly related to the amount of calcium phosphate deposition on the fibres prior to cell seeding. Expression of osteogenic markers osteopontin, alkaline phosphatase (ALP), osteocalcin and type 1 collagen increased gradually with calcium phosphate deposition on GLU-NF microsheets. Results demonstrate that surface modification of aligned synthetic nanofibres with EEGGC peptide dramatically affects nucleation and growth of calcium phosphate crystals on the fibres leading to increased osteogenic differentiation of marrow stromal cells and mineralization. Copyright © 2013 John Wiley & Sons, Ltd.

  1. Maize Bioactive Peptides against Cancer

    Science.gov (United States)

    Díaz-Gómez, Jorge L.; Castorena-Torres, Fabiola; Preciado-Ortiz, Ricardo E.; García-Lara, Silverio

    2017-06-01

    Cancer is one of the main chronic degenerative diseases worldwide. In recent years, consumption of whole-grain cereals and their derived food products has been associated with reduction risks of various types of cancer. Cereals main biomolecules includes proteins, peptides, and amino acids present in different quantities within the grain. The nutraceutical properties associated with peptides exerts biological functions that promote health and prevent this disease. In this review, we report the current status and advances on maize peptides regarding bioactive properties that have been reported such as antioxidant, antihypertensive, hepatoprotective, and anti-tumour activities. We also highlighted its biological potential through which maize bioactive peptides exert anti-cancer activity. Finally, we analyse and emphasize the possible areas of application for maize peptides.

  2. Peptide nucleic acid probe for protein affinity purification based on biotin-streptavidin interaction and peptide nucleic acid strand hybridization.

    Science.gov (United States)

    Tse, Jenny; Wang, Yuanyuan; Zengeya, Thomas; Rozners, Eriks; Tan-Wilson, Anna

    2015-02-01

    We describe a new method for protein affinity purification that capitalizes on the high affinity of streptavidin for biotin but does not require dissociation of the biotin-streptavidin complex for protein retrieval. Conventional reagents place both the selectively reacting group (the "warhead") and the biotin on the same molecule. We place the warhead and the biotin on separate molecules, each linked to a short strand of peptide nucleic acid (PNA), synthetic polymers that use the same bases as DNA but attached to a backbone that is resistant to attack by proteases and nucleases. As in DNA, PNA strands with complementary base sequences hybridize. In conditions that favor PNA duplex formation, the warhead strand (carrying the tagged protein) and the biotin strand form a complex that is held onto immobilized streptavidin. As in DNA, the PNA duplex dissociates at moderately elevated temperature; therefore, retrieval of the tagged protein is accomplished by a brief exposure to heat. Using iodoacetate as the warhead, 8-base PNA strands, biotin, and streptavidin-coated magnetic beads, we demonstrate retrieval of the cysteine protease papain. We were also able to use our iodoacetyl-PNA:PNA-biotin probe for retrieval and identification of a thiol reductase and a glutathione transferase from soybean seedling cotyledons. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Structure, Content, and Bioactivity of Food-Derived Peptides in the Body.

    Science.gov (United States)

    Sato, Kenji

    2018-03-28

    Orally administered peptides are assumed to be degraded into amino acids in the body. However, our recent studies revealed some food-derived prolyl and pyroglutamyl peptides with 2-3 amino acid residues in the blood of humans and animals, while most of the peptides in the endoproteinase digest of food protein are degraded by exopeptidase. Some food-derived dipeptides in the body display in vitro and in vivo biological activities. These facts indicate that the biological activities of food-derived peptides in the body rather than those in food are crucial to understanding the mechanism of the beneficial effects of orally administered peptides.

  4. Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

    KAUST Repository

    Rydberg, Hanna A

    2014-04-18

    Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

  5. Peptide-membrane interactions of arginine-tryptophan peptides probed using quartz crystal microbalance with dissipation monitoring.

    KAUST Repository

    Rydberg, Hanna A; Kunze, Angelika; Carlsson, Nils; Altgä rde, Noomi; Svedhem, Sofia; Nordé n, Bengt

    2014-01-01

    Membrane-active peptides include peptides that can cross cellular membranes and deliver macromolecular cargo as well as peptides that inhibit bacterial growth. Some of these peptides can act as both transporters and antibacterial agents. It is desirable to combine the knowledge from these two different fields of membrane-active peptides into design of new peptides with tailored actions, as transporters of cargo or as antibacterial substances, targeting specific membranes. We have previously shown that the position of the amino acid tryptophan in the peptide sequence of three arginine-tryptophan peptides affects their uptake and intracellular localization in live mammalian cells, as well as their ability to inhibit bacterial growth. Here, we use quartz crystal microbalance with dissipation monitoring to assess the induced changes caused by binding of the three peptides to supported model membranes composed of POPC, POPC/POPG, POPC/POPG/cholesterol or POPC/lactosyl PE. Our results indicate that the tryptophan position in the peptide sequence affects the way these peptides interact with the different model membranes and that the presence of cholesterol in particular seems to affect the membrane interaction of the peptide with an even distribution of tryptophans in the peptide sequence. These results give mechanistic insight into the function of these peptides and may aid in the design of membrane-active peptides with specified cellular targets and actions.

  6. pDeep: Predicting MS/MS Spectra of Peptides with Deep Learning.

    Science.gov (United States)

    Zhou, Xie-Xuan; Zeng, Wen-Feng; Chi, Hao; Luo, Chunjie; Liu, Chao; Zhan, Jianfeng; He, Si-Min; Zhang, Zhifei

    2017-12-05

    In tandem mass spectrometry (MS/MS)-based proteomics, search engines rely on comparison between an experimental MS/MS spectrum and the theoretical spectra of the candidate peptides. Hence, accurate prediction of the theoretical spectra of peptides appears to be particularly important. Here, we present pDeep, a deep neural network-based model for the spectrum prediction of peptides. Using the bidirectional long short-term memory (BiLSTM), pDeep can predict higher-energy collisional dissociation, electron-transfer dissociation, and electron-transfer and higher-energy collision dissociation MS/MS spectra of peptides with >0.9 median Pearson correlation coefficients. Further, we showed that intermediate layer of the neural network could reveal physicochemical properties of amino acids, for example the similarities of fragmentation behaviors between amino acids. We also showed the potential of pDeep to distinguish extremely similar peptides (peptides that contain isobaric amino acids, for example, GG = N, AG = Q, or even I = L), which were very difficult to distinguish using traditional search engines.

  7. Amphipathic DNA polymers exhibit antiviral activity against systemic Murine Cytomegalovirus infection

    Directory of Open Access Journals (Sweden)

    Juteau Jean-Marc

    2009-12-01

    Full Text Available Abstract Background Phosphorothioated oligonucleotides (PS-ONs have a sequence-independent, broad spectrum antiviral activity as amphipathic polymers (APs and exhibit potent in vitro antiviral activity against a broad spectrum of herpesviruses: HSV-1, HSV-2, HCMV, VZV, EBV, and HHV-6A/B, and in vivo activity in a murine microbiocide model of genital HSV-2 infection. The activity of these agents against animal cytomegalovirus (CMV infections in vitro and in vivo was therefore investigated. Results In vitro, a 40 mer degenerate AP (REP 9 inhibited both murine CMV (MCMV and guinea pig CMV (GPCMV with an IC50 of 0.045 μM and 0.16 μM, respectively, and a 40 mer poly C AP (REP 9C inhibited MCMV with an IC50 of 0.05 μM. Addition of REP 9 to plaque assays during the first two hours of infection inhibited 78% of plaque formation whereas addition of REP 9 after 10 hours of infection did not significantly reduce the number of plaques, indicating that REP 9 antiviral activity against MCMV occurs at early times after infection. In a murine model of CMV infection, systemic treatment for 5 days significantly reduced virus replication in the spleens and livers of infected mice compared to saline-treated control mice. REP 9 and REP 9C were administered intraperitoneally for 5 consecutive days at 10 mg/kg, starting 2 days prior to MCMV infection. Splenomegaly was observed in infected mice treated with REP 9 but not in control mice or in REP 9 treated, uninfected mice, consistent with mild CpG-like activity. When REP 9C (which lacks CpG motifs was compared to REP 9, it exhibited comparable antiviral activity as REP 9 but was not associated with splenomegaly. This suggests that the direct antiviral activity of APs is the predominant therapeutic mechanism in vivo. Moreover, REP 9C, which is acid stable, was effective when administered orally in combination with known permeation enhancers. Conclusion These studies indicate that APs exhibit potent, well tolerated

  8. Sum Frequency Generation Vibrational Spectroscopy of Adsorbed Amino Acids, Peptides and Proteins of Hydrophilic and Hydrophobic Solid-Water Interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Holinga IV, George Joseph [Univ. of California, Berkeley, CA (United States)

    2010-09-01

    Sum frequency generation (SFG) vibrational spectroscopy was used to investigate the interfacial properties of several amino acids, peptides, and proteins adsorbed at the hydrophilic polystyrene solid-liquid and the hydrophobic silica solid-liquid interfaces. The influence of experimental geometry on the sensitivity and resolution of the SFG vibrational spectroscopy technique was investigated both theoretically and experimentally. SFG was implemented to investigate the adsorption and organization of eight individual amino acids at model hydrophilic and hydrophobic surfaces under physiological conditions. Biointerface studies were conducted using a combination of SFG and quartz crystal microbalance (QCM) comparing the interfacial structure and concentration of two amino acids and their corresponding homopeptides at two model liquid-solid interfaces as a function of their concentration in aqueous solutions. The influence of temperature, concentration, equilibration time, and electrical bias on the extent of adsorption and interfacial structure of biomolecules were explored at the liquid-solid interface via QCM and SFG. QCM was utilized to quantify the biological activity of heparin functionalized surfaces. A novel optical parametric amplifier was developed and utilized in SFG experiments to investigate the secondary structure of an adsorbed model peptide at the solid-liquid interface.

  9. Complete amino acid sequence of a Lolium perenne (perennial rye grass) pollen allergen, Lol p II.

    Science.gov (United States)

    Ansari, A A; Shenbagamurthi, P; Marsh, D G

    1989-07-05

    The complete amino acid sequence of a Lolium perenne (rye grass) pollen allergen, Lol p II was determined by automated Edman degradation of the protein and selected fragments. Cleavage of the protein by enzymatic and chemical techniques established an unambiguous sequence for the protein. Lol p II contains 97 amino acid residues, with a calculated molecular weight of 10,882. The protein lacks cysteine and glutamine and shows no evidence of glycosylation. Theoretical predictions by Fraga's (Fraga, S. (1982) Can. J. Chem. 60, 2606-2610) and Hopp and Woods' (Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 3824-3828) methods indicate the presence of four hydrophilic regions, which may contribute to sequential or parts of conformational B-cell epitopes. Analysis of amphipathic regions by Berzofsky's method indicates the presence of a highly amphipathic region, which may contain, or contribute to, an Ia/T-cell epitope. This latter segment of Lol p II was found to be highly homologous with an antibody-binding segment of the major rye allergen Lol p I and may explain why immune responsiveness to both the allergens is associated with HLA-DR3.

  10. Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo

    International Nuclear Information System (INIS)

    Grossmann, Michael E.; Madden, Benjamin J.; Gao, Fan; Pang, Yuan-Ping; Carpenter, John E.; McCormick, Daniel; Young, Charles Y.F.

    2004-01-01

    Heat shock protein 70 (Hsp70) binds peptide and has several functions that include protein folding, protein trafficking, and involvement with immune function. However, endogenous Hsp70-binding peptides had not previously been identified. Therefore, we eluted and identified several hundred endogenously bound peptides from Hsp70 using liquid chromatography ion trap mass spectrophotometry (LC-ITMS). Our work shows that the peptides are capable of binding Hsp70 as previously described. They are generally 8-26 amino acids in length and correspond to specific regions of many proteins. Through computationally assisted analysis of peptides eluted from Hsp70 we determined variable amino acid sequences, including a 5 amino acid core sequence that Hsp70 favorably binds. We also developed a computer algorithm that predicts Hsp70 binding within proteins. This work helps to define what peptides are bound by Hsp70 in vivo and suggests that Hsp70 facilitates peptide selection by aiding a funneling mechanism that is flexible but allows only a limited number of peptides to be processed

  11. Protection efficacy of the Brucella abortus ghost vaccine candidate lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36) in murine models.

    Science.gov (United States)

    Kwon, Ae Jeong; Moon, Ja Young; Kim, Won Kyong; Kim, Suk; Hur, Jin

    2016-11-01

    Brucella abortus cells were lysed by the N-terminal 24-amino acid fragment (GI24) of the 36-amino acid peptide PMAP-36 (porcine myeloid antimicrobial peptide 36). Next, the protection efficacy of the lysed fragment as a vaccine candidate was evaluated. Group A mice were immunized with sterile PBS, group B mice were intraperitoneally (ip) immunized with 3 × 10 8 colony-forming units (CFUs) of B. abortus strain RB51, group C mice were immunized ip with 3 × 10 8 cells of the B. abortus vaccine candidate, and group D mice were orally immunized with 3 × 10 9 cells of the B. abortus vaccine candidate. Brucella lipopolysaccharide (LPS)-specific serum IgG titers were considerably higher in groups C and D than in group A. The levels of interleukin (IL)-4, IL-10, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were significantly higher in groups B-D than in group A. After an ip challenge with B. abortus 544, only group C mice showed a significant level of protection as compared to group A. Overall, these results show that ip immunization with a vaccine candidate lysed by GI24 can effectively protect mice from systemic infection with virulent B. abortus.

  12. Morintides: cargo-free chitin-binding peptides from Moringa oleifera.

    Science.gov (United States)

    Kini, Shruthi G; Wong, Ka H; Tan, Wei Liang; Xiao, Tianshu; Tam, James P

    2017-03-31

    Hevein-like peptides are a family of cysteine-rich and chitin-binding peptides consisting of 29-45 amino acids. Their chitin-binding property is essential for plant defense against fungi. Based on the number of cysteine residues in their sequences, they are divided into three sub-families: 6C-, 8C- and 10C-hevein-like peptides. All three subfamilies contain a three-domain precursor comprising a signal peptide, a mature hevein-like peptide and a C-terminal domain comprising a hinge region with protein cargo in 8C- and 10C-hevein-like peptides. Here we report the isolation and characterization of two novel 8C-hevein-like peptides, designated morintides (mO1 and mO2), from the drumstick tree Moringa oleifera, a drought-resistant tree belonging to the Moringaceae family. Proteomic analysis revealed that morintides comprise 44 amino acid residues and are rich in cysteine, glycine and hydrophilic amino acid residues such as asparagine and glutamine. Morintides are resistant to thermal and enzymatic degradation, able to bind to chitin and inhibit the growth of phyto-pathogenic fungi. Transcriptomic analysis showed that they contain a three-domain precursor comprising an endoplasmic reticulum (ER) signal sequence, a mature peptide domain and a C-terminal domain. A striking feature distinguishing morintides from other 8C-hevein-like peptides is a short and protein-cargo-free C-terminal domain. Previously, a similar protein-cargo-free C-terminal domain has been observed only in ginkgotides, the 8C-hevein-like peptides from a gymnosperm Ginkgo biloba. Thus, morintides, with a cargo-free C-terminal domain, are a stand-alone class of 8C-hevein-like peptides from angiosperms. Our results expand the existing library of hevein-like peptides and shed light on molecular diversity within the hevein-like peptide family. Our work also sheds light on the anti-fungal activity and stability of 8C-hevein-like peptides.

  13. Biomimetic peptide-based models of [FeFe]-hydrogenases: utilization of phosphine-containing peptides

    Energy Technology Data Exchange (ETDEWEB)

    Roy, Souvik [Department of Chemistry and Biochemistry; Arizona State University; Tempe, USA; Nguyen, Thuy-Ai D. [Department of Chemistry and Biochemistry; Arizona State University; Tempe, USA; Gan, Lu [Department of Chemistry and Biochemistry; Arizona State University; Tempe, USA; Jones, Anne K. [Department of Chemistry and Biochemistry; Arizona State University; Tempe, USA

    2015-01-01

    Peptide based models for [FeFe]-hydrogenase were synthesized utilizing unnatural phosphine-amino acids and their electrocatalytic properties were investigated in mixed aqueous-organic solvents.

  14. Peptide array-based screening of human mesenchymal stem cell-adhesive peptides derived from fibronectin type III domain

    International Nuclear Information System (INIS)

    Okochi, Mina; Nomura, Shigeyuki; Kaga, Chiaki; Honda, Hiroyuki

    2008-01-01

    Human mesenchymal stem cell-adhesive peptides were screened based on the amino acid sequence of fibronectin type III domain 8-11 (FN-III 8-11 ) using a peptide array synthesized by the Fmoc-chemistry. Using hexameric peptide library of FN-III 8-11 scan, we identified the ALNGR (Ala-Leu-Asn-Gly-Arg) peptide that induced cell adhesion as well as RGDS (Arg-Gly-Asp-Ser) peptide. After incubation for 2 h, approximately 68% of inoculated cells adhere to the ALNGR peptide disk. Adhesion inhibition assay with integrin antibodies showed that the ALNGR peptide interacts with integrin β1 but not with αvβ3, indicating that the receptors for ALNGR are different from RGDS. Additionally, the ALNGR peptide expressed cell specificities for adhesion: cell adhesion was promoted for fibroblasts but not for keratinocytes or endotherial cells. The ALNGR peptide induced cell adhesion and promoted cell proliferation without changing its property. It is therefore useful for the construction of functional biomaterials

  15. C-terminal peptide extension via gas-phase ion/ion reactions

    Science.gov (United States)

    Peng, Zhou; McLuckey, Scott A.

    2015-01-01

    The formation of peptide bonds is of great importance from both a biological standpoint and in routine organic synthesis. Recent work from our group demonstrated the synthesis of peptides in the gas-phase via ion/ion reactions with sulfo-NHS reagents, which resulted in conjugation of individual amino acids or small peptides to the N-terminus of an existing ‘anchor’ peptide. Here, we demonstrate a complementary approach resulting in the C-terminal extension of peptides. Individual amino acids or short peptides can be prepared as reagents by incorporating gas phase-labile protecting groups to the reactive C-terminus and then converting the N-terminal amino groups to the active ketenimine reagent. Gas-phase ion/ion reactions between the anionic reagents and doubly protonated “anchor” peptide cations results in extension of the “anchor” peptide with new amide bond formation at the C-terminus. We have demonstrated that ion/ion reactions can be used as a fast, controlled, and efficient means for C-terminal peptide extension in the gas phase. PMID:26640400

  16. Modulation of the conductance of a 2,2′-bipyridine-functionalized peptidic ion channel by Ni2+

    Science.gov (United States)

    Pilz, Claudia S.

    2008-01-01

    An α-helical amphipathic peptide with the sequence H2N-(LSSLLSL)3-CONH2 was obtained by solid phase synthesis and a 2,2′-bipyridine was coupled to its N-terminus, which allows complexation of Ni2+. Complexation of the 2,2′-bipyridine residues was proven by UV/Vis spectroscopy. The peptide helices were inserted into lipid bilayers (nano black lipid membranes, nano-BLMs) that suspend the pores of porous alumina substrates with a pore diameter of 60 nm by applying a potential difference. From single channel recordings, we were able to distinguish four distinct conductance states, which we attribute to an increasing number of peptide helices participating in the conducting helix bundle. Addition of Ni2+ in micromolar concentrations altered the conductance behaviour of the formed ion channels in nano-BLMs considerably. The first two conductance states appear much more prominent demonstrating that the complexation of bipyridine by Ni2+ results in a considerable confinement of the observed multiple conductance states. However, the conductance levels were independent of the presence of Ni2+. Moreover, from a detailed analysis of the open lifetimes of the channels, we conclude that the complexation of Ni2+ diminishes the frequency of channel events with larger open times. Electronic supplementary material The online version of this article (doi:10.1007/s00249-008-0298-8) contains supplementary material, which is available to authorized users. PMID:18347789

  17. Amino-acid- and peptide-directed synthesis of chiral plasmonic gold nanoparticles.

    Science.gov (United States)

    Lee, Hye-Eun; Ahn, Hyo-Yong; Mun, Jungho; Lee, Yoon Young; Kim, Minkyung; Cho, Nam Heon; Chang, Kiseok; Kim, Wook Sung; Rho, Junsuk; Nam, Ki Tae

    2018-04-01

    Understanding chirality, or handedness, in molecules is important because of the enantioselectivity that is observed in many biochemical reactions 1 , and because of the recent development of chiral metamaterials with exceptional light-manipulating capabilities, such as polarization control 2-4 , a negative refractive index 5 and chiral sensing 6 . Chiral nanostructures have been produced using nanofabrication techniques such as lithography 7 and molecular self-assembly 8-11 , but large-scale and simple fabrication methods for three-dimensional chiral structures remain a challenge. In this regard, chirality transfer represents a simpler and more efficient method for controlling chiral morphology 12-18 . Although a few studies 18,19 have described the transfer of molecular chirality into micrometre-sized helical ceramic crystals, this technique has yet to be implemented for metal nanoparticles with sizes of hundreds of nanometres. Here we develop a strategy for synthesizing chiral gold nanoparticles that involves using amino acids and peptides to control the optical activity, handedness and chiral plasmonic resonance of the nanoparticles. The key requirement for achieving such chiral structures is the formation of high-Miller-index surfaces ({hkl}, h ≠ k ≠ l ≠ 0) that are intrinsically chiral, owing to the presence of 'kink' sites 20-22 in the nanoparticles during growth. The presence of chiral components at the inorganic surface of the nanoparticles and in the amino acids and peptides results in enantioselective interactions at the interface between these elements; these interactions lead to asymmetric evolution of the nanoparticles and the formation of helicoid morphologies that consist of highly twisted chiral elements. The gold nanoparticles that we grow display strong chiral plasmonic optical activity (a dis-symmetry factor of 0.2), even when dispersed randomly in solution; this observation is supported by theoretical calculations and direct

  18. Enteroendocrine-derived glucagon-like peptide-2 controls intestinal amino acid transport.

    Science.gov (United States)

    Lee, Jennifer; Koehler, Jacqueline; Yusta, Bernardo; Bahrami, Jasmine; Matthews, Dianne; Rafii, Mahroukh; Pencharz, Paul B; Drucker, Daniel J

    2017-03-01

    Glucagon-like peptide-2 (GLP-2) is co-secreted with GLP-1 from gut endocrine cells, and both peptides act as growth factors to expand the surface area of the mucosal epithelium. Notably, GLP-2 also enhances glucose and lipid transport in enterocytes; however, its actions on control of amino acid (AA) transport remain unclear. Here we examined the mechanisms linking gain and loss of GLP-2 receptor (GLP-2R) signaling to control of intestinal amino acid absorption in mice. Absorption, transport, and clearance of essential AAs, specifically lysine, were measured in vivo by Liquid Chromatography triple quadrupole Mass Spectrometry (LC-MS/MS) and ex vivo with Ussing chambers using intestinal preparations from Glp2 r +/+ and Glp2r - / - mice. Immunoblotting determined jejunal levels of protein components of signaling pathways (PI3K-AKT, and mTORC1-pS6-p4E-BP1) following administration of GLP-2, protein gavage, and rapamycin to fasted Glp2 r +/+ and Glp2r - / - mice. Expression of AA transporters from full thickness jejunum and 4F2hc from brush border membrane vesicles (BBMVs) was measured by real-time PCR and immunoblotting, respectively. Acute administration of GLP-2 increased basal AA absorption in vivo and augmented basal lysine transport ex vivo . GLP-2-stimulated lysine transport was attenuated by co-incubation with wortmannin, rapamycin, or tetrodotoxin ex vivo . Phosphorylation of mTORC1 effector proteins S6 and 4E-BP1 was significantly increased in wild-type mice in response to GLP-2 alone, or when co-administered with protein gavage, and abolished following oral gavage of rapamycin. In contrast, activation of GLP-1R signaling did not enhance S6 phosphorylation. Disruption of GLP-2 action in Glp2r -/- mice reduced lysine transport ex vivo and attenuated the phosphorylation of S6 and 4E-BP1 in response to oral protein. Moreover, the expression of cationic AA transporter slc7a9 in response to refeeding, and the abundance of 4F2hc in BBMVs following protein

  19. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

    Science.gov (United States)

    Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

    2017-08-01

    One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

  20. Mass spectrometric differentiation of linear peptides composed of L-amino acids from isomers containing one D-amino acid residue.

    Science.gov (United States)

    Serafin, Scott V; Maranan, Rhonda; Zhang, Kangling; Morton, Thomas Hellman

    2005-09-01

    MS/MS of electrosprayed ions is shown to have the capacity to discriminate between peptides that differ by configuration about their alpha-carbons. It is not necessary for the peptides to possess tertiary structures that are affected by stereochemistry, since five epimers of the pentapeptide, H2N-Gly-Leu-Ser-Phe-Ala-OH (GLSFA) all display different collisionally activated dissociation (CAD) patterns of their protonated parent ions. The figure of merit, r, is a ratio of ratios of fragment ion abundances between stereoisomers, where r = 1 corresponds to no stereochemical effect. Values of r as high as 3.8 are seen for diastereomer pairs. Stereochemical effects are also seen for the diprotonated dodecapeptide H2N-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-OH (LVFFAEDVGSNK), a tryptic fragment from the amyloid beta-protein. Triply charged complexes of the protonated dodecapeptide with cobalt(II) ions undergo CAD at lower collision energies than do doubly protonated LVFFAEDVGSNK ions. Statistically significant (p < 0.01) differences between the all-L-dodecapeptide and the ones containing a d-serine or a D-aspartic acid are observed.

  1. Comprehensive computational design of ordered peptide macrocycles

    Science.gov (United States)

    Hosseinzadeh, Parisa; Bhardwaj, Gaurav; Mulligan, Vikram Khipple; Shortridge, Matthew D.; Craven, Timothy W.; Pardo-Avila, Fátima; Rettie, Stephen A.; Kim, David E.; Silva, Daniel-Adriano; Ibrahim, Yehia M.; Webb, Ian K.; Cort, John R.; Adkins, Joshua N.; Varani, Gabriele; Baker, David

    2018-01-01

    Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with L-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of L- and D-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods. PMID:29242347

  2. Isolation and characterisation of in vitro and cellular free radical scavenging peptides from corn peptide fractions.

    Science.gov (United States)

    Wang, Liying; Ding, Long; Wang, Ying; Zhang, Yan; Liu, Jingbo

    2015-02-16

    Corn gluten meal, a corn processing industry by-product, is a good source for the preparation of bioactive peptides due to its special amino acid composition. In the present study, the in vitro and cellular free radical scavenging activities of corn peptide fractions (CPFs) were investigated. Results indicated that CPF1 (molecular weight less than 1 kDa) and CPF2 (molecular weight between 1 and 3 kDa) exhibited good hydroxyl radical, superoxide anion radical and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) diammonium salt (ABTS) radical scavenging activity and oxygen radical absorbance capacity (ORAC). Meanwhile, the in vitro radical scavenging activity of CPF1 was slightly higher than that of CPF2. Both CPF1 and CPF2 also exhibited significant cytoprotective effects and intracellular reactive oxygen species scavenging activity in Caco-2 cells exposed to hydrogen peroxide (H2O2). The amino acid composition analysis revealed that the CPF were rich in hydrophobic amino acids, which comprised of more than 45% of total amino acids. An antioxidant peptide sequence of Tyr-Phe-Cys-Leu-Thr (YFCLT) was identified from CPF1 using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI TOF/TOF MS). The YFCLT exhibited excellent ABTS radical scavenging activity with a 50% effective concentration (EC50) value of 37.63 µM, which was much lower than that of Trolox. In conclusion, corn gluten meal might be a good source to prepare antioxidant peptides.

  3. Acquisition and Assimilation of Nitrogen as Peptide-Bound and D-Enantiomers of Amino Acids by Wheat

    Science.gov (United States)

    Hill, Paul W.; Quilliam, Richard S.; DeLuca, Thomas H.; Farrar, John; Farrell, Mark; Roberts, Paula; Newsham, Kevin K.; Hopkins, David W.; Bardgett, Richard D.; Jones, David L.

    2011-01-01

    Nitrogen is a key regulator of primary productivity in many terrestrial ecosystems. Historically, only inorganic N (NH4 + and NO3 -) and L-amino acids have been considered to be important to the N nutrition of terrestrial plants. However, amino acids are also present in soil as small peptides and in D-enantiomeric form. We compared the uptake and assimilation of N as free amino acid and short homopeptide in both L- and D-enantiomeric forms. Sterile roots of wheat (Triticum aestivum L.) plants were exposed to solutions containing either 14C-labelled L-alanine, D-alanine, L-trialanine or D-trialanine at a concentration likely to be found in soil solution (10 µM). Over 5 h, plants took up L-alanine, D-alanine and L-trialanine at rates of 0.9±0.3, 0.3±0.06 and 0.3±0.04 µmol g−1 root DW h−1, respectively. The rate of N uptake as L-trialanine was the same as that as L-alanine. Plants lost ca.60% of amino acid C taken up in respiration, regardless of the enantiomeric form, but more (ca.80%) of the L-trialanine C than amino acid C was respired. When supplied in solutions of mixed N form, N uptake as D-alanine was ca.5-fold faster than as NO3 -, but slower than as L-alanine, L-trialanine and NH4 +. Plants showed a limited capacity to take up D-trialanine (0.04±0.03 µmol g−1 root DW h−1), but did not appear to be able to metabolise it. We conclude that wheat is able to utilise L-peptide and D-amino acid N at rates comparable to those of N forms of acknowledged importance, namely L-amino acids and inorganic N. This is true even when solutes are supplied at realistic soil concentrations and when other forms of N are available. We suggest that it may be necessary to reconsider which forms of soil N are important in the terrestrial N cycle. PMID:21541281

  4. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E

    2012-01-01

    have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA......Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...

  5. Antimicrobial Peptides for Therapeutic Applications: A Review

    Directory of Open Access Journals (Sweden)

    Tsogbadrakh Mishig-Ochir

    2012-10-01

    Full Text Available Antimicrobial peptides (AMPs have been considered as potential therapeutic sources of future antibiotics because of their broad-spectrum activities and different mechanisms of action compared to conventional antibiotics. Although AMPs possess considerable benefits as new generation antibiotics, their clinical and commercial development still have some limitations, such as potential toxicity, susceptibility to proteases, and high cost of peptide production. In order to overcome those obstacles, extensive efforts have been carried out. For instance, unusual amino acids or peptido-mimetics are introduced to avoid the proteolytic degradation and the design of short peptides retaining antimicrobial activities is proposed as a solution for the cost issue. In this review, we focus on small peptides, especially those with less than twelve amino acids, and provide an overview of the relationships between their three-dimensional structures and antimicrobial activities. The efforts to develop highly active AMPs with shorter sequences are also described.

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

    Directory of Open Access Journals (Sweden)

    Nabila Belhaj

    2013-10-01

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

  7. Use of synthetic peptide libraries for the H-2Kd binding motif identification.

    Science.gov (United States)

    Quesnel, A; Casrouge, A; Kourilsky, P; Abastado, J P; Trudelle, Y

    1995-01-01

    To identify Kd-binding peptides, an approach based on small peptide libraries has been developed. These peptide libraries correspond to all possible single-amino acid variants of a particular Kd-binding peptide, SYIPSAEYI, an analog of the Plasmodium berghei 252-260 antigenic peptide SYIPSAEKI. In the parent sequence, each position is replaced by all the genetically encoded amino acids (except cysteine). The multiple analog syntheses are performed either by the Divide Couple and Recombine method or by the Single Resin method and generate mixtures containing 19 peptides. The present report deals with the synthesis, the purification, the chemical characterization by amino acid analysis and electrospray mass spectrometry (ES-MS), and the application of such mixtures in binding tests with a soluble, functionally empty, single-chain H-2Kd molecule denoted SC-Kd. For each mixture, bound peptides were eluted and analyzed by sequencing. Since the binding tests were realized in noncompetitive conditions, our results show that a much broader set of peptides bind to Kd than expected from previous studies. This may be of practical importance when looking for low affinity peptides such as tumor peptides capable of eliciting protective immune response.

  8. Contribution of the Tyr-1 in Plantaricin149a to Disrupt Phospholipid Model Membranes

    Directory of Open Access Journals (Sweden)

    Georgina Tonarelli

    2013-06-01

    Full Text Available Plantaricin149a (Pln149a is a cationic antimicrobial peptide, which was suggested to cause membrane destabilization via the carpet mechanism. The mode of action proposed to this antimicrobial peptide describes the induction of an amphipathic α-helix from Ala7 to Lys20, while the N-terminus residues remain in a coil conformation after binding. To better investigate this assumption, the purpose of this study was to determine the contributions of the Tyr1 in Pln149a in the binding to model membranes to promote its destabilization. The Tyr to Ser substitution increased the dissociation constant (KD of the antimicrobial peptide from the liposomes (approximately three-fold higher, and decreased the enthalpy of binding to anionic vesicles from −17.2 kcal/mol to −10.2 kcal/mol. The peptide adsorption/incorporation into the negatively charged lipid vesicles was less effective with the Tyr1 substitution and peptide Pln149a perturbed the liposome integrity more than the analog, Pln149S. Taken together, the peptide-lipid interactions that govern the Pln149a antimicrobial activity are found not only in the amphipathic helix, but also in the N-terminus residues, which take part in enthalpic contributions due to the allocation at a lipid-aqueous interface.

  9. Molecular architecture with carbohydrate functionalized β-peptides adopting 314-helical conformation

    Directory of Open Access Journals (Sweden)

    Nitin J. Pawar

    2014-04-01

    Full Text Available Carbohydrate recognition is essential in cellular interactions and biological processes. It is characterized by structural diversity, multivalency and cooperative effects. To evaluate carbohydrate interaction and recognition, the structurally defined attachment of sugar units to a rigid template is highly desired. β-Peptide helices offer conformationally stable templates for the linear presentation of sugar units in defined distances. The synthesis and β-peptide incorporation of sugar-β-amino acids are described providing the saccharide units as amino acid side chain. The respective sugar-β-amino acids are accessible by Michael addition of ammonia to sugar units derivatized as α,β-unsaturated esters. Three sugar units were incorporated in β-peptide oligomers varying the sugar (glucose, galactose, xylose and sugar protecting groups. The influence of sugar units and the configuration of sugar-β-amino acids on β-peptide secondary structure were investigated by CD spectroscopy.

  10. Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands.

    Science.gov (United States)

    Auvynet, Constance; Seddiki, Nabila; Dunia, Irene; Nicolas, Pierre; Amiche, Mohamed; Lacombe, Claire

    2006-01-01

    The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.

  11. Common and divergent structural features of a series of corticotropin releasing factor-related peptides.

    Science.gov (United States)

    Grace, Christy Rani R; Perrin, Marilyn H; Cantle, Jeffrey P; Vale, Wylie W; Rivier, Jean E; Riek, Roland

    2007-12-26

    Members of the corticoliberin family include the corticotropin releasing factors (CRFs), sauvagine, the urotensins, and urocortin 1 (Ucn1), which bind to both the CRF receptors CRF-R1 and CRF-R2, and the urocortins 2 (Ucn2) and 3 (Ucn3), which are selective agonists of CRF-R2. Structure activity relationship studies led to several potent and long-acting analogues with selective binding to either one of the receptors. NMR structures of six ligands of this family (the antagonists astressin B and astressin2-B, the agonists stressin1, and the natural ligands human Ucn1, Ucn2, and Ucn3) were determined in DMSO. These six peptides show differences in binding affinities, receptor-selectivity, and NMR structure. Overall, their backbones are alpha-helical, with a small kink or a turn around residues 25-27, resulting in a helix-loop-helix motif. The C-terminal helices are of amphipathic nature, whereas the N-terminal helices vary in their amphipathicity. The C-terminal helices thereby assume a conformation very similar to that of astressin bound to the ECD1 of CRF-R2 recently reported by our group.1 On the basis of an analysis of the observed 3D structures and relative potencies of [Ala]-substituted analogues, it is proposed that both helices could play a crucial role in receptor binding and selectivity. In conclusion, the C-terminal helices may interact along their hydrophobic faces with the ECD1, whereas the entire N-terminal helical surface may be involved in receptor activation. On the basis of the common and divergent features observed in the 3D structures of these ligands, multiple binding models are proposed that may explain their plurality of actions.

  12. Mechanisms Underlying the Anti-Aging and Anti-Tumor Effects of Lithocholic Bile Acid

    Directory of Open Access Journals (Sweden)

    Anthony Arlia-Ciommo

    2014-09-01

    Full Text Available Bile acids are cholesterol-derived bioactive lipids that play essential roles in the maintenance of a heathy lifespan. These amphipathic molecules with detergent-like properties display numerous beneficial effects on various longevity- and healthspan-promoting processes in evolutionarily distant organisms. Recent studies revealed that lithocholic bile acid not only causes a considerable lifespan extension in yeast, but also exhibits a substantial cytotoxic effect in cultured cancer cells derived from different tissues and organisms. The molecular and cellular mechanisms underlying the robust anti-aging and anti-tumor effects of lithocholic acid have emerged. This review summarizes the current knowledge of these mechanisms, outlines the most important unanswered questions and suggests directions for future research.

  13. δ-Peptides from RuAAC-Derived 1,5-Disubstituted Triazole Units

    KAUST Repository

    Johansson, Johan R.

    2014-02-14

    Non-natural peptides with structures and functions similar to natural peptides have emerged lately in biomedical as well as nanotechnological contexts. They are interesting for pharmaceutical applications since they can adopt structures with new targeting potentials and because they are generally not prone to degradation by proteases. We report here a new set of peptidomimetics derived from δ-peptides, consisting of n units of a 1,5-disubstituted 1,2,3-triazole amino acid (5Tzl). The monomer was prepared using ruthenium-catalyzed azide-alkyne cycloaddition (RuAAC) chemistry using [RuCl2Cp]x as the catalyst, allowing for simpler purification and resulting in excellent yields. This achiral monomer was used to prepare peptide oligomers that are water soluble independent of peptide chain length. Conformational analysis and structural investigations of the oligomers were performed by 2D NOESY NMR experiments, and by quantum chemical calculations using the ωB97X-D functional. These data indicate that several conformations may co-exist with slight energetic differences. Together with their increased hydrophilicity, this feature of homo-5Tzl may prove essential for mimicking natural peptides composed of α-amino acids, where the various secondary structures are achieved by side chain effects and not by the rigidity of the peptide backbone. The improved synthetic method allows for facile variation of the 5Tzl amino acid side chains, further increasing the versatility of these compounds. A new set of non-natural peptides composed of 1,5-disubstituted 1,2,3-triazole amino acids is presented. These peptides benefit from: a) modular synthesis of the monomers, allowing variation of the side chains; b) increased solubility of the oligomers in water, irrespective of peptide length; c) flexibility of the backbone allowing these foldamers to adopt several conformations. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Optimization of Reversed-Phase Peptide Liquid Chromatography Ultraviolet Mass Spectrometry Analyses Using an Automated Blending Methodology

    Science.gov (United States)

    Chakraborty, Asish B.; Berger, Scott J.

    2005-01-01

    The balance between chromatographic performance and mass spectrometric response has been evaluated using an automated series of experiments where separations are produced by the real-time automated blending of water with organic and acidic modifiers. In this work, the concentration effects of two acidic modifiers (formic acid and trifluoroacetic acid) were studied on the separation selectivity, ultraviolet, and mass spectrometry detector response, using a complex peptide mixture. Peptide retention selectivity differences were apparent between the two modifiers, and under the conditions studied, trifluoroacetic acid produced slightly narrower (more concentrated) peaks, but significantly higher electrospray mass spectrometry suppression. Trifluoroacetic acid suppression of electrospray signal and influence on peptide retention and selectivity was dominant when mixtures of the two modifiers were analyzed. Our experimental results indicate that in analyses where the analyzed components are roughly equimolar (e.g., a peptide map of a recombinant protein), the selectivity of peptide separations can be optimized by choice and concentration of acidic modifier, without compromising the ability to obtain effective sequence coverage of a protein. In some cases, these selectivity differences were explored further, and a rational basis for differentiating acidic modifier effects from the underlying peptide sequences is described. PMID:16522853

  15. Lipid-peptide-polymer conjugates and nanoparticles thereof

    Science.gov (United States)

    Xu, Ting; Dong, He; Shu, Jessica

    2015-06-02

    The present invention provides a conjugate having a peptide with from about 10 to about 100 amino acids, wherein the peptide adopts a helical structure. The conjugate also includes a first polymer covalently linked to the peptide, and a hydrophobic moiety covalently linked to the N-terminus of the peptide, wherein the hydrophobic moiety comprises a second polymer or a lipid moiety. The present invention also provides helix bundles form by self-assembling the conjugates, and particles formed by self-assembling the helix bundles. Methods of preparing the helix bundles and particles are also provided.

  16. Deep Subseafloor Fungi as an Untapped Reservoir of Amphipathic Antimicrobial Compounds.

    Science.gov (United States)

    Navarri, Marion; Jégou, Camille; Meslet-Cladière, Laurence; Brillet, Benjamin; Barbier, Georges; Burgaud, Gaëtan; Fleury, Yannick

    2016-03-10

    The evolving global threat of antimicrobial resistance requires a deep renewal of the antibiotic arsenal including the isolation and characterization of new drugs. Underexplored marine ecosystems may represent an untapped reservoir of novel bioactive molecules. Deep-sea fungi isolated from a record-depth sediment core of almost 2000 m below the seafloor were investigated for antimicrobial activities. This antimicrobial screening, using 16 microbial targets, revealed 33% of filamentous fungi synthesizing bioactive compounds with activities against pathogenic bacteria and fungi. Interestingly, occurrence of antimicrobial producing isolates was well correlated with the complexity of the habitat (in term of microbial richness), as higher antimicrobial activities were obtained at specific layers of the sediment core. It clearly highlights complex deep-sea habitats as chemical battlefields where synthesis of numerous bioactive compounds appears critical for microbial competition. The six most promising deep subseafloor fungal isolates were selected for the production and extraction of bioactive compounds. Depending on the fungal isolates, antimicrobial compounds were only biosynthesized in semi-liquid or solid-state conditions as no antimicrobial activities were ever detected using liquid fermentation. An exception was made for one fungal isolate, and the extraction procedure designed to extract amphipathic compounds was successful and highlighted the amphiphilic profile of the bioactive metabolites.

  17. Chemo-enzymatic peptide synthesis : bioprocess engineering aspects

    NARCIS (Netherlands)

    Vossenberg, P.

    2012-01-01

    Peptides, in particular oligopeptides, play an important role in the fields of health care, nutrition and cosmetics. Chemical synthesis is currently the most mature technique for the synthesis of peptides that range in length from 5 to 80 amino acids. Chemical synthesis is, however,

  18. Cloning of precursors for two MIH/VIH-related peptides in the prawn, Macrobrachium rosenbergii.

    Science.gov (United States)

    Yang, W J; Rao, K R

    2001-11-30

    Two cDNA clones (634 and 1366 bp) encoding MIH/VIH (molt-inhibiting hormone/vitellogenesis-inhibiting hormone)-related peptides were isolated and sequenced from a Macrobrachium rosenbergii eyestalk ganglia cDNA library. The clones contain a 360 and 339 bp open-reading frame, and their conceptually translated peptides consist of a 41 and 34 amino acid signal peptide, respectively, and a 78 amino acid residue mature peptide hormone. The amino acid sequences of the peptides exhibit higher identities with other known MIHs and VIH (44-69%) than with CHHs (28-33%). This is the first report describing the cloning and sequencing of two MIH/VIH-related peptides in a single crustacean species. Transcription of these mRNAs was detected in the eyestalk ganglia, but not in the thoracic ganglia, hepatopancreas, gut, gill, heart, or muscle.

  19. Peptide ligands for targeting the extracellular domain of EGFR: Comparison between linear and cyclic peptides.

    Science.gov (United States)

    Williams, Tyrslai M; Sable, Rushikesh; Singh, Sitanshu; Vicente, Maria Graca H; Jois, Seetharama D

    2018-02-01

    Colorectal cancer (CRC) is the third most common solid internal malignancy among cancers. Early detection of cancer is key to increasing the survival rate of colorectal cancer patients. Overexpression of the EGFR protein is associated with CRC. We have designed a series of peptides that are highly specific for the extracellular domain of EGFR, based on our earlier studies on linear peptides. The previously reported linear peptide LARLLT, known to bind to EGFR, was modified with the goals of increasing its stability and its specificity toward EGFR. Peptide modifications, including D-amino acid substitution, cyclization, and chain reversal, were investigated. In addition, to facilitate labeling of the peptide with a fluorescent dye, an additional lysine residue was introduced onto the linear (KLARLLT) and cyclic peptides cyclo(KLARLLT) (Cyclo.L1). The lysine residue was also converted into an azide group in both a linear and reversed cyclic peptide sequences cyclo(K(N3)larllt) (Cyclo.L1.1) to allow for subsequent "click" conjugation. The cyclic peptides showed enhanced binding to EGFR by SPR. NMR and molecular modeling studies suggest that the peptides acquire a β-turn structure in solution. In vitro stability studies in human serum show that the cyclic peptide is more stable than the linear peptide. © 2017 John Wiley & Sons A/S.

  20. Radiation chemistry of amino acids and peptides in aqueous solutions

    International Nuclear Information System (INIS)

    Simic, M.G.

    1978-01-01

    Radiation chemistry relevant to radiation preservation of high protein foods is reviewed. Some conclusions concerning the chemistry of irradiated amino acids, peptides, and proteins have been derived from product analysis of γ-irradiated solutions while the main mechanistic considerations result from the chemistry and kinetics of free radical intermediates observed by pulse radiolysis. The precursors of chemistry in not too concentrated solutions ( - , OH, and H. Their reactivity with molecules and their preference for characteristic groups within the molecule are discussed. The reviewed reactions of the model systems are accountable for a variety of radiolytic products found in irradiated foods. From detailed understanding of radiation chemistry in aqueous and frozen systems formation of many classes of compounds can be predicted or entirely eliminated in order to corroborate and extend the conclusions reached from the animal feeding experiments concerning the formation of toxic, mutagenic, and carcinogenic compounds and/or reduction of the nutritional value of foods

  1. Multifunctional hybrid networks based on self assembling peptide sequences

    Science.gov (United States)

    Sathaye, Sameer

    The overall aim of this dissertation is to achieve a comprehensive correlation between the molecular level changes in primary amino acid sequences of amphiphilic beta-hairpin peptides and their consequent solution-assembly properties and bulk network hydrogel behavior. This has been accomplished using two broad approaches. In the first approach, amino acid substitutions were made to peptide sequence MAX1 such that the hydrophobic surfaces of the folded beta-hairpins from the peptides demonstrate shape specificity in hydrophobic interactions with other beta-hairpins during the assembly process, thereby causing changes to the peptide nanostructure and bulk rheological properties of hydrogels formed from the peptides. Steric lock and key complementary hydrophobic interactions were designed to occur between two beta-hairpin molecules of a single molecule, LNK1 during beta-sheet fibrillar assembly of LNK1. Experimental results from circular dichroism, transmission electron microscopy and oscillatory rheology collectively indicate that the molecular design of the LNK1 peptide can be assigned the cause of the drastically different behavior of the networks relative to MAX1. The results indicate elimination or significant reduction of fibrillar branching due to steric complementarity in LNK1 that does not exist in MAX1, thus supporting the original hypothesis. As an extension of the designed steric lock and key complementarity between two beta-hairpin molecules of the same peptide molecule. LNK1, three new pairs of peptide molecules LP1-KP1, LP2-KP2 and LP3-KP3 that resemble complementary 'wedge' and 'trough' shapes when folded into beta-hairpins were designed and studied. All six peptides individually and when blended with their corresponding shape complement formed fibrillar nanostructures with non-uniform thickness values. Loose packing in the assembled structures was observed in all the new peptides as compared to the uniform tight packing in MAX1 by SANS analysis. This

  2. Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in E. coli

    DEFF Research Database (Denmark)

    Ghosal, Anubrata; Vitali, Ally; Stach, James E M

    2013-01-01

    Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA......-peptide conjugates had disruptions in the region of sbmA, a gene encoding an inner membrane peptide transporter. The wild type sensitivity to the PNA conjugates was re-established in the resistance mutants by complementation with sbmA. Furthermore, deletion of sbmA in E. coli AS19, a strain that is sensitive...

  3. What peptides these deltorphins be.

    Science.gov (United States)

    Lazarus, L H; Bryant, S D; Cooper, P S; Salvadori, S

    1999-02-01

    The deltorphins are a class of highly selective delta-opioid heptapeptides from the skin of the Amazonian frogs Phyllomedusa sauvagei and P. bicolor. The first of these fascinating peptides came to light in 1987 by cloning of the cDNA of from frog skins, while the other members of this family were identified either by cDNA or isolation of the peptides. The distinctive feature of deltorphins is the presence of a naturally occurring D-enantiomer at the second position in their common N-terminal sequence, Tyr-D-Xaa-Phe, comparable to dermorphin, which is the prototype of a group of mu-selective opioids from the same source. The D-amino acid and the anionic residues, either Glu or Asp, as well as their unique amino acid compositions are responsible for the remarkable biostability, high delta-receptor affinity, bioactivity and peptide conformation. This review summarizes a decade of research from many laboratories that defined which residues and substituents in the deltorphins interact with the delta-receptor and characterized pharmacological and physiological activities in vitro and in vivo. It begins with a historical description of the topic and presents general schema for the synthesis of peptide analogues of deltorphins A, B and C as a means to document the methods employed in producing a myriad of analogues. Structure activity studies of the peptides and their pharmacological activities in vitro are detailed in abundantly tabulated data. A brief compendium of the current level of knowledge of the delta-receptor assists the reader to appreciate the rationale for the design of these analogues. Discussion of the conformation of these peptides addresses how structure leads to further hypotheses regarding ligand receptor interaction. The review ends with a broad discussion of the potential applications of these peptides in clinical and therapeutic settings.

  4. Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity.

    Science.gov (United States)

    Joseph, Christine G; Wilczynski, Andrzej; Holder, Jerry R; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Haskell-Luevano, Carrie

    2003-12-01

    Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111-113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7-9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle4-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH2 peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.

  5. Structure-activity studies and therapeutic potential of host defense peptides of human thrombin.

    Science.gov (United States)

    Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Mörgelin, Matthias; Albiger, Barbara; Malmsten, Martin; Schmidtchen, Artur

    2011-06-01

    Peptides of the C-terminal region of human thrombin are released upon proteolysis and identified in human wounds. In this study, we wanted to investigate minimal determinants, as well as structural features, governing the antimicrobial and immunomodulating activity of this peptide region. Sequential amino acid deletions of the peptide GKYGFYTHVFRLKKWIQKVIDQFGE (GKY25), as well as substitutions at strategic and structurally relevant positions, were followed by analyses of antimicrobial activity against the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, the Gram-positive bacterium Staphylococcus aureus, and the fungus Candida albicans. Furthermore, peptide effects on lipopolysaccharide (LPS)-, lipoteichoic acid-, or zymosan-induced macrophage activation were studied. The thrombin-derived peptides displayed length- and sequence-dependent antimicrobial as well as immunomodulating effects. A peptide length of at least 20 amino acids was required for effective anti-inflammatory effects in macrophage models, as well as optimal antimicrobial activity as judged by MIC assays. However, shorter (>12 amino acids) variants also displayed significant antimicrobial effects. A central K14 residue was important for optimal antimicrobial activity. Finally, one peptide variant, GKYGFYTHVFRLKKWIQKVI (GKY20) exhibiting improved selectivity, i.e., low toxicity and a preserved antimicrobial as well as anti-inflammatory effect, showed efficiency in mouse models of LPS shock and P. aeruginosa sepsis. The work defines structure-activity relationships of C-terminal host defense peptides of thrombin and delineates a strategy for selecting peptide epitopes of therapeutic interest.

  6. Development of novel radiogallium-labeled bone imaging agents using oligo-aspartic acid peptides as carriers.

    Directory of Open Access Journals (Sweden)

    Kazuma Ogawa

    Full Text Available (68Ga (T 1/2 = 68 min, a generator-produced nuclide has great potential as a radionuclide for clinical positron emission tomography (PET. Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting (68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Aspn (n = 2, 5, 8, 11, or 14 with easy-to-handle (67Ga, with the previously described (67Ga-DOTA complex conjugated bisphosphonate, (67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Aspn by a Fmoc-based solid-phase method, complexes were formed with (67Ga, resulting in (67Ga-DOTA-(Aspn with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of (67Ga-DOTA-(Aspn increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, (67Ga-DOTA-(Asp8, (67Ga-DOTA-(Asp11, and (67Ga-DOTA-(Asp14 showed high accumulation in bone (10.5 ± 1.5, 15.1 ± 2.6, and 12.8 ± 1.7% ID/g, respectively but were barely observed in other tissues at 60 min after injection. Although bone accumulation of (67Ga-DOTA-(Aspn was lower than that of (67Ga-DOTA-Bn-SCN-HBP, blood clearance of (67Ga-DOTA-(Aspn was more rapid. Accordingly, the bone/blood ratios of (67Ga-DOTA-(Asp11 and (67Ga-DOTA-(Asp14 were comparable with those of (67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of (68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

  7. Antioxidative Peptides Derived from Enzyme Hydrolysis of Bone Collagen after Microwave Assisted Acid Pre-Treatment and Nitrogen Protection

    Directory of Open Access Journals (Sweden)

    Jin Sun

    2010-11-01

    Full Text Available This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid. The highest degree of hydrolysis (DH was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain, with an optimum condition of: (1 ratio of enzyme and substrate, 4760 U/g; (2 concentration of substrate, 4%; (3 reaction temperature, 55 °C and (4 pH 7.0. At 4 h, DH increased significantly (P < 0.01 under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen.

  8. Novel phosphine-peptide hybrids as selective catalysts

    DEFF Research Database (Denmark)

    Nygaard, David

    (His(Trt), Gln, Gln(Trt), Cys(tBu), Thr(OtBu), azido- Dab, Asp(OtBu), Arg(Pmc))) yielding a range of novel modified peptides. Peptides containing one secondary amine were phosphinylated and captured as either phosphine-boranes or oxides. Both borane and oxide protection of phosphine-peptide hybrids...... was discovered and the compounds were structurally elucidated via NMR and mass spectroscopy. Two of these compounds were incorporated into peptides. An existing method of obtaining peptides containing secondary amines in the peptide backbone have been expanded for incorporation of functional amino acids as well...... palladium chloride dimer did not yield an observable phosphine-palladium complex. A peptide containing two secondary amine sites was synthesized, phosphinylated and complexed to respectively palladium and copper. The palladium complex was utilized successfully as a palladium catalyst in a model Sonogashira...

  9. A versatile palindromic amphipathic repeat coding sequence horizontally distributed among diverse bacterial and eucaryotic microbes

    Directory of Open Access Journals (Sweden)

    Glass John I

    2010-07-01

    Full Text Available Abstract Background Intragenic tandem repeats occur throughout all domains of life and impart functional and structural variability to diverse translation products. Repeat proteins confer distinctive surface phenotypes to many unicellular organisms, including those with minimal genomes such as the wall-less bacterial monoderms, Mollicutes. One such repeat pattern in this clade is distributed in a manner suggesting its exchange by horizontal gene transfer (HGT. Expanding genome sequence databases reveal the pattern in a widening range of bacteria, and recently among eucaryotic microbes. We examined the genomic flux and consequences of the motif by determining its distribution, predicted structural features and association with membrane-targeted proteins. Results Using a refined hidden Markov model, we document a 25-residue protein sequence motif tandemly arrayed in variable-number repeats in ORFs lacking assigned functions. It appears sporadically in unicellular microbes from disparate bacterial and eucaryotic clades, representing diverse lifestyles and ecological niches that include host parasitic, marine and extreme environments. Tracts of the repeats predict a malleable configuration of recurring domains, with conserved hydrophobic residues forming an amphipathic secondary structure in which hydrophilic residues endow extensive sequence variation. Many ORFs with these domains also have membrane-targeting sequences that predict assorted topologies; others may comprise reservoirs of sequence variants. We demonstrate expressed variants among surface lipoproteins that distinguish closely related animal pathogens belonging to a subgroup of the Mollicutes. DNA sequences encoding the tandem domains display dyad symmetry. Moreover, in some taxa the domains occur in ORFs selectively associated with mobile elements. These features, a punctate phylogenetic distribution, and different patterns of dispersal in genomes of related taxa, suggest that the

  10. 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...... than 5 amino acids showed binding and a clear correlation with hydrophobicity was demonstrated for oligomers of different hydrophobic amino acids. Insertion of hydrophilic amino acids in a hydrophobic sequence diminished or abolished binding. In conclusion our results show that calreticulin has...

  11. Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: implications for therapy of Alzheimer disease

    NARCIS (Netherlands)

    Bana, Laura; Minniti, Stefania; Salvati, Elisa; Sesana, Silvia; Zambelli, Vanessa; Cagnotto, Alfredo; Orlando, Antonina; Cazzaniga, Emanuela; Zwart, Rob; Scheper, Wiep; Masserini, Massimo; Re, Francesca

    2014-01-01

    Targeting amyloid-β peptide (Aβ) within the brain is a strategy actively sought for therapy of Alzheimer's disease (AD). We investigated the ability of liposomes bi-functionalized with phosphatidic acid and with a modified ApoE-derived peptide (mApoE-PA-LIP) to affect Aβ aggregation/disaggregation

  12. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

    Science.gov (United States)

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, Jongone; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic `fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

  13. The Suzuki–Miyaura Cross-Coupling as a Versatile Tool for Peptide Diversification and Cyclization

    Directory of Open Access Journals (Sweden)

    Tom Willemse

    2017-02-01

    Full Text Available The (site-selective derivatization of amino acids and peptides represents an attractive field with potential applications in the establishment of structure–activity relationships and labeling of bioactive compounds. In this respect, bioorthogonal cross-coupling reactions provide valuable means for ready access to peptide analogues with diversified structure and function. Due to the complex and chiral nature of peptides, mild reaction conditions are preferred; hence, a suitable cross-coupling reaction is required for the chemical modification of these challenging substrates. The Suzuki reaction, involving organoboron species, is appropriate given the stability and environmentally benign nature of these reactants and their amenability to be applied in (partial aqueous reaction conditions, an expected requirement upon the derivatization of peptides. Concerning the halogenated reaction partner, residues bearing halogen moieties can either be introduced directly as halogenated amino acids during solid-phase peptide synthesis (SPPS or genetically encoded into larger proteins. A reversed approach building in boron in the peptidic backbone is also possible. Furthermore, based on this complementarity, cyclic peptides can be prepared by halogenation, and borylation of two amino acid side chains present within the same peptidic substrate. Here, the Suzuki–Miyaura reaction is a tool to induce the desired cyclization. In this review, we discuss diverse amino acid and peptide-based applications explored by means of this extremely versatile cross-coupling reaction. With the advent of peptide-based drugs, versatile bioorthogonal conversions on these substrates have become highly valuable.

  14. Dissociation Behavior of a TEMPO-Active Ester Cross-Linker for Peptide Structure Analysis by Free Radical Initiated Peptide Sequencing (FRIPS) in Negative ESI-MS.

    Science.gov (United States)

    Hage, Christoph; Ihling, Christian H; Götze, Michael; Schäfer, Mathias; Sinz, Andrea

    2017-01-01

    We have synthesized a homobifunctional amine-reactive cross-linking reagent, containing a TEMPO (2,2,6,6-tetramethylpiperidine-1-oxy) and a benzyl group (Bz), termed TEMPO-Bz-linker, to derive three-dimensional structural information of proteins. The aim for designing this novel cross-linker was to facilitate the mass spectrometric analysis of cross-linked products by free radical initiated peptide sequencing (FRIPS). In an initial study, we had investigated the fragmentation behavior of TEMPO-Bz-derivatized peptides upon collision activation in (+)-electrospray ionization collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) experiments. In addition to the homolytic NO-C bond cleavage FRIPS pathway delivering the desired odd-electron product ions, an alternative heterolytic NO-C bond cleavage, resulting in even-electron product ions mechanism was found to be relevant. The latter fragmentation route clearly depends on the protonation of the TEMPO-Bz-moiety itself, which motivated us to conduct (-)-ESI-MS, CID-MS/MS, and MS 3 experiments of TEMPO-Bz-cross-linked peptides to further clarify the fragmentation behavior of TEMPO-Bz-peptide molecular ions. We show that the TEMPO-Bz-linker is highly beneficial for conducting FRIPS in negative ionization mode as the desired homolytic cleavage of the NO-C bond is the major fragmentation pathway. Based on characteristic fragments, the isomeric amino acids leucine and isoleucine could be discriminated. Interestingly, we observed pronounced amino acid side chain losses in cross-linked peptides if the cross-linked peptides contain a high number of acidic amino acids. Graphical Abstract ᅟ.

  15. Sungsanpin, a lasso peptide from a deep-sea streptomycete.

    Science.gov (United States)

    Um, Soohyun; Kim, Young-Joo; Kwon, Hyuknam; Wen, He; Kim, Seong-Hwan; Kwon, Hak Cheol; Park, Sunghyouk; Shin, Jongheon; Oh, Dong-Chan

    2013-05-24

    Sungsanpin (1), a new 15-amino-acid peptide, was discovered from a Streptomyces species isolated from deep-sea sediment collected off Jeju Island, Korea. The planar structure of 1 was determined by 1D and 2D NMR spectroscopy, mass spectrometry, and UV spectroscopy. The absolute configurations of the stereocenters in this compound were assigned by derivatizations of the hydrolysate of 1 with Marfey's reagents and 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate, followed by LC-MS analysis. Careful analysis of the ROESY NMR spectrum and three-dimensional structure calculations revealed that sungsanpin possesses the features of a lasso peptide: eight amino acids (-Gly(1)-Phe-Gly-Ser-Lys-Pro-Ile-Asp(8)-) that form a cyclic peptide and seven amino acids (-Ser(9)-Phe-Gly-Leu-Ser-Trp-Leu(15)) that form a tail that loops through the ring. Sungsanpin is thus the first example of a lasso peptide isolated from a marine-derived microorganism. Sungsanpin displayed inhibitory activity in a cell invasion assay with the human lung cancer cell line A549.

  16. A Novel Soluble Peptide with pH-Responsive Membrane Insertion.

    Science.gov (United States)

    Nguyen, Vanessa P; Alves, Daiane S; Scott, Haden L; Davis, Forrest L; Barrera, Francisco N

    2015-11-03

    Several diseases, such as cancer, are characterized by acidification of the extracellular environment. Acidosis can be employed as a target to specifically direct therapies to the diseased tissue. We have used first principles to design an acidity-triggered rational membrane (ATRAM) peptide with high solubility in solution that is able to interact with lipid membranes in a pH-dependent fashion. Biophysical studies show that the ATRAM peptide binds to the surface of lipid membranes at pH 8.0. However, acidification leads to the peptide inserting into the lipid bilayer as a transmembrane α-helix. The insertion of ATRAM into membranes occurs at a moderately acidic pH (with a pK of 6.5), similar to the extracellular pH found in solid tumors. Studies with human cell lines showed a highly efficient pH-dependent membrane targeting, without causing toxicity. Here we show that it is possible to rationally design a soluble peptide that selectively targets cell membranes in acidic environments.

  17. Effects of Rice Bran, Flax Seed, and Sunflower Seed on Growth Performance, Carcass Characteristics, Fatty Acid Composition, Free Amino Acid and Peptide Contents, and Sensory Evaluations of Native Korean Cattle (Hanwoo

    Directory of Open Access Journals (Sweden)

    Chang Bon Choi

    2016-02-01

    Full Text Available This study was conducted to evaluate the effect of dietary supplementation with rice bran, flax seed, or sunflower seed to finishing native Korean cattle (Hanwoo on growth performances, carcass characteristics, fatty acid composition, free amino acid and peptide contents, and sensory evaluations of Longissimus muscle (LM. A total of 39 Hanwoo steers (average age of 22.2 mo and average body weight (BW of 552.2 kg were randomly divided into Control, rice bran (RB, flax seed (FS, or Sunflower seed (SS groups. The steers were group fed for 273 d until they reached an average age of 31.2 mo. Final BW was 768.2, 785.8, 786.2, and 789.0 kg, and average daily gain was 0.79, 0.85, 0.82, and 0.84 kg for the Control, RS, FS, and SS groups, respectively (p>0.05. Fat thickness of the FS group (19.8 mm was greater (p0.05 scores for flavor, umami, and overall palatability in sensory evaluations. In conclusion, supplementation of flax seed to diets of finishing Hanwoo steers improved sensory evaluations which might have been caused by increases in flavor related amino acids such as methionine, glutamic acid and α-AAA and peptides, anserine and carnosine, and their complex reactions.

  18. Reduction of free fatty acids by acipimox enhances the growth hormone (GH) responses to GH-releasing peptide 2 in elderly men

    NARCIS (Netherlands)

    Smid, HEC; de Vries, WR; Niesink, M; Bolscher, E; Waasdorp, EJ; Dieguez, C; Casanueva, FF; Koppeschaar, HPF

    2000-01-01

    GH release is increased by reducing circulating free fatty acids (FFAs). Aging is associated with decreased plasma GH concentrations. We evaluated GH releasing capacity in nine healthy elderly men after administration of GH-releasing peptide 2 (GHRP-2), with or without pretreatment with the

  19. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Eysturskard, Jonhard; Nielsen, Peter E

    2010-01-01

    ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human ca...

  20. Metabolism and pharmacokinetic of cyclo-peptides and peptides. Use of radioelement and stable isotopes

    International Nuclear Information System (INIS)

    Aninat, C.

    2003-10-01

    More and more peptides and proteins are used in therapeutic. Three mainly techniques are used for pharmacokinetic and metabolism studies: immunoassay, radioactively labeled molecules and mass spectrometry. In the first part of this work, we have used uniformly labelled peptides (C-peptide and insulin) with stables ( 13 C, 15 N, and 13 C/ 15 N) or radioactive ( 14 C) isotopes to investigated these kind of studies. These works are based on isotope dilution mass spectrometry assay. In a second time we have investigated the metabolism of a particular cyclo-peptides families composed of two amino acids: the diketo-piperazine. These compounds are found in mammals and in microorganisms. There are not recognized by proteolytic enzymes. We have estimated if the main enzymes implicated in the metabolism of xenobiotics, the P450 cytochrome mono-oxygenases, were able to recognized them

  1. Structures of peptide families by nuclear magnetic resonance spectroscopy and distance geometry

    Energy Technology Data Exchange (ETDEWEB)

    Pease, J.H.

    1989-12-01

    The three dimensional structures of several small peptides were determined using a combination of {sup 1}H nuclear magnetic resonance (NMR) and distance geometry calculations. These techniques were found to be particularly helpful for analyzing structural differences between related peptides since all of the peptides' {sup 1}H NMR spectra are very similar. The structures of peptides from two separate classes are presented. Peptides in the first class are related to apamin, an 18 amino acid peptide toxin from honey bee venom. The {sup 1}H NMR assignments and secondary structure determination of apamin were done previously. Quantitative NMR measurements and distance geometry calculations were done to calculate apamin's three dimensional structure. Peptides in the second class are 48 amino acid toxins from the sea anemone Radianthus paumotensis. The {sup 1}H NMR assignments of toxin II were done previously. The {sup 1}H NMR assignments of toxin III and the distance geometry calculations for both peptides are presented.

  2. Novel ZnO-binding peptides obtained by the screening of a phage display peptide library

    Energy Technology Data Exchange (ETDEWEB)

    Golec, Piotr [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Molecular Biology (affiliated with the University of Gdansk) (Poland); Karczewska-Golec, Joanna [University of Gdansk and Medical University of Gdansk, Laboratory of Molecular Bacteriology, Intercollegiate Faculty of Biotechnology (Poland); Los, Marcin; Wegrzyn, Grzegorz, E-mail: wegrzyn@biotech.univ.gda.pl [University of Gdansk, Department of Molecular Biology (Poland)

    2012-11-15

    Zinc oxide (ZnO) is a semiconductor compound with a potential for wide use in various applications, including biomaterials and biosensors, particularly as nanoparticles (the size range of ZnO nanoparticles is from 2 to 100 nm, with an average of about 35 nm). Here, we report isolation of novel ZnO-binding peptides, by screening of a phage display library. Interestingly, amino acid sequences of the ZnO-binding peptides reported in this paper and those described previously are significantly different. This suggests that there is a high variability in sequences of peptides which can bind particular inorganic molecules, indicating that different approaches may lead to discovery of different peptides of generally the same activity (e.g., binding of ZnO) but having various detailed properties, perhaps crucial under specific conditions of different applications.

  3. Peptide-Loaded Solid Lipid Nanoparticles Prepared through Coacervation Technique

    Directory of Open Access Journals (Sweden)

    Marina Gallarate

    2011-01-01

    Full Text Available Stearic acid solid lipid nanoparticles were prepared according to a new technique, called coacervation. The main goal of this experimental work was the entrapment of peptide drugs into SLN, which is a difficult task, since their chemical characteristics (molecular weight, hydrophilicity, and stability hamper peptide-containing formulations. Insulin and leuprolide, chosen as model peptide drugs, were encapsulated within nanoparticles after hydrophobic ion pairing with anionic surfactants. Peptide integrity was maintained after encapsulation, and nanoparticles can act in vitro as a sustained release system for peptide.

  4. Protein antigenic structures recognized by T cells: potential applications to vaccine design.

    Science.gov (United States)

    Berzofsky, J A; Cease, K B; Cornette, J L; Spouge, J L; Margalit, H; Berkower, I J; Good, M F; Miller, L H; DeLisi, C

    1987-08-01

    In summary, our results using the model protein antigen myoglobin indicated, in concordance with others, that helper T lymphocytes recognize a limited number of immunodominant antigenic sites of any given protein. Such immunodominant sites are the focus of a polyclonal response of a number of different T cells specific for distinct but overlapping epitopes. Therefore, the immunodominance does not depend on the fine specificity of any given clone of T cells, but rather on other factors, either intrinsic or extrinsic to the structure of the antigen. A major extrinsic factor is the MHC of the responding individual, probably due to a requirement for the immunodominant peptides to bind to the MHC of presenting cells in that individual. In looking for intrinsic factors, we noted that both immunodominant sites of myoglobin were amphipathic helices, i.e., helices having hydrophilic and hydrophobic residues on opposite sides. Studies with synthetic peptides indicated that residues on the hydrophilic side were necessary for T-cell recognition. However, unfolding of the native protein was shown to be the apparent goal of processing of antigen, presumably to expose something not already exposed on the native molecule, such as the hydrophobic sides of these helices. We propose that such exposure is necessary to interact with something on the presenting cell, such as MHC or membrane, where we have demonstrated the presence of antigenic peptides by blocking of presentation of biotinylated peptide with avidin. The membrane may serve as a short-term memory of peptides from antigens encountered by the presenting cell, for dynamic sampling by MHC molecules to be available for presentation to T cells. These ideas, together with the knowledge that T-cell recognition required only short peptides and therefore had to be based only on primary or secondary structure, not tertiary folding of the native protein, led us to propose that T-cell immunodominant epitopes may tend to be amphipathic

  5. Rapid identification of Staphylococcus aureus and methicillin resistance by flow cytometry using a peptide nucleic acid probe.

    Science.gov (United States)

    Shrestha, Nabin K; Scalera, Nikole M; Wilson, Deborah A; Brehm-Stecher, Byron; Procop, Gary W

    2011-09-01

    A total of 56 Staphylococcus aureus isolates incubated for 2 h in the presence or absence of oxacillin were analyzed by flow cytometry after labeling with an S. aureus-specific peptide nucleic acid (PNA) probe. Two defined ratios, the paired signal count ratio (PSCR) and the gate signal count ratio (GSCR), differentiated methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) with sensitivities of 100% each and specificities of 96% and 100%, respectively.

  6. Purification and Characterization of Antioxidant Peptide from Sunflower Protein Hydrolysate

    Directory of Open Access Journals (Sweden)

    Xi-Qun Zheng

    2010-01-01

    Full Text Available Sunflower proteins were hydrolyzed with Flavourzyme for the production of antioxidant peptide. DEAE-Sepharose Fast Flow, Sephadex G-25 gel filtration chromatography and reversed-phase HPLC were consecutively employed to purify a novel sunflower antioxidant peptide, and the ability to inhibit the autoxidation of pyrogallol was expressed as the antioxidative activity of the peptide. The amino acid sequence was identified as Ala-Cys-Ala-His-Asp-Lys-Val by a Q-Tof2 mass spectrometer. This novel peptide exhibited a high antioxidative activity of 79.42 U/mL, which is expected to protect against oxidative damage in living systems in relation to aging and carcinogenesis. Higher antioxidative activities were presumed mainly due to the presence of hydrophobic amino acids in its sequence.

  7. Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

    International Nuclear Information System (INIS)

    Isenman, L.D.; Dice, J.F.

    1989-01-01

    We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [ 3 H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides

  8. Solvent polarity controls the helical conformation of short peptides rich in Calpha-tetrasubstituted amino acids.

    Science.gov (United States)

    Bellanda, Massimo; Mammi, Stefano; Geremia, Silvano; Demitri, Nicola; Randaccio, Lucio; Broxterman, Quirinus B; Kaptein, Bernard; Pengo, Paolo; Pasquato, Lucia; Scrimin, Paolo

    2007-01-01

    The two peptides, rich in C(alpha)-tetrasubstituted amino acids, Ac-[Aib-L-(alphaMe)Val-Aib](2)-L-His-NH(2) (1) and Ac-[Aib-L-(alphaMe)Val-Aib](2)-O-tBu (2 a) are prevalently helical. They present the unique property of changing their conformation from the alpha- to the 3(10)-helix as a function of the polarity of the solvent: alpha in more polar solvents, 3(10) in less polar ones. Conclusive evidence of this reversible change of conformation is reported on the basis of the circular dichroism (CD) spectra and a detailed two-dimensional NMR analysis in two solvents (trifluoroethanol and methanol) refined with molecular dynamics calculations. The X-ray diffractometric analysis of the crystals of both peptides reveals that they assume a prevalent 3(10)-helix conformation in the solid state. This conformation is practically superimposable on that obtained from the NMR analysis of 1 in methanol. The NMR results further validate the reported CD signature of the 3(10)-helix and the use of the CD technique for its assessment.

  9. UV laser-induced cross-linking in peptides

    Science.gov (United States)

    Leo, Gabriella; Altucci, Carlo; Bourgoin-Voillard, Sandrine; Gravagnuolo, Alfredo M.; Esposito, Rosario; Marino, Gennaro; Costello, Catherine E.; Velotta, Raffaele; Birolo, Leila

    2013-01-01

    RATIONALE The aim of this study was to demonstrate, and to characterize by high resolution mass spectrometry, that it is possible to preferentially induce covalent cross-links in peptides by using high energy femtosecond UV laser pulses. The cross-link is readily formed only when aromatic amino acids are present in the peptide sequence. METHODS Three peptides, xenopsin, angiotensin I, interleukin, individually or in combination, were exposed to high energy femtosecond UV laser pulses, either alone or in the presence of spin trapping molecules, the reaction products being characterized by high resolution mass spectrometry. RESULTS High resolution mass spectrometry and spin trapping strategies showed that cross-linking occurs readily, proceeds via a radical mechanism, and is the highly dominant reaction, proceeding without causing significant photo-damage in the investigated range of experimental parameters. CONCLUSIONS High energy femtosecond UV laser pulses can be used to induce covalent cross-links between aromatic amino acids in peptides, overcoming photo-oxidation processes, that predominate as the mean laser pulse intensity approaches illumination conditions achievable with conventional UV light sources. PMID:23754800

  10. Chiral Symmetry Breaking in Peptide Systems During Formation of Life on Earth

    Science.gov (United States)

    Konstantinov, Konstantin K.; Konstantinova, Alisa F.

    2018-03-01

    Chiral symmetry breaking in complex chemical systems with a large number of amino acids and a large number of similar reactions was considered. It was shown that effective averaging over similar reaction channels may result in very weak effective enantioselectivity of forward reactions, which does not allow most of the known models to result in chiral symmetry breaking during formation of life on Earth. Models with simple and catalytic synthesis of a single amino acid, formation of peptides up to length five, and sedimentation of insoluble pair of substances were considered. It was shown that depending on the model and the values of the parameters, chiral symmetry breaking may occur in up to about 10% out of all possible unique insoluble pair combinations even in the absence of any catalytic synthesis and that minimum total number of amino acids in the pair is 5. If weak enantioselective forward catalytic synthesis of amino acids is present, then the number of possible variants, in which chiral symmetry breaking may occur, increases substantially. It was shown that that the most interesting catalysts have zero or one amino acid of "incorrect" chirality. If the parameters of the model are adjusted in such a way to result in an increase of concentration of longer peptides, then catalysts with two amino acids of incorrect chirality start to appear at peptides of length five. Models of chiral symmetry breaking in the presence of epimerization were considered for peptides up to length three. It was shown that the range of parameters in which chiral symmetry breaking could occur significantly shrinks in comparison to previously considered models with peptides up to length two. An experiment of chiral symmetry breaking was proposed. The experiment consists of a three-step cycle: reversible catalytic synthesis of amino acids, reversible synthesis of peptides, and irreversible sedimentation of insoluble substances.

  11. Membrane Disruption Mechanism of a Prion Peptide (106-126) Investigated by Atomic Force Microscopy, Raman and Electron Paramagnetic Resonance Spectroscopy.

    Science.gov (United States)

    Pan, Jianjun; Sahoo, Prasana K; Dalzini, Annalisa; Hayati, Zahra; Aryal, Chinta M; Teng, Peng; Cai, Jianfeng; Rodriguez Gutierrez, Humberto; Song, Likai

    2017-05-18

    A fragment of the human prion protein spanning residues 106-126 (PrP106-126) recapitulates many essential properties of the disease-causing protein such as amyloidogenicity and cytotoxicity. PrP106-126 has an amphipathic characteristic that resembles many antimicrobial peptides (AMPs). Therefore, the toxic effect of PrP106-126 could arise from a direct association of monomeric peptides with the membrane matrix. Several experimental approaches are employed to scrutinize the impacts of monomeric PrP106-126 on model lipid membranes. Porous defects in planar bilayers are observed by using solution atomic force microscopy. Adding cholesterol does not impede defect formation. A force spectroscopy experiment shows that PrP106-126 reduces Young's modulus of planar lipid bilayers. We use Raman microspectroscopy to study the effect of PrP106-126 on lipid atomic vibrational dynamics. For phosphatidylcholine lipids, PrP106-126 disorders the intrachain conformation, while the interchain interaction is not altered; for phosphatidylethanolamine lipids, PrP106-126 increases the interchain interaction, while the intrachain conformational order remains similar. We explain the observed differences by considering different modes of peptide insertion. Finally, electron paramagnetic resonance spectroscopy shows that PrP106-126 progressively decreases the orientational order of lipid acyl chains in magnetically aligned bicelles. Together, our experimental data support the proposition that monomeric PrP106-126 can disrupt lipid membranes by using similar mechanisms found in AMPs.

  12. Conjugation of cell-penetrating peptides with poly(lactic-co-glycolic acid-polyethylene glycol nanoparticles improves ocular drug delivery

    Directory of Open Access Journals (Sweden)

    Vasconcelos A

    2015-01-01

    Full Text Available Aimee Vasconcelos,1 Estefania Vega,2 Yolanda Pérez,3 María J Gómara,1 María Luisa García,2 Isabel Haro1 1Unit of Synthesis and Biomedical Applications of Peptides, Department of Biomedical Chemistry, Institute for Advanced Chemistry of Catalonia, Consejo Superior de Investigaciones Científicas (IQAC-CSIC, 2Department of Physical Chemistry, Institute of Nanoscience and Nanotechnology, Faculty of Pharmacy, University of Barcelona, 3Nuclear Magnetic Resonance Unit, IQAC-CSIC, Barcelona, Spain Abstract: In this work, a peptide for ocular delivery (POD and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid (PGLA–polyethylene glycol (PEG-nanoparticles (NPs in an attempt to improve ocular drug bioavailability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide; the other involved self-assembly of PLGA-PEG and the PLGA-PEG-peptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confirmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flurbiprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance were studied. In vivo anti-inflammatory efficacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment efficiency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective prevention of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation

  13. 2D-QSAR in hydroxamic acid derivatives as peptide deformylase inhibitors and antibacterial agents.

    Science.gov (United States)

    Gupta, Manish K; Mishra, Pradeep; Prathipati, Philip; Saxena, Anil K

    2002-12-01

    Peptide deformylase catalyzes the removal of N-formyl group from the N-formylmethionine of ribosome synthesized polypeptide in eubacteria. Quantitative structure-activity relationship (QSAR) studies have been carried out in a series of beta-sulfonyl and beta-sulfinyl hydroxamic acid derivatives for their PDF enzyme inhibitory and antibacterial activities against Escherichia coli DC2 and Moraxella catarrhalis RA21 which demonstrate that the PDF inhibitory activity in cell free and whole cell system increases with increase in molar refractivity and hydrophobicity. The comparison of the QSARs between the cell free and whole cell system indicate that the active binding sites in PDF isolated from E. coli and in M. catarrhalis RA21 are similar and the whole cell antibacterial activity is mainly due to the inhibition of PDF. Apart from this the QSARs on some matrixmetelloproteins (COL-1, COL-3, MAT and HME) and natural endopeptidase (NEP) indicate the possibilities of introducing selectivity in these hydroxamic acid derivatives for their PDF inhibitory activity.

  14. Structure, antimicrobial activities and mode of interaction with membranes of novel [corrected] phylloseptins from the painted-belly leaf frog, Phyllomedusa sauvagii.

    Directory of Open Access Journals (Sweden)

    Zahid Raja

    Full Text Available Transcriptomic and peptidomic analysis of skin secretions from the Painted-belly leaf frog Phyllomedusa sauvagii led to the identification of 5 novel phylloseptins (PLS-S2 to -S6 and also of phylloseptin-1 (PSN-1, here renamed PLS-S1, the only member of this family previously isolated in this frog. Synthesis and characterization of these phylloseptins revealed differences in their antimicrobial activities. PLS-S1, -S2, and -S4 (79-95% amino acid sequence identity; net charge  = +2 were highly potent and cidal against Gram-positive bacteria, including multidrug resistant S. aureus strains, and killed the promastigote stage of Leishmania infantum, L. braziliensis and L. major. By contrast, PLS-S3 (95% amino acid identity with PLS-S2; net charge  = +1 and -S5 (net charge  = +2 were found to be almost inactive against bacteria and protozoa. PLS-S6 was not studied as this peptide was closely related to PLS-S1. Differential scanning calorimetry on anionic and zwitterionic multilamellar vesicles combined with circular dichroism spectroscopy and membrane permeabilization assays on bacterial cells indicated that PLS-S1, -S2, and -S4 are structured in an amphipathic α-helix that disrupts the acyl chain packing of anionic lipid bilayers. As a result, regions of two coexisting phases could be formed, one phase rich in peptide and the other lipid-rich. After reaching a threshold peptide concentration, the disruption of lipid packing within the bilayer may lead to local cracks and disintegration of the microbial membrane. Differences in the net charge, α-helical folding propensity, and/or degree of amphipathicity between PLS-S1, -S2 and -S4, and between PLS-S3 and -S5 appear to be responsible for their marked differences in their antimicrobial activities. In addition to the detailed characterization of novel phylloseptins from P. sauvagii, our study provides additional data on the previously isolated PLS-S1 and on the mechanism of action of

  15. Gastrin-releasing peptide in the porcine pancreas

    DEFF Research Database (Denmark)

    Holst, J J; Poulsen, Steen Seier

    1987-01-01

    to consist of one main form, namely the 27-amino acid peptide originally extracted from porcine stomach, and small amounts of a C-terminal fragment identical with the C-terminal 10-amino acid peptide. Gastrin-releasing peptide-like immunoreactivity released from the isolated perfused porcine pancreas during...... electrical vagal stimulation was shown by gel filtration to consist of the same two forms. By use of immunocytochemical techniques employing an antiserum directed against its N terminus, GRP was localized to varicose nerve fibers in close association with the exocrine tissue of the porcine pancreas...... in particular. Some fibers were found penetrating into pancreatic islets also. Immunoreactive nerve cell bodies as well as fibers were found within intrapancreatic ganglia. The potency of GRP in stimulating exocrine as well as endocrine secretion from the porcine pancreas, its presence in close contact...

  16. Peptide and protein delivery using new drug delivery systems.

    Science.gov (United States)

    Jain, Ashish; Jain, Aviral; Gulbake, Arvind; Shilpi, Satish; Hurkat, Pooja; Jain, Sanjay K

    2013-01-01

    Pharmaceutical and biotechnological research sorts protein drug delivery systems by importance based on their various therapeutic applications. The effective and potent action of the proteins/peptides makes them the drugs of choice for the treatment of numerous diseases. Major research issues in protein delivery include the stabilization of proteins in delivery devices and the design of appropriate target-specific protein carriers. Many efforts have been made for effective delivery of proteins/peptidal drugs through various routes of administrations for successful therapeutic effects. Nanoparticles made of biodegradable polymers such as poly lactic acid, polycaprolactone, poly(lactic-co-glycolic acid), the poly(fumaric-co-sebacic) anhydride chitosan, and modified chitosan, as well as solid lipids, have shown great potential in the delivery of proteins/peptidal drugs. Moreover, scientists also have used liposomes, PEGylated liposomes, niosomes, and aquasomes, among others, for peptidal drug delivery. They also have developed hydrogels and transdermal drug delivery systems for peptidal drug delivery. A receptor-mediated delivery system is another attractive strategy to overcome the limitation in drug absorption that enables the transcytosis of the protein across the epithelial barrier. Modification such as PEGnology is applied to various proteins and peptides of the desired protein and peptides also increases the circulating life, solubility and stability, pharmacokinetic properties, and antigenicity of protein. This review focuses on various approaches for effective protein/peptidal drug delivery, with special emphasis on insulin delivery.

  17. Cloning of cDNAs encoding new peptides of the dermaseptin-family.

    Science.gov (United States)

    Wechselberger, C

    1998-10-14

    Dermaseptins are a group of basic (lysine-rich) peptides, 27-34 amino acids in length and involved in the defense of frog skin against microbial invasion. By using a degenerated oligonucleotide primer binding to the 5'-untranslated region of previously characterized cDNAs of these peptides, it was possible to identify new members of the dermaseptin family in the South American frogs Agalychnis annae and Pachymedusa dacnicolor. Amino acid alignment and secondary structure prediction reveals, that only five of the deduced peptides can be supposed to be also functional homologs to the known dermaseptins from Phyllomedusa bicolor and Phyllomedusa sauvagei. The remaining six peptides described in this paper have not been isolated and characterized yet.

  18. Antimicrobial peptides from the skins of North American frogs.

    Science.gov (United States)

    Conlon, J Michael; Kolodziejek, Jolanta; Nowotny, Norbert

    2009-08-01

    North America is home to anuran species belonging to the families Bufonidae, Eleutherodactylidae, Hylidae, Leiopelmatidae, Ranidae, and Scaphiopodidae but antimicrobial peptides have been identified only in skin secretions and/or skin extracts of frogs belonging to the Leiopelmatidae ("tailed frogs") and Ranidae ("true frogs"). Eight structurally-related cationic alpha-helical peptides with broad-spectrum antibacterial activity, termed ascaphins, have been isolated from specimens of Ascaphus truei (Leiopelmatidae) occupying a coastal range. Characterization of orthologous antimicrobial peptides from Ascaphus specimens occupying an inland range supports the proposal that this population should be regarded as a separate species A. montanus. Ascaphin-8 shows potential for development into a therapeutically valuable anti-infective agent. Peptides belonging to the brevinin-1, esculentin-1, esculentin-2, palustrin-1, palustrin-2, ranacyclin, ranatuerin-1, ranatuerin-2, and temporin families have been isolated from North American ranids. It is proposed that "ranalexins" represent brevinin-1 peptides that have undergone a four amino acid residue internal deletion. Current taxonomic recommendations divide North American frogs from the family Ranidae into two genera: Lithobates and Rana. Cladistic analysis based upon the amino acid sequences of the brevinin-1 peptides provides strong support for this assignment.

  19. Structure of genes for dermaseptins B, antimicrobial peptides from frog skin. Exon 1-encoded prepropeptide is conserved in genes for peptides of highly different structures and activities.

    Science.gov (United States)

    Vouille, V; Amiche, M; Nicolas, P

    1997-09-01

    We cloned the genes of two members of the dermaseptin family, broad-spectrum antimicrobial peptides isolated from the skin of the arboreal frog Phyllomedusa bicolor. The dermaseptin gene Drg2 has a 2-exon coding structure interrupted by a small 137-bp intron, wherein exon 1 encoded a 22-residue hydrophobic signal peptide and the first three amino acids of the acidic propiece; exon 2 contained the 18 additional acidic residues of the propiece plus a typical prohormone processing signal Lys-Arg and a 32-residue dermaseptin progenitor sequence. The dermaseptin genes Drg2 and Drg1g2 have conserved sequences at both untranslated ends and in the first and second coding exons. In contrast, Drg1g2 comprises a third coding exon for a short version of the acidic propiece and a second dermaseptin progenitor sequence. Structural conservation between the two genes suggests that Drg1g2 arose recently from an ancestral Drg2-like gene through amplification of part of the second coding exon and 3'-untranslated region. Analysis of the cDNAs coding precursors for several frog skin peptides of highly different structures and activities demonstrates that the signal peptides and part of the acidic propieces are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The organization of the genes that belong to this family, with the signal peptide and the progenitor sequence on separate exons, permits strikingly different peptides to be directed into the secretory pathway. The recruitment of such a homologous 'secretory' exon by otherwise non-homologous genes may have been an early event in the evolution of amphibian.

  20. Chemical methods for peptide and protein production.

    Science.gov (United States)

    Chandrudu, Saranya; Simerska, Pavla; Toth, Istvan

    2013-04-12

    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 α-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.

  1. A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

    Science.gov (United States)

    Fang, Wei-Jie; Yakovleva, Tatyana; Aldrich, Jane V.

    2014-01-01

    Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the PAL-PEG-PS (Peptide Amide Linker-polyethylene glycol-polystyrene) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. PMID:22252422

  2. Homologies between the amino acid sequences of some vertebrate peptide hormones and peptides isolated from invertebrate sources.

    Science.gov (United States)

    De Loof, A; Schoofs, L

    1990-01-01

    1. The 4K-prothoracicotropic hormone (PTTH) or bombyxin and the melanization-reddish coloration hormone of the silkworm Bombyx mori resemble insulin and insulin-like growth factors. 2. The family of adipokinetic/red pigment concentrating hormones has some similarity with glucagon. 3. Members of the FMRFamide family are found in vertebrates as well as in invertebrates. 4. In Locusta, a molecule immunologically and biologically related to amphibian melanophore stimulating hormone has been partially characterized. 5. Enkephalins and enkephalin-related peptides occur in insects and other invertebrates. 6. Peptides belonging to the tachykinin family have been isolated from molluscan (Octopus) salivary glands and from insect nervous tissue (Locusta migratoria). 7. Invertebrate arginine-vasotocin homologs have been isolated from an insect (Locusta migratoria) and from a mollusc (Conus). 8. In Leucophaea, Locusta and Drosophila, peptides resembling those of the vertebrate gastrin/cholecystokinin family have been identified. 9. As the number of different neuro-/gut peptides with possible function(s) as hormone, neurotransmitter or neuromodulator is now estimated to be of the order of a few hundred, more similarities will probably show up in the near future.

  3. Optimization of simultaneous saccharification and fermentation conditions with amphipathic lignin derivatives for concentrated bioethanol production.

    Science.gov (United States)

    Cheng, Ningning; Koda, Keiichi; Tamai, Yutaka; Yamamoto, Yoko; Takasuka, Taichi E; Uraki, Yasumitsu

    2017-05-01

    Amphipathic lignin derivatives (A-LDs) prepared from the black liquor of soda pulping of Japanese cedar are strong accelerators for bioethanol production under a fed-batch simultaneous enzymatic saccharification and fermentation (SSF) process. To improve the bioethanol production concentration, conditions such as reaction temperature, stirring program, and A-LDs loadings were optimized in both small scale and large scale fed-batch SSF. The fed-batch SSF in the presence of 3.0g/L A-LDs at 38°C gave the maximum ethanol production and a high enzyme recovery rate. Furthermore, a jar-fermenter equipped with a powerful mechanical stirrer was designed for 1.5L-scale fed-batch SSF to achieve rigorous mixing during high substrate loading. Finally, the 1.5L fed-batch SSF with a substrate loading of 30% (w/v) produced a high ethanol concentration of 87.9g/L in the presence of A-LDs under optimized conditions. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. B-Type Natriuretic Peptide: From Posttranslational Processing to Clinical Measurement

    DEFF Research Database (Denmark)

    Goetze, Jens P

    2012-01-01

    BACKGROUND:Plasma cardiac natriuretic peptides and peptide fragments from their molecular precursors are markers of heart disease. Clinical studies have defined the current diagnostic utility of these markers, whereas biochemical elucidation of peptide structure and posttranslational processing has...... revealed new plasma peptide forms of potential clinical use.CONTENT:Natriuretic propeptide structures undergo variable degrees of endo- and exoproteolytic cleavages as well as amino acid modifications, which leave the plasma phase of the peptides highly heterogeneous and dependent on cardiac......-atrial natriuretic peptide and pro-B-type natriuretic peptide are useful plasma markers in heart failure. New data have defined cardiac myocytes as competent endocrine cells in posttranslational processing and cellular secretion....

  5. Design, synthesis, and actions of a novel chimeric natriuretic peptide: CD-NP.

    Science.gov (United States)

    Lisy, Ondrej; Huntley, Brenda K; McCormick, Daniel J; Kurlansky, Paul A; Burnett, John C

    2008-07-01

    Our aim was to design, synthesize and test in vivo and in vitro a new chimeric peptide that would combine the beneficial properties of 2 distinct natriuretic peptides with a biological profile that goes beyond native peptides. Studies have established the beneficial vascular and antiproliferative properties of C-type natriuretic peptide (CNP). While lacking renal actions, CNP is less hypotensive than the cardiac peptides atrial natriuretic peptide and B-type natriuretic peptide but unloads the heart due to venodilation. Dendroaspis natriuretic peptide is a potent natriuretic and diuretic peptide that is markedly hypotensive and functions via a separate guanylyl cyclase receptor compared with CNP. Here we engineered a novel chimeric peptide CD-NP that represents the fusion of the 22-amino acid peptide CNP together with the 15-amino acid linear C-terminus of Dendroaspis natriuretic peptide. We also determined in vitro in cardiac fibroblasts cyclic guanosine monophosphate-activating and antiproliferative properties of CD-NP. Our studies demonstrate in vivo that CD-NP is natriuretic and diuretic, glomerular filtration rate enhancing, cardiac unloading, and renin inhibiting. CD-NP also demonstrates less hypotensive properties when compared with B-type natriuretic peptide. In addition, CD-NP in vitro activates cyclic guanosine monophosphate and inhibits cardiac fibroblast proliferation. The current findings advance an innovative design strategy in natriuretic peptide drug discovery and development to create therapeutic peptides with favorable properties that may be preferable to those associated with native natriuretic peptides.

  6. Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Robaczewska, Magdalena; Narayan, Ramamurthy; Seigneres, Beatrice

    2005-01-01

    BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT...... in primary duck hepatocytes (PDH). RESULTS: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT...

  7. Amino acid substitutions in the melanoma antigen recognized by T cell 1 peptide modulate cytokine responses in melanoma-specific T cells

    DEFF Research Database (Denmark)

    Nielsen, M B; Kirkin, A F; Loftus, D

    2000-01-01

    enhances the production of mRNA for interleukin (IL)-5, IL-10, IL-13, IL-15, and interferon-gamma and significantly enhances release of IL-13 and IL-10 from anti-MART-1 cytotoxic T cells. Another heteroclitic peptide, 1L, with an A to L substitution in MART-1(27-35), also enhances the tyrosine...... phosphorylation response in anti-MART-1 cytotoxic CD8+ T cells. Yet, 1L does not enhance the production of T helper cell type 2-like cytokines (IL-10 and IL-13). Together these data show that minor amino acid modifications of immunodominant melanoma peptides profoundly influence the cytokine response in melanoma...

  8. Reactions of tritium atoms with amino acids, deuterated amino acids and mixtures of amino acids. Additivity property and isotope effect

    International Nuclear Information System (INIS)

    Badun, G.A.; Filatov, Eh.S.

    1988-01-01

    Interaction of tritium atoms with glycine (1) and leucine (2) amino acids, deuterated amino acids, their mixtures and glycylleucine (3) peptide in the 77-300 K temperature range is studied in isothermal and gradient regimes. Tagged amino acids were separated from targets after conducting the reaction. At T 150 K are associated with intermolecular transmission of free valence in the mixture of amino acids. Regularities of the reaction found for the mixture of amino acids are conserved for (3) as well, i.e. the peptide bond does not essentially affect the reaction of isotopic exchange conditioned by atomic tritium

  9. Thermal behavior of potato starch and water-vaporization behavior of its paste controlled with amino acid and peptide-rich food materials.

    Science.gov (United States)

    Sakauchi, Satoshi; Hattori, Makoto; Yoshida, Tadashi; Yagishita, Takahiro; Ito, Koichi; Akemitsu, Shin-Ichi; Takahashi, Koji

    2010-03-01

    The particular effect of 4 kinds of amino acid and peptide-rich food material (APRM) containing different charged amino acid contents on the gelatinization and retrogradation behavior of potato starch granules and on the water-vaporization behavior was analyzed by differential scanning calorimetry, rapid viscoanalysis, x-ray diffractometry, thermal gravimetry-differential thermal analysis, and pulsed NMR. APRM with a high-charged amino acid content produced unique gelatinization and retrogradation behavior in terms of an elevated gelatinization temperature, reduced viscosity, higher setback, and lower retrograded starch melting enthalpy. The recovered x-ray diffraction intensity decreased with increasing charged amino acid content. APRM with high-charged amino acid content could provide an improved paste having easy vaporization of external water in the swollen starch granules due to the reduced swelling.

  10. Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles

    DEFF Research Database (Denmark)

    Norregaard, Kamilla; Andersson, Magnus; Nielsen, Peter Eigil

    2014-01-01

    This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation...... of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti......-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less...

  11. Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides

    Science.gov (United States)

    Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

    2011-01-01

    The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides. Methods Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice. Results DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively. Conclusion The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic

  12. new caerin-like antibacterial peptide from the venom gland of the ...

    African Journals Online (AJOL)

    Scorpion venom consists of different types of peptides and proteins which are encoded by individual genes. A full length cDNA consisting of 238 base pair nucleotides and encoding 74 amino acids peptide was isolated from the venom gland of the Iranian scorpion Mesobuthus eupeus (Buthidae family). This peptide named ...

  13. Hexagonally Ordered Arrays of α-Helical Bundles Formed from Peptide-Dendron Hybrids

    Energy Technology Data Exchange (ETDEWEB)

    Barkley, Deborah A. [Department; Rokhlenko, Yekaterina [Department; Marine, Jeannette E. [Department; David, Rachelle [Department; Sahoo, Dipankar [Department; Watson, Matthew D. [Department; Koga, Tadanori [Department; Department; Osuji, Chinedum O. [Department; Rudick, Jonathan G. [Department

    2017-10-24

    Combining monodisperse building blocks that have distinct folding properties serves as a modular strategy for controlling structural complexity in hierarchically organized materials. We combine an α-helical bundle-forming peptide with self-assembling dendrons to better control the arrangement of functional groups within cylindrical nanostructures. Site-specific grafting of dendrons to amino acid residues on the exterior of the α-helical bundle yields monodisperse macromolecules with programmable folding and self-assembly properties. The resulting hybrid biomaterials form thermotropic columnar hexagonal mesophases in which the peptides adopt an α-helical conformation. Bundling of the α-helical peptides accompanies self-assembly of the peptide-dendron hybrids into cylindrical nanostructures. The bundle stoichiometry in the mesophase agrees well with the size found in solution for α-helical bundles of peptides with a similar amino acid sequence.

  14. Efficient molecular mechanics simulations of the folding, orientation, and assembly of peptides in lipid bilayers using an implicit atomic solvation model

    Science.gov (United States)

    Bordner, Andrew J.; Zorman, Barry; Abagyan, Ruben

    2011-10-01

    Membrane proteins comprise a significant fraction of the proteomes of sequenced organisms and are the targets of approximately half of marketed drugs. However, in spite of their prevalence and biomedical importance, relatively few experimental structures are available due to technical challenges. Computational simulations can potentially address this deficit by providing structural models of membrane proteins. Solvation within the spatially heterogeneous membrane/solvent environment provides a major component of the energetics driving protein folding and association within the membrane. We have developed an implicit solvation model for membranes that is both computationally efficient and accurate enough to enable molecular mechanics predictions for the folding and association of peptides within the membrane. We derived the new atomic solvation model parameters using an unbiased fitting procedure to experimental data and have applied it to diverse problems in order to test its accuracy and to gain insight into membrane protein folding. First, we predicted the positions and orientations of peptides and complexes within the lipid bilayer and compared the simulation results with solid-state NMR structures. Additionally, we performed folding simulations for a series of host-guest peptides with varying propensities to form alpha helices in a hydrophobic environment and compared the structures with experimental measurements. We were also able to successfully predict the structures of amphipathic peptides as well as the structures for dimeric complexes of short hexapeptides that have experimentally characterized propensities to form beta sheets within the membrane. Finally, we compared calculated relative transfer energies with data from experiments measuring the effects of mutations on the free energies of translocon-mediated insertion of proteins into lipid bilayers and of combined folding and membrane insertion of a beta barrel protein.

  15. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II

    DEFF Research Database (Denmark)

    Schneider, Tanja; Kruse, Thomas; Wimmer, Reinhard

    2010-01-01

    Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show th...

  16. Expression of a wolf spider toxin in tobacco inhibits the growth of microbes and insects

    Science.gov (United States)

    Abstract Lycotoxin I, from the wolf spider (Lycosa carolinensis), is an amphipathic pore-forming peptide that has antimicrobial and anti-insect activity. Constitutive expression of a lycotoxin I odified for oral toxicity to insects in tobacco (Nicotiana abacum) conferred significantly enhanced resis...

  17. Lumazine Peptides from the Marine-Derived Fungus Aspergillus terreus

    Directory of Open Access Journals (Sweden)

    Minjung You

    2015-03-01

    Full Text Available Terrelumamides A (1 and B (2, two new lumazine-containing peptides, were isolated from the culture broth of the marine-derived fungus Aspergillus terreus. From the results of combined spectroscopic and chemical analyses, the structures of these compounds were determined to be linear assemblies of 1-methyllumazine-6-carboxylic acid, an amino acid residue and anthranilic acid methyl ester connected by peptide bonds. These new compounds exhibited pharmacological activity by improving insulin sensitivity, which was evaluated in an adipogenesis model using human bone marrow mesenchymal stem cells. In addition, the compounds exhibited fluorescence changes upon binding to DNA, demonstrating their potential applications to DNA sequence recognition.

  18. Using X-PEEM to study biomaterials: Protein and peptide adsorption to a polystyrene-poly(methyl methacrylate)-b-polyacrylic acid blend

    Energy Technology Data Exchange (ETDEWEB)

    Leung, Bonnie O. [Chemistry and Chemical Biology, BIMR, McMaster University, Hamilton, ON, Canada L8S 4M1 (Canada); Hitchcock, Adam P., E-mail: aph@mcmaster.ca [Chemistry and Chemical Biology, BIMR, McMaster University, Hamilton, ON, Canada L8S 4M1 (Canada); Cornelius, Rena M.; Brash, John L. [School of Biomedical Engineering, McMaster University, Hamilton, ON, Canada L8S 4M1 (Canada); Scholl, Andreas; Doran, Andrew [Advanced Light Source, Berkeley Lab, Berkeley, CA 94720 (United States)

    2012-10-15

    Highlights: Black-Right-Pointing-Pointer We review applications of synchrotron X-PEEM to biomaterials. Black-Right-Pointing-Pointer We report characterization of a PS/PMMA-b-PAA blend surface by AFM and X-PEEM. Black-Right-Pointing-Pointer We report quantitative mapping of protein (HSA) and peptide adsorption on PS/PMMA-b-PAA. Black-Right-Pointing-Pointer We report how this adsorption changes with pH. -- Abstract: Recent synchrotron-based soft X-ray photoemission electron microscopy (X-PEEM) studies of protein and peptide interaction with phase segregated and patterned polymer surfaces in the context of optimization of candidate biomaterials are reviewed and a study of a new system is reported. X-PEEM and atomic force microscopy (AFM) were used to investigate the morphology of a phase-segregated thin film of a polystyrene/poly(methyl methacrylate)-b-polyacrylic acid (PS/PMMA-PAA) blend, and its interactions with negatively charged human serum albumin (HSA) and positively charged SUB-6 (a cationic antimicrobial peptide, RWWKIWVIRWWR-NH{sub 2}) at several pHs. At neutral pH, where the polymer surface is partially negatively charged, HSA and SUB-6 peptide showed contrasting adsorption behavior which is interpreted in terms of differences in their electrostatic interactions with the polymer surface.

  19. Peptide Macrocycles Featuring a Backbone Secondary Amine: A Convenient Strategy for the Synthesis of Lipidated Cyclic and Bicyclic Peptides on Solid Support

    DEFF Research Database (Denmark)

    Oddo, Alberto; Münzker, Lena; Hansen, Paul Robert

    2015-01-01

    A convenient strategy for the on-resin synthesis of macrocyclic peptides (3- to 13-mers) via intramolecular halide substitution by a diamino acid is described. The method is compatible with standard Fmoc/tBu SPPS and affords a tail-to-side-chain macrocyclic peptide featuring an endocyclic secondary...

  20. Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary

    DEFF Research Database (Denmark)

    Fenger, M; Johnsen, A H

    1988-01-01

    Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well...... (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders...... amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests...

  1. Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy.

    Science.gov (United States)

    Das, Priyadip; Duanias-Assaf, Tal; Reches, Meital

    2017-03-06

    The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

  2. Prediction of binding modes between protein L-isoaspartyl (D-aspartyl) O-methyltransferase and peptide substrates including isomerized aspartic acid residues using in silico analytic methods for the substrate screening.

    Science.gov (United States)

    Oda, Akifumi; Noji, Ikuhiko; Fukuyoshi, Shuichi; Takahashi, Ohgi

    2015-12-10

    Because the aspartic acid (Asp) residues in proteins are occasionally isomerized in the human body, not only l-α-Asp but also l-β-Asp, D-α-Asp and D-β-Asp are found in human proteins. In these isomerized aspartic acids, the proportion of D-β-Asp is the largest and the proportions of l-β-Asp and D-α-Asp found in human proteins are comparatively small. To explain the proportions of aspartic acid isomers, the possibility of an enzyme able to repair l-β-Asp and D-α-Asp is frequently considered. The protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PIMT) is considered one of the possible repair enzymes for l-β-Asp and D-α-Asp. Human PIMT is an enzyme that recognizes both l-β-Asp and D-α-Asp, and catalyzes the methylation of their side chains. In this study, the binding modes between PIMT and peptide substrates containing l-β-Asp or D-α-Asp residues were investigated using computational protein-ligand docking and molecular dynamics simulations. The results indicate that carboxyl groups of both l-β-Asp and D-α-Asp were recognized in similar modes by PIMT and that the C-terminal regions of substrate peptides were located in similar positions on PIMT for both the l-β-Asp and D-α-Asp peptides. In contrast, for peptides containing l-α-Asp or D-β-Asp residues, which are not substrates of PIMT, the computationally constructed binding modes between PIMT and peptides greatly differed from those between PIMT and substrates. In the nonsubstrate peptides, not inter- but intra-molecular hydrogen bonds were observed, and the conformations of peptides were more rigid than those of substrates. Thus, the in silico analytical methods were able to distinguish substrates from nonsubstrates and the computational methods are expected to complement experimental analytical methods. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Acylation of Therapeutic Peptides

    DEFF Research Database (Denmark)

    Trier, Sofie; Henriksen, Jonas Rosager; Jensen, Simon Bjerregaard

    ) , which promotes intestinal growth and is used to treat bowel disorders such as inflammatory bowel diseases and short bowel syndrome, and the 32 amino acid salmon calcitonin (sCT), which lowers blood calcium and is employed in the treatment of post-menopausal osteoporosis and hypercalcemia. The two...... peptides are similar in size and structure, but oppositely charged at physiological pH. Both peptides were acylated with linear acyl chains of systematically increasing length, where sCT was furthermore acylated at two different positions on the peptide backbone. For GLP-2, we found that increasing acyl...... remained optimal overall. The results indicate that rational acylation of GLP-2 can increase its in vitro intestinal absorption, alone or in combination with permeation enhancers, and are consistent with the initial project hypothesis. For sCT, an unpredicted effect of acylation largely superseded...

  4. Cloning and heterologous expression of the antibiotic peptide (ABP) genes from Rhizopus oligosporus NBRC 8631.

    Science.gov (United States)

    Yamada, Osamu; Sakamoto, Kazutoshi; Tominaga, Mihoko; Nakayama, Tasuku; Koseki, Takuya; Fujita, Akiko; Akita, Osamu

    2005-03-01

    We carried out protein sequencing of purified Antibiotic Peptide (ABP), and cloned two genes encoding this peptide as abp1 and abp2, from Rhizopus oligosporus NBRC 8631. Both genes contain an almost identical 231-bp segment, with only 3 nucleotide substitutions, encoding a 77 amino acid peptide. The abp gene product comprises a 28 amino acid signal sequence and a 49 amino acid mature peptide. Northern blot analysis showed that at least one of the abp genes is transcribed in R. oligosporus NBRC 8631. A truncated form of abp1 encoding only the mature peptide was fused with the alpha-factor signal peptide and engineered for expression in Pichia pastoris SMD1168H. Culture broth of the recombinant Pichia displayed ABP activity against Bacillus subtilis NBRC 3335 after induction of heterologous gene expression. This result indicates that mature ABP formed the active structure without the aid of other factors from R. oligosporus, and was secreted.

  5. Oxidation of protein tyrosine or methionine residues: From the amino acid to the peptide

    Energy Technology Data Exchange (ETDEWEB)

    Berges, J [Universite Pierre et Marie Curie, UMR 7616, Laboratoire de Chimie Theorique, 75005 Paris (France); Trouillas, P [EA 4021 Faculte de Pharmacie, 2 Rue du Dr. Marcland, 87025 Limoges Cedex (France); Houee-Levin, C, E-mail: jb@lct.jussieu.fr, E-mail: patrick.trouillas@unilim.fr, E-mail: chantal.houee@u-psud.fr [Universite Paris Sud, UMR 8000, Laboratoire de Chimie Physique, 91405 Orsay (France) (France)

    2011-01-01

    Methionine and tyrosine are competing targets of oxidizing free radicals in peptides or proteins. The first step is the addition of OH radicals either on the sulphur atom of methionine, followed by OH{sup -} elimination, or on the aromatic cycle of tyrosine. The next step can be stabilization of methionine radical cation by a two centre-three electron bond, or intramolecular electron transfer from tyrosine to the methionine radical cation. In this latter case a tyrosine radical is formed, which appears deprotonated. In a first step we have compared the stability of the OH radical adducts on Methionine or on Tyrosine. In agreement with experimental results, the thermodynamical data indicate that the OH adduct on Tyrosine and the radical cation are more stable than those on methionine. In a second step we have investigated the stabilization of the radical cations of Methionine by formation of intramolecular S:X two-center three-electron bond (X=S, N, O). Finally we have compared the spin densities on separated amino acids to that in a radical pentapeptide, methionine enkephalin. One observes a delocalisation of the orbital of the odd electron on the sulfur atom of Met and on the cycle of Tyr. The peptidic chain is also concerned.

  6. Oxidation of protein tyrosine or methionine residues: From the amino acid to the peptide

    International Nuclear Information System (INIS)

    Berges, J; Trouillas, P; Houee-Levin, C

    2011-01-01

    Methionine and tyrosine are competing targets of oxidizing free radicals in peptides or proteins. The first step is the addition of OH radicals either on the sulphur atom of methionine, followed by OH - elimination, or on the aromatic cycle of tyrosine. The next step can be stabilization of methionine radical cation by a two centre-three electron bond, or intramolecular electron transfer from tyrosine to the methionine radical cation. In this latter case a tyrosine radical is formed, which appears deprotonated. In a first step we have compared the stability of the OH radical adducts on Methionine or on Tyrosine. In agreement with experimental results, the thermodynamical data indicate that the OH adduct on Tyrosine and the radical cation are more stable than those on methionine. In a second step we have investigated the stabilization of the radical cations of Methionine by formation of intramolecular S:X two-center three-electron bond (X=S, N, O). Finally we have compared the spin densities on separated amino acids to that in a radical pentapeptide, methionine enkephalin. One observes a delocalisation of the orbital of the odd electron on the sulfur atom of Met and on the cycle of Tyr. The peptidic chain is also concerned.

  7. Recent studies on the antimicrobial peptides lactoferricin and lactoferrampin.

    Science.gov (United States)

    Yin, C; Wong, J H; Ng, T B

    2014-01-01

    Lactoferricin and lactoferrampin, peptides derived from the whey protein lactoferrin, are antimicrobial agents with a promising prospect and are currently one of the research focuses. In this review, a basic introduction including location and solution structures of these two peptides is given. Their biological activities encompassing antiviral, antibacterial, antifungal and anti-inflammatory activities with possible mechanisms are mentioned. In terms of modification studies, research about identification of their active derivatives and crucial amino acid residues is also discussed. Various attempts at modification of lactoferricin and lactoferrampin such as introducing big hydrophobic side-chains; employing special amino acids for synthesis; N-acetylization, amidation, cyclization and peptide chimera are summarized. The studies on lactoferricin-lactoferrampin chimera are discussed in detail. Future prospects of lactoferricin and lactoferrampin are covered.

  8. InverPep: A database of invertebrate antimicrobial peptides.

    Science.gov (United States)

    Gómez, Esteban A; Giraldo, Paula; Orduz, Sergio

    2017-03-01

    The aim of this work was to construct InverPep, a database specialised in experimentally validated antimicrobial peptides (AMPs) from invertebrates. AMP data contained in InverPep were manually curated from other databases and the scientific literature. MySQL was integrated with the development platform Laravel; this framework allows to integrate programming in PHP with HTML and was used to design the InverPep web page's interface. InverPep contains 18 separated fields, including InverPep code, phylum and species source, peptide name, sequence, peptide length, secondary structure, molar mass, charge, isoelectric point, hydrophobicity, Boman index, aliphatic index and percentage of hydrophobic amino acids. CALCAMPI, an algorithm to calculate the physicochemical properties of multiple peptides simultaneously, was programmed in PERL language. To date, InverPep contains 702 experimentally validated AMPs from invertebrate species. All of the peptides contain information associated with their source, physicochemical properties, secondary structure, biological activity and links to external literature. Most AMPs in InverPep have a length between 10 and 50 amino acids, a positive charge, a Boman index between 0 and 2 kcal/mol, and 30-50% hydrophobic amino acids. InverPep includes 33 AMPs not reported in other databases. Besides, CALCAMPI and statistical analysis of InverPep data is presented. The InverPep database is available in English and Spanish. InverPep is a useful database to study invertebrate AMPs and its information could be used for the design of new peptides. The user-friendly interface of InverPep and its information can be freely accessed via a web-based browser at http://ciencias.medellin.unal.edu.co/gruposdeinvestigacion/prospeccionydisenobiomoleculas/InverPep/public/home_en. Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

  9. 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.

  10. A multiple multicomponent approach to chimeric peptide-peptoid podands.

    Science.gov (United States)

    Rivera, Daniel G; León, Fredy; Concepción, Odette; Morales, Fidel E; Wessjohann, Ludger A

    2013-05-10

    The success of multi-armed, peptide-based receptors in supramolecular chemistry traditionally is not only based on the sequence but equally on an appropriate positioning of various peptidic chains to create a multivalent array of binding elements. As a faster, more versatile and alternative access toward (pseudo)peptidic receptors, a new approach based on multiple Ugi four-component reactions (Ugi-4CR) is proposed as a means of simultaneously incorporating several binding and catalytic elements into organizing scaffolds. By employing α-amino acids either as the amino or acid components of the Ugi-4CRs, this multiple multicomponent process allows for the one-pot assembly of podands bearing chimeric peptide-peptoid chains as appended arms. Tripodal, bowl-shaped, and concave polyfunctional skeletons are employed as topologically varied platforms for positioning the multiple peptidic chains formed by Ugi-4CRs. In a similar approach, steroidal building blocks with several axially-oriented isocyano groups are synthesized and utilized to align the chimeric chains with conformational constrains, thus providing an alternative to the classical peptido-steroidal receptors. The branched and hybrid peptide-peptoid appendages allow new possibilities for both rational design and combinatorial production of synthetic receptors. The concept is also expandable to other multicomponent reactions. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Preservation of peptide moieties in three Spanish sulfur-rich Tertiary kerogens

    Energy Technology Data Exchange (ETDEWEB)

    Rio, J.C. del [Consejo Superior de Investigaciones Cientificas, Seville (Spain). Inst. de Recursos Naturales y Agrobiologia; Olivella, M.A.; Heras, F.X.D. de las [Escola Universitaria Politecnica de Manresa, Catalonia (Spain); Knicker, H. [Technische Universitaet Muenchen (Germany). Lehrstuhl fuer Bodenkunde

    2004-09-01

    Thermochemolysis with tetramethylammonium hydroxide (TMAH) and solid-state {sup 15}N NMR were utilized for the characterization of refractory organic nitrogen in Tertiary Spanish kerogens. The samples included sulfur-rich oil shales from the Ribesalbes (Serravallian), Libros (Tortonian) and Cerdanya (Tortonian) basins. Analysis using solid state {sup 15}N NMR showed that part of the refractory nitrogen in the kerogens corresponds to amide groups. Moreover, the release of amino acid derivatives after pyrolysis in the presence of TMAH indicated that this amide-N arose from peptide moieties. The amino acids released from the kerogens were dominated by high amounts of glycine and alanine. Minor amounts of aspartic acid, serine, {alpha}-aminobutyric acid and other unidentified amino acids were also detected. Because proteinaceous structures, including small peptides, are generally considered as being highly sensitive to diagenetic degradation, encapsulation of labile peptide material into aliphatic structures in S-rich kerogens (probably via lipid sulfurization) has been proposed to explain the survival of these moieties. Substantial amounts of fatty acids (as methyl esters) were also released from all the kerogens after pyrolysis/TMAH, indicating their highly aliphatic character. The production of both fatty acids and amino acids from the kerogens supports the encapsulation process. (author)

  12. Whey Peptide-Based Formulas With ω-3 Fatty Acids Are Protective in Lipopolysaccharide-Mediated Sepsis.

    Science.gov (United States)

    Tsutsumi, Rie; Horikawa, Yousuke T; Kume, Katsuyoshi; Tanaka, Katsuya; Kasai, Asuka; Kadota, Takako; Tsutsumi, Yasuo M

    2015-07-01

    Sepsis and septic shock syndrome are among the leading causes of death in critically ill patients. Lipopolysaccharide (LPS) released by bacteria within the colon may translocate across a compromised epithelium, leading to oxidative stress, inflammation, sepsis, and eventually death. We examined the effects of a whey-based enteral formula high in cysteine (antioxidant precursor) and the addition of ω-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), against a mouse model of LPS-induced sepsis. Mice were fed either a whey-based diet with EPA-DHA (PAF), a whey-based diet without EPA-DHA (PSTD), or a casein-based control diet (CONT). Mice fed PAF or PSTD were protected against LPS-induced weight loss. Whey-based diets suppressed inflammatory cytokine release and oxidative stress damage. Furthermore, PAF and PSTD were able to inhibit autophagy, a mechanism in which the cell recycles damaged organelles. These anti-inflammatory and antioxidative effects of PSTD and PAF resulted in decreased liver inflammation and intestinal damage and promoted protective microbiota within the intestines. These data suggest a clinical role for whey peptide-based diets in promoting healing and recovery in critically ill patients. © 2014 American Society for Parenteral and Enteral Nutrition.

  13. Use of the 2-chlorotrityl chloride resin for microwave-assisted solid phase peptide synthesis.

    Science.gov (United States)

    Ieronymaki, Matthaia; Androutsou, Maria Eleni; Pantelia, Anna; Friligou, Irene; Crisp, Molly; High, Kirsty; Penkman, Kirsty; Gatos, Dimitrios; Tselios, Theodore

    2015-09-01

    A fast and efficient microwave (MW)-assisted solid-phase peptide synthesis protocol using the 2-chlorotrityl chloride resin and the Fmoc/tBu methodology, has been developed. The established protocol combines the advantages of MW irradiation and the acid labile 2-chlorotrityl chloride resin. The effect of temperature during the MW irradiation, the degree of resin substitution during the coupling of the first amino acids and the rate of racemization for each amino acid were evaluated. The suggested solid phase methodology is applicable for orthogonal peptide synthesis and for the synthesis of cyclic peptides. © 2015 Wiley Periodicals, Inc.

  14. Side Chain Cyclized Aromatic Amino Acids

    DEFF Research Database (Denmark)

    Van der Poorten, Olivier; Knuhtsen, Astrid; Sejer Pedersen, Daniel

    2016-01-01

    Constraining the conformation of flexible peptides is a proven strategy to increase potency, selectivity, and metabolic stability. The focus has mostly been on constraining the backbone dihedral angles; however, the correct orientation of the amino acid side chains (χ-space) that constitute...... the peptide pharmacophore is equally important. Control of χ-space utilizes conformationally constrained amino acids that favor, disfavor, or exclude the gauche (-), the gauche (+), or the trans conformation. In this review we focus on cyclic aromatic amino acids in which the side chain is connected...... to the peptide backbone to provide control of χ(1)- and χ(2)-space. The manifold applications for cyclized analogues of the aromatic amino acids Phe, Tyr, Trp, and His within peptide medicinal chemistry are showcased herein with examples of enzyme inhibitors and ligands for G protein-coupled receptors....

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

    Science.gov (United States)

    Elliott, Tim; Williams, Anthony

    2005-10-01

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

  16. Degradation of peptides by gamma-irradiation, 2

    Energy Technology Data Exchange (ETDEWEB)

    Oku, Tadatake; Yoshida, Shigeki; Kondo, Mitsumasa; Ishida, Tomoharu; Fukui, Manabu; Ito, Teiichiro (Nihon Univ., Tokyo (Japan). Coll. of Agriculture and Veterinary Medicine)

    1990-10-01

    The radiolytic products of two kinds of dipeptides containing aromatic amino acid, gly-L-tyr and L-tyr-gly in 1 mM aqueous solution in the presence of air were examined by gamma-irradiation at doses of about 6, 12 and 25 kGy. Peptide samples in aqueous solution were analyzed by HPLC and GC after gamma-irradiation. Amides which the amounts of formation was very small, were collected several times by an amino acid autoanalyzer and isolated by HPLC. The ninhydrin-positive products from gly-L-tyr were detected gly, tyr, dopa, asp, ammonia, methylamine, ethylamine and glycinamide. The products from L-tyr-gly were tyr, gly, dopa, asp, ammonia, methylamine and ethylamine, but tyrosinamide was not confirmed. The total amounts of ninhydrin-positive products formed were less than the decreasing amount of each peptide at every irradiation dose. Methanal and ethanal were detected in both peptides. A radiolytic pathway of gly-L-tyr and L-tyr-gly was estimated from these results. (author).

  17. Summarization on the synthesis and radionuclide-labeling of peptide nucleic acid for an oligonucleotide analogue

    International Nuclear Information System (INIS)

    Song, Hongtao; Zhang, Huaming; Gao, Hui

    2009-04-01

    Peptide nucleic acid (PNA), which is one kind of antisense nucleic acid compounds and an oligonucleotide analogue that binds strongly to DNA and RNA in a sequence specific manner, has its unique advantages in the field of molecular diagnostics and treatment of diseases. Now, people gradually attach more importance to PNA. To optimize the application of PNA in genetic re- search and therapy, a great number of backbone modifications on the newly- type structures of PNA were synthesized to improve its physicochemical proper- ties, such as hybridization speciality, solubility in biofluid, or cell permeability. The modified PNA labeled with radionuclides, which can obtain the aim at specific target and minimal non-target trauma, has important role in research and application of tumorous genitherapy. Here a review on the basic synthesis idea and several primary synthetic methods of PNA analogs was given, and also correlative studies and expectation on the compounds belonging to PNA series labeled with radionuclides were included. (authors)

  18. Effect of sequence and stereochemistry reversal on p53 peptide mimicry.

    Directory of Open Access Journals (Sweden)

    Alessio Atzori

    Full Text Available Peptidomimetics effective in modulating protein-protein interactions and resistant to proteolysis have potential in therapeutic applications. An appealing yet underperforming peptidomimetic strategy is to employ D-amino acids and reversed sequences to mimic a lead peptide conformation, either separately or as the combined retro-inverso peptide. In this work, we examine the conformations of inverse, reverse and retro-inverso peptides of p53(15-29 using implicit solvent molecular dynamics simulation and circular dichroism spectroscopy. In order to obtain converged ensembles for the peptides, we find enhanced sampling is required via the replica exchange molecular dynamics method. From these replica exchange simulations, the D-peptide analogues of p53(15-29 result in a predominantly left-handed helical conformation. When the parent sequence is reversed sequence as either the L-peptide and D-peptide, these peptides display a greater helical propensity, feature reflected by NMR and CD studies in TFE/water solvent. The simulations also indicate that, while approximately similar orientations of the side-chains are possible by the peptide analogues, their ability to mimic the parent peptide is severely compromised by backbone orientation (for D-amino acids and side-chain orientation (for reversed sequences. A retro-inverso peptide is disadvantaged as a mimic in both aspects, and further chemical modification is required to enable this concept to be used fruitfully in peptidomimetic design. The replica exchange molecular simulation approach adopted here, with its ability to provide detailed conformational insights into modified peptides, has potential as a tool to guide structure-based design of new improved peptidomimetics.

  19. Selenium as an alternative peptide label - comparison to fluorophore-labelled penetratin

    DEFF Research Database (Denmark)

    Hyrup Møller, Laura; Bahnsen, Jesper Søborg; Nielsen, Hanne Mørck

    2015-01-01

    lysates, primarily the intact peptide (PenMSe, TAMRA-PenMSe or TAMRA-Pen) was observed. Selenium labelling caused minimal alteration of the physicochemical properties of the peptide and allowed for absolute quantitative determination of cellular uptake by inductively coupled plasma mass spectrometry......In the present study, the impact on peptide properties of labelling peptides with the fluorophore TAMRA or the selenium (Se) containing amino acid SeMet was evaluated. Three differently labelled variants of the cell-penetrating peptide (CPP) penetratin (Pen) were synthesized, PenMSe, TAMRA....... Selenium is thus proposed as a promising alternative label for quantification of peptides in general, altering the properties of the peptide to a minor extent as compared to commonly used peptide labels....

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

    Science.gov (United States)

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

    2018-05-01

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

  1. Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins.

    Science.gov (United States)

    Amiche, M; Ducancel, F; Mor, A; Boulain, J C; Menez, A; Nicolas, P

    1994-07-08

    The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family

  2. Peptides derivatized with bicyclic quaternary ammonium ionization tags. Sequencing via tandem mass spectrometry.

    Science.gov (United States)

    Setner, Bartosz; Rudowska, Magdalena; Klem, Ewelina; Cebrat, Marek; Szewczuk, Zbigniew

    2014-10-01

    Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1-azabicyclo[2.2.2]octane (ABCO) or 1,4-diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI-MS) and longer retention times on the reverse-phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision-induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a- and b-type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision-induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI-MS/MS sequencing of peptides. Copyright © 2014 John Wiley & Sons, Ltd.

  3. Anionic magnetite nanoparticle conjugated with pyrrolidinyl peptide nucleic acid for DNA base discrimination

    International Nuclear Information System (INIS)

    Khadsai, Sudarat; Rutnakornpituk, Boonjira; Vilaivan, Tirayut; Nakkuntod, Maliwan; Rutnakornpituk, Metha

    2016-01-01

    Magnetite nanoparticles (MNPs) were surface modified with anionic poly(N-acryloyl glycine) (PNAG) and streptavidin for specific interaction with biotin-conjugated pyrrolidinyl peptide nucleic acid (PNA). Hydrodynamic size (D h ) of PNAG-grafted MNPs varied from 334 to 496 nm depending on the loading ratio of the MNP to NAG in the reaction. UV–visible and fluorescence spectrophotometries were used to confirm the successful immobilization of streptavidin and PNA on the MNPs. About 291 pmol of the PNA/mg MNP was immobilized on the particle surface. The PNA-functionalized MNPs were effectively used as solid supports to differentiate between fully complementary and non-complementary/single-base mismatch DNA using the PNA probe. These novel anionic MNPs can be efficiently applicable for use as a magnetically guidable support for DNA base discrimination.Graphical Abstract

  4. Application of biomimetic HPLC to estimate lipophilicity, protein and phospholipid binding of potential peptide therapeutics

    Directory of Open Access Journals (Sweden)

    Klara Livia Valko

    2018-06-01

    Full Text Available Peptide therapeutics are new modalities offering several challenges to drug discovery. They are generally less stable and permeable in vivo. The characterization of their lipophilicity cannot be carried out using the traditional in silico or wet octanol/water partition coefficients. The prediction of their in vivo distribution and permeability is also challenging. In this paper, it is demonstrated that the biomimetic properties such as lipophilicity, protein and phospholipid binding can be easily assessed by HPLC using chemically bonded protein and immobilized artificial membrane (IAM stationary phases. The obtained properties for a set of potential therapeutic peptides with 3 to 33 amino acids have been analysed and it was found that similar characteristics of the properties could be observed as for small molecule drugs. The albumin binding showed correlation with their measured lipophilicity on the C-18 stationary phase with acidic peptides showing stronger than expected albumin binding. The (IAM chromatography revealed peptide membrane affinity, which was stronger for positively charged peptides (containing arginine and showed correlation to the alpha-1-acid glycoprotein (AGP binding, which was also stronger for positively charged compounds. The in vivo volume of distribution and drug efficiency of the peptides have been estimated using the models developed for small molecules. One of the candidate linear peptides has been assessed in various cellular and in vivo assays and the results have confirmed the estimated cell partition and brain to plasma ratio. It can be demonstrated, that up to 21 amino acids, the peaks of the peptides obtained on the protein phase were symmetrical and narrow. The interaction of larger peptides with the protein stationary phases resulted in wide peaks showing multiple equilibrium processes with slow kinetics during chromatography. The larger peptides showed narrow and symmetrical peaks on the IAM column enabling

  5. Enhancement of thermo-stability and product tolerance of Pseudomonas putida nitrile hydratase by fusing with self-assembling peptide.

    Science.gov (United States)

    Liu, Yi; Cui, Wenjing; Liu, Zhongmei; Cui, Youtian; Xia, Yuanyuan; Kobayashi, Michihiko; Zhou, Zhemin

    2014-09-01

    Self-assembling amphipathic peptides (SAPs) are the peptides that can spontaneously assemble into ordered nanostructures. It has been reported that the attachment of SAPs to the N- or C-terminus of an enzyme can benefit the thermo-stability of the enzyme. Here, we discovered that the thermo-stability and product tolerance of nitrile hydratase (NHase) were enhanced by fusing with two of the SAPs (EAK16 and ELK16). When the ELK16 was fused to the N-terminus of β-subunit, the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16 fused NHase in the N-terminus of β-subunit (SAP-NHase-1) and ELK16 fused NHase in the C-terminus of β-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the deactivation of the wild-type NHase after 30 min incubation at 50°C, SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 45%, 30% and 50% activity; after treatment in the buffer containing 10% acrylamide, the wild-type retained 30% activity, while SAP-NHase-1, SAP-NHase-2 and SAP-NHase-10 retained 52%, 42% and 55% activity. These SAP-NHases with enhanced thermo-stability and product tolerance would be helpful for further industrial applications of the NHase. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. A Maple Program That Illustrates the Effect of pH on Peptide Charge

    Science.gov (United States)

    Sokolik, Charles W.

    1998-11-01

    One topic covered early in an introductory biochemistry course is acid-base chemistry and the Henderson-Hasselbalch equation (buffer equation). Using this equation a biochemistry student can determine the partial charges of amino acids in a peptide chain. This is an important concept to master for a student who is learning the structure-function relationship in proteins. The program described in this paper, written for Maple V, release 3 (Waterloo Maple Software, Waterloo, ON, Canada), uses the Henderson-Hasselbalch equation to calculate the partial charges of individual amino acids and the net charge of a peptide over the pH range 0 to 14. The amino acid sequence of a peptide is entered and an animated histogram is displayed illustrating the partial charge of the amino acids over the pH range. A graph showing the net charge of the peptide from pH 0 to 14 is also given. The program has been used with success in an introductory biochemistry course as an in-class demonstration as well as for individual homework assignments. The program is available through the Web page of the Journal of Chemical Education.

  7. On-resin conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) congeners.

    Science.gov (United States)

    Mullen, Daniel G; Weigel, Benjamin; Barany, George; Distefano, Mark D

    2010-05-01

    The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg(2+)) that are orthogonal to the acidic milieu used for global deprotection in Fmoc-based solid-phase peptide synthesis. This use of a toxic heavy metal for deprotection has limited the usefulness of Acm in peptide synthesis. The Acm group may be converted to the Scm derivative that can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive conditions to generate unprotected cysteine. Conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on-resin Acm to Scm conversion that allows the preparation of Cys(Scm)-containing peptides under conditions that do not modify other amino acids. (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.

  8. Rapid Identification of Staphylococcus aureus and Methicillin Resistance by Flow Cytometry Using a Peptide Nucleic Acid Probe ▿

    Science.gov (United States)

    Shrestha, Nabin K.; Scalera, Nikole M.; Wilson, Deborah A.; Brehm-Stecher, Byron; Procop, Gary W.

    2011-01-01

    A total of 56 Staphylococcus aureus isolates incubated for 2 h in the presence or absence of oxacillin were analyzed by flow cytometry after labeling with an S. aureus-specific peptide nucleic acid (PNA) probe. Two defined ratios, the paired signal count ratio (PSCR) and the gate signal count ratio (GSCR), differentiated methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) with sensitivities of 100% each and specificities of 96% and 100%, respectively. PMID:21795508

  9. Phage display peptide libraries: deviations from randomness and correctives

    Science.gov (United States)

    Ryvkin, Arie; Ashkenazy, Haim; Weiss-Ottolenghi, Yael; Piller, Chen; Pupko, Tal; Gershoni, Jonathan M

    2018-01-01

    Abstract Peptide-expressing phage display libraries are widely used for the interrogation of antibodies. Affinity selected peptides are then analyzed to discover epitope mimetics, or are subjected to computational algorithms for epitope prediction. A critical assumption for these applications is the random representation of amino acids in the initial naïve peptide library. In a previous study, we implemented next generation sequencing to evaluate a naïve library and discovered severe deviations from randomness in UAG codon over-representation as well as in high G phosphoramidite abundance causing amino acid distribution biases. In this study, we demonstrate that the UAG over-representation can be attributed to the burden imposed on the phage upon the assembly of the recombinant Protein 8 subunits. This was corrected by constructing the libraries using supE44-containing bacteria which suppress the UAG driven abortive termination. We also demonstrate that the overabundance of G stems from variant synthesis-efficiency and can be corrected using compensating oligonucleotide-mixtures calibrated by mass spectroscopy. Construction of libraries implementing these correctives results in markedly improved libraries that display random distribution of amino acids, thus ensuring that enriched peptides obtained in biopanning represent a genuine selection event, a fundamental assumption for phage display applications. PMID:29420788

  10. Lack of effects of a single high-fat meal enriched with vegetable n-3 or a combination of vegetable and marine n-3 fatty acids on intestinal peptide release and adipokines in healthy female subjects

    Directory of Open Access Journals (Sweden)

    Ingunn Naverud

    2016-08-01

    Full Text Available Peptides released from the small intestine and colon regulate short-term food intake by suppressing appetite and inducing satiety. Intake of marine omega-3 (n-3 fatty acids from fish and fish oils is associated with beneficial health effects, whereas the relation between intake of the vegetable n-3 fatty acid α-linolenic acid and diseases is less clear. The aim of the present study was to investigate the postprandial effects of a single high-fat meal enriched with vegetable n-3 or a combination of vegetable and marine n-3 fatty acids with their different unsaturated fatty acid composition on intestinal peptide release and the adipose tissue. Fourteen healthy lean females consumed three test meals with different fat quality in a fixed order. The test meal consisted of three cakes enriched with coconut fat, linseed oil and a combination of linseed and cod liver oil. The test days were separated by two weeks. Fasting and postprandial blood samples at three and six hours after intake were analysed. A significant postprandial effect was observed for cholecystokinin, peptide YY, glucose-dependent insulinotropic polypeptide, amylin and insulin which increased, while leptin decreased postprandially independent of the fat composition in the high-fat meal. In conclusion, in healthy, young, lean females, an intake of a high-fat meal enriched with n-3 fatty acids from different origin stimulates intestinal peptide release without any difference between the different fat compositions.

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

    International Nuclear Information System (INIS)

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

    2002-01-01

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

  12. Tumor-Triggered Geometrical Shape Switch of Chimeric Peptide for Enhanced in Vivo Tumor Internalization and Photodynamic Therapy.

    Science.gov (United States)

    Han, Kai; Zhang, Jin; Zhang, Weiyun; Wang, Shibo; Xu, Luming; Zhang, Chi; Zhang, Xianzheng; Han, Heyou

    2017-03-28

    Geometrical shape of nanoparticles plays an important role in cellular internalization. However, the applicability in tumor selective therapeutics is still scarcely reported. In this article, we designed a tumor extracellular acidity-responsive chimeric peptide with geometrical shape switch for enhanced tumor internalization and photodynamic therapy. This chimeric peptide could self-assemble into spherical nanoparticles at physiological condition. While at tumor extracellular acidic microenvironment, chimeric peptide underwent detachment of acidity-sensitive 2,3-dimethylmaleic anhydride groups. The subsequent recovery of ionic complementarity between chimeric peptides resulted in formation of rod-like nanoparticles. Both in vitro and in vivo studies demonstrated that this acidity-triggered geometrical shape switch endowed chimeric peptide with accelerated internalization in tumor cells, prolonged accumulation in tumor tissue, enhanced photodynamic therapy, and minimal side effects. Our results suggested that fusing tumor microenvironment with geometrical shape switch should be a promising strategy for targeted drug delivery.

  13. Synthesis of Functional Polyester Based on Polylactic Acid and Its Effect on PC12 Cells after Coupling with Small Peptides

    Directory of Open Access Journals (Sweden)

    Na Qiang

    2016-01-01

    Full Text Available Polyesters containing functional groups are a suitable candidate matrix for cell culture in tissue engineering. Three types of semicrystalline copolymer poly(L-lactide-co-β-malic acid [P(LA-co-BMD] with pendent carboxyl groups were synthesized in this study. The functional monomer 3(S-[(benzyloxycarbonylmethyl]-1,4-dioxane-2,5-dione (BMD was synthesized using L-aspartic acid. The copolymer P(LA-co-BMD was then synthesized through ring-opening copolymerization of L-LA and BMD, with dodecanol as initiator and stannous octoate as catalyst. Copolymer structure was characterized by 1H nuclear magnetic resonance (1H NMR, gel permeation chromatography (GPC, and differential scanning calorimetry (DSC analyses. Results of 1H NMR and GPC analyses showed that the copolymers were synthesized successfully. DSC curves showed that the crystal melting peak and enthalpy decreased with increased BMD. The crystallinity of the copolymer was destroyed by the presence of the functional monomer. After deprotection, carboxyl groups were coupled with the isoleucine-lysine-valine-alanine-valine peptide through N-hydroxysuccinimide/dicyclohexylcarbodiimide method. The small peptide was beneficial to the axon growth of PC12 cells.

  14. Synthesis and evaluation of amphiphilic peptides as nanostructures and drug delivery tools

    Science.gov (United States)

    Sayeh, Naser Ali

    Intracellular delivery of cell-impermeable compounds in a variety cells using delivery systems have been extensively studied in recent years. Obtaining desirable cellular uptake levels often requires the administration of high quantities of drugs to achieve the expected intracellular biological effect. Thus, improving the translocation process across the plasma membrane will significantly reduce the quantity of required administered drug and consequently minimize the side effects in most of the cases. Efficient delivery of these molecules to the cells and tissues is a difficult challenge. Compounds with low cellular permeability are commonly considered to be of limited therapeutic value. Over the past few decades, several biomedical carriers, such as polymers, nanospheres, nanocapsules, liposomes, micelles, peptides and dendrimers have been widely used to deliver therapeutic and diagnostic agents to the cells. Biomaterials generated from nano-scale compounds have shown some promising data for delivery of many compounds in a number of diseases, such as viral infections, cancer, and genetic disorders. Although much progress has been achieved in this field, many challenges still remain, such as toxicity and limited stability. Liposomes suffer from poor stability in the bloodstream and leakage during storage. They tend to aggregate and fuse with or leak entrapped drugs, especially highly hydrophilic small molecules. For solid lipid nanoparticles (SLNs), drug expulsion after polymorphic transition during storage, inadequate loading capacity, and relatively high water content of the dispersions have been observed. Poly(lactic-coglycolic acid (PLGA) degrades in the body producing its original monomers of lactic acid and glycolic acid, which are the by-products of various metabolic pathways. However, this acidic microenvironment that occurs during degradation could negatively affect the stability of the loaded compound. Dendrimers can carry drugs as complexes or as

  15. Gold-catalyzed direct alkynylation of tryptophan in peptides using TIPS-EBX

    Directory of Open Access Journals (Sweden)

    Gergely L. Tolnai

    2016-04-01

    Full Text Available The selective functionalization of peptides containing only natural amino acids is important for the modification of biomolecules. In particular, the installation of an alkyne as a useful handle for bioconjugation is highly attractive, but the use of a carbon linker is usually required. Herein, we report the gold-catalyzed direct alkynylation of tryptophan in peptides using the hypervalent iodine reagent TIPS-EBX (1-[(triisopropylsilylethynyl]-1,2-benziodoxol-3(1H-one. The reaction proceeded in 50–78% yield under mild conditions and could be applied to peptides containing other nucleophilic and aromatic amino acids, such as serine, phenylalanine or tyrosine.

  16. Synthesis of a Hoechst 32258 Analogue Amino Acid Building Block for Direct Incorporation of a Fluorescent High-Affinity DNA Binding Motif into Peptides

    DEFF Research Database (Denmark)

    Harrit, Niels; Behrens, Carsten; Nielsen, P. E.

    2001-01-01

    The synthesis of a new versatile "Hoechst 33258-like" Boc-protected amino acid building block for peptide synthesis is described. It is demonstrated that this new ligand is an effective mimic of Hoechst 33258 in terms of DNA affinity and sequence specificity. Furthermore, this minor groove binder...

  17. A specific scenario for the origin of life and the genetic code based on peptide/oligonucleotide interdependence.

    Science.gov (United States)

    Griffith, Robert W

    2009-12-01

    Among various scenarios that attempt to explain how life arose, the RNA world is currently the most widely accepted scientific hypothesis among biologists. However, the RNA world is logistically implausible and doesn't explain how translation arose and DNA became incorporated into living systems. Here I propose an alternative hypothesis for life's origin based on cooperation between simple nucleic acids, peptides and lipids. Organic matter that accumulated on the prebiotic Earth segregated into phases in the ocean based on density and solubility. Synthesis of complex organic monomers and polymerization reactions occurred within a surface hydrophilic layer and at its aqueous and atmospheric interfaces. Replication of nucleic acids and translation of peptides began at the emulsified interface between hydrophobic and aqueous layers. At the core of the protobiont was a family of short nucleic acids bearing arginine's codon and anticodon that added this amino acid to pre-formed peptides. In turn, the survival and replication of nucleic acid was aided by the peptides. The arginine-enriched peptides served to sequester and transfer phosphate bond energy and acted as cohesive agents, aggregating nucleic acids and keeping them at the interface.

  18. Postprandial fate of amino acids: adaptation to molecular forms

    NARCIS (Netherlands)

    Nolles, J.A.

    2006-01-01

    During the postprandial phase dietary proteins are digested to peptides and amino acids and absorbed. Once absorbed the peptides are further hydrolyzed to amino acids and transported to the tissues. These amino acids are largely incorporated into body proteins. Not all amino acids are, however,

  19. Treating autoimmune disorders with venom-derived peptides.

    Science.gov (United States)

    Shen, Bingzheng; Cao, Zhijian; Li, Wenxin; Sabatier, Jean-Marc; Wu, Yingliang

    2017-09-01

    The effective treatment of autoimmune diseases remains a challenge. Voltage-gated potassium Kv1.3 channels, which are expressed in lymphocytes, are a new therapeutic target for treating autoimmune disease. Consequently, Kv1.3 channel-inhibiting venom-derived peptides are a prospective resource for new drug discovery and clinical application. Area covered: Preclinical and clinical studies have produced a wealth of information on Kv1.3 channel-inhibiting venom-derived peptides, especially from venomous scorpions and sea anemones. This review highlights the advances in screening and design of these peptides with diverse structures and potencies. It focuses on representative strategies for improving peptide selectivity and discusses the preclinical research on those venom-derived peptides as well as their clinical developmental status. Expert opinion: Encouraging results indicate that peptides isolated from the venom of venomous animals are a large resource for discovering immunomodulators that act on Kv1.3 channels. Since the structural diversity of venom-derived peptides determines the variety of their pharmacological activities, the design and optimization of venom-peptides for improved Kv1.3 channel-specificity has been advanced through some representative strategies, such as peptide chemical modification, amino acid residue truncation and binding interface modulation. These advances should further accelerate research, development and the future clinical application of venom-derived peptides selectively targeting Kv1.3 channels.

  20. Synthesis, analysis, and cytotoxic effects of novel SMAC-based peptides

    Czech Academy of Sciences Publication Activity Database

    Georgieva, M.; Dzimbova, T.; Sázelová, Petra; Detcheva, R.; Kašička, Václav; Momekov, G.; Pajpanova, T.

    2015-01-01

    Roč. 47, č. 8 (2015), s. 1687 ISSN 0939-4451. [International Congress on Amino Acids , Peptides and Proteins /14./. 03.08.2015-07.08.2015, Vienna] Institutional support: RVO:61388963 Keywords : apoptotic peptides * SMAC-mimetics * capillary electrophoresis Subject RIV: CB - Analytical Chemistry, Separation

  1. Peptide inhibitors of appressorium development in Glomerella cingulata.

    Science.gov (United States)

    Al-Samarrai, Taha H; Sullivan, Patrick A; Templeton, Matthew D; Farley, Peter C

    2002-04-09

    The phytopathogen Glomerella cingulata (anamorph: Colletotrichum gloeosporioides) infects host tissue by means of a specialised infection structure, the appressorium. The Saccharomyces cerevisiae alpha-mating factor pheromone, the Saccharomyces kluyveri alpha-mating factor pheromone and a hendecapeptide produced by G. cingulata inhibit appressorium development. The amino acid sequence of the G. cingulata peptide, GYFSYPHGNLF, is different from that of the mature pheromone peptides of other filamentous fungi. The peptide has sequence similarity with the Saccharomyces alpha-mating factor pheromones, but is unable to elicit growth arrest in S. cerevisiae.

  2. Co-assembly of Peptide Amphiphiles and Lipids into Supramolecular Nanostructures Driven by Anion-π Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Zhilin; Erbas, Aykut; Tantakitti, Faifan; Palmer, Liam C.; Jackman, Joshua A.; Olvera de la Cruz, Monica; Cho, Nam-Joon; Stupp, Samuel I. (Nanyang); (NWU)

    2017-06-01

    Co-assembly of binary systems driven by specific non-covalent interactions can greatly expand the structural and functional space of supramolecular nanostructures. We report here on the self-assembly of peptide amphiphiles and fatty acids driven primarily by anion-π interactions. The peptide sequences investigated were functionalized with a perfluorinated phenylalanine residue to promote anion-π interactions with carboxylate headgroups in fatty acids. These interactions were verified here by NMR and circular dichroism experiments as well as investigated using atomistic simulations. Positioning the aromatic units close to the N-terminus of the peptide backbone near the hydrophobic core of cylindrical nanofibers leads to strong anion-π interactions between both components. With a low content of dodecanoic acid in this position, the cylindrical morphology is preserved. However, as the aromatic units are moved along the peptide backbone away from the hydrophobic core, the interactions with dodecanoic acid transform the cylindrical supramolecular morphology into ribbon-like structures. Increasing the ratio of dodecanoic acid to PA leads to either the formation of large vesicles in the binary systems where the anion-π interactions are strong, or a heterogeneous mixture of assemblies when the peptide amphiphiles associate weakly with dodecanoic acid. Our findings reveal how co-assembly involving designed specific interactions can drastically change supramolecular morphology and even cross from nano to micro scales.

  3. Designing of peptides with desired half-life in intestine-like environment

    KAUST Repository

    Sharma, Arun

    2014-08-20

    Background: In past, a number of peptides have been reported to possess highly diverse properties ranging from cell penetrating, tumor homing, anticancer, anti-hypertensive, antiviral to antimicrobials. Owing to their excellent specificity, low-toxicity, rich chemical diversity and availability from natural sources, FDA has successfully approved a number of peptide-based drugs and several are in various stages of drug development. Though peptides are proven good drug candidates, their usage is still hindered mainly because of their high susceptibility towards proteases degradation. We have developed an in silico method to predict the half-life of peptides in intestine-like environment and to design better peptides having optimized physicochemical properties and half-life.Results: In this study, we have used 10mer (HL10) and 16mer (HL16) peptides dataset to develop prediction models for peptide half-life in intestine-like environment. First, SVM based models were developed on HL10 dataset which achieved maximum correlation R/R2 of 0.57/0.32, 0.68/0.46, and 0.69/0.47 using amino acid, dipeptide and tripeptide composition, respectively. Secondly, models developed on HL16 dataset showed maximum R/R2 of 0.91/0.82, 0.90/0.39, and 0.90/0.31 using amino acid, dipeptide and tripeptide composition, respectively. Furthermore, models that were developed on selected features, achieved a correlation (R) of 0.70 and 0.98 on HL10 and HL16 dataset, respectively. Preliminary analysis suggests the role of charged residue and amino acid size in peptide half-life/stability. Based on above models, we have developed a web server named HLP (Half Life Prediction), for predicting and designing peptides with desired half-life. The web server provides three facilities; i) half-life prediction, ii) physicochemical properties calculation and iii) designing mutant peptides.Conclusion: In summary, this study describes a web server \\'HLP\\' that has been developed for assisting scientific

  4. Amphipaths Differentially Modulate Membrane Surface Deformation in Rat Peritoneal Mast Cells During Exocytosis

    Directory of Open Access Journals (Sweden)

    Itsuro Kazama

    2013-04-01

    Full Text Available Background/Aims: Salicylate and chlorpromazine exert differential effects on the chemokine release from mast cells. Since these drugs are amphiphilic and preferentially partitioned into the lipid bilayers of the plasma membranes, they would induce some morphological changes in mast cells and thus affect the process of exocytosis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of salicylate and chlorpromazine on the membrane capacitance (Cm during exocytosis in rat peritoneal mast cells. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on plasma membrane deformation of the cells. Results: Salicylate dramatically accelerated the GTP-γ-S-induced increase in the Cm immediately after its application, whereas chlorpromazine significantly suppressed the increase. Treatment with salicylate increased the trapping of the dye on the cell surface, while treatment with chlorpromazine completely washed it out, indicating that both drugs induced membrane surface deformation in mast cells. Conclusion: This study demonstrated for the first time that membrane amphipaths, such as salicylate and chlorpromazine, may oppositely modulate the process of exocytosis in mast cells, as detected by the changes in the Cm. The plasma membrane deformation induced by the drugs was thought to be responsible for their differential effects.

  5. Complete sequences of glucagon-like peptide-1 from human and pig small intestine

    DEFF Research Database (Denmark)

    Orskov, C; Bersani, M; Johnsen, A H

    1989-01-01

    intestine of the proglucagon precursor were determined by pairs of basic amino acid residues flanking the two peptides. Earlier studies have shown that synthetic glucagon-like peptide-1 (GLP-1) synthesized according to the proposed structure (proglucagon 71-108 or because residue 108 is Gly, 72-107 amide......) had no physiological effects, whereas a truncated from of GLP-1, corresponding to proglucagon 78-107 amide, strongly stimulated insulin secretion and depressed glucagon secretion. To determine the amino acid sequence of the naturally occurring peptide we isolated GLP-1 from human small intestine...

  6. The gastric acid secretagogue gastrin-releasing peptide and the inhibitor oxyntomodulin do not exert their effect directly on the parietal cell in the rat

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Holst, J J

    1988-01-01

    in vitro by measuring [14C]-aminopyrine accumulation, a reliable index of H+ generation, in isolated rat parietal cells. However, neither gastrin-releasing peptide nor oxyntomodulin influenced basal acid secretion or histamine-stimulated gastric acid secretion. Electron-microscopic studies of unstimulated...... and histamine-stimulated parietal cells confirmed that the cells retained the normal morphology of intracellular organelles and that the cells responded to physiological stimulation by marked expansion of the intracellular canaliculi....

  7. Effect of a novel amphipathic ionic liquid on lithium deposition in gel polymer electrolytes

    International Nuclear Information System (INIS)

    Choi, Nam-Soon; Koo, Bonjae; Yeon, Jin-Tak; Lee, Kyu Tae; Kim, Dong-Won

    2011-01-01

    Highlights: · Synthesis of a dimeric ionic liquid. · Gel polymer electrolytes providing uniform lithium deposit pathway. · An amphipathic ionic liquid locates at the interface between an electrolyte-rich phase and a polymer matrix in a gel polymer electrolyte. · The presence of PDMITFSI ionic liquid leads to the suppression of dendritic lithium formation on a lithium metal electrode. - Abstract: A novel dimeric ionic liquid based on imidazolium cation and bis(trifluoromethanesulfonyl) imide (TFSI) anion has been synthesized through a metathesis reaction. Its chemical shift values and thermal properties are identified via 1 H nuclear magnetic resonance (NMR) imaging and differential scanning calorimetry (DSC). The effect of the synthesized dimeric ionic liquid on the interfacial resistance of gel polymer electrolytes is described. Differences in the SEM images of lithium electrodes after lithium deposition with and without the 1,1'-pentyl-bis(2,3-dimethylimidazolium) bis(trifluoromethane-sulfonyl)imide (PDMITFSI) ionic liquid in gel polymer electrolytes are clearly discernible. This occurs because the PDMITFSI ionic liquid with hydrophobic moieties and polar groups modulates lithium deposit pathways onto the lithium metal anode. Moreover, high anodic stability for a gel polymer electrolyte with the PDMITFSI ionic liquid was clearly observed.

  8. BIOACTIVE PEPTIDES OF THE COW MILK WHEY PROTEINS (Bos taurus

    Directory of Open Access Journals (Sweden)

    A. V. Iukalo

    2013-10-01

    Full Text Available Data on the biological functions of milk whey proteins, which are implemented at the level of their proteolytic degradation products — bioactive peptides have been reviewed. The main functions of these proteins is to provide the amino acid nutrition of mammals in the early stages of development, as well as the transport of fatty acids, retinol, involved in the synthesis of lactose, ions of calcium and iron, immune protection, antimicrobial action, etc. However, in recent years, it has been found that milk proteins like casein are precursors of biologically active peptides. Аngiotensin — converting enzyme, opioid peptides which are opiate receptor agonists, anti–microbial peptides, peptides with immunomodulatory and hypocholesterolemic action, and peptides affecting motility have been found among the products of proteolytic degradation of ?-lactoglobulin, ?-laktoalbumin, lactoferrin and milk whey albumin. Also data on the possible participation of peptides from milk whey proteins in the implementation of the biological functions of both the assimilation of calcium, antioxidant effect, the regulation of appetite, anticarcinogenic are provided. The authors assume that the phenomenon of bioactive peptides formation could be considered as an additional function of natural food proteins, which gives advantages to the mammals and has a positive effect on their development in the postnatal period. Ways of bioactive peptides formation, their resistance to action of proteolytic enzymes, the ability to cross into the bloodstream and have biological effects have been also discussed. Up to date, only a few products with bioactive peptides from milk whey proteins are obtained. Further studies of their structure, mechanism of action, ways of formation and methods of isolation are required for their wider use. Formation of functional products based on bioactive peptides from milk whey proteins will allow efficient use of milk whey, which is often a

  9. Effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of 111In-labeled lactam bridge-cyclized alpha-MSH peptides.

    Science.gov (United States)

    Guo, Haixun; Yang, Jianquan; Gallazzi, Fabio; Miao, Yubin

    2011-04-01

    The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma-targeting and pharmacokinetic properties of (111)In-labeled lactam bridge-cyclized DOTA-[X]-CycMSH(hex) {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH(2); X = GGNle, GENle, or NleGE; GG = -Gly-Gly- and GE = -Gly-Glu-} peptides. Three novel peptides (DOTA-GGNle-CycMSH(hex), DOTA-GENle-CycMSH(hex), and DOTA-NleGE-CycMSH(hex)) were designed and synthesized. The melanocortin-1 (MC1) receptor-binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma-targeting and pharmacokinetic properties of (111)In-DOTA-GGNle-CycMSH(hex) and (111)In-DOTA-GENle-CycMSH(hex) were determined in B16/F1 melanoma-bearing C57 mice. DOTA-GGNle-CycMSH(hex) and DOTA-GENle-CycMSH(hex) displayed 2.1 and 11.5 nM MC1 receptor-binding affinities, whereas DOTA-NleGE-CycMSH(hex) showed 873.4 nM MC1 receptor-binding affinity. The introduction of the -GG- linker maintained high melanoma uptake while decreasing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex). The tumor uptake of (111)In-DOTA-GGNle-CycMSH(hex) was 19.05 ± 5.04 and 18.6 ± 3.56 percentage injected dose per gram at 2 and 4 h after injection, respectively. (111)In-DOTA-GGNle-CycMSH(hex) exhibited 28%, 32%, and 42% less kidney uptake than (111)In-DOTA-Nle-CycMSH(hex) we reported previously, and 61%, 65%, and 68% less liver uptake than (111)In-DOTA-Nle-CycMSH(hex) at 2, 4, and 24 h after injection, respectively. The amino acid linkers exhibited profound effects on the melanoma-targeting and pharmacokinetic properties of the (111)In-labeled lactam bridge-cyclized α-melanocyte-stimulating hormone peptides. Introduction of the -GG- linker maintained high melanoma uptake while reducing kidney and liver uptake of (111)In-DOTA-GGNle-CycMSH(hex), highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide

  10. Encoded libraries of chemically modified peptides.

    Science.gov (United States)

    Heinis, Christian; Winter, Greg

    2015-06-01

    The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries. Copyright © 2015. Published by Elsevier Ltd.

  11. Highly selective enrichment of phosphorylated peptides using titanium dioxide

    DEFF Research Database (Denmark)

    Thingholm, Tine; Jørgensen, Thomas J D; Jensen, Ole N

    2006-01-01

    -column. Although phosphopeptide enrichment can be achieved by using TFA and acetonitrile alone, the selectivity is dramatically enhanced by adding DHB or phthalic acid since these compounds, in conjunction with the low pH caused by TFA, prevent binding of nonphosphorylated peptides to TiO2. Using an alkaline...... a protocol for selective phosphopeptide enrichment using titanium dioxide (TiO2) chromatography. The selectivity toward phosphopeptides is obtained by loading the sample in a 2,5-dihydroxybenzoic acid (DHB) or phthalic acid solution containing acetonitrile and trifluoroacetic acid (TFA) onto a TiO2 micro...... solution (pH > or = 10.5) both monophosphorylated and multiphosphorylated peptides are eluted from the TiO2 beads. This highly efficient method for purification of phosphopeptides is well suited for the characterization of phosphoproteins from both in vitro and in vivo studies in combination with mass...

  12. Bioorthogonal Diversification of Peptides through Selective Ruthenium(II)-Catalyzed C-H Activation.

    Science.gov (United States)

    Schischko, Alexandra; Ren, Hongjun; Kaplaneris, Nikolaos; Ackermann, Lutz

    2017-02-01

    Methods for the chemoselective modification of amino acids and peptides are powerful techniques in biomolecular chemistry. Among other applications, they enable the total synthesis of artificial peptides. In recent years, significant momentum has been gained by exploiting palladium-catalyzed cross-coupling for peptide modification. Despite major advances, the prefunctionalization elements on the coupling partners translate into undesired byproduct formation and lengthy synthetic operations. In sharp contrast, we herein illustrate the unprecedented use of versatile ruthenium(II)carboxylate catalysis for the step-economical late-stage diversification of α- and β-amino acids, as well as peptides, through chemo-selective C-H arylation under racemization-free reaction conditions. The ligand-accelerated C-H activation strategy proved water-tolerant and set the stage for direct fluorescence labelling as well as various modes of peptide ligation with excellent levels of positional selectivity in a bioorthogonal fashion. The synthetic utility of our approach is further demonstrated by twofold C-H arylations for the complexity-increasing assembly of artificial peptides within a multicatalytic C-H activation manifold. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Practical multipeptide synthesis: dedicated software for the definition of multiple, overlapping peptides covering polypeptide sequences.

    Science.gov (United States)

    Heegaard, P M; Holm, A; Hagerup, M

    1993-01-01

    A personal computer program for the conversion of linear amino acid sequences to multiple, small, overlapping peptide sequences has been developed. Peptide lengths and "jumps" (the distance between two consecutive overlapping peptides) are defined by the user. To facilitate the use of the program for parallel solid-phase chemical peptide syntheses for the synchronous production of multiple peptides, amino acids at each acylation step are laid out by the program in a convenient standard multi-well setup. Also, the total number of equivalents, as well as the derived amount in milligrams (depend-ending on user-defined equivalent weights and molar surplus), of each amino acid are given. The program facilitates the implementation of multipeptide synthesis, e.g., for the elucidation of polypeptide structure-function relationships, and greatly reduces the risk of introducing mistakes at the planning step. It is written in Pascal and runs on any DOS-based personal computer. No special graphic display is needed.

  14. Antimicrobial Peptides from Plants

    Directory of Open Access Journals (Sweden)

    James P. Tam

    2015-11-01

    Full Text Available Plant antimicrobial peptides (AMPs have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic, lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.

  15. Anionic magnetite nanoparticle conjugated with pyrrolidinyl peptide nucleic acid for DNA base discrimination

    Energy Technology Data Exchange (ETDEWEB)

    Khadsai, Sudarat; Rutnakornpituk, Boonjira [Naresuan University, Department of Chemistry and Center of Excellence in Biomaterials, Faculty of Science (Thailand); Vilaivan, Tirayut [Chulalongkorn University, Department of Chemistry, Organic Synthesis Research Unit, Faculty of Science (Thailand); Nakkuntod, Maliwan [Naresuan University, Department of Biology, Faculty of Science (Thailand); Rutnakornpituk, Metha, E-mail: methar@nu.ac.th [Naresuan University, Department of Chemistry and Center of Excellence in Biomaterials, Faculty of Science (Thailand)

    2016-09-15

    Magnetite nanoparticles (MNPs) were surface modified with anionic poly(N-acryloyl glycine) (PNAG) and streptavidin for specific interaction with biotin-conjugated pyrrolidinyl peptide nucleic acid (PNA). Hydrodynamic size (D{sub h}) of PNAG-grafted MNPs varied from 334 to 496 nm depending on the loading ratio of the MNP to NAG in the reaction. UV–visible and fluorescence spectrophotometries were used to confirm the successful immobilization of streptavidin and PNA on the MNPs. About 291 pmol of the PNA/mg MNP was immobilized on the particle surface. The PNA-functionalized MNPs were effectively used as solid supports to differentiate between fully complementary and non-complementary/single-base mismatch DNA using the PNA probe. These novel anionic MNPs can be efficiently applicable for use as a magnetically guidable support for DNA base discrimination.Graphical Abstract.

  16. δ-Peptides from RuAAC-Derived 1,5-Disubstituted Triazole Units

    KAUST Repository

    Johansson, Johan R.; Hermansson, Elin; Nordé n, Bengt; Kann, Nina; Beke-Somfai, Tamá s

    2014-01-01

    of non-natural peptides composed of 1,5-disubstituted 1,2,3-triazole amino acids is presented. These peptides benefit from: a) modular synthesis of the monomers, allowing variation of the side chains; b) increased solubility of the oligomers in water

  17. Double quick, double click reversible peptide "stapling".

    Science.gov (United States)

    Grison, Claire M; Burslem, George M; Miles, Jennifer A; Pilsl, Ludwig K A; Yeo, David J; Imani, Zeynab; Warriner, Stuart L; Webb, Michael E; Wilson, Andrew J

    2017-07-01

    The development of constrained peptides for inhibition of protein-protein interactions is an emerging strategy in chemical biology and drug discovery. This manuscript introduces a versatile, rapid and reversible approach to constrain peptides in a bioactive helical conformation using BID and RNase S peptides as models. Dibromomaleimide is used to constrain BID and RNase S peptide sequence variants bearing cysteine (Cys) or homocysteine ( h Cys) amino acids spaced at i and i + 4 positions by double substitution. The constraint can be readily removed by displacement of the maleimide using excess thiol. This new constraining methodology results in enhanced α-helical conformation (BID and RNase S peptide) as demonstrated by circular dichroism and molecular dynamics simulations, resistance to proteolysis (BID) as demonstrated by trypsin proteolysis experiments and retained or enhanced potency of inhibition for Bcl-2 family protein-protein interactions (BID), or greater capability to restore the hydrolytic activity of the RNAse S protein (RNase S peptide). Finally, use of a dibromomaleimide functionalized with an alkyne permits further divergent functionalization through alkyne-azide cycloaddition chemistry on the constrained peptide with fluorescein, oligoethylene glycol or biotin groups to facilitate biophysical and cellular analyses. Hence this methodology may extend the scope and accessibility of peptide stapling.

  18. A tetrameric peptide derived from bovine lactoferricin as a potential therapeutic tool for oral squamous cell carcinoma: A preclinical model.

    Science.gov (United States)

    Solarte, Víctor Alfonso; Conget, Paulette; Vernot, Jean-Paul; Rosas, Jaiver Eduardo; Rivera, Zuly Jenny; García, Javier Eduardo; Arango-Rodríguez, Martha Ligia

    2017-01-01

    Oral squamous cell carcinoma is the fifth most common epithelial cancer in the world, and its current clinical treatment has both low efficiency and poor selectivity. Cationic amphipathic peptides have been proposed as new drugs for the treatment of different types of cancer. The main goal of the present work was to determine the potential of LfcinB(20-25)4, a tetrameric peptide based on the core sequence RRWQWR of bovine lactoferricin LfcinB(20-25), for the treatment of OSCC. In brief, OSCC was induced in the buccal pouch of hamsters by applying 7,12-Dimethylbenz(a)anthracene, and tumors were treated with one of the following peptides: LfcinB(20-25)4, LfcinB(20-25), or vehicle (control). Lesions were macroscopically evaluated every two days and both histological and serum IgG assessments were conducted after 5 weeks. The size of the tumors treated with LfcinB(20-25)4 and LfcinB(20-25) was smaller than that of the control group (46.16±4.41 and 33.92±2.74 mm3 versus 88.77±10.61 mm3, respectively). Also, LfcinB(20-25)4 caused acellularity in the parenchymal tumor compared with LfcinB(20-25) and vehicle treatments. Furthermore, our results demonstrated that both LfcinB(20-25)4 and LfcinB(20-25) induced higher degree of apoptosis relative to the untreated tumors (75-86% vs 8%, respectively). Moreover, although the lowest inflammatory response was achieved when LfcinB(20-25)4 was used, this peptide appeared to induce higher levels of IgG antibodies relative to the vehicle and LfcinB(20-25). In addition the cellular damage and selectivity of the LfcinB(20-25)4 peptide was evaluated in vitro. These assays showed that LfcinB(20-25)4 triggers a selective necrotic effect in the carcinoma cell line. Cumulatively, these data indicate that LfcinB(20-25)4 could be considered as a new therapeutic agent for the treatment of OSCC.

  19. A tetrameric peptide derived from bovine lactoferricin as a potential therapeutic tool for oral squamous cell carcinoma: A preclinical model.

    Directory of Open Access Journals (Sweden)

    Víctor Alfonso Solarte

    Full Text Available Oral squamous cell carcinoma is the fifth most common epithelial cancer in the world, and its current clinical treatment has both low efficiency and poor selectivity. Cationic amphipathic peptides have been proposed as new drugs for the treatment of different types of cancer. The main goal of the present work was to determine the potential of LfcinB(20-254, a tetrameric peptide based on the core sequence RRWQWR of bovine lactoferricin LfcinB(20-25, for the treatment of OSCC. In brief, OSCC was induced in the buccal pouch of hamsters by applying 7,12-Dimethylbenz(aanthracene, and tumors were treated with one of the following peptides: LfcinB(20-254, LfcinB(20-25, or vehicle (control. Lesions were macroscopically evaluated every two days and both histological and serum IgG assessments were conducted after 5 weeks. The size of the tumors treated with LfcinB(20-254 and LfcinB(20-25 was smaller than that of the control group (46.16±4.41 and 33.92±2.74 mm3 versus 88.77±10.61 mm3, respectively. Also, LfcinB(20-254 caused acellularity in the parenchymal tumor compared with LfcinB(20-25 and vehicle treatments. Furthermore, our results demonstrated that both LfcinB(20-254 and LfcinB(20-25 induced higher degree of apoptosis relative to the untreated tumors (75-86% vs 8%, respectively. Moreover, although the lowest inflammatory response was achieved when LfcinB(20-254 was used, this peptide appeared to induce higher levels of IgG antibodies relative to the vehicle and LfcinB(20-25. In addition the cellular damage and selectivity of the LfcinB(20-254 peptide was evaluated in vitro. These assays showed that LfcinB(20-254 triggers a selective necrotic effect in the carcinoma cell line. Cumulatively, these data indicate that LfcinB(20-254 could be considered as a new therapeutic agent for the treatment of OSCC.

  20. Linkers, resins, and general procedures for solid-phase peptide synthesis

    DEFF Research Database (Denmark)

    Shelton, Anne Pernille Tofteng; Jensen, Knud Jørgen

    2013-01-01

    and linkers for solid-phase synthesis is a key parameter for successful peptide synthesis. This chapter provides an overview of the most common and useful resins and linkers for the synthesis of peptides with C-terminal amides, carboxylic acids, and more. The chapter finishes with robust protocols for general...

  1. Extraction of aggrecan-peptide from cartilage by tissue autolysis.

    Science.gov (United States)

    Nakano, Takuo; Srichamroen, Anchalee; Ozimek, Lech

    2014-01-01

    Aggrecan is a cartilage specific proteoglycan containing chondroitin sulfate (CS) and keratan sulfate (KS). CS is an acidic polysaccharide having wide range of applications in pharmaceutical, cosmetic, and food industries. CS is extracted from cartilage by tissue proteolysis with an exogenous proteinase or by activating endogenous proteinases (autolysis) to release aggrecan-peptides from the tissue. This review is focused on the latter technique. Bovine nasal and tracheal cartilages, and broiler chicken sternum cartilage have been used for autolysis studies. To extract aggrecan-peptide, cartilage tissues are cut into small pieces, and incubated in a monovalent or divalent salt solution (e.g., 0.1 M sodium or calcium acetate) at pH 4.5 and 37 °C for 7 - 24 h. Most (~80% or more) of total tissue uronic acid, a constituent sugar of aggrecan, is extracted and released into the salt solution during incubation. Reextraction of the tissue residue results in release of a small amount of uronic acid. Aggrecan-peptides purified using anion exchange chromatography are large compounds containing CS and KS. On gel chromatography, they are excluded from the column of Sephacryl S-300. Chemical composition analysis demonstrated that aggrecan-peptides from either bovine or chicken cartilage contain >90% CS with small amount (autolysis has been used as a plate coating antigen in enzyme- linked immunosorbent assay (ELISA) to determine KS.

  2. Theoretical study for volume changes associated with the helix-coil transition of peptides.

    Science.gov (United States)

    Imai, T; Harano, Y; Kovalenko, A; Hirata, F

    2001-12-01

    We calculate the partial molar volumes and their changes associated with the coil(extended)-to-helix transition of two types of peptide, glycine-oligomer and glutamic acid-oligomer, in aqueous solutions by using the Kirkwood-Buff solution theory coupled with the three-dimensional reference interaction site model (3D-RISM) theory. The volume changes associated with the transition are small and positive. The volume is analyzed by decomposing it into five contributions following the procedure proposed by Chalikian and Breslauer: the ideal volume, the van der Waals volume, the void volume, the thermal volume, and the interaction volume. The ideal volumes and the van der Waals volumes do not change appreciably upon the transition. In the both cases of glycine-peptide and glutamic acid-peptide, the changes in the void volumes are positive, while those in the thermal volumes are negative, and tend to balance those in the void volumes. The change in the interaction volume of glycine-peptide does not significantly contribute, while that of glutamic acid-peptide makes a negative contribution. Copyright 2001 John Wiley & Sons, Inc. Biopolymers 59: 512-519, 2001

  3. Lipopolysaccharide interactions of C-terminal peptides from human thrombin.

    Science.gov (United States)

    Singh, Shalini; Kalle, Martina; Papareddy, Praveen; Schmidtchen, Artur; Malmsten, Martin

    2013-05-13

    Interactions with bacterial lipopolysaccharide (LPS), both in aqueous solution and in lipid membranes, were investigated for a series of amphiphilic peptides derived from the C-terminal region of human thrombin, using ellipsometry, dual polarization interferometry, fluorescence spectroscopy, circular dichroism (CD), dynamic light scattering, and z-potential measurements. The ability of these peptides to block endotoxic effects caused by LPS, monitored through NO production in macrophages, was compared to peptide binding to LPS and its endotoxic component lipid A, and to size, charge, and secondary structure of peptide/LPS complexes. While the antiendotoxic peptide GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE) displayed significant binding to both LPS and lipid A, so did two control peptides with either selected D-amino acid substitutions or with maintained composition but scrambled sequence, both displaying strongly attenuated antiendotoxic effects. Hence, the extent of LPS or lipid A binding is not the sole discriminant for the antiendotoxic effect of these peptides. In contrast, helix formation in peptide/LPS complexes correlates to the antiendotoxic effect of these peptides and is potentially linked to this functionality. Preferential binding to LPS over lipid membrane was furthermore demonstrated for these peptides and preferential binding to the lipid A moiety within LPS inferred.

  4. [Transduction peptides, the useful face of a new signaling mechanism].

    Science.gov (United States)

    Joliot, Alain; Prochiantz, Alain

    2005-03-01

    Transduction peptides that cross the plasma membrane of live cells are commonly used for the in vitro and in vivo targeting of hydrophilic drugs into the cell interior. Although this family of peptides has recently increased and will probably continue to do so, the two mainly used peptides are derived from transcription factors. Indeed, TAT is a 12 amino acid long arginine-rich peptide present in the HIV transcription factor, and penetratin - or its variants - corresponds to 16 amino acids that define the highly conserved third helix of the DNA-binding domain (homeodomain) of homeoprotein transcription factors. In this review, we shall recall the different steps that have led to the discovery of transduction peptides and present the most likely hypotheses concerning the mechanisms involved in their internalization. At the risk of being incomplete or, even, biased, we shall concentrate on penetratins and TAT. The reason is that these peptides have been studied for over ten years leading to the edification of robust knowledge regarding their properties. This attitude will not preclude comparisons with other peptides, if necessary. Our goal is to describe the mode of action of these transduction peptides, their range of activity in term of cell types that accept them and cargoes that they can transport, and, also, some of the limitations that one can encounter in their use. Finally, based on the idea that peptide transduction is the technological face of a physiological property of some transcription factors, we shall discuss the putative physiological function of homeoprotein transduction, and, as a consequence, the possibility to use these factors as therapeutic proteins.

  5. Short peptides allowing preferential detection of Candida albicans hyphae.

    Science.gov (United States)

    Kaba, Hani E J; Pölderl, Antonia; Bilitewski, Ursula

    2015-09-01

    Whereas the detection of pathogens via recognition of surface structures by specific antibodies and various types of antibody mimics is frequently described, the applicability of short linear peptides as sensor molecules or diagnostic tools is less well-known. We selected peptides which were previously reported to bind to recombinant S. cerevisiae cells, expressing members of the C. albicans Agglutinin-Like-Sequence (ALS) cell wall protein family. We slightly modified amino acid sequences to evaluate peptide sequence properties influencing binding to C. albicans cells. Among the selected peptides, decamer peptides with an "AP"-N-terminus were superior to shorter peptides. The new decamer peptide FBP4 stained viable C. albicans cells more efficiently in their mature hyphal form than in their yeast form. Moreover, it allowed distinction of C. albicans from other related Candida spp. and could thus be the basis for the development of a useful tool for the diagnosis of invasive candidiasis.

  6. Peptide bond formation of alanine on silica and alumina surfaces as a catalyst

    Science.gov (United States)

    Sánchez Arenillas, M.; Mateo-Martí, E.

    2012-09-01

    Polymerization of amino acids has been important for the origin of life because the peptides may have been the first self-replicating systems. The amino acid concentrations in the oceans may have been too diluted in the early phases of the Earth. The formation of the biopolymers could have been due to the catalytic action of various minerals (such as silica or alumina). Our work is based on the comparison between alumina and silica minerals with and without prior activation of their silanol groups for the formation of peptide bonds using alanina like amino acid which it is the simplest quiral amino acid.

  7. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Science.gov (United States)

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-01-01

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry. PMID:23435052

  8. Label-Free Potentiometry for Detecting DNA Hybridization Using Peptide Nucleic Acid and DNA Probes

    Directory of Open Access Journals (Sweden)

    Yuji Miyahara

    2013-02-01

    Full Text Available Peptide nucleic acid (PNA has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  9. Functional Modification of Thioether Groups in Peptides, Polypeptides, and Proteins.

    Science.gov (United States)

    Deming, Timothy J

    2017-03-15

    Recent developments in the modification of methionine and other thioether-containing residues in peptides, polypeptides, and proteins are reviewed. Properties and potential applications of the resulting functionalized products are also discussed. While much of this work is focused on natural Met residues, modifications at other side-chain residues have also emerged as new thioether-containing amino acids have been incorporated into peptidic materials. Functional modification of thioether-containing amino acids has many advantages and is a complementary methodology to the widely utilized methods for modification at cysteine residues.

  10. PEGylated nanoparticles bind to and alter amyloid-beta peptide conformation

    DEFF Research Database (Denmark)

    Brambilla, Davide; Verpillot, Romain; Le Droumaguet, Benjamin

    2012-01-01

    We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aß(1-42)) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs on the int......We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aß(1-42)) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs...

  11. Increased electrical conductivity of peptides through annealing process

    Directory of Open Access Journals (Sweden)

    Seok Daniel Namgung

    2017-08-01

    Full Text Available Biocompatible biologically occurring polymer is suggested as a component of human implantable devices since conventional inorganic materials are apt to trigger inflammation and toxicity problem within human body. Peptides consisting of aromatic amino acid, tyrosine, are chosen, and enhancement on electrical conductivity is studied. Annealing process gives rise to the decrease on resistivity of the peptide films and the growth of the carrier concentration is a plausible reason for such a decrease on resistivity. The annealed peptides are further applied to an active layer of field effect transistor, in which low on/off current ratio (∼10 is obtained.

  12. A readily applicable strategy to convert peptides to peptoid-based therapeutics.

    Directory of Open Access Journals (Sweden)

    Minyoung Park

    Full Text Available Incorporation of unnatural amino acids and peptidomimetic residues into therapeutic peptides is highly efficacious and commonly employed, but generally requires laborious trial-and-error approaches. Previously, we demonstrated that C20 peptide has the potential to be a potential antiviral agent. Herein we report our attempt to improve the biological properties of this peptide by introducing peptidomimetics. Through combined alanine, proline, and sarcosine scans coupled with a competitive fluorescence polarization assay developed for identifying antiviral peptides, we enabled to pinpoint peptoid-tolerant peptide residues within C20 peptide. The synergistic benefits of combining these (and other commonly employed methods could lead to a easily applicable strategy for designing and refining therapeutically-attractive peptidomimetics.

  13. 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...

  14. Developing a Dissociative Nanocontainer for Peptide Drug Delivery

    Directory of Open Access Journals (Sweden)

    Patrick Kelly

    2015-10-01

    Full Text Available The potency, selectivity, and decreased side effects of bioactive peptides have propelled these agents to the forefront of pharmacological research. Peptides are especially promising for the treatment of neurological disorders and pain. However, delivery of peptide therapeutics often requires invasive techniques, which is a major obstacle to their widespread application. We have developed a tailored peptide drug delivery system in which the viral capsid of P22 bacteriophage is modified to serve as a tunable nanocontainer for the packaging and controlled release of bioactive peptides. Recent efforts have demonstrated that P22 nanocontainers can effectively encapsulate analgesic peptides and translocate them across blood-brain-barrier (BBB models. However, release of encapsulated peptides at their target site remains a challenge. Here a Ring Opening Metathesis Polymerization (ROMP reaction is applied to trigger P22 nanocontainer disassembly under physiological conditions. Specifically, the ROMP substrate norbornene (5-Norbornene-2-carboxylic acid is conjugated to the exterior of a loaded P22 nanocontainer and Grubbs II Catalyst is used to trigger the polymerization reaction leading to nanocontainer disassembly. Our results demonstrate initial attempts to characterize the ROMP-triggered release of cargo peptides from P22 nanocontainers. This work provides proof-of-concept for the construction of a triggerable peptide drug delivery system using viral nanocontainers.

  15. Chimeric peptide beacons: a direct polypeptide analog of DNA molecular beacons†

    OpenAIRE

    Oh, Kenneth J.; Cash, Kevin J.; Lubin, Arica A.; Plaxco, Kevin W.

    2007-01-01

    We have developed a new biosensor architecture, which is comprised of a polypeptide–peptide nucleic acid tri-block copolymer and which we have termed chimeric peptide beacons (CPB), that generates an optical output via a mechanism analogous to that employed in DNA-based molecular beacons.

  16. Manipulation of charged residues within the two-peptide lantibiotic lacticin 3147

    NARCIS (Netherlands)

    Deegan, Lucy H.; Suda, Srinivas; Lawton, Elaine M.; Draper, Lorraine A.; Hugenholtz, Floor; Peschel, Andreas; Hill, Colin; Cotter, Paul D.; Ross, R. Paul

    2010-01-01

    Lantibiotics are antimicrobial peptides which contain a high percentage of post-translationally modified residues. While most attention has been paid to the role of these critical structural features, evidence continues to emerge that charged amino acids also play a key role in these peptides. Here

  17. Metabolic fate of lactoferricin-based antimicrobial peptides: effect of truncation and incorporation of amino acid analogs on the in vitro metabolic stability.

    Science.gov (United States)

    Svenson, Johan; Vergote, Valentijn; Karstad, Rasmus; Burvenich, Christian; Svendsen, John S; De Spiegeleer, Bart

    2010-03-01

    A series of promising truncated antibacterial tripeptides derived from lactoferricin has been prepared, and their in vitro metabolic stability in the main metabolic compartments, plasma, liver, kidney, stomach, duodenum, and brain, has been investigated for the first time. The potential stabilizing effect of truncation, C-terminal capping, and introduction of the bulky synthetic amino acid biphenylalanine is also investigated. The drug-like peptides displayed large differences in half-lives in the different matrixes ranging from 4.2 min in stomach and duodenum to 355.9 min in liver. Kinetic analysis of the metabolites revealed that several different degrading enzymes simultaneously target the different peptide bonds and that the outcome of the tested strategies to increase the stability is clearly enzyme-specific. Some of the metabolic enzymes even prefer the synthetic modifications incorporated over the natural counterparts. Collectively, it is shown that the necessary antibacterial pharmacophore generates compounds that are not only potent antibacterial peptides, but excellent substrates for the main degrading enzymes. All the amide bonds are thus rapidly targeted by different enzymes despite the short peptidic sequences of the tested compounds. Hence, our results illustrate that several structural changes are needed before these compounds can be considered for oral administration. Strategies to overcome such metabolic challenges are discussed.

  18. Antimicrobial Peptide-PNA Conjugates Selectively Targeting Bacterial Genes

    Science.gov (United States)

    2013-07-22

    antibacterial therapy. Initial publications suggest that conjugates of cell penetrating peptides and PNA’s can overcome the barrier in transporting ...Zhou, Y., Hou, Z., Meng, J., and Luo, X. Targeting RNA polymerase primary σ70 as a therapeutic strategy against methicillin - resistant ... Staphylococcus aureus by antisense peptide nucleic acid. PLoS One. 2012; 7(1):e29886. 2. Good, L., Sandberg, R., Larsson, O., Nielsen, P.E., and Wahlestedt, C

  19. Maize EMBRYO SAC family peptides interact differentially with pollen tubes and fungal cells.

    Science.gov (United States)

    Woriedh, Mayada; Merkl, Rainer; Dresselhaus, Thomas

    2015-08-01

    EMBRYO SAC1-4 (ES1-4) peptides belong to the defensin subgroup of cysteine-rich peptides known to mediate pollen tube burst in Zea mays (maize). ES1-4 are reported here to also be capable of inhibiting germination and growth of the maize fungal pathogens Fusarium graminearum and Ustilago maydis at higher concentrations. Dividing the peptides into smaller pieces showed that a 15-amino-acid peptide located in a highly variable loop region lacking similarity to other defensins or defensin-like peptides binds to maize pollen tube surfaces, causing swelling prior to burst. This peptide fragment and a second conserved neighbouring fragment showed suppression of fungal germination and growth. The two peptides caused swelling of fungal cells, production of reactive oxygen species, and finally the formation of big vacuoles prior to burst at high peptide concentration. Furthermore, peptide fragments were found to bind differently to fungal cells. In necrotrophic F. graminearum, a peptide fragment named ES-d bound only at cell surfaces whereas the peptide ES-c bound at cell surfaces and also accumulated inside cells. Conversely, in biotrophic U. maydis, both peptide fragments accumulated inside cells, but, if applied at higher concentration, ES-c but not ES-d accumulated mainly in vacuoles. Mapping of peptide interaction sites identified amino acids differing in pollen tube burst and fungal response reactions. In summary, these findings indicate that residues targeting pollen tube burst in maize are specific to the ES family, while residues targeting fungal growth are conserved within defensins and defensin-like peptides. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  20. Fischer carbene mediated covalent grafting of a peptide nucleic acid on gold surfaces and IR optical detection of DNA hybridization with a transition metalcarbonyl label

    Czech Academy of Sciences Publication Activity Database

    Srivastava, P.; Ghasemi, M.; Ray, N.; Sarkar, A.; Kocábová, Jana; Lachmanová, Štěpánka; Hromadová, Magdaléna; Boujday, S.; Cauteruccio, S.; Thakare, P.; Licandro, E.; Fosse, C.; Salmain, M.

    2016-01-01

    Roč. 385, NOV 2016 (2016), s. 47-55 ISSN 0169-4332 R&D Projects: GA ČR(CZ) GA14-05180S Institutional support: RVO:61388955 Keywords : peptide nucleic acid * self-assembled monolayer * genosensor Subject RIV: CG - Electrochemistry Impact factor: 3.387, year: 2016

  1. Genetics of the proteolytic system of lactic acid bacteria

    NARCIS (Netherlands)

    Kok, Jan

    1990-01-01

    The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have

  2. Studies on lactoferricin-derived Escherichia coli membrane-active peptides reveal differences in the mechanism of N-acylated versus nonacylated peptides.

    Science.gov (United States)

    Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

    2011-06-17

    To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis.

  3. Studies on Lactoferricin-derived Escherichia coli Membrane-active Peptides Reveal Differences in the Mechanism of N-Acylated Versus Nonacylated Peptides*

    Science.gov (United States)

    Zweytick, Dagmar; Deutsch, Günter; Andrä, Jörg; Blondelle, Sylvie E.; Vollmer, Ekkehard; Jerala, Roman; Lohner, Karl

    2011-01-01

    To improve the low antimicrobial activity of LF11, an 11-mer peptide derived from human lactoferricin, mutant sequences were designed based on the defined structure of LF11 in the lipidic environment. Thus, deletion of noncharged polar residues and strengthening of the hydrophobic N-terminal part upon adding a bulky hydrophobic amino acid or N-acylation resulted in enhanced antimicrobial activity against Escherichia coli, which correlated with the peptides' degree of perturbation of bacterial membrane mimics. Nonacylated and N-acylated peptides exhibited different effects at a molecular level. Nonacylated peptides induced segregation of peptide-enriched and peptide-poor lipid domains in negatively charged bilayers, although N-acylated peptides formed small heterogeneous domains resulting in a higher degree of packing defects. Additionally, only N-acylated peptides perturbed the lateral packing of neutral lipids and exhibited increased permeability of E. coli lipid vesicles. The latter did not correlate with the extent of improvement of the antimicrobial activity, which could be explained by the fact that elevated binding of N-acylated peptides to lipopolysaccharides of the outer membrane of Gram-negative bacteria seems to counteract the elevated membrane permeabilization, reflected in the respective minimal inhibitory concentration for E. coli. The antimicrobial activity of the peptides correlated with an increase of membrane curvature stress and hence bilayer instability. Transmission electron microscopy revealed that only the N-acylated peptides induced tubular protrusions from the outer membrane, whereas all peptides caused detachment of the outer and inner membrane of E. coli bacteria. Viability tests demonstrated that these bacteria were dead before onset of visible cell lysis. PMID:21515687

  4. Effects of prepartum fat supplementation on plasma concentrations of glucagon-like peptide-1, peptide YY, adropin, insulin, and leptin in periparturient dairy cows.

    Science.gov (United States)

    Zapata, Rizaldy C; Salehi, Reza; Ambrose, Divakar J; Chelikani, Prasanth K

    2015-10-01

    Dietary fat supplementation during the periparturient period is one strategy to increase energy intake and attenuate the degree of negative energy balance during early lactation; however, little is known of the underlying hormonal an