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Sample records for acidic amphipathic peptides

  1. Formulation of nucleic acid with pH-responsive amphipathic peptides for pulmonary delivery

    OpenAIRE

    Liang, Wanling; 梁婉玲

    2014-01-01

    Nucleic acids could be used as therapeutic agents for the treatment of many different diseases, but poor delivery limits their clinical application. A series of pH-responsive amphipathic peptides containing histidine or 2,3-diaminopropionic acid (Dap) derivatives, LAH and LADap peptides, were investigated in this study as nucleic acid carriers for the treatment of respiratory infectious disease. LAH and LADap peptides are cationic, amphipathic pH-responsive peptides. The major attractive ...

  2. Anisotropic Membrane Curvature Sensing by Amphipathic Peptides.

    Science.gov (United States)

    Gómez-Llobregat, Jordi; Elías-Wolff, Federico; Lindén, Martin

    2016-01-01

    Many proteins and peptides have an intrinsic capacity to sense and induce membrane curvature, and play crucial roles for organizing and remodeling cell membranes. However, the molecular driving forces behind these processes are not well understood. Here, we describe an approach to study curvature sensing by simulating the interactions of single molecules with a buckled lipid bilayer. We analyze three amphipathic antimicrobial peptides, a class of membrane-associated molecules that specifically target and destabilize bacterial membranes, and find qualitatively different sensing characteristics that would be difficult to resolve with other methods. Our findings provide evidence for direction-dependent curvature sensing mechanisms in amphipathic peptides and challenge existing theories of hydrophobic insertion. The buckling approach is generally applicable to a wide range of curvature-sensing molecules, and our results provide strong motivation to develop new experimental methods to track position and orientation of membrane proteins. PMID:26745422

  3. Tryptophan rotamer distributions in amphipathic peptides at a lipid surface.

    OpenAIRE

    Clayton, A H; Sawyer, W. H.

    1999-01-01

    The fluorescence decay of tryptophan is a sensitive indicator of its local environment within a peptide or protein. We describe the use of frequency domain fluorescence spectroscopy to determine the conformational and environmental changes associated with the interaction of single tryptophan amphipathic peptides with a phospholipid surface. The five 18-residue peptides studied are based on a class A amphipathic peptide known to associate with lipid bilayers. The peptides contain a single tryp...

  4. Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

    Indian Academy of Sciences (India)

    Chandrasekaran Sivakamasundari; Ramakrishnan Nagaraj

    2009-06-01

    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 conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide–membrane interactions.

  5. Importance of Tryptophan in Transforming an Amphipathic Peptide into a Pseudomonas aeruginosa-Targeted Antimicrobial Peptide.

    Directory of Open Access Journals (Sweden)

    Xin Zhu

    Full Text Available Here, we found that simple substitution of amino acids in the middle position of the hydrophobic face of an amphipathic peptide RI16 with tryptophan (T9W considerably transformed into an antimicrobial peptide specifically targeting Pseudomonas aeruginosa. Minimal inhibitory concentration (MIC results demonstrated that T9W had a strong and specifically antimicrobial activity against P. aeruginosa, including antibiotic-resistant strains, but was not active against Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Staphyfococcus epidermidis. Fluorescent spectroscopic assays indicated that T9W interacted with the membrane of P. aeruginosa, depolarizing the outer and the inner membrane of bacterial cells. Salt susceptibility assay showed that T9W still maintained its strong anti-pseudomonas activity in the presence of salts at physiological concentrations, and in hemolytic and MTT assays T9W also showed no toxicity against human blood cells and macrophages. In vivo assay demonstrated that T9W also displayed no toxicity to Chinese Kun Ming (KM mice. Furthermore, the strong antibiofilm activity was also observed with the peptide T9W, which decreased the percentage of biomass formation in a dose-dependent manner. Overall, these findings indicated that design of single-pathogen antimicrobial agents can be achieved by simple amino acid mutation in naturally occurring peptide sequences and this study suggested a model of optimization/design of anti-pseudomonas drugs in which the tryptophan residue was a conserved element.

  6. Effects of Single Amino Acid Substitution on the Biophysical Properties and Biological Activities of an Amphipathic α-Helical Antibacterial Peptide Against Gram-Negative Bacteria

    Directory of Open Access Journals (Sweden)

    Juanjuan Tan

    2014-07-01

    Full Text Available An antimicrobial peptide, known as V13K, was utilized as the framework to study the effects of charge, hydrophobicity and helicity on the biophysical properties and biological activities of α-helical peptides. Six amino acids (Lys, Glu, Gly, Ser, Ala, and Leu were individually used to substitute the original hydrophobic valine at the selected sixteenth location on the non-polar face of V13K. The results showed that the single amino acid substitutions changed the hydrophobicity of peptide analogs as monitored by RP-HPLC, but did not cause significant changes on peptide secondary structures both in a benign buffer and in a hydrophobic environment. The biological activities of the analogs exhibited a hydrophobicity-dependent behavior. The mechanism of peptide interaction with the outer membrane and cytoplasmic membrane of Gram-negative bacteria was investigated. We demonstrated that this single amino acid substitution method has valuable potential for the rational design of antimicrobial peptides with enhanced activities.

  7. Mixed α/β-Peptides as a Class of Short Amphipathic Peptide Hydrogelators with Enhanced Proteolytic Stability.

    Science.gov (United States)

    Mangelschots, Jeroen; Bibian, Mathieu; Gardiner, James; Waddington, Lynne; Van Wanseele, Yannick; Van Eeckhaut, Ann; Acevedo, Maria M Diaz; Van Mele, Bruno; Madder, Annemieke; Hoogenboom, Richard; Ballet, Steven

    2016-02-01

    Peptide hydrogels are a highly promising class of materials for biomedical application, albeit facing many challenges with regard to stability and tunability. Here, we report a new class of amphipathic peptide hydrogelators, namely mixed α/β-peptide hydrogelators. These mixed α/β-gelators possess good rheological properties (high storage moduli) and form transparent self-supporting gels with shear-thinning behavior. Infrared spectroscopy indicates the presence of β-sheets as the underlying secondary structure. Interestingly, self-assembled nanofibers of the mixed α/β-peptides display unique structural morphologies with alteration of the C-terminus (acid vs amide) playing a key role in the fiber formation and gelation properties of the resulting hydrogels. The incorporation of β3-homoamino acid residues within the mixed α/β-peptide gelators led to an increase in proteolytic stability of the peptides under nongelating conditions (in solution) as well as gelating conditions (as hydrogel). Under diluted conditions, degradation of mixed α/β-peptides in the presence of elastase was slowed down 120-fold compared to that of an α-peptide, thereby demonstrating beneficial enzymatic resistance for hydrogel applications in vivo. In addition, increased half-life values were obtained for the mixed α/β-peptides in human blood plasma, as compared to corresponding α-peptides. It was also found that the mixed α/β-peptides were amenable to injection via needles used for subcutaneous administrations. The preformed peptide gels could be sheared upon injection and were found to quickly reform to a state close to that of the original hydrogel. The shown properties of enhanced proteolytic stability and injectability hold great promise for the use of these novel mixed α/β-peptide hydrogels for applications in the areas of tissue engineering and drug delivery. PMID:26741458

  8. Improving membrane binding as a design strategy for amphipathic peptide hormones

    DEFF Research Database (Denmark)

    Pedersen, Søren Ljungberg; Bhatia, Vikram Kjøller; Jurt, Simon;

    2012-01-01

    It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3-36 is believed to perform its appetite-suppressing actions through binding to hypothalamic Y2 receptors. It has...... potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2-helix variant of PYY3-36 will be useful as a tool compound for studying peptide-membrane interactions....

  9. Oxyopinins, large amphipathic peptides isolated from the venom of the wolf spider Oxyopes kitabensis with cytolytic properties and positive insecticidal cooperativity with spider neurotoxins.

    Science.gov (United States)

    Corzo, Gerardo; Villegas, Elba; Gómez-Lagunas, Froylan; Possani, Lourival D; Belokoneva, Olga S; Nakajima, Terumi

    2002-06-28

    Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially alpha-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.

  10. Membrane Thinning and Thickening Induced by Membrane-Active Amphipathic Peptides.

    Science.gov (United States)

    Grage, Stephan L; Afonin, Sergii; Kara, Sezgin; Buth, Gernot; Ulrich, Anne S

    2016-01-01

    Membrane thinning has been discussed as a fundamental mechanism by which antimicrobial peptides can perturb cellular membranes. To understand which factors play a role in this process, we compared several amphipathic peptides with different structures, sizes and functions in their influence on the lipid bilayer thickness. PGLa and magainin 2 from X. laevis were studied as typical representatives of antimicrobial cationic amphipathic α-helices. A 1:1 mixture of these peptides, which is known to possess synergistically enhanced activity, allowed us to evaluate whether and how this synergistic interaction correlates with changes in membrane thickness. Other systems investigated here include the α-helical stress-response peptide TisB from E. coli (which forms membrane-spanning dimers), as well as gramicidin S from A. migulanus (a natural antibiotic), and BP100 (designer-made antimicrobial and cell penetrating peptide). The latter two are very short, with a circular β-pleated and a compact α-helical structure, respectively. Solid-state (2)H-NMR and grazing incidence small angle X-ray scattering (GISAXS) on oriented phospholipid bilayers were used as complementary techniques to access the hydrophobic thickness as well as the bilayer-bilayer repeat distance including the water layer in between. This way, we found that magainin 2, gramicidin S, and BP100 induced membrane thinning, as expected for amphiphilic peptides residing in the polar/apolar interface of the bilayer. PGLa, on the other hand, decreased the hydrophobic thickness only at very high peptide:lipid ratios, and did not change the bilayer-bilayer repeat distance. TisB even caused an increase in the hydrophobic thickness and repeat distance. When reconstituted as a mixture, PGLa and magainin 2 showed a moderate thinning effect which was less than that of magainin 2 alone, hence their synergistically enhanced activity does not seem to correlate with a modulation of membrane thickness. Overall, the absence of

  11. Differential neuroprotective potential of CRMP2 peptide aptamers conjugated to cationic, hydrophobic, and amphipathic cell penetrating peptides

    Directory of Open Access Journals (Sweden)

    Aubin eMoutal

    2015-01-01

    Full Text Available The microtubule-associated axonal specification collapsin response mediator protein 2 (CRMP2 is a novel target for neuroprotection. A CRMP2 peptide (TAT-CBD3 conjugated to the HIV transactivator of transcription (TAT protein’s cationic cell penetrating peptide motif (CPP protected neurons in the face of toxic levels of Ca2+ influx leaked in via N-methyl-D-aspartate receptor (NMDAR hyperactivation. Here we tested whether replacing the hydrophilic TAT motif with alternative cationic (nona-arginine (R9, hydrophobic (membrane transport sequence (MTS of k-fibroblast growth factor or amphipathic (model amphipathic peptide (MAP CPPs could be superior to the neuroprotection bestowed by TAT-CBD3. In giant plasma membrane vesicles (GPMVs derived from cortical neurons, the peptides translocated across plasma membranes with similar efficiencies. Cortical neurons, acutely treated with peptides prior to a toxic glutamate challenge, demonstrated enhanced efflux of R9-CBD3 compared to others. R9-CBD3 inhibited N-methyl-D-aspartate (NMDA-evoked Ca2+ influx to a similar extent as TAT-CBD3 while MTS-CBD3 was ineffective which correlated with the ability of R9- and TAT-CBD3, but not MTS-CBD3, to block NMDAR interaction with CRMP2. Unrestricted Ca2+ influx through NMDARs leading to delayed calcium dysregulation and neuronal cell death was blocked by all peptides but MAP-CBD3. When applied acutely for 10 minutes, R9-CBD3 was more effective than TAT-CBD3 at neuroprotection while MTS- and MAP-CBD3 were ineffective. In contrast, long-term (> 24 hours treatment with MTS-CBD3 conferred neuroprotection where TAT-CBD3 failed. Neither peptide altered surface trafficking of NMDARs. Neuroprotection conferred by MTS-CBD3 peptide is likely due to its increased uptake coupled with decreased efflux when compared to TAT-CBD3. Overall, our results demonstrate that altering CPPs can bestow differential neuroprotective potential onto the CBD3 cargo.

  12. 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.

  13. 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....

  14. 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....

  15. 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....

  16. 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....

  17. 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...

  18. 两亲性结构修饰的非天然氨基酸及二肽的合成%Synthesis of Unusual Amino Acids and Dipeptides Modified with Amphipathic Betaine

    Institute of Scientific and Technical Information of China (English)

    吕玉健; 周宁; 刘克良

    2011-01-01

    目的 合成两亲性结构修饰的非天然氨基酸和二肽.方法 将具有两亲性结构特点的甜菜碱的羧基活化成酰氯,然后与对氨基苯丙氨酸的侧链氨基反应,制备甜菜碱修饰的非天然氨基酸构建单元和目标二肽,并进行油水分配系数的测定.结果 得到了1个甜菜碱修饰的非天然氨基酸和2个甜菜碱修饰的二肽.结论 甜菜碱修饰的二肽具有更强的两亲性,为增强药物两亲性,改善药学性质提供了一种新的选择.%Objective To synthesize unusual amino acid and dipeptides modified with an amphipathic structure. Methods The amphipathic betaine was activated as N-chlorobetainyl chloride and then coupled with to-a-mino of 4-amino phenylalanine to synthesize the target compound. The peptides were synthesized in the sold phase. Results One betaine-modified non-natural amino acid and two dipeptides were obtained. Conclusion One unusual amino acid and two dipeptides modified with betaine were synthesized. The carboxyl of betaine was activated with acyl chlorides and the reactivity of betaine was greatly increased. Betaine could smoothly couple with w-amino of amino acid, which provided a novel alternative for the amphipathic modification of peptides.

  19. The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix

    Science.gov (United States)

    Barneda, David; Planas-Iglesias, Joan; Gaspar, Maria L; Mohammadyani, Dariush; Prasannan, Sunil; Dormann, Dirk; Han, Gil-Soo; Jesch, Stephen A; Carman, George M; Kagan, Valerian; Parker, Malcolm G; Ktistakis, Nicholas T; Klein-Seetharaman, Judith; Dixon, Ann M; Henry, Susan A; Christian, Mark

    2015-01-01

    Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue, and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs, which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD–LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat. DOI: http://dx.doi.org/10.7554/eLife.07485.001 PMID:26609809

  20. General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides.

    Science.gov (United States)

    Dathe, Margitta; Meyer, Jana; Beyermann, Michael; Maul, Björn; Hoischen, Christian; Bienert, Michael

    2002-02-01

    Model compounds of modified hydrophobicity (Eta), hydrophobic moment (mu) and angle subtended by charged residues (Phi) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. Eta and mu influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Phi, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of mu and Phi on both lipid bilayers and cell membranes.

  1. 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....

  2. Peptide Nucleic Acids Complexes of Two Peptide Nucleic Acid Strands and One

    DEFF Research Database (Denmark)

    1999-01-01

    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...

  3. 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....

  4. 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....

  5. 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 of nat...

  6. The effect of glycine replacement with flexible ω-amino acids on the antimicrobial and haemolytic activity of an amphipathic cyclic heptapeptide

    DEFF Research Database (Denmark)

    Oddo, Alberto; Nyberg, Nils; Frimodt-Møller, Niels;

    2015-01-01

    residue with a variety of flexible residues (i.e. ω-amino and α,ω-diamino acids). The resulting library has been tested for antimicrobial activity against a wide range of clinically relevant pathogens as well as for toxicity to red blood cells. Circular dichroism and molecular modelling have been used......Although cyclic peptide structures are usually investigated as highly constrained scaffolds, cyclic antimicrobial peptides of natural origin often feature flexible residues. Hereby we report our findings concerning a structure-activity study conducted on a model sequence by replacing a glycine...... to study changes in conformation. Increments as high as 16-fold in antimicrobial activity (as effective as lipidation) and >2-fold in haemolytic EC50 values were observed. Interestingly, secondary structures can be stabilized by increasing, rather than decreasing, ring flexibility....

  7. 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.

  8. An introduction to peptide nucleic acid

    DEFF Research Database (Denmark)

    Nielsen, P E; Egholm, M

    1999-01-01

    Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful...

  9. 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...

  10. Structural characterization of the model amphipathic peptide Ac-LKKLLKLLKKLLKL-NH2 in aqueous solution and with 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoroisopropanol

    Energy Technology Data Exchange (ETDEWEB)

    Buchko, Garry W.; Jain, Avijita; Reback, Matthew L.; Shaw, Wendy J.

    2013-06-03

    Short-chain amphiphilic peptides are promising components in the new generation of engineered biomaterials with many potential applications. The 14-residue leucine-lysine peptide Ac-LKKLLKLLKKLLKL-NH2 (LKα) is one such amphiphilic peptide. The periodic distribution of hydrophobic and hydrophilic amino acid residues in the sequence of LKα has been shown to promote α-helix formation in an ionic environment and at high peptide concentrations (> ~0.5 mM, no salt). Here, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy is used to demonstrate that LKα, in the absence of salt and at concentrations < 0.5 mM, readily adopts a helical structure in the presence of the structure stabilizing agents 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP). Maximal helical character, as monitored by negative bands with double minima at 222 and 208-210 nm in the CD spectrum, was observed in 20% TFE and 10% HFIP (volume percent). The helical character suggested by the CD data was corroborated with amide to alpha proton, long range, 1HN(i) to 1Hα(i-3) NOEs characteristic of an α-helical structure. In unbuffered water in the absence of a flouronated alcohol and at low peptide concentrations, LKα was essentially unstructured in solution. These observations confirm that LKα has a predisposition to adopt a helical structure that may be maximized with minimal amounts of fluorinated alcohol. This characterization of the structural and physical properties of LKα will assist the design of future biomaterials containing amphiphilic peptides.

  11. Nucleic Acid Backbone Structure Variations: Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    Nielsen, Peter E.

    2010-01-01

    Synthetic analogues and mimics of the natural genetic material deoxyribonucleic acid (DNA) are potential gene therapeutic (antisense or antigene) drugs. One of these mimics, peptide nucleic acids (PNAs), are chemically closer to peptides and proteins than to DNA, but nonetheless have retained many...... of the structural properties of DNA. These molecules have found applications as probes in genetic diagnostics and are also being developed into antisense (RNA (ribonucleic acid) interference) gene therapeutic drugs, targeting selected genes through sequence-specific recognition of (messenger or micro......)RNA and in the future also antigene applications targeting the double-stranded DNA of the genes themselves leading to gene silencing or guiding specific gene repair. Finally, the special chemical and structural properties of PNA suggest that these or similar molecules might have played a role in the prebiotic origin...

  12. Neutron diffraction studies of amphipathic helices in phospholipid bilayers

    International Nuclear Information System (INIS)

    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

  13. 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.

  14. Molecular design and genetic optimization of antimicrobial peptides containing unnatural amino acids against antibiotic-resistant bacterial infections.

    Science.gov (United States)

    He, Yongkang; He, Xiaofeng

    2016-09-01

    Antimicrobial peptides (AMPs) have been the focus of intense research towards the finding of a viable alternative to current small-molecule antibiotics, owing to their commonly observed and naturally occurring resistance against pathogens. However, natural peptides have many problems such as low bioavailability and high allergenicity that largely limit the clinical applications of AMPs. In the present study, an integrative protocol that combined chemoinformatics modeling, molecular dynamics simulations, and in vitro susceptibility test was described to design AMPs containing unnatural amino acids (AMP-UAAs). To fulfill this, a large panel of synthetic AMPs with determined activity was collected and used to perform quantitative structure-activity relationship (QSAR) modeling. The obtained QSAR predictors were then employed to direct genetic algorithm (GA)-based optimization of AMP-UAA population, to which a number of commercially available, structurally diverse unnatural amino acids were introduced during the optimization process. Subsequently, several designed AMP-UAAs were confirmed to have high antibacterial potency against two antibiotic-resistant strains, i.e. multidrug-resistant Pseudomonas aeruginosa (MDRPA) and methicillin-resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentration (MIC) < 10 μg/ml. Structural dynamics characterizations revealed that the most potent AMP-UAA peptide is an amphipathic helix that can spontaneously embed into an artificial lipid bilayer and exhibits a strong destructuring tendency associated with the embedding process. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 746-756, 2016.

  15. Penetration Depth of Surfactant Peptide KL4 into Membranes Is Determined by Fatty Acid Saturation

    OpenAIRE

    Antharam, Vijay C.; Elliott, Douglas W.; Mills, Frank D.; Farver, R. Suzanne; Sternin, Edward; Long, Joanna R.

    2009-01-01

    KL4 is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL4 binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL4 is dep...

  16. Penetration depth of surfactant peptide KL4 into membranes is determined by fatty acid saturation.

    Science.gov (United States)

    Antharam, Vijay C; Elliott, Douglas W; Mills, Frank D; Farver, R Suzanne; Sternin, Edward; Long, Joanna R

    2009-05-20

    KL(4) is a 21-residue functional peptide mimic of lung surfactant protein B, an essential protein for lowering surface tension in the alveoli. Its ability to modify lipid properties and restore lung compliance was investigated with circular dichroism, differential scanning calorimetry, and solid-state NMR spectroscopy. KL(4) binds fluid lamellar phase PC/PG lipid membranes and forms an amphipathic helix that alters lipid organization and acyl chain dynamics. The binding and helicity of KL(4) is dependent on the level of monounsaturation in the fatty acid chains. At physiologic temperatures, KL(4) is more peripheral and dynamic in fluid phase POPC/POPG MLVs but is deeply inserted into fluid phase DPPC/POPG vesicles, resulting in immobilization of the peptide. Substantial increases in the acyl chain order are observed in DPPC/POPG lipid vesicles with increasing levels of KL(4), and POPC/POPG lipid vesicles show small decreases in the acyl chain order parameters on addition of KL(4). Additionally, a clear effect of KL(4) on the orientation of the fluid phase PG headgroups is observed, with similar changes in both lipid environments. Near the phase transition temperature of the DPPC/POPG lipid mixtures, which is just below the physiologic temperature of lung surfactant, KL(4) causes phase separation with the DPPC remaining in a gel phase and the POPG partitioned between gel and fluid phases. The ability of KL(4) to differentially partition into lipid lamellae containing varying levels of monounsaturation and subsequent changes in curvature strain suggest a mechanism for peptide-mediated lipid organization and trafficking within the dynamic lung environment. PMID:19450480

  17. 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 cons...

  18. An Amphipathic Undecapeptide with All d-Amino Acids Shows Promising Activity against Colistin-Resistant Strains of Acinetobacter baumannii and a Dual Mode of Action.

    Science.gov (United States)

    Oddo, Alberto; Thomsen, Thomas T; Kjelstrup, Susanne; Gorey, Ciara; Franzyk, Henrik; Frimodt-Møller, Niels; Løbner-Olesen, Anders; Hansen, Paul R

    2016-01-01

    Multiple strains of Acinetobacter baumannii have developed multidrug resistance (MDR), leaving colistin as the only effective treatment. The cecropin-α-melittin hybrid BP100 (KKLFKKILKYL-NH2) and its analogs have previously shown activity against a wide array of plant and human pathogens. In this study, we investigated the in vitro antibacterial activities of 18 BP100 analogs (four known and 14 new) against the MDR A. baumannii strain ATCC BAA-1605, as well as against a number of other clinically relevant human pathogens. Selected peptides were further evaluated against strains of A. baumannii that acquired resistance to colistin due to mutations of the lpxC, lpxD, pmrA, and pmrB genes. The novel analogue BP214 showed antimicrobial activity at 1 to 2 μM and a hemolytic 50% effective concentration (EC50) of >150 μM. The lower activity of its enantiomer suggests a dual, specific and nonspecific mode of action. Interestingly, colistin behaved antagonistically to BP214 when pmrAB and lpxC mutants were challenged. PMID:26574005

  19. Fatty acid conjugation enhances the activities of antimicrobial peptides.

    Science.gov (United States)

    Li, Zhining; Yuan, Penghui; Xing, Meng; He, Zhumei; Dong, Chuanfu; Cao, Yongchang; Liu, Qiuyun

    2013-04-01

    Antimicrobial peptides are small molecules that play a crucial role in innate immunity in multi-cellular organisms, and usually expressed and secreted constantly at basal levels to prevent infection, but local production can be augmented upon an infection. The clock is ticking as rising antibiotic abuse has led to the emergence of many drug resistance bacteria. Due to their broad spectrum antibiotic and antifungal activities as well as anti-viral and anti-tumor activities, efforts are being made to develop antimicrobial peptides into future microbial agents. This article describes some of the recent patents on antimicrobial peptides with fatty acid conjugation. Potency and selectivity of antimicrobial peptide can be modulated with fatty acid tails of variable length. Interaction between membranes and antimicrobial peptides was affected by fatty acid conjugation. At concentrations above the critical miscelle concentration (CMC), propensity of solution selfassembly hampered binding of the peptide to cell membranes. Overall, fatty acid conjugation has enhanced the activities of antimicrobial peptides, and occasionally it rendered inactive antimicrobial peptides to be bioactive. Antimicrobial peptides can not only be used as medicine but also as food additives.

  20. Hyaluronic acid functional amphipathic and redox-responsive polymer particles for the co-delivery of doxorubicin and cyclopamine to eradicate breast cancer cells and cancer stem cells

    Science.gov (United States)

    Hu, Kelei; Zhou, Huige; Liu, Ying; Liu, Zhu; Liu, Jing; Tang, Jinglong; Li, Jiayang; Zhang, Jiakun; Sheng, Wang; Zhao, Yuliang; Wu, Yan; Chen, Chunying

    2015-04-01

    Cancer stem cells (CSCs) have the ability to transform into bulk cancer cells, to promote tumor growth and establish tumor metastasis. To effectively inhibit tumor growth and prevent metastasis, treatments with conventional chemotherapy drugs should be combined with CSC targeted drugs. In this study, we describe the synthesis and characterization of a new amphiphilic polymer, hyaluronic acid-cystamine-polylactic-co-glycolic acid (HA-SS-PLGA), composed of a hydrophobic PLGA head and a hydrophilic HA segment linked by a bioreducible disulfide bond. With a double emulsion method, a nano delivery system was constructed to deliver doxorubicin (DOX) and cyclopamine (CYC, a primary inhibitor of the hedgehog signaling pathway of CSCs) to both a CD44-overexpressing breast CSC subpopulation and bulk breast cancer cells and allow an on-demand release. The resulting drug-loaded NPs exhibited a redox-responsive drug release profile. Dual drug-loaded particles potently diminished the number and size of tumorspheres and HA showed a targeting effect towards breast CSCs. In vivo combination therapy further demonstrated a remarkable synergistic anti-tumor effect and prolonged survival compared to mono-therapy using the orthotopic mammary fat pad tumor growth model. The co-delivery of drug and the CSC specific inhibitor towards targeted cancer chemotherapeutics provides an insight into anticancer strategy with facile control and high efficacy.Cancer stem cells (CSCs) have the ability to transform into bulk cancer cells, to promote tumor growth and establish tumor metastasis. To effectively inhibit tumor growth and prevent metastasis, treatments with conventional chemotherapy drugs should be combined with CSC targeted drugs. In this study, we describe the synthesis and characterization of a new amphiphilic polymer, hyaluronic acid-cystamine-polylactic-co-glycolic acid (HA-SS-PLGA), composed of a hydrophobic PLGA head and a hydrophilic HA segment linked by a bioreducible disulfide bond

  1. The Cytolytic Amphipathic β(2,2-Amino Acid LTX-401 Induces DAMP Release in Melanoma Cells and Causes Complete Regression of B16 Melanoma.

    Directory of Open Access Journals (Sweden)

    Liv-Marie Eike

    Full Text Available In the present study we examined the ability of the amino acid derivative LTX-401 to induce cell death in cancer cell lines, as well as the capacity to induce regression in a murine melanoma model. Mode of action studies in vitro revealed lytic cell death and release of danger-associated molecular pattern molecules, preceded by massive cytoplasmic vacuolization and compromised lysosomes in treated cells. The use of a murine melanoma model demonstrated that the majority of animals treated with intratumoural injections of LTX-401 showed complete and long-lasting remission. Taken together, these results demonstrate the potential of LTX-401 as an immunotherapeutic agent for the treatment of solid tumors.

  2. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    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 a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic aci...

  3. 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.

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

    OpenAIRE

    Claude Daneault; Saïd Barazzouk

    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, th...

  5. 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....

  6. 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...

  7. 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"...

  8. Metal-based chemosensors for amino acids, peptides, and nucleotides

    OpenAIRE

    Buryak, Andrey

    2007-01-01

    An organometallic 4d transition metal complex [Cp*RhCl2]2, together with commercially available dyes, was used to construct indicator displacement assays (IDAs) for the detection of peptides, amino acids, and nucleotides. The combination of the Cp*Rh complex with the dye azophloxine was found to form a chemosensing ensemble for the sequence-selective detection of histidine- and methionine-containing peptides in water at neutral pH. A strong interaction of the rhodium complex with peptides bea...

  9. Metal-based chemosensors for amino acids, peptides, and nucleotides

    OpenAIRE

    Buryak, Andrey; Severin, Kay

    2008-01-01

    An organometallic 4d transition metal complex [Cp*RhCl2]2, together with commercially available dyes, was used to construct indicator displacement assays (IDAs) for the detection of peptides, amino acids, and nucleotides. The combination of the Cp*Rh complex with the dye azophloxine was found to form a chemosensing ensemble for the sequence-selective detection of histidine- and methionine-containing peptides in water at neutral pH. A strong interaction of the rhodium complex with peptides bea...

  10. Design and Characterization of an Acid-Activated Antimicrobial Peptide

    OpenAIRE

    Li, Lina; He, Jian; Eckert, Randal; Yarbrough, Daniel; Lux, Renate; Anderson, Maxwell; Shi, Wenyuan

    2009-01-01

    Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/ remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals creation of an acidic environment favors growth of acid enduring and acid generating species, which causes further reduction in the plaque pH. In this study we developed a prototype antimicrobial peptide ...

  11. How amino acids and peptides shaped the RNA world.

    Science.gov (United States)

    van der Gulik, Peter T S; Speijer, Dave

    2015-01-01

    The "RNA world" hypothesis is seen as one of the main contenders for a viable theory on the origin of life. Relatively small RNAs have catalytic power, RNA is everywhere in present-day life, the ribosome is seen as a ribozyme, and rRNA and tRNA are crucial for modern protein synthesis. However, this view is incomplete at best. The modern protein-RNA ribosome most probably is not a distorted form of a "pure RNA ribosome" evolution started out with. Though the oldest center of the ribosome seems "RNA only", we cannot conclude from this that it ever functioned in an environment without amino acids and/or peptides. Very small RNAs (versatile and stable due to basepairing) and amino acids, as well as dipeptides, coevolved. Remember, it is the amino group of aminoacylated tRNA that attacks peptidyl-tRNA, destroying the bond between peptide and tRNA. This activity of the amino acid part of aminoacyl-tRNA illustrates the centrality of amino acids in life. With the rise of the "RNA world" view of early life, the pendulum seems to have swung too much towards the ribozymatic part of early biochemistry. The necessary presence and activity of amino acids and peptides is in need of highlighting. In this article, we try to bring the role of the peptide component of early life back into focus. We argue that an RNA world completely independent of amino acids never existed. PMID:25607813

  12. Conjugation of fatty acids with different lengths modulates the antibacterial and antifungal activity of a cationic biologically inactive peptide.

    Science.gov (United States)

    Malina, Amir; Shai, Yechiel

    2005-09-15

    Many studies have shown that an amphipathic structure and a threshold of hydrophobicity of the peptidic chain are crucial for the biological function of AMPs (antimicrobial peptides). However, the factors that dictate their cell selectivity are not yet clear. In the present study, we show that the attachment of aliphatic acids with different lengths (10, 12, 14 or 16 carbon atoms) to the N-terminus of a biologically inactive cationic peptide is sufficient to endow the resulting lipopeptides with lytic activity against different cells. Mode-of-action studies were performed with model phospholipid membranes mimicking those of bacterial, mammalian and fungal cells. These include determination of the structure in solution and membranes by using CD and ATR-FTIR (attenuated total reflectance Fourier-transform infrared) spectroscopy, membrane leakage experiments and by visualizing bacterial and fungal damage via transmission electron microscopy. The results obtained reveal that: (i) the short lipopeptides (10 and 12 carbons atoms) are non-haemolytic, active towards both bacteria and fungi and monomeric in solution. (ii) The long lipopeptides (14 and 16 carbons atoms) are highly antifungal, haemolytic only at concentrations above their MIC (minimal inhibitory concentration) values and aggregate in solution. (iii) All the lipopeptides adopt a partial alpha-helical structure in 1% lysophosphatidylcholine and bacterial and mammalian model membranes. However, the two short lipopeptides contain a significant fraction of random coil in fungal membranes, in agreement with their reduced antifungal activity. (iv) All the lipopeptides have a membranolytic effect on all types of cells assayed. Overall, the results reveal that the length of the aliphatic chain is sufficient to control the pathogen specificity of the lipopeptides, most probably by controlling both the overall hydrophobicity and the oligomeric state of the lipopeptides in solution. Besides providing us with basic

  13. Design and characterization of an acid-activated antimicrobial peptide.

    Science.gov (United States)

    Li, Lina; He, Jian; Eckert, Randal; Yarbrough, Daniel; Lux, Renate; Anderson, Maxwell; Shi, Wenyuan

    2010-01-01

    Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals, creation of an acidic environment favors growth of acid-enduring and acid-generating species, which causes further reduction in the plaque pH. In this study, we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype 'acid-activated peptides' (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity, which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries. PMID:19878192

  14. Engineering Dehydrated Amino Acid Residues in the Antimicrobial Peptide Nisin

    NARCIS (Netherlands)

    Kuipers, Oscar P.; Rollema, Harry S.; Yap, Wyanda M.G.J.; Boot, Hein J.; Siezen, Roland J.; Vos, Willem M. de

    1992-01-01

    The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role i

  15. Non-protein amino acids in peptide design

    Indian Academy of Sciences (India)

    S Aravinda; N Shamala; Rituparna S Roy; P Balaram

    2003-10-01

    An overview of the use of non-protein amino acids in the design of conformationally well-defined peptides, based on work from the author’s laboratory, is discussed. The crystal structures of several designed oligopeptides illustrate the use -aminoisobutyric acid (Aib) in the construction of helices, D-amino acids in the design of helix termination segments and DPro-Xxx segments for nucleating of -hairpin structures. - and -amino acid residues have been used to expand the range of designed polypeptide structures.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge....... 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...

  17. Acid-catalyzed oxygen-18 labeling of peptides.

    Science.gov (United States)

    Niles, Richard; Witkowska, H Ewa; Allen, Simon; Hall, Steven C; Fisher, Susan J; Hardt, Markus

    2009-04-01

    In enzymatic (18)O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as method for introducing (18)O label into peptides. Several synthetic peptides were dissolved in dilute acid containing 50% (v/v) H(2)(18)O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). (18)O-incorporation ratios were determined by linear regression analysis that allowed for multiple stable-isotope incorporations. At low pH, peptides exchanged their carboxyl oxygen atoms with the aqueous solvent. The isotope patterns gradually shifted to higher masses until they reached the expected binomial distribution at equilibrium after approximately 11 days. Reaction rates were residue- and sequence-specific. Due to its slow nature, the acid-catalyzed back exchange is expected to minimally interfere with enzymatic (18)O-labeling studies provided that storage and analysis conditions minimize low-pH exposure times. On its own, acid-catalyzed (18)O labeling is a general tagging strategy that is an alternative to the chemical, metabolic, and enzymatic isotope-labeling schemes currently used in quantitative proteomics. PMID:19243188

  18. Biological activity and biotechnological aspects of peptide nucleic acid.

    Science.gov (United States)

    Lundin, Karin E; Good, Liam; Strömberg, Roger; Gräslund, Astrid; Smith, C I Edvard

    2006-01-01

    During the latest decades a number of different nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized. An example of this is peptide nucleic acid (PNA), a DNA mimic with a noncyclic peptide-like backbone, which was first synthesized in 1991. Owing to its flexible and neutral backbone PNA displays very good hybridization properties also at low-ion concentrations and has subsequently attracted large interest both in biotechnology and biomedicine. Numerous modifications have been made, which could be of value for particular settings. However, the original PNA does so far perform well in many diverse applications. The high biostability makes it interesting for in vivo use, although the very limited diffusion over lipid membranes requires further modifications in order to make it suitable for treatment in eukaryotic cells. The possibility to use this nucleic acid analog for gene regulation and gene editing is discussed. Peptide nucleic acid is now also used for specific genetic detection in a number of diagnostic techniques, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA, areas that are also discussed in this chapter.

  19. Lycotoxin-1 insecticidal peptide optimized by amino acid scanning mutagenesis and expressed as a coproduct in an ethanologenic Saccharomyces cerevisiae strain.

    Science.gov (United States)

    Hughes, Stephen R; Dowd, Patrick F; Hector, Ronald E; Panavas, Tadas; Sterner, David E; Qureshi, Nasib; Bischoff, Kenneth M; Bang, Sookie S; Mertens, Jeffrey A; Johnson, Eric T; Li, Xin-Liang; Jackson, John S; Caughey, Robert J; Riedmuller, Steven B; Bartolett, Scott; Liu, Siqing; Rich, Joseph O; Farrelly, Philip J; Butt, Tauseef R; Labaer, Joshua; Cotta, Michael A

    2008-09-01

    New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin alpha-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction.

  20. Synthesis and photoactivity of phenylazonaphthalene peptide nucleic acid monomers

    Institute of Scientific and Technical Information of China (English)

    Jin Du Li; Miao Chen; Sheng Liu; Hao Bo Zhang; Zhi Lu Liu

    2008-01-01

    Phenylazonaphthalene peptide nucleic acid (PNA) monomers were successfully synthesized,and their photoisomerization was examined.The new PNA monomers showed reversible trans-cis isomerization with UV and visible light irradiation,which might be the foundation of photo-regulating the hybridization between PNA containing phenylazonaphthalene unit and DNA.Simultaneously,the fluorescence of the new PNA monomers might make them especially useful as structural probes.

  1. Recent Advances in Chemical Modification of Peptide Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Eriks Rozners

    2012-01-01

    Full Text Available Peptide nucleic acid (PNA has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification.

  2. The interaction of amino acids, peptides, and proteins with DNA.

    Science.gov (United States)

    Solovyev, Andrey Y; Tarnovskaya, Svetlana I; Chernova, Irina A; Shataeva, Larisa K; Skorik, Yury A

    2015-01-01

    Amino acids that carry charges on their side groups can bind to double stranded DNA (dsDNA) and change the strength of the double helix. Measurement of the DNA melting temperature (Tm) confirmed that acidic amino acids (Glu, Asp) weaken the H-bonds between DNA strands, whereas basic amino acids (Arg, Lys) strengthen the interaction between the strands. A rank correlation exists between the amino acid isoelectric points and the observed changes in Tm. A similar dependence of the hyperchromic effect on the isoelectric point of a protein (pepsin, insulin, cortexin, and protamine) was observed for DNA-protein complexes at room temperature. Short peptides (KE, AEDG, and KEDP) containing a mixture of acidic and basic amino acid residues also affect Tm and the stability of the double helix. A model for binding Glu and Lys to dsDNA was explored by a docking simulation. The model shows that Glu, in an untwisted shape, binds to dsDNA in its major groove and disrupts three H-bonds between the strands, thereby destabilizing the double helix. Lys, in an untwisted shape, binds to the external side of the dsDNA and forms two bonds with O atoms of neighboring phosphodiester groups, thereby strengthening the DNA helix.

  3. Structural Characterization of de Novo Designed L5K5W Model Peptide Isomers with Potent Antimicrobial and Varied Hemolytic Activities

    Directory of Open Access Journals (Sweden)

    Sung-Jean Park

    2013-01-01

    Full Text Available In an effort to develop short antimicrobial peptides with simple amino acid compositions, we generated a series of undecapeptide isomers having the L5K5W formula. Amino acid sequences were designed to be perfectly amphipathic when folded into a helical conformation by converging leucines onto one side and lysines onto the other side of the helical axis. The single tryptophans, whose positions were varied in the primary structures, were located commonly at the critical amphipathic interface in the helical wheel projection. Helical conformations and the tryptophanyl environments of the 11 L5K5W peptides were confirmed and characterized by circular dichroism, fluorescence and nuclear magnetic resonance spectroscopy. All of the isomers exhibited a potent, broad-spectrum of antibacterial activity with just a slight variance in individual potency, whereas their hemolytic activities against human erythrocytes were significantly diversified. Interestingly, helical dispositions and fluorescence blue shifts of the peptides in aqueous trifluoroethanol solutions, rather than in detergent micelles, showed a marked linear correlation with their hemolytic potency. These results demonstrate that our de novo design strategy for amphipathic helical model peptides is effective for developing novel antimicrobial peptides and their hemolytic activities can be estimated in correlation with structural parameters.

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

    DEFF Research Database (Denmark)

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

    1994-01-01

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

  5. Synthesis of New Chrial Building Blocks for Novel Peptide Nucleic Acids

    Institute of Scientific and Technical Information of China (English)

    WU,Jie; XU,Xiao-Yu; LIU,Ke-Liang

    2003-01-01

    N-Boc protected amino acids of analogues of peptide nucleic acid (PNA),which are a class of conformationally constrained building blocks based on 4-aminoproline backbone with chirality at 2-c and 4-c,have been synthesized.Those monomers can be used for the construction of novel peptide nucleic acid analogues.

  6. Manipulating lipid bilayer material properties using biologically active amphipathic molecules

    Energy Technology Data Exchange (ETDEWEB)

    Ashrafuzzaman, Md [Department of Physiology and Biophysics, Weill Medical College of University of Cornell, New York, NY 10021 (United States); Lampson, M A [Department of Physiology and Biophysics, Weill Medical College of University of Cornell, New York, NY 10021 (United States); Greathouse, D V [Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701 (United States); II, R E Koeppe [Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701 (United States); Andersen, O S [Department of Physiology and Biophysics, Weill Medical College of University of Cornell, New York, NY 10021 (United States)

    2006-07-19

    Lipid bilayers are elastic bodies with properties that can be manipulated/controlled by the adsorption of amphipathic molecules. The resulting changes in bilayer elasticity have been shown to regulate integral membrane protein function. To further understand the amphiphile-induced modulation of bilayer material properties (thickness, intrinsic monolayer curvature and elastic moduli), we examined how an enantiomeric pair of viral anti-fusion peptides (AFPs)-Z-Gly-D-Phe and Z-Gly-Phe, where Z denotes a benzyloxycarbonyl group, as well as Z-Phe-Tyr and Z-D-Phe-Phe-Gly-alters the function of enantiomeric pairs of gramicidin channels of different lengths in planar bilayers. For both short and long channels, the channel lifetimes and appearance frequencies increase as linear functions of the aqueous AFP concentration, with no apparent effect on the single-channel conductance. These changes in channel function do not depend on the chirality of the channels or the AFPs. At pH 7.0, the relative changes in channel lifetimes do not vary when the channel length is varied, indicating that these compounds exert their effects primarily by causing a positive-going change in the intrinsic monolayer curvature. At pH 4.0, the AFPs are more potent than at pH 7.0 and have greater effects on the shorter channels, indicating that these compounds now change the bilayer elastic moduli. When AFPs of different anti-fusion potencies are compared, the rank order of the anti-fusion activity and the channel-modifying activity is similar, but the relative changes in anti-fusion potency are larger than the changes in channel-modifying activity. We conclude that gramicidin channels are useful as molecular force transducers to probe the influence of small amphiphiles upon lipid bilayer material properties.

  7. 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.

  8. Cell-Penetrating Recombinant Peptides for Potential Use in Agricultural Pest Control Applications

    Directory of Open Access Journals (Sweden)

    Eric T. Johnson

    2012-09-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2016-05-01

    As one of the greatest threats facing the 21st century, antibiotic resistance is now a major public health concern. Host-defense peptides (HDPs) offer an alternative approach to combat emerging multi-drug-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 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. PMID:27030636

  10. 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...

  11. An expanded set of amino acid analogs for the ribosomal translation of unnatural peptides.

    Directory of Open Access Journals (Sweden)

    Matthew C T Hartman

    Full Text Available BACKGROUND: The application of in vitro translation to the synthesis of unnatural peptides may allow the production of extremely large libraries of highly modified peptides, which are a potential source of lead compounds in the search for new pharmaceutical agents. The specificity of the translation apparatus, however, limits the diversity of unnatural amino acids that can be incorporated into peptides by ribosomal translation. We have previously shown that over 90 unnatural amino acids can be enzymatically loaded onto tRNA. METHODOLOGY/PRINCIPAL FINDINGS: We have now used a competition assay to assess the efficiency of tRNA-aminoacylation of these analogs. We have also used a series of peptide translation assays to measure the efficiency with which these analogs are incorporated into peptides. The translation apparatus tolerates most side chain derivatives, a few alpha,alpha disubstituted, N-methyl and alpha-hydroxy derivatives, but no beta-amino acids. We show that over 50 unnatural amino acids can be incorporated into peptides by ribosomal translation. Using a set of analogs that are efficiently charged and translated we were able to prepare individual peptides containing up to 13 different unnatural amino acids. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that a diverse array of unnatural building blocks can be translationally incorporated into peptides. These building blocks provide new opportunities for in vitro selections with highly modified drug-like peptides.

  12. An amphipathic undecapeptide with all D-amino acids shows promising activity against colistin-resistant strains of Acinetobacter baumannii and a dual mode of action

    DEFF Research Database (Denmark)

    Oddo, Alberto; Thomsen, Thomas Thyge; Kjelstrup, Susanne;

    2016-01-01

    Multiple strains of Acinetobacter baumannii have developed multidrug resistance (MDR), leaving colistin as the only effective treatment. The cecropin-α-melittin hybrid BP100 (KKLFKKILKYL-NH2) and its analogs have previously shown activity against a wide array of plant and human pathogens. In this...... study, we investigated the in vitro antibacterial activities of 18 BP100 analogs (four known and 14 new) against the MDR A. baumannii strain ATCC BAA-1605, as well as against a number of other clinically relevant human pathogens. Selected peptides were further evaluated against strains of A. baumannii...... that acquired resistance to colistin due to mutations of the lpxC, lpxD, pmrA, and pmrB genes. The novel analogue BP214 showed antimicrobial activity at 1 to 2 μM and a hemolytic 50% effective concentration (EC50) of >150 μM. The lower activity of its enantiomer suggests a dual, specific and...

  13. How curved membranes recruit amphipathic helices and protein anchoring motifs

    DEFF Research Database (Denmark)

    Hatzakis, Nikos; Bhatia, Vikram Kjøller; Larsen, Jannik;

    2009-01-01

    Lipids and several specialized proteins are thought to be able to sense the curvature of membranes (MC). Here we used quantitative fluorescence microscopy to measure curvature-selective binding of amphipathic motifs on single liposomes 50-700 nm in diameter. Our results revealed that sensing is p...

  14. Mutual Amino Acid Catalysis in Salt-Induced Peptide Formation Supports this Mechanism's Role in Prebiotic Peptide Evolution

    Science.gov (United States)

    Suwannachot, Yuttana; Rode, Bernd M.

    1999-10-01

    The presence of some amino acids and dipeptides under the conditions of the salt-induced peptide formation reaction (aqueous solution at 85 °C, Cu(II) and NaCl) has been found to catalyze the formation of homopeptides of other amino acids, which are otherwise produced only in traces or not at all by this reaction. The condensation of Val, Leu and Lys to form their homodipeptides can occur to a considerable extent due to catalytic effects of other amino acids and related compounds, among which glycine, histidine, diglycine and diketopiperazine exhibit the most remarkable activity. These findings also lead to a modification of the table of amino acid sequences preferentially formed by the salt-induced peptide formation (SIPF) reaction, previously used for a comparison with the sequence preferences in membrane proteins of primitive organisms

  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

    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...

  16. A Peptide & Peptide Nucleic Acid Synthesis Technology for Transporter Molecules and Theranostics - The SPPS

    OpenAIRE

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and functi...

  17. Biomimetic PEG hydrogels crosslinked with minimal plasmin-sensitive tri-amino acid peptides

    OpenAIRE

    Jo, Suk Jo; Rizzi, Simone C.; Ehrbar, Martin; Weber, Franz E.; Hubbell, Jeffrey A.; Lutolf, Matthias P.

    2010-01-01

    Semi-synthetic, proteolytically degradable polymer hydrogels have proven effective as scaffolds to augment bone and skin regeneration in animals. However, high costs due to expensive peptide building blocks pose a significant hurdle towards broad clinical usage of these materials. Here we demonstrate that tri-amino acid peptides bearing lysine (or arginine), flanked by two cysteine residues for crosslinking, are adequate as minimal plasmin-sensitive peptides in poly(ethylene glycol)-based hyd...

  18. Peptide nucleic acid (PNA) binding-mediated gene regulation

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a singlestranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The Dloops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.

  19. Self-assembling properties of all γ-cyclic peptides containing sugar amino acid residues.

    Science.gov (United States)

    Guerra, Arcadio; Brea, Roberto J; Amorín, Manuel; Castedo, Luis; Granja, Juan R

    2012-11-28

    In this study, a novel dimer-forming cyclic peptide composed exclusively by cyclic γ-amino acids with a saccharide-like outer surface is described. The antiparallel β-sheet type hydrogen bonding interactions responsible for the large association constant in non-polar solvents constitute a suitable model for a novel class of self-assembling peptide nanotubes.

  20. A peptide & peptide nucleic acid synthesis technology for transporter molecules and theranostics--the SPPS.

    Science.gov (United States)

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here. PMID:24843319

  1. A Peptide & Peptide Nucleic Acid Synthesis Technology for Transporter Molecules and Theranostics - The SPPS

    Science.gov (United States)

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here. PMID:24843319

  2. 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.

  3. Peptide nucleic acid: a new artificial biomacromolecular with great potential applications in molecular biology and biomedicine

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-ke; LU Zu-hong; HE Nong-yue

    2001-01-01

    @@ Peptide nucleic acid (PNA) is a DNA mimic that was originally developed by Peter E Nielsen in 1991 as a reagent for sequence-specific recognition of double stranded (ds) DNA by a conventional triple helix type principle.

  4. 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.

  5. The 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 p......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...

  6. A Novel Lactic Acid Bacteria Growth-stimulating Peptide from Broad Bean (Vicia faba .) Protein Hydrolysates

    OpenAIRE

    Ping Xiao; Yuan Liu; Rizwan-ur-Rehman; Ran Kang; Yanping Wang

    2015-01-01

    In this study, broad bean protein hydrolysates (BPH) produced by alcalase with strong-stimulating activity for lactic acid bacteria (LAB) was first time reported. In order to obtain the key peptide that have growth-stimulating activity for lactic acid bacteria (LAB), gel filtration chromatography and Reverse Phase High Performance Liquid Chromatography (RP-HPLC) were applied to isolate and purify the peptides from BPH. Finally, F4-2 elicited the highest activity for LAB, corresponding to amin...

  7. Gamma-aminoadamantane carboxylic acids : orientating building blocks in peptide chemistry

    OpenAIRE

    Wanka, Lukas

    2007-01-01

    The present thesis deals with gamma-aminoadamantane carboxylic acids. Allthough the simplest of these structural analogues of GABA were known for decades, no significant peptide chemistry has been disclosed. Therefore, improved syntheses of these compounds were worked out that allowed for facilitated syntheses of already known as well as new members of gamma-aminoadamantane carboxylic acids. Fmoc-/tert. butyl protective group chemistry was utilized to synthesize peptides incorporating the gam...

  8. 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....... The modified electrode described here opens up new possibilities for future applications in early stage diagnoses of diseases where cells over-express folate receptors, such as in cancer or leishmaniasis disease....

  9. The impact of α-hydrazino acids embedded in short fluorescent peptides on peptide interactions with DNA and RNA.

    Science.gov (United States)

    Suć, Josipa; Tumir, Lidija-Marija; Glavaš-Obrovac, Ljubica; Jukić, Marijana; Piantanida, Ivo; Jerić, Ivanka

    2016-06-01

    A series of novel hydrazino-based peptidomimetics and analogues comprising N-terminal lysine and C-terminal phenanthridinyl-l-alanine were prepared. The presented results demonstrate the up to now unknown possibility to finely modulate peptide interactions with DNA/RNA by α-hydrazino group insertion and how the different positioning of two α-hydrazino groups in peptides controls binding to various double stranded and single stranded DNA and RNA. All peptidomimetics bind with 1-10 micromolar affinity to ds-DNA/RNA, whereby the binding mode is a combination of electrostatic interactions and hydrophobic interactions within DNA/RNA grooves. Insertion of the α-hydrazino group into the peptide systematically decreased its fluorimetric response to DNA/RNA binding in the order: mono-hydrazino peptide sequence. Particularly interesting was the interaction of two sequential α-hydrazino acids-peptidomimetic with poly rG, characterised by a specific strong increase of CD bands, while all other peptide/ssRNA combinations gave only a CD-band decrease. All mentioned interactions could also be reversibly controlled by adjusting the pH, due to the protonation of the fluorophore.

  10. Analysis and prediction of the critical regions of antimicrobial peptides based on conditional random fields.

    Directory of Open Access Journals (Sweden)

    Kuan Y Chang

    Full Text Available Antimicrobial peptides (AMPs are potent drug candidates against microbes such as bacteria, fungi, parasites, and viruses. The size of AMPs ranges from less than ten to hundreds of amino acids. Often only a few amino acids or the critical regions of antimicrobial proteins matter the functionality. Accurately predicting the AMP critical regions could benefit the experimental designs. However, no extensive analyses have been done specifically on the AMP critical regions and computational modeling on them is either non-existent or settled to other problems. With a focus on the AMP critical regions, we thus develop a computational model AMPcore by introducing a state-of-the-art machine learning method, conditional random fields. We generate a comprehensive dataset of 798 AMPs cores and a low similarity dataset of 510 representative AMP cores. AMPcore could reach a maximal accuracy of 90% and 0.79 Matthew's correlation coefficient on the comprehensive dataset and a maximal accuracy of 83% and 0.66 MCC on the low similarity dataset. Our analyses of AMP cores follow what we know about AMPs: High in glycine and lysine, but low in aspartic acid, glutamic acid, and methionine; the abundance of α-helical structures; the dominance of positive net charges; the peculiarity of amphipathicity. Two amphipathic sequence motifs within the AMP cores, an amphipathic α-helix and an amphipathic π-helix, are revealed. In addition, a short sequence motif at the N-terminal boundary of AMP cores is reported for the first time: arginine at the P(-1 coupling with glycine at the P1 of AMP cores occurs the most, which might link to microbial cell adhesion.

  11. Cysteine-containing peptides having antioxidant properties

    Science.gov (United States)

    Bielicki, John K.

    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.

  12. Between peptides and bile acids: self-assembly of phenylalanine substituted cholic acids.

    Science.gov (United States)

    Travaglini, Leana; D'Annibale, Andrea; di Gregorio, Maria Chiara; Schillén, Karin; Olsson, Ulf; Sennato, Simona; Pavel, Nicolae V; Galantini, Luciano

    2013-08-01

    Biocompatible molecules that undergo self-assembly are of high importance in biological and medical applications of nanoscience. Peptides and bile acids are among the most investigated due to their ability to self-organize into many different, often stimuli-sensitive, supramolecular structures. With the aim of preparing molecules mixing the aggregation properties of bile acid and amino acid-based molecules, we report on the synthesis and self-association behavior of two diastereomers obtained by substituting a hydroxyl group of cholic acid with a l-phenylalanine residue. The obtained molecules are amphoteric, and we demonstrate that they show a pH-dependent self-assembly. Both molecules aggregate in globular micelles at high pH, whereas they form tubular superstructures under acid conditions. Unusual narrow nanotubes with outer and inner cross-section diameters of about 6 and 3 nm are formed by the derivatives. The diasteroisomer with α orientation of the substituent forms in addition a wider tubule (17 nm cross-section diameter). The ability to pack in supramolecular tubules is explained in terms of a wedge-shaped bola-form structure of the derivatives. Parallel or antiparallel face-to-face dimers are hypothesized as fundamental building blocks for the formation of the narrow and wide nanotubes, respectively.

  13. Formation of Amino Acid Thioesters for Prebiotic Peptide Synthesis: Catalysis By Amino Acid Products

    Science.gov (United States)

    Weber, Arthur L.; DeVincenzi, Donald L. (Technical Monitor)

    1999-01-01

    The origin of life can be described as a series of events in which a prebiotic chemical process came increasingly under the control of its catalytic products. In our search for this prebiotic process that yielded catalytic takeover products (such as polypeptides), we have been investigating a reaction system that generates peptide-forming amino acid thioesters from formaldehyde, glycolaldehyde, and ammonia in the presence of thiols. As shown below, this model process begins by aldol condensation of formaldehyde and glycolaldehyde to give trioses and releases. These sugars then undergo beta-dehydration yielding their respective alpha-ketoaldehydes. Addition of ammonia to the alpha-ketoaldehydes yields imines which can either: (a) rearrange in the presence of thesis to give amino acid thioesters or (be react with another molecule of aldehyde to give imidazoles. This 'one-pot' reaction system operates under mild aqueous conditions, and like modem amino acid biosynthesis, uses sugar intermediates which are converted to products by energy-yielding redox reactions. Recently, we discovered that amino acids, such as the alanine reaction product, catalyze the first and second steps of the process. In the presence of ammonia the process also generates other synthetically useful products, like the important biochemical -- pyruvic acid.

  14. Synthesis and biological properties of amino acids and peptides containing a tetrazolyl moiety

    Science.gov (United States)

    Popova, E. A.; Trifonov, R. E.

    2015-09-01

    Literature data published mainly in the last 15 years on the synthesis and biological properties of amino acid analogues and derivatives containing tetrazolyl moieties are analyzed. Tetrazolyl analogues and derivatives of amino acids and peptides are shown to be promising for medicinal chemistry. Being polynitrogen heterocyclic systems comprising four endocyclic nitrogen atoms, tetrazoles can behave as acids and bases and form strong hydrogen bonds with proton donors (more rarely, with acceptors). They have high metabolic stability and are able to penetrate biological membranes. The review also considers the synthesis and properties of linear and cyclic peptides based on modified amino acids incorporating a tetrazolyl moiety. A special issue is the discussion of the biological properties of tetrazole-containing amino acids and peptides, which exhibit high biological activity and can be used to design new drugs. The bibliography includes 200 references.

  15. Endogenous flow of amino acids in the avian ileum as influenced by increasing dietary peptide concentrations.

    Science.gov (United States)

    Ravindran, Velmurugu; Morel, Patrick C H; Rutherfurd, Shane M; Thomas, Donald V

    2009-03-01

    The aim of the present study was to establish whether feeding broiler chickens with diets containing increasing dietary peptide concentrations would cause increases in ileal endogenous amino acid flow. The flow of N and most amino acids increased quadratically (P < 0.05 to 0.001) with increasing dietary concentrations of peptides. The exceptions were the flow of threonine, serine, glycine, tyrosine and cystine, which increased linearly (P < 0.001) with dietary peptide levels. Another notable exception to the general trend was the flow of proline, which was significantly higher (P < 0.01) in birds fed the protein-free diet. The amino acid profile of endogenous protein, expressed as proportion of crude protein, indicated that the ratios of threonine, glutamic acid, proline, glycine, leucine, histidine, arginine and cystine were influenced (P < 0.05) with increasing dietary peptide concentrations. In general, compared with the protein-free diet, the ratios of threonine and arginine in endogenous protein were lower (P < 0.05) and those of glutamic acid, glycine and histidine were greater (P < 0.05) in diets with high concentrations of peptides. The ratio of proline was found to decrease (P < 0.05) with increasing dietary peptide concentrations. These changes in the amino acid profile of endogenous protein are probably reflective of changes in the output of one or more of the components of endogenous protein. Overall, the present results demonstrated that increasing dietary peptide concentrations increased the flow of endogenous amino acid flow at the terminal ileum of broiler chickens in a dose-dependent manner and also caused changes in the composition of endogenous protein. The observed changes in endogenous amino flow will influence the maintenance requirements for amino acids and also have implications for the calculation of true digestibility coefficient of feedstuffs. PMID:18662428

  16. The Prebiotic Synthesis of Ethylenediamine Monoacetic Acid, The Repeating Unit of Peptide Nucleic Acids

    Science.gov (United States)

    Nelson, Kevin E.; Miller, Stanley L.

    1992-01-01

    The polymerization of ribonucleic acids or their precursors constitutes an important event in prebiotic chemistry. The various problems using ribonucleotides to make RNA suggest that there may have been a precursor. An attractive possibility are the peptide nucleic acids (PNA). PNAs are nucleotide analogs that make use of a polymer of ethylenediamine monoacetic acid (EDMA or 2-amninoethyl glycine) with the bases attached by an acetic acid. EDMA is an especially attractive alternative to the ribose phosphate or deoxyribose phosphate backbone because it contains no chiral centers and is potentially prebiotic, but there is no reported prebiotic synthesis. We have synthesized both EDMA and ethylenediamine diacetic acid (EDDA) from the prebiotic compounds ethylenediamine, formaldehyde, and hydrogen cyanide. The yields of EDMA range from 11 to 79% along with some sEDDA and uEDDA. These reactions work with concentrations of 10(exp -1)M and as low as 10(exp -4)M, and the reaction is likely to be effective at even lower concentrations. Ethylenediamine is a likely prebiotic compound, but it has not yet been demonstrated, although compounds such as ethanolamine and cysteamine have been proven to be prebiotic. Under neutral pH and heating at l00 C, EDMA is converted to the lactam, monoketopiperazine (MKP). The cyclization occurs and has an approximate ratio of MKP/EDMA = 3 at equilibrium. We have measured the solubilities of EDMA center dot H20 as 6.4 m, EDMA center dot HCl center dot H20 as 13.7 m, and EDMA center dot 2HCl center dot H20 as 3.4 m. These syntheses together with the high solubility of EDMA suggest that EDMA would concentrate in drying lagoons and might efficiently form polymers. Given the instability of ribose and the poor polymerizability of nucleotides, the prebiotic presence of EDMA and the possibility of its polymerization raises the possibility that PNAs are the progenitors of present day nucleic acids. A pre-RNA world may have existed in which PNAs or

  17. Topical Delivery of Hyaluronic Acid into Skin using SPACE-peptide Carriers

    OpenAIRE

    Chen, Ming; Gupta, Vivek; Anselmo, Aaron C.; Muraski, John A.; Mitragotri, Samir

    2013-01-01

    Topical penetration of macromolecules into skin is limited by their low permeability. Here, we report the use of a skin penetrating peptide, SPACE peptide, to enhance topical delivery of a macromolecule, hyaluronic acid (HA, MW: 200–325 kDa). The peptide was conjugated to phospholipids and used to prepare an ethosomal carrier system (~110 nm diameter), encapsulating HA. The SPACE-ethosomal system (SES) enhanced HA penetration into porcine skin in vitro by 7.8+/−1.1-fold compared to PBS. The s...

  18. Synthesis and application of acid labile anchor groups for the synthesis of peptide amides by Fmoc-solid-phase peptide synthesis.

    Science.gov (United States)

    Breipohl, G; Knolle, J; Stüber, W

    1989-10-01

    The preparation and application of a new linker for the synthesis of peptide amides using a modified Fmoc-method is described. The new anchor group was developed based on our experience with 4,4'-dimethoxybenzhydryl (Mbh)-protecting group for amides. Lability towards acid treatment was increased dramatically and results in an easy cleavage procedure for the preparation of peptide amides. The synthesis of N-9-fluorenylmethoxycarbonyl- ([5-carboxylatoethyl-2.4-dimethoxyphenyl)- 4'-methoxyphenyl]-methylamin is reported in detail. This linker was coupled to a commercially available aminomethyl polystyrene resin. Peptide synthesis proceeded smoothly using HOOBt esters of Fmoc-amino acids. Release of the peptide amide and final cleavage of the side chain protecting groups was accomplished by treatment with trifluoroacetic acid-dichloromethane mixtures in the presence of scavengers. The synthesis of peptide amides such as LHRH and C-terminal hexapeptide of secretin are given as examples.

  19. 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.

  20. 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.

  1. HPLC monitoring of spontaneous non-linear peptidization dynamics of selected amino acids in solution.

    Science.gov (United States)

    Godziek, Agnieszka; Maciejowska, Anna; Sajewicz, Mieczysław; Kowalska, Teresa

    2015-03-01

    This is our new study in a series of publications devoted to exploration of applicability of high-performance liquid chromatography (HPLC) to providing answers to difficult questions from the area of the reaction kinetics and mechanisms with non-linear reactions. Although an excellent analytical performance of HPLC is an indisputable fact, so far its performance as a tool in the kinetic and mechanistic studies has been tested to a lesser extent. In our earlier studies, spontaneous peptidization dynamics of amino acids in solution was demonstrated by means of HPLC upon a few amino acid examples, and on that basis a theoretical model has been developed, anticipating an interdependence of dynamics on chemical structures of amino acids involved. In order to expand the spectrum of experimentally investigated amino acid cases, in this study we present the results valid for three novel amino acids of significant life sciences importance, which differ in terms of peptidization dynamics. Experimental evidence originates from the achiral HPLC with the evaporative light scattering detection and MS detection. A conclusion is drawn that different spontaneous peptidization dynamics of amino acids may significantly influence chemical composition of proteins encountered in living organisms. Hence, a need emerges for systematic physicochemical studies on spontaneous non-linear peptidization dynamics of proteinogenic amino acids in liquid abiotic (but also in the biotic) systems.

  2. A Novel Lactic Acid Bacteria Growth-stimulating Peptide from Broad Bean (Vicia faba . Protein Hydrolysates

    Directory of Open Access Journals (Sweden)

    Ping Xiao

    2015-03-01

    Full Text Available In this study, broad bean protein hydrolysates (BPH produced by alcalase with strong-stimulating activity for lactic acid bacteria (LAB was first time reported. In order to obtain the key peptide that have growth-stimulating activity for lactic acid bacteria (LAB, gel filtration chromatography and Reverse Phase High Performance Liquid Chromatography (RP-HPLC were applied to isolate and purify the peptides from BPH. Finally, F4-2 elicited the highest activity for LAB, corresponding to amino acid sequence Ser-Ala-Gln (304.10Da was identified by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS/MS. Thus, this study shows that broad bean peptide is a good source to promote the LAB growth and this function is reported for the first time.

  3. Incorporation of noncanonical amino acids into Rosetta and use in computational protein-peptide interface design.

    Directory of Open Access Journals (Sweden)

    P Douglas Renfrew

    Full Text Available Noncanonical amino acids (NCAAs can be used in a variety of protein design contexts. For example, they can be used in place of the canonical amino acids (CAAs to improve the biophysical properties of peptides that target protein interfaces. We describe the incorporation of 114 NCAAs into the protein-modeling suite Rosetta. We describe our methods for building backbone dependent rotamer libraries and the parameterization and construction of a scoring function that can be used to score NCAA containing peptides and proteins. We validate these additions to Rosetta and our NCAA-rotamer libraries by showing that we can improve the binding of a calpastatin derived peptides to calpain-1 by substituting NCAAs for native amino acids using Rosetta. Rosetta (executables and source, auxiliary scripts and code, and documentation can be found at (http://www.rosettacommons.org/.

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

    International Nuclear Information System (INIS)

    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)

  5. Membrane-targeted self-assembling cyclic peptide nanotubes.

    Science.gov (United States)

    Rodríguez-Vázquez, Nuria; Ozores, H Lionel; Guerra, Arcadio; González-Freire, Eva; Fuertes, Alberto; Panciera, Michele; Priegue, Juan M; Outeiral, Juan; Montenegro, Javier; Garcia-Fandino, Rebeca; Amorin, Manuel; Granja, Juan R

    2014-01-01

    Peptide nanotubes are novel supramolecular nanobiomaterials that have a tubular structure. The stacking of cyclic components is one of the most promising strategies amongst the methods described in recent years for the preparation of nanotubes. This strategy allows precise control of the nanotube surface properties and the dimensions of the tube diameter. In addition, the incorporation of 3- aminocycloalkanecarboxylic acid residues in the nanotube-forming peptides allows control of the internal properties of the supramolecular tube. The research aimed at the application of membrane-interacting self-assembled cyclic peptide nanotubes (SCPNs) is summarized in this review. The cyclic peptides are designed to interact with phospholipid bilayers to induce nanotube formation. The properties and orientation of the nanotube can be tuned by tailoring the peptide sequence. Hydrophobic peptides form transmembrane pores with a hydrophilic orifice, the nature of which has been exploited to transport ions and small molecules efficiently. These synthetic ion channels are selective for alkali metal ions (Na(+), K(+) or Cs(+)) over divalent cations (Ca(2+)) or anions (Cl(-)). Unfortunately, selectivity was not achieved within the series of alkali metal ions, for which ion transport rates followed the diffusion rates in water. Amphipathic peptides form nanotubes that lie parallel to the membrane. Interestingly, nanotube formation takes place preferentially on the surface of bacterial membranes, thus making these materials suitable for the development of new antimicrobial agents.

  6. Calcium Binding to Amino Acids and Small Glycine Peptides in Aqueous Solution: Toward Peptide Design for Better Calcium Bioavailability.

    Science.gov (United States)

    Tang, Ning; Skibsted, Leif H

    2016-06-01

    Deprotonation of amino acids as occurs during transfer from stomach to intestines during food digestion was found by comparison of complex formation constants as determined electrochemically for increasing pH to increase calcium binding (i) by a factor of around 6 for the neutral amino acids, (ii) by a factor of around 4 for anions of the acidic amino acids aspartic and glutamic acid, and (iii) by a factor of around 5.5 for basic amino acids. Optimized structures of the 1:1 complexes and ΔHbinding for calcium binding as calculated by density functional theory (DFT) confirmed in all complexes a stronger calcium binding and shorter calcium-oxygen bond length in the deprotonated form. In addition, the stronger calcium binding was also accompanied by a binding site shift from carboxylate binding to chelation by α-amino group and carboxylate oxygen for leucine, aspartate, glutamate, alanine, and asparagine. For binary amino acid mixtures, the calcium-binding constant was close to the predicted geometric mean of the individual amino acid binding constants indicating separate binding of calcium to two amino acids when present together in solution. At high pH, corresponding to conditions for calcium absorption, the binding affinity increased in the order Lys < Arg < Cys < Gln < Gly ∼ Ala < Asn < His < Leu < Glu< Asp. In a series of glycine peptides, calcium-binding affinity was found to increase in the order Gly-Leu ∼ Gly-Gly < Ala-Gly < Gly-His ∼ Gly-Lys-Gly < Glu-Cys-Gly < Gly-Glu, an ordering confirmed by DFT calculations for the dipeptides and which also accounted for large synergistic effects in calcium binding for up to 6 kJ/mol when compared to the corresponding amino acid mixtures. PMID:27159329

  7. 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

    The excipient citric acid (CA) has been reported to improve oral absorption of peptides by different mechanisms. The balance between its related properties of calcium chelation and permeation enhancement compared to a proteolysis inhibition was examined. A predictive model of CA's calcium chelation...... 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...

  8. Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support

    Directory of Open Access Journals (Sweden)

    Leclerc Mario

    2005-04-01

    Full Text Available Abstract Background Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. Results Using surface-bound peptide nucleic acids (PNA probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. Conclusion This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

  9. Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

    Science.gov (United States)

    Liutkus, Mantas; Fraser, Samuel A; Caron, Karine; Stigers, Dannon J; Easton, Christopher J

    2016-05-17

    Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis. PMID:26918308

  10. A Study on Anti-oxidative Activity of Soybean Peptides with Linoleic Acid Peroxidation Systems

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Soybean bioactive peptides(SBPs) were prepared from the isolated soybean protein by proteolysis with an alkaline protease, alcalase, at 50 ℃ and pH = 8. 0. The dependence of hydrolysis time on hydrolysis degree and molecular weight distribution were examined. The hydrolysate was fractionated on a Sephadex G-25 column and the anti-oxidative activities of the fractions were detected by the method of pyrogallol auto-oxidation. The average chain length of soybean peptides that have anti-oxidative activity was estimated to be about 7. The anti-oxidative properties of the soybean peptide were also studied by using linoleic acid peroxidation systems. The optimal condition of the peroxidation system was set up, Vc/Cu2 + as the inducer at pH = 7.4 and 25 ℃. In addition, soybean peptides show higher antioxidative activity compared with GSH.

  11. 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

    2016-09-01

    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.

  12. Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk.

    Science.gov (United States)

    Arakawa, K; Matsunaga, K; Takihiro, S; Moritoki, A; Ryuto, S; Kawai, Y; Masuda, T; Miyamoto, T

    2015-03-01

    Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious

  13. Lactobacillus gasseri requires peptides, not proteins or free amino acids, for growth in milk.

    Science.gov (United States)

    Arakawa, K; Matsunaga, K; Takihiro, S; Moritoki, A; Ryuto, S; Kawai, Y; Masuda, T; Miyamoto, T

    2015-03-01

    Lactobacillus gasseri is a widespread commensal lactic acid bacterium inhabiting human mucosal niches and has many beneficial effects as a probiotic. However, L. gasseri is difficult to grow in milk, which hurts usability for the food industry. It had been previously reported that supplementation with yeast extract or proteose peptone, including peptides, enables L. gasseri to grow well in milk. In this study, our objective was to confirm peptide requirement of L. gasseri and evaluate efficacy of peptide release by enzymatic proteolysis on growth of L. gassei in milk. Three strains of L. gasseri did not grow well in modified DeMan, Rogosa, Sharpe broth without any nitrogen sources (MRS-N), but addition of a casein-derived peptide mixture, tryptone, promoted growth. In contrast, little effect was observed after adding casein or a casein-derived amino acid mixture, casamino acids. These results indicate that L. gasseri requires peptides, not proteins or free amino acids, among milk-derived nitrogen sources for growth. Lactobacillus gasseri JCM 1131T hardly had growth capacity in 6 kinds of milk-based media: bovine milk, human milk, skim milk, cheese whey, modified MRS-N (MRSL-N) supplemented with acid whey, and MRSL-N supplemented with casein. Moreover, treatment with digestive proteases, particularly pepsin, to release peptides made it grow well in each milk-based medium. The pepsin treatment was the most effective for growth of strain JCM 1131T in skim milk among the tested food-grade proteases such as trypsin, α-chymotrypsin, calf rennet, ficin, bromelain, and papain. As well as strain JCM 1131T, pepsinolysis of milk improved growth of other L. gasseri strains and some strains of enteric lactobacilli such as Lactobacillus crispatus, Lactobacillus gallinarum, Lactobacillus johnsonii, and Lactobacillus reuteri. These results suggest that some relatives of L. gasseri also use peptides as desirable nitrogen sources, and that milk may be a good supplier of nutritious

  14. 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.

  15. 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...

  16. One-pot nanoparticulation of potentially bioactive peptides and gallic acid encapsulation.

    Science.gov (United States)

    Nourbakhsh, Himan; Madadlou, Ashkan; Emam-Djomeh, Zahra; Wang, Yi-Cheng; Gunasekaran, Sundaram

    2016-11-01

    Whey protein isolate was hydrolyzed to an in vitro antioxidative hydrolysate, followed by transglutaminase-induced cross-linking and microemulsification in an oil phase. The obtained microemulsion was then dispersed in a gallic acid-rich model wastewater which caused gallic acid transportation into internal nanodroplets. Whey peptides were consequently gelled, yielding nanoparticles. Electrophoresis showed that β-lactoglobulin and low molecular weight peptides were cross-linked by transglutaminase. Protein hydrolysis and subsequent enzymatic cross-linking increased the ζ-potential value. Microscopic investigation indicated that most particles were non-spherical. Non-cross-linked and cross-linked peptides underwent a form of heat-triggered self-assembly in the dry state, while nanoparticles did not show such behavior. Peptide crystallites size was increased by cross-linking and acid-induced particle formation. The latter also caused a reduction in intensity of C-H stretching and C-N bending peaks in infra-red spectrum. Gallic acid release from particles to simulated gastrointestinal fluids was through diffusion from swollen particles, and reached almost 70% release. PMID:27211653

  17. Phytochemicals that modulate amino acid and peptide catabolism by caprine rumen microbes

    Science.gov (United States)

    Background: Microbe-derived ionophores and macrolide antibiotics are often added to ruminant diets, and growth promotion and feed efficiency are among the benefits. One mechanism is inhibition of microbes that catabolize amino acids or peptides and produce ammonia. Plants also produce antimicrobial ...

  18. Design and Synthesis of Novel Peptide Nucleic Acid Monomers

    Institute of Scientific and Technical Information of China (English)

    白金泉; 李英; 刘克良

    2001-01-01

    All of the four nucleobases in DNA have replaced the 4-hydroxy group of N-[2-(tert-butoxycarbonylaminomethyl)-trams-4-hydroxy]tetrahydropyrrole acetic acid methyl ester with cis-stereochemistry. An efficient route for the synthesis of N-[2-(tert-butoxycarbonylaminomethyl)-trans-4-hydroxy]-tetrahydropyrrole acetic acid methyl ester has been developed.Starting with this intermediate, the protected monmers were synthesized by the Mitsunobu reaction or via its tosylate.

  19. Antimicrobial peptides targeting Gram-negative pathogens, produced and delivered by lactic acid bacteria.

    Science.gov (United States)

    Volzing, Katherine; Borrero, Juan; Sadowsky, Michael J; Kaznessis, Yiannis N

    2013-11-15

    We present results of tests with recombinant Lactococcus lactis that produce and secrete heterologous antimicrobial peptides with activity against Gram-negative pathogenic Escherichia coli and Salmonella . In an initial screening, the activities of numerous candidate antimicrobial peptides, made by solid state synthesis, were assessed against several indicator pathogenic E. coli and Salmonella strains. Peptides A3APO and Alyteserin were selected as top performers based on high antimicrobial activity against the pathogens tested and on significantly lower antimicrobial activity against L. lactis . Expression cassettes containing the signal peptide of the protein Usp45 fused to the codon-optimized sequence of mature A3APO and Alyteserin were cloned under the control of a nisin-inducible promoter PnisA and transformed into L. lactis IL1403. The resulting recombinant strains were induced to express and secrete both peptides. A3APO- and Alyteserin-containing supernatants from these recombinant L. lactis inhibited the growth of pathogenic E. coli and Salmonella by up to 20-fold, while maintaining the host's viability. This system may serve as a model for the production and delivery of antimicrobial peptides by lactic acid bacteria to target Gram-negative pathogenic bacteria populations.

  20. Overexpression of Antimicrobial, Anticancer, and Transmembrane Peptides in Escherichia coli through a Calmodulin-Peptide Fusion System.

    Science.gov (United States)

    Ishida, Hiroaki; Nguyen, Leonard T; Gopal, Ramamourthy; Aizawa, Tomoyasu; Vogel, Hans J

    2016-09-01

    In recent years, the increasing number of antibiotic-resistant bacteria has become a serious health concern. Antimicrobial peptides (AMPs) are an important component of the innate immune system of most organisms. A better understanding of their structures and mechanisms of action would lead to the design of more potent and safer AMPs as alternatives for current antibiotics. For detailed investigations, effective recombinant production which allows the facile modification of the amino acid sequence, the introduction of unnatural amino acids, and labeling with stable isotopes for nuclear magnetic resonance (NMR) studies is desired. Several expression strategies have been introduced in previous reports; however, their effectiveness has been limited to a select few AMPs. Here, we have studied calmodulin (CaM) as a more universal carrier protein to express many types of AMPs in E. coli. We have discovered that the unique architecture of CaM, consisting of two independent target binding domains with malleable methionine-rich interaction surfaces, can accommodate numerous amino acid sequences containing basic and hydrophobic residues. This effectively masks the toxic antimicrobial activities of many amphipathic AMPs and protects them from degradation during expression and purification. Here, we demonstrate the expression of various AMPs using a CaM-fusion expression system, including melittin, fowlicidin-1, tritrpticin, indolicidin, puroindoline A peptide, magainin II F5W, lactoferrampin B, MIP3α51-70, and human β-defensin 3 (HBD-3), the latter requiring three disulfide bonds for proper folding. In addition, our approach was extended to the transmembrane domain of the cell adhesion protein l-selectin. We propose the use of the CaM-fusion system as a universal approach to express many cationic amphipathic peptides that are normally toxic and would kill the bacterial host cells. PMID:27502305

  1. Novel amino acids: synthesis of furoxan and sydnonimine containing amino acids and peptides as potential nitric oxide releasing motifs.

    Science.gov (United States)

    Nortcliffe, Andrew; Botting, Nigel P; O'Hagan, David

    2013-07-28

    The incorporation of furoxan and sydnonimine ring systems into amino acid side chains is demonstrated with the preparation of four novel amino acids which carry these nitric oxide-releasing motifs. N-((4-Nitrophenoxy)carbonyl)-3-phenylsydnonimine 9 and bis(phenylsulfonyl)furoxan 10 are the key intermediates for introducing the heterocycle side chains onto appropriate amine and alcohol functionalities respectively. Furoxan 5 and 7 both displayed NO release based on determination of nitrite production. Orthogonal amino acid protecting group strategies were deployed to demonstrate that the amino acids could be incorporated into peptide frameworks. By way of demonstration the amino acids were placed centrally into several tripeptide motifs. Griess test assays showed that these amino acids released NO in the presence of γ-glutathione (GST). PMID:23753002

  2. Question 1: Peptide Nucleic Acids and the Origin and Homochirality of Life

    Science.gov (United States)

    Nielsen, Peter E.

    2007-10-01

    The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality.

  3. Question 1: Peptide nucleic acids and the origin and homochirality of life.

    Science.gov (United States)

    Nielsen, Peter E

    2007-10-01

    The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality.

  4. Acidity-Mediated, Electrostatic Tuning of Asymmetrically Charged Peptides Interactions with Protein Nanopores.

    Science.gov (United States)

    Asandei, Alina; Chinappi, Mauro; Kang, Hee-Kyoung; Seo, Chang Ho; Mereuta, Loredana; Park, Yoonkyung; Luchian, Tudor

    2015-08-01

    Despite success in probing chemical reactions and dynamics of macromolecules on submillisecond time and nanometer length scales, a major impasse faced by nanopore technology is the need to cheaply and controllably modulate macromolecule capture and trafficking across the nanopore. We demonstrate herein that tunable charge separation engineered at the both ends of a macromolecule very efficiently modulates the dynamics of macromolecules capture and traffic through a nanometer-size pore. In the proof-of-principle approach, we employed a 36 amino acids long peptide containing at the N- and C-termini uniform patches of glutamic acids and arginines, flanking a central segment of asparagines, and we studied its capture by the α-hemolysin (α-HL) and the mean residence time inside the pore in the presence of a pH gradient across the protein. We propose a solution to effectively control the dynamics of peptide interaction with the nanopore, with both association and dissociation reaction rates of peptide-α-HL interactions spanning orders of magnitude depending upon solution acidity on the peptide addition side and the transmembrane electric potential, while preserving the amplitude of the blockade current signature. PMID:26144534

  5. Enhanced cellular delivery of cell-penetrating peptide-peptide nucleic acid conjugates by photochemical internalization

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    )) or tetraphenylporphyrin tetrasulfonic acid (TPPS). Cellular uptake of the PNA conjugates were evaluated by using a sensitive cellular method with HeLa pLuc705 cells based on the splicing correction of luciferase gene by targeting antisense oligonucleotides to a cryptic splice site of the mutated luciferase gene....... The cellular efficacy of CPP conjugates were evaluated by measuring luciferase activity as a result of splicing correction and was also confirmed by RT-PCR analysis of luciferase pre-mRNA....

  6. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    Science.gov (United States)

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  7. 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.

  8. Inspiration from the mirror: D-amino acid containing peptides in biomedical approaches.

    Science.gov (United States)

    Feng, Zhaoqianqi; Xu, Bing

    2016-06-01

    D-amino acids, the enantiomers of naturally abundant L-amino acids, bear unique stereochemistry properties that lead to the resistance towards most of the endogenous enzymes. Previous works have demonstrated applications of D-amino acids in therapeutic development with the aid of mirror-image phage display and retro-inverso peptide synthesis. In this review, we highlight the recent progress and challenges in the exploration of D-amino acids at the interface of chemistry and life science. First, we will introduce some progress made in traditional application of D-amino acids to enhance biostability of peptide therapeutics. Then, we discuss some works that explore the relatively underexplored interactions between the enzyme and D-amino acids and enzymatic reactions of D-amino acids. To highlight the enzymatic reactions of D-amino acids, we will describe several emerging works on the enzyme-instructed self-assembly (EISA) and their potential application in selective anti-inflammatory or anticancer therapies. At the end, we briefly mention the challenges and possible future directions. PMID:27159920

  9. Interaction of actinides with amino acids: from peptides to proteins

    International Nuclear Information System (INIS)

    Structural information on complexes of actinides with molecules of biological interest is required to better understand the mechanisms of actinides transport in living organisms, and can contribute to develop new decorporation treatments. Our study is about Th(IV), Np(IV), Pu(IV) and uranyl(VI) cations, which have a high affinity for some protein domains, and Fe(III), which is the natural cation of these biological systems. In this work, chelation of actinides has been brought to light with UV-visible-Near Infra Red spectroscopy, NMR, EPR, and ultrafiltration. Determination of the structure of the complexation site has been undertaken with Exafs measurements, and of the tertiary structure of the protein with SANS measurements. The first approach was to describe the interaction modes between actinides and essential chemical functions of proteins. Thus, the Ac-AspAspProAspAsp-NH2 peptide was studied as a possible chelate of actinides. Polynuclear species with μ-oxo or μ-hydroxo bridges were identified. The iron complex is binuclear, and the actinide ones have a higher nuclearity. The second approach was to study a real case of complexation of actinide with a protein: transferrin. Results show that around physiological ph a mononuclear complex is formed with Np(IV) and Pu(IV), while transferrin does not complex Th(IV) in the same conditions. Characteristic distances of M-transferrin complexes (M = Fe, Np, Pu) were determined. Moreover, the protein seems to be in its close conformation with Pu(IV), and in its open form with Np(IV) and UO22+. (author)

  10. Acid-Catalyzed Oxygen-18 Labeling of Peptides for Proteomics Applications

    OpenAIRE

    Niles, Richard; Witkowska, H. Ewa; Allen, Simon; Hall, Steven C.; Fisher, Susan J.; Hardt, Markus

    2009-01-01

    In enzymatic 18O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as an alternative method for introducing 18O label into peptides. Several synthetic amino acid sequences were dissolved in dilute acid containing 50% (v/v) H218O and incubated at room temperature. Aliquots were removed over a per...

  11. Proteins, Peptides and Amino Acids: Role in Infant Nutrition.

    Science.gov (United States)

    Nutten, Sophie

    2016-01-01

    Proteins are polymers composed of 30 or more amino acids; some of them are essential dietary components, since they are not synthetized by human metabolic processes. They are crucial for healthy growth and development and influence major functions of the body. The infant's first year is a critical time of rapid growth and development, which must be supported by a high rate of protein synthesis. Breast milk, as a single specific food source in the first months of life, is providing the total protein and essential amino acids required. Infant formulas have been designed for infants who cannot be breastfed. They should be similar to breast milk in their composition and their functional outcomes, insuring appropriate growth, optimal development, maturation of the immune system, easy digestion and healthy metabolic programming. By modifying their protein components, specific infant formulas have also been developed for specific needs. For example, partially hydrolyzed (prevention of atopic dermatitis) and extensively hydrolyzed or amino-acid-based infant formulas (reduction in allergy symptoms) have been designed for the management of cow's milk protein allergy. In conclusion, proteins provided via breast milk or infant formula are essential components of the infant's diet; therefore, the specific quality, quantity and conformation of proteins are of utmost importance for healthy growth and development.

  12. Proteins, Peptides and Amino Acids: Role in Infant Nutrition.

    Science.gov (United States)

    Nutten, Sophie

    2016-01-01

    Proteins are polymers composed of 30 or more amino acids; some of them are essential dietary components, since they are not synthetized by human metabolic processes. They are crucial for healthy growth and development and influence major functions of the body. The infant's first year is a critical time of rapid growth and development, which must be supported by a high rate of protein synthesis. Breast milk, as a single specific food source in the first months of life, is providing the total protein and essential amino acids required. Infant formulas have been designed for infants who cannot be breastfed. They should be similar to breast milk in their composition and their functional outcomes, insuring appropriate growth, optimal development, maturation of the immune system, easy digestion and healthy metabolic programming. By modifying their protein components, specific infant formulas have also been developed for specific needs. For example, partially hydrolyzed (prevention of atopic dermatitis) and extensively hydrolyzed or amino-acid-based infant formulas (reduction in allergy symptoms) have been designed for the management of cow's milk protein allergy. In conclusion, proteins provided via breast milk or infant formula are essential components of the infant's diet; therefore, the specific quality, quantity and conformation of proteins are of utmost importance for healthy growth and development. PMID:27336588

  13. A parallel method for enumerating amino acid compositions and masses of all theoretical peptides

    Directory of Open Access Journals (Sweden)

    Nefedov Alexey V

    2011-11-01

    Full Text Available Abstract Background Enumeration of all theoretically possible amino acid compositions is an important problem in several proteomics workflows, including peptide mass fingerprinting, mass defect labeling, mass defect filtering, and de novo peptide sequencing. Because of the high computational complexity of this task, reported methods for peptide enumeration were restricted to cover limited mass ranges (below 2 kDa. In addition, implementation details of these methods as well as their computational performance have not been provided. The increasing availability of parallel (multi-core computers in all fields of research makes the development of parallel methods for peptide enumeration a timely topic. Results We describe a parallel method for enumerating all amino acid compositions up to a given length. We present recursive procedures which are at the core of the method, and show that a single task of enumeration of all peptide compositions can be divided into smaller subtasks that can be executed in parallel. The computational complexity of the subtasks is compared with the computational complexity of the whole task. Pseudocodes of processes (a master and workers that are used to execute the enumerating procedure in parallel are given. We present computational times for our method executed on a computer cluster with 12 Intel Xeon X5650 CPUs (72 cores running Windows HPC Server. Our method has been implemented as a 32- and 64-bit Windows application using Microsoft Visual C++ and the Message Passing Interface. It is available for download at https://ispace.utmb.edu/users/rgsadygo/Proteomics/ParallelMethod. Conclusion We describe implementation of a parallel method for generating mass distributions of all theoretically possible amino acid compositions.

  14. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    Science.gov (United States)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  15. Heteropoly acids triggered self-assembly of cationic peptides into photo- and electro-chromic gels.

    Science.gov (United States)

    Li, Jingfang; Xu, Jing; Li, Xiaodong; Gao, Wenmei; Wang, Liyan; Wu, Lixin; Lee, Myongsoo; Li, Wen

    2016-07-01

    A series of cationic peptides with alternating lysines and hydrophobic residues were designed and synthesized. These kinds of short peptides with protonated lysines can complex with anionic heteropoly acids (HPAs) to form a stable gel in water/ethanol mixed solution. Circular dichroism spectroscopy showed that the short peptides adopted a mixed conformation (β-sheet and random-coil) within the gel matrix. Scanning and transmission electron microscopy revealed that the heteropoly acids, acting as nanosized cross-linkers, first initiated the self-assembly of the cationic peptides into spherical nanostructures. Then these nanospheres accumulated with each other through hydrogen bonds and hydrophobic interactions to form large sheet-like assemblies, which further interconnected with each other forming continuous 3D network structures. Fourier-transform infrared spectroscopy showed that the structural integrity of the HPAs was maintained during the gelation process. The resultant hybrid gels showed reversible photo- and elecrtro-chromic properties. X-ray photoelectron spectroscopy revealed that the hybrid gels, capable of persistent and reversible changes of their colour, are attributed to the intervalence charge-transfer transition of the HPAs. Reversible information writing and erasing were demonstrated through a repeated photo-lithograph or electric stimuli without significant loss of the gel performance. PMID:27240759

  16. Anhydrous trifluoroacetic acid pretreatment converts insoluble polyglutamine peptides to soluble monomers

    Directory of Open Access Journals (Sweden)

    Gunasekhar Burra

    2015-12-01

    Full Text Available The data provided in this article are related to the research article entitled “Unaided trifluoroacetic acid pretreatment solubilizes polyglutamine (polyGln peptides and retains their biophysical properties of aggregation” by Burra and Thakur (in press [1]. This research article reports data from size exclusion chromatography (SEC, reversed phase-high performance liquid chromatography (RP-HPLC and mass spectrometry (MS assays. This data show that trifluoroacetic acid (TFA has the ability to convert insoluble polyGln peptides to soluble monomers. The data also clarify the possibility of trifluoroacetylation modification caused due to TFA. We hope the data presented here will enhance the understanding of polyGln disaggregation and solubilization. For more insightful and useful discussions, see the research article published in Analytical Biochemistry: Methods in the Biological Sciences (Burra and Thakur, in press [1].

  17. Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope.

    OpenAIRE

    Xu, W; Ellington, A. D.

    1996-01-01

    In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of huma...

  18. Lactic acid bacteria and their antimicrobial peptides : Induction,detection,partial characterization, and potential applications

    OpenAIRE

    Abbas Hilmi, Hanan

    2010-01-01

    Bacteriocin-producing lactic acid bacteria and their isolated peptide bacteriocins are of value to control pathogens and spoiling microorganisms in foods and feed. Nisin is the only bacteriocin that is commonly accepted as a food preservative and has a broad spectrum of activity against Gram-positive organisms including spore forming bacteria. In this study nisin induction was studied from two perspectives, induction from inside of the cell and selection of nisin inducible strains with increa...

  19. Recognition of Double Stranded RNA by Guanidine-Modified Peptide Nucleic Acids (GPNA)

    OpenAIRE

    Gupta, Pankaj; Muse, Oluwatoyosi; Rozners, Eriks

    2011-01-01

    Double helical RNA has become an attractive target for molecular recognition because many non-coding RNAs play important roles in control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double helical RNA via triple helix formation. Herein we tested if the molecular recognition of RNA can be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-worker...

  20. 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.

  1. Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins.

    Science.gov (United States)

    Peitzsch, R M; McLaughlin, S

    1993-10-01

    We studied the binding of fatty acids and acylated peptides to phospholipid vesicles by making electrophoretic mobility and equilibrium dialysis measurements. The binding energies of the anionic form of the fatty acids and the corresponding acylated glycines were identical; the energies increased by 0.8 kcal/mol per number of carbons in the acyl chain (Ncarbon = 10, 12, 14, 16), a value identical to that for the classical entropy-driven hydrophobic effect discussed by Tanford [The Hydrophobic Effect (1980) Wiley, New York]. The unitary Gibbs free binding energy, delta Gou, of myristoylated glycine, 8 kcal/mol, is independent of the nature of the electrically neutral lipids used to form the vesicles. Similar binding energies were obtained with other myristoylated peptides (e.g., Gly-Ala, Gly-Ala-Ala). The 8 kcal/mol, which corresponds to an effective dissociation constant of 10(-4) M for myristoylated peptides with lipids, provides barely enough energy to attach a myristoylated protein in the cytoplasm to the plasma membrane. Thus, other factors that reduce (e.g., hydrophobic interaction of myristate with the covalently attached protein) or enhance (e.g., electrostatic interactions of basic residues with acidic lipids; protein-protein interactions with intrinsic receptor proteins) the interaction of myristoylated proteins with membranes are likely to be important and may cause reversible translocation of these proteins to the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Slow peptide bond formation by proline and other N-alkylamino acids in translation

    OpenAIRE

    Pavlov, Michael Y; Watts, Richard E.; Tan, Zhongping; Cornish, Virginia W; Ehrenberg, Måns; Forster, Anthony C.

    2008-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid (“imino acid”), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala...

  3. DESIGN AND SYNTHESIS OF 4-[2’-(5’- NITRO] IMIDAZOLYL BENZOYL (N-METHYL AMINO ACIDS AND PEPTIDES

    Directory of Open Access Journals (Sweden)

    PARAMITA DAS

    2010-06-01

    Full Text Available In the past two decades, a wide variety of bioactive peptides have been discovered. Condensation of heterocyclic moieties viz nicotinic acid, thiazole coumarin, quinolin, furan, imidazole etc. with amino acids and peptides resulted in compounds with potent biological activities. Many of the heterocyclic found to exhibit antifungal, antibacterial, cytotoxic, antineoplastic, insectisidal, antiinflammatory, anthelmintic, tyrosinase inhibitory and melanin production inhibitory activities. Metronidazole, serconidazole, flucanazole are well known marketed drugs. Introduction of D-amino acids and N-methylation of amino acids like tyrosine, valine, alanine etc enhanced antimicrobial activity. Hence an attempt is made towards the synthesis of 5-nitroimidazolyl-benzoic acid derivative of N-methylamino acids and peptide using solution phase technique of peptide synthesis. The method includes the introduction of tert-butyloxy carboxyl group (Boc to amino acids to protect the amino group forming Boc-amino acids .The protection of carboxyl group was done by converting the amino acids into corresponding methyl ester. The protected amino acids were coupled using diisopropylcarbodimide and triethylamine to get protected dipeptides. N-methylation was done by treating with methyl iodide and sodium hydride. The ester group was then removed by lithium hydroxide. The Boc(N-methyldipetide were coupled to amino acids or Boc(Nmethyl dipeptide were coupled to 4-[2-(5-nitroimidazoly]benzoic acids.

  4. Acid-Catalyzed Oxygen-18 Labeling of Peptides for Proteomics Applications

    Science.gov (United States)

    Niles, Richard; Witkowska, H. Ewa; Allen, Simon; Hall, Steven C.; Fisher, Susan J.; Hardt, Markus

    2010-01-01

    In enzymatic 18O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as an alternative method for introducing 18O label into peptides. Several synthetic amino acid sequences were dissolved in dilute acid containing 50% (v/v) H218O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). 18O-incorporation ratios were determined by linear regression analysis that allowed for multiple stable isotope incorporations. At low pH, peptides exchanged their carboxyl oxygen atoms with the aqueous solvent. The isotope patterns gradually shifted to higher masses until they reached the expected binomial distribution at equilibrium after ~11 days. Reaction rates were residue and sequence specific. Due to its slow nature, the acid-based back exchange is expected to minimally interfere with enzymatic 18O-labeling studies provided that storage and analysis conditions minimize low pH exposure times. On its own, acid-catalyzed 18O labeling is a general tagging strategy that is an alternative to the chemical, metabolic and enzymatic isotope-labeling schemes currently used in quantitative proteomics. PMID:19243188

  5. Amino acid absorption and homeostasis in mice lacking the intestinal peptide transporter PEPT1.

    Science.gov (United States)

    Nässl, Anna-Maria; Rubio-Aliaga, Isabel; Fenselau, Henning; Marth, Mena Katharina; Kottra, Gabor; Daniel, Hannelore

    2011-07-01

    The intestinal peptide transporter PEPT1 mediates the uptake of di- and tripeptides derived from dietary protein breakdown into epithelial cells. Whereas the transporter appears to be essential to compensate for the reduced amino acid delivery in patients with mutations in amino acid transporter genes, such as in cystinuria or Hartnup disease, its physiological role in overall amino acid absorption is still not known. To assess the quantitative importance of PEPT1 in overall amino acid absorption and metabolism, PEPT1-deficient mice were studied by using brush border membrane vesicles, everted gut sacs, and Ussing chambers, as well as by transcriptome and proteome analysis of intestinal tissue samples. Neither gene expression nor proteome profiling nor functional analysis revealed evidence for any compensatory changes in the levels and/or function of transporters for free amino acids in the intestine. However, most plasma amino acid levels were increased in Pept1(-/-) compared with Pept1(+/+) animals, suggesting that amino acid handling is altered. Plasma appearance rates of (15)N-labeled amino acids determined after intragastric administration of a low dose of protein remained unchanged, whereas administration of a large protein load via gavage revealed marked differences in plasma appearance of selected amino acids. PEPT1 seems, therefore, important for overall amino acid absorption only after high dietary protein intake when amino acid transport processes are saturated and PEPT1 can provide additional absorption capacity. Since renal amino acid excretion remained unchanged, elevated basal concentrations of plasma amino acids in PEPT1-deficient animals seem to arise mainly from alterations in hepatic amino acid metabolism. PMID:21350187

  6. 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.

  7. Effect of amino acid substitution in the hydrophobic face of amphiphilic peptides on membrane curvature and perturbation: N-terminal helix derived from adenovirus internal protein VI as a model.

    Science.gov (United States)

    Murayama, Tomo; Pujals, Sílvia; Hirose, Hisaaki; Nakase, Ikuhiko; Futaki, Shiroh

    2016-11-01

    The N-terminal amphipathic helical segment of adenovirus internal protein VI (AdVpVI) plays a critical role in viral infection. Here, we report that the peptide segment corresponding to AdVpVI (positions 33-55) can induce positive membrane curvature together with membrane perturbation. The enhanced perturbation ability of the peptide was observed for membranes containing negatively charged phospholipids. Based on the liposome leakage assay, substitution of leucine at position 40 to other aliphatic (isoleucine) and aromatic (phenylalanine and tryptophan) residues yielded a similar degree of membrane perturbation by the peptides, which was considerably diminished by the substitution to glutamine. Further studies using the wild-type AdVpVI (33-55) (WT) and phenylalanine-substituted peptides (L40F) demonstrated that both peptides have positive membrane-curvature-inducing ability. These peptides showed higher binding affinity to 50-nm large unilamellar vesicles (LUVs) than to 200-nm LUVs. However, no enhanced perturbation by these peptides was observed for 50-nm LUVs compared to 200-nm LUVs, suggesting that both the original membrane curvature and the additional strain due to peptide insertion affect the membrane perturbation ability of these peptides. In the case of L40F, this peptide rather had a lower membrane perturbation ability for 50-nm LUVs than for 200-nm LUVs, which can be attributed to possible shallower binding of L40F on membranes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 430-439, 2016. PMID:27271816

  8. Derivation of an amino acid similarity matrix for peptide:MHC binding and its application as a Bayesian prior

    Directory of Open Access Journals (Sweden)

    Sette Alessandro

    2009-11-01

    Full Text Available Abstract Background Experts in peptide:MHC binding studies are often able to estimate the impact of a single residue substitution based on a heuristic understanding of amino acid similarity in an experimental context. Our aim is to quantify this measure of similarity to improve peptide:MHC binding prediction methods. This should help compensate for holes and bias in the sequence space coverage of existing peptide binding datasets. Results Here, a novel amino acid similarity matrix (PMBEC is directly derived from the binding affinity data of combinatorial peptide mixtures. Like BLOSUM62, this matrix captures well-known physicochemical properties of amino acid residues. However, PMBEC differs markedly from existing matrices in cases where residue substitution involves a reversal of electrostatic charge. To demonstrate its usefulness, we have developed a new peptide:MHC class I binding prediction method, using the matrix as a Bayesian prior. We show that the new method can compensate for missing information on specific residues in the training data. We also carried out a large-scale benchmark, and its results indicate that prediction performance of the new method is comparable to that of the best neural network based approaches for peptide:MHC class I binding. Conclusion A novel amino acid similarity matrix has been derived for peptide:MHC binding interactions. One prominent feature of the matrix is that it disfavors substitution of residues with opposite charges. Given that the matrix was derived from experimentally determined peptide:MHC binding affinity measurements, this feature is likely shared by all peptide:protein interactions. In addition, we have demonstrated the usefulness of the matrix as a Bayesian prior in an improved scoring-matrix based peptide:MHC class I prediction method. A software implementation of the method is available at: http://www.mhc-pathway.net/smmpmbec.

  9. Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile

    DEFF Research Database (Denmark)

    Czerwiec, Eva; Kalume, Dario E; Roepstorff, Peter;

    2006-01-01

    -containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans......-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding...... the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-Tx...

  10. The Perseus Exobiology Mission on MIR: Behaviour of Amino Acids and Peptides in Earth Orbit

    Science.gov (United States)

    Boillot, F.; Chabin, A.; Buré, C.; Venet, M.; Belsky, A.; Bertrand-Urbaniak, M.; Delmas, A.; Brack, A.; Barbier, B.

    2002-08-01

    Leucine, α-methyl leucine and two peptides were exposed to space conditions on board the MIR station during the Perseus-Exobiology mission. This long duration space mission was aimed at testing the delivery of prebiotic building blocks. During this mission, two amino acids (leucine and α-methyl leucine) and two peptides (leucine-diketopiperazine and trileucine thioethylester) were exposed in Earth orbit for three months. Basalt, clay and meteorite powder were also mixed with the samples in order to simulate the effects of potential meteorite protection. Analysis of the material after the flight did not reveal any racemization or polymerisation but did provide information regarding photochemical pathways for the degradation of leucine and of the tripeptide. Amino acids appeared to be more sensitive to UV radiation than peptides, the cyclic dipeptide being found to be as particularly resistant. Meteorite powder which exhibits the highest absorption in Vacuum UltraViolet (VUV) afforded the best protection to the organic molecules whereas montmorillonite clay, almost transparent in VUV, was the least efficient. By varying the thickness of the meteorite, we found that the threshold for efficient protection against radiation was about 5 μm. The possible exogenous origin of biological building blocks is discussed with respect to the stability to the molecules and the nature of the associated minerals.

  11. Orientation of the peptide formation of N-phosphoryl amino acids in solution

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The peptide formation of N-phosphoryl aminoacids with amino acids proceeds in aqueous solution withoutany coupling reagents. After being separated in sephadex gelcolumn, the phosphoryl dipeptides were analyzed by theelectrospray ionization tandem mass spectrometry (ESIMS/MS). The result demonstrates that phosphoryl dipeptides were detected in all the reaction systems. It is found that theformation of N-phosphoryl dipeptides is oriented: theN-terminal amino acid residues of the N-phosphoryl dipep-tides are from N-phosphoryl amino acids, and the peptideelongation happened at the C-terminal. Only (-dipeptide, no(-dipeptide, is formed in the N-phosphoryl dipeptides,showing that ?-carboxylic group is activated selectively byN-phosphorylation. Theoretical calculation shows that the peptide formation of N-phosphoryl amino acids might hap-pen through a penta-coordinate carboxylic-phosphoric in-termediate in solution. These results might give some clues tothe study on the origin of proteins and protein biosynthesis.

  12. Slow peptide bond formation by proline and other N-alkylamino acids in translation.

    Science.gov (United States)

    Pavlov, Michael Y; Watts, Richard E; Tan, Zhongping; Cornish, Virginia W; Ehrenberg, Måns; Forster, Anthony C

    2009-01-01

    Proteins are made from 19 aa and, curiously, one N-alkylamino acid ("imino acid"), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA(Phe) depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA(Pro) isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the "tRNA abundance" hypothesis. PMID:19104062

  13. Electrostatic Binding and Hydrophobic Collapse of Peptide-Nucleic Acid Aggregates Quantified Using Force Spectroscopy

    CERN Document Server

    Camunas-Soler, Joan; Bizarro, Cristiano V; de Loreno, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramon; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2014-01-01

    Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g. Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide which contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interact...

  14. Thioacetic acid/NaSH-mediated synthesis of N-protected amino thioacids and their utility in peptide synthesis.

    Science.gov (United States)

    Mali, Sachitanand M; Gopi, Hosahudya N

    2014-03-21

    Thioacids are recently gaining momentum due to their versatile reactivity. The reactivity of thioacids has been widely explored in the selective amide/peptide bond formation. Thioacids are generally synthesized from the reaction between activated carboxylic acids such as acid chlorides, active esters, etc., and Na2S, H2S, or NaSH. We sought to investigate whether the versatile reactivity of the thioacids can be tuned for the conversion of carboxylic acids into corresponding thioacids in the presence of NaSH. Herein, we report that thioacetic acid- and NaSH-mediated synthesis of N-protected amino thioacids from the corresponding N-protected amino acids, oxidative dimerization of thioacids, crystal conformations of thioacid oxidative dimers, and the utility of thioacids and oxidative dimers in peptide synthesis. Our results suggest that peptides can be synthesized without using standard coupling agents.

  15. Synthesis of stable C-linked ferrocenyl amino acids and their use in solution-phase peptide synthesis.

    Science.gov (United States)

    Philip, Anijamol T; Chacko, Shibin; Ramapanicker, Ramesh

    2015-12-01

    Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post-synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C-linked side chain are potentially useful building units for the synthesis of ferrocene-containing peptides. We report here an efficient route to synthesize ferrocene-containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2-ferrocenyl-1,3-dithiane and iodides derived from aspartic acid or glutamic acid using n-butyllithium leads to the incorporation of a ferrocenyl unit to the δ-position or ε-position of an α-amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C-terminus and N-terminus of tripeptides in solution phase.

  16. Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot ( Scophthalmus maximus L.)

    Science.gov (United States)

    Xu, Dandan; He, Gen; Mai, Kangsen; Zhou, Huihui; Xu, Wei; Song, Fei

    2016-04-01

    Turbot ( Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B0-type amino acid transporter 1 (B0AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y+L-type amino acid transporter 1 (y+LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y+L-type amino acid transporter 2 (y+LAT2) abundantly expressed in stomach. In addition, system b0,+ transporters (rBAT and b0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.

  17. 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 conjuga...

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

    International Nuclear Information System (INIS)

    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)

  19. Interfacial self-assembly of amino acids and peptides: Scanning tunneling microscopy investigation

    Science.gov (United States)

    Xu, Li-Ping; Liu, Yibiao; Zhang, Xueji

    2011-12-01

    Proteins play important roles in human daily life. To take advantage of the lessons learned from nature, it is essential to investigate the self-assembly of subunits of proteins, i.e., amino acids and polypeptides. Due to its high resolution and versatility of working environment, scanning tunneling microscopy (STM) has become a powerful tool for studying interfacial molecular assembly structures. This review is intended to reflect the progress in studying interfacial self-assembly of amino acids and peptides by STM. In particular, we focus on environment-induced polymorphism, chiral recognition, and coadsorption behavior with molecular templates. These studies would be highly beneficial to research endeavors exploring the mechanism and nanoscale-controlling molecular assemblies of amino acids and polypeptides on surfaces, understanding the origin of life, unravelling the essence of disease at the molecular level and deeming what is necessary for the ``bottom-up'' nanofabrication of molecular devices and biosensors being constructed with useful properties and desired performance.

  20. The chain length of biologically produced (R)-3-hydroxyalkanoic acid affects biological activity and structure of anti-cancer peptides.

    Science.gov (United States)

    Szwej, Emilia; Devocelle, Marc; Kenny, Shane; Guzik, Maciej; O'Connor, Stephen; Nikodinovic-Runic, Jasmina; Radivojevic, Jelena; Maslak, Veselin; Byrne, Annete T; Gallagher, William M; Zulian, Qun Ren; Zinn, Manfred; O'Connor, Kevin E

    2015-06-20

    Conjugation of DP18L peptide with (R)-3-hydroxydecanoic acid, derived from the biopolymer polyhydroxyalkanoate, enhances its anti-cancer activity (O'Connor et al., 2013. Biomaterials 34, 2710-2718). However, it is unknown if other (R)-3-hydroxyalkanoic acids (R3HAs) can enhance peptide activity, if chain length affects enhancement, and what effect R3HAs have on peptide structure. Here we show that the degree of enhancement of peptide (DP18L) anti-cancer activity by R3HAs is carbon chain length dependent. In all but one example the R3HA conjugated peptides were more active against cancer cells than the unconjugated peptides. However, R3HAs with 9 and 10 carbons were most effective at improving DP18L activity. DP18L peptide variant DP17L, missing a hydrophobic amino acid (leucine residue 4) exhibited lower efficacy against MiaPaCa cells. Circular dichroism analysis showed DP17L had a lower alpha helix content and the conjugation of any R3HA ((R)-3-hydroxyhexanoic acid to (R)-3-hydroxydodecanoic acid) to DP17L returned the helix content back to levels of DP18L. However (R)-3-hydroxyhexanoic did not enhance the anti-cancer activity of DP17L and at least 7 carbons were needed in the R3HA to enhance activity of D17L. DP17L needs a longer chain R3HA to achieve the same activity as DP18L conjugated to an R3HA. As a first step to assess the synthetic potential of polyhydroxyalkanoate derived R3HAs, (R)-3-hydroxydecanoic acid was synthetically converted to (±)3-chlorodecanoic acid, which when conjugated to DP18L improved its antiproliferative activity against MiaPaCa cells. PMID:25820126

  1. Aliphatic acid-conjugated antimicrobial peptides--potential agents with anti-tumor, multidrug resistance-reversing activity and enhanced stability.

    Science.gov (United States)

    Deng, Xin; Qiu, Qianqian; Ma, Ke; Wang, Xuekun; Huang, Wenlong; Qian, Hai

    2015-07-28

    Compared with traditional therapeutics, antimicrobial peptides as novel anti-tumor agents have prominent advantages of higher specificity and circumvention of multi-drug resistance. In a previous study, we found that B1, an antimicrobial peptide derived from Cathelicidin-BF15, presented specific anti-tumor activity against several tumor cells. Since aliphatic chain-conjugated peptides have shown ameliorative activity and stability, we conjugated aliphatic acids with different lengths to the amino terminal of B1. All the conjugated peptides exhibited improved anti-tumor activity over B1. Further investigations revealed that the peptides were capable of disrupting the cell membrane, stimulating cytochrome c release into the cytosol, which results in apoptosis. The peptides also acted against multidrug resistant cells and had multidrug resistance-reversing effects. Additionally, conjugation of aliphatic acid enhanced the peptide stability in plasma. In summary, aliphatic acid-modified peptides might be promising anti-tumor agents in the future. PMID:26083110

  2. Synthesis and antimicrobial activity in vitro of new amino acids and peptides containing thiazole and oxazole moieties.

    Science.gov (United States)

    Stanchev, M; Tabakova, S; Videnov, G; Golovinsky, E; Jung, G

    1999-09-01

    2-(Pyrrolidinyl)thiazole-4-carboxylic acid 5d, 2-(1-aminoalkyl)thiazole-4-carboxamides and hydrazides 8, 10 have been synthesized using alanine, valine, and proline as educts. In addition oxazole amino acids derived from leucine 20a and alanine 20b and some peptides 13, 14, 16 containing the 5-ring heterocyclic backbone modifications have been prepared. The thiazole and oxazole containing amino acids and peptides showed moderate antibacterial activity in vitro against various Gram-positive (Staphylococcus aureus, Bacillus cereus, etc.) and Gram-negative (Escherichia coli, Protens vulgaris, etc.) bacteria, fungi (Candida albicans), and yeast (Saccharomyces cerevisae, etc.). PMID:10520298

  3. Topical Delivery of Hyaluronic Acid into Skin using SPACE-peptide Carriers

    Science.gov (United States)

    Chen, Ming; Gupta, Vivek; Anselmo, Aaron C.; Muraski, John A.; Mitragotri, Samir

    2014-01-01

    Topical penetration of macromolecules into skin is limited by their low permeability. Here, we report the use of a skin penetrating peptide, SPACE peptide, to enhance topical delivery of a macromolecule, hyaluronic acid (HA, MW: 200–325 kDa). The peptide was conjugated to phospholipids and used to prepare an ethosomal carrier system (~110 nm diameter), encapsulating HA. The SPACE-ethosomal system (SES) enhanced HA penetration into porcine skin in vitro by 7.8+/−1.1-fold compared to PBS. The system also enhanced penetration of HA in human skin in vitro, penetrating deep into the epidermis and dermis in skin of both species. In vivo experiments performed using SKH1 hairless mice also confirmed increased dermal penetration of HA using the delivery system; a 5-fold enhancement in penetration was found compared to PBS control. Concentrations of HA in skin were about 1000-fold higher than those in blood; confirming the localized nature of HA delivery into skin. The SPACE-ethosomal delivery system provides a formulation for topical delivery of macromolecules that are otherwise difficult to deliver into skin. PMID:24129342

  4. Reaction of the hydrated electron with amino acids, peptides, and proteins in aqueous solutions

    Energy Technology Data Exchange (ETDEWEB)

    Faraggi, M. (Ohio State Univ., Columbus); Bettelheim, A.

    1977-08-01

    The reaction rate constants of e/sup -//sub aq/ with glycyl-histidine (Gly-His) and ..beta..-alanylhistidine (Carnosine, ..beta..-Ala-His) were determined and compared to those of ..beta..-alanylalanine (..beta..-Ala-Ala), alanyl-alanine ((Ala)/sub 2/), and histidine (His). The rate constants were found to be pH dependent. Below the pK value of the imidazole ring, the rate constants of the histidyl peptides are similar to that of His. This indicates that the main site of the e/sup -//sub aq/ reaction is the protonated ring. Above this pK value the pH dependent rate constants were less in the His amino acids than in the His peptides. This difference was attributed to the presence of the carbonyl grup in the peptides. This group, which is known to react quite rapidly with e/sup -//sub aq/, exhibits its presence when the imidazole ring loses its reactivity after deprotonation. The difference in reactivity toward e/sup -//sub aq/ between the ..cap alpha.. and ..beta.. His peptides is explained by the relative position of the protonated amino groups with respect to the carbonyl groups. A similar difference was also found in (Ala)/sub 2/ and ..beta..-Ala-Ala. The transient absorption spectra resulting from the reaction of e/sup -//sub aq/ with the His peptides were recorded and examined with respect to peptide concentration and pH dependence. Here again, at pH values below the pK of the imidazole, the transient absorption spectra are similar to that of histidine. In alkaline solutions, however, proper experimental conditions could be attained only for Gly-His. In His and ..beta..-Ala-His the interference of the OH radical reaction was observed. In Gly/sup -/His it was found that the band characterizing the imidazole transient (lambda/sub max/ = 360 nm) disappears with a simultaneous appearance of a band at lambda/sub max/ similarly ordered 410 nm.

  5. 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

  6. Antimicrobial Peptides as Mediators of Innate Immunity in Teleosts

    Directory of Open Access Journals (Sweden)

    Barbara A. Katzenback

    2015-09-01

    Full Text Available Antimicrobial peptides (AMPs have been identified throughout the metazoa suggesting their evolutionarily conserved nature and their presence in teleosts is no exception. AMPs are short (18–46 amino acids, usually cationic, amphipathic peptides. While AMPs are diverse in amino acid sequence, with no two AMPs being identical, they collectively appear to have conserved functions in the innate immunity of animals towards the pathogens they encounter in their environment. Fish AMPs are upregulated in response to pathogens and appear to have direct broad-spectrum antimicrobial activity towards both human and fish pathogens. However, an emerging role for AMPs as immunomodulatory molecules has become apparent—the ability of AMPs to activate the innate immune system sheds light onto the multifaceted capacity of these small peptides to combat pathogens through direct and indirect means. Herein, this review focuses on the role of teleost AMPs as modulators of the innate immune system and their regulation in response to pathogens or other exogenous molecules. The capacity to regulate AMP expression by exogenous factors may prove useful in modulating AMP expression in fish to prevent disease, particularly in aquaculture settings where crowded conditions and environmental stress pre-dispose these fish to infection.

  7. Antimicrobial Peptides as Mediators of Innate Immunity in Teleosts.

    Science.gov (United States)

    Katzenback, Barbara A

    2015-09-25

    Antimicrobial peptides (AMPs) have been identified throughout the metazoa suggesting their evolutionarily conserved nature and their presence in teleosts is no exception. AMPs are short (18-46 amino acids), usually cationic, amphipathic peptides. While AMPs are diverse in amino acid sequence, with no two AMPs being identical, they collectively appear to have conserved functions in the innate immunity of animals towards the pathogens they encounter in their environment. Fish AMPs are upregulated in response to pathogens and appear to have direct broad-spectrum antimicrobial activity towards both human and fish pathogens. However, an emerging role for AMPs as immunomodulatory molecules has become apparent-the ability of AMPs to activate the innate immune system sheds light onto the multifaceted capacity of these small peptides to combat pathogens through direct and indirect means. Herein, this review focuses on the role of teleost AMPs as modulators of the innate immune system and their regulation in response to pathogens or other exogenous molecules. The capacity to regulate AMP expression by exogenous factors may prove useful in modulating AMP expression in fish to prevent disease, particularly in aquaculture settings where crowded conditions and environmental stress pre-dispose these fish to infection.

  8. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

    Science.gov (United States)

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  9. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines.

    Science.gov (United States)

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain

  10. 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

    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...... 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...

  11. Preparation of surfactant-stabilized gold nanoparticle-peptide nucleic acid conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Duy, Janice, E-mail: janice.duy@umit.maine.ed [University of Maine, Graduate School of Biomedical Sciences (United States); Connell, Laurie B. [University of Maine, School of Marine Sciences (United States); Eck, Wolfgang [University of Heidelberg, Applied Physical Chemistry (Germany); Collins, Scott D. [University of Maine, Department of Chemistry (United States); Smith, Rosemary L. [University of Maine, Electrical and Computer Engineering Department (United States)

    2010-09-15

    A simple, two-step method of producing stable and functional peptide nucleic acid (PNA)-conjugated gold nanoparticles using a surfactant stabilization step is presented. PNA are DNA analogs with superior chemical stability and target discrimination, but their use in metallic nanoparticle systems has been limited by the difficulty of producing stable colloids of nanoparticle-PNA conjugates. In this work, the nonionic surfactant Tween 20 (polyoxyethylene (20) sorbitan monolaurate) was used to sterically shield gold surfaces prior to the addition of thiolated PNA, producing conjugates which remain dispersed in solution and retain the ability to hybridize to complementary nucleic acid sequences. The conjugates were characterized using transmission electron microscopy, dynamic light scattering, and UV-visible absorbance spectroscopy. PNA attachment to gold nanoparticles was confirmed with an enzyme-linked immunoassay, while the ability of nanoparticle-bound PNA to hybridize to its complement was demonstrated using labeled DNA.

  12. Summarization on the synthesis and radionuclide-labeling of peptide nucleic acid for an oligonucleotide analogue

    International Nuclear Information System (INIS)

    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)

  13. In situ synthesis of peptide nucleic acids in porous silicon for drug delivery and biosensing.

    Science.gov (United States)

    Beavers, Kelsey R; Mares, Jeremy W; Swartz, Caleb M; Zhao, Yiliang; Weiss, Sharon M; Duvall, Craig L

    2014-07-16

    Peptide nucleic acids (PNA) are a unique class of synthetic molecules that have a peptide backbone and can hybridize with nucleic acids. Here, a versatile method has been developed for the automated, in situ synthesis of PNA from a porous silicon (PSi) substrate for applications in gene therapy and biosensing. Nondestructive optical measurements were performed to monitor single base additions of PNA initiated from (3-aminopropyl)triethoxysilane attached to the surface of PSi films, and mass spectrometry was conducted to verify synthesis of the desired sequence. Comparison of in situ synthesis to postsynthesis surface conjugation of the full PNA molecules showed that surface mediated, in situ PNA synthesis increased loading 8-fold. For therapeutic proof-of-concept, controlled PNA release from PSi films was characterized in phosphate buffered saline, and PSi nanoparticles fabricated from PSi films containing in situ grown PNA complementary to micro-RNA (miR) 122 generated significant anti-miR activity in a Huh7 psiCHECK-miR122 cell line. The applicability of this platform for biosensing was also demonstrated using optical measurements that indicated selective hybridization of complementary DNA target molecules to PNA synthesized in situ on PSi films. These collective data confirm that we have established a novel PNA-PSi platform with broad utility in drug delivery and biosensing.

  14. New Descriptors of Amino Acids and Its Applications to Peptide Quantitative Structure-activity Relationship

    Institute of Scientific and Technical Information of China (English)

    SHU Mao; HUO Dan-Qun; MEI Hua; LIANG Gui-Zhao; ZHANG Mei; LI Zhi-Liang

    2008-01-01

    A new set of descriptors, HSEHPCSV (component score vector of hydrophobic, steric, and electronic properties together with hydrogen bonding contributions), were derived from principal component analyses of 95 physicochemical variables of 20 natural amino acids separately according to different kinds of properties described, namely, hydrophobic, steric, and electronic properties as well as hydrogen bonding contributions. HSEHPCSV scales were then employed to express structures of angiotensin-converting enzyme inhibitors, bitter tasting thresholds and bactericidal 18 peptide, and to construct QSAR models based on partial least square (PLS). The results obtained are as follows: the multiple correlation coefficient (R2cum) of 0.846, 0.917 and 0.993, leave-one-out cross validated Q2cum of 0.835, 0.865 and 0.899, and root-mean-square error for estimated error (RMSEE) of 0.396, 0.187and 0.22, respectively. Satisfactory results showed that, as new amino acid scales, data of HSEHPCSV may be a useful structural expression methodology for the studies on peptide QSAR (quantitative structure-activity relationship) due to many advantages such as plentiful structural information, definite physical and chemical meaning and easy interpretation.

  15. Antioxidant Activity of Peptides from Fermented Milk with Mix Culture of Lactic Acid Bacteria and Yeast

    Directory of Open Access Journals (Sweden)

    Yun Li

    2015-02-01

    Full Text Available The aim of the present study is to investigate the production of antioxidant peptides during milk fermentation with co-culture of Lactic Acid Bacteria (LAB and yeast. Five LAB strains, previously screened with higher hydrolysis activity and Debaryomyces hansenii H2 which isolated from Tibet kefir were used in the study. The peptides separated from fermented milk were analysed antioxidant activity with DPPH radical scavenging, hydroxyl radical scavenging, chelation of metal ions and reducing power assays. The growth of Streptococcus. thermophilus Lactobacillus. delbrueckii ssp. bulgaricus and Lactococcus. lactis was enhanced with co-cultures and L. acidophilus was inhibited in co-culture with yeast. In co-culture with yeast, a significant decrease of the acidity was observed among all the fermentation and the pH reached higher values than in single LAB cultures. Except for L.delbrueckii ssp. bulgaricus, there was no significant difference of protein hydrolysis with other test LAB strains between co-culture and single culture. The co-incubation of LAB with the yeast developed a stronger antioxidant activity in DPPH radical and hydroxyl radical scavenging and no significant (p>0.05 difference in chelation of metal ions. The reducing power of L.delbrueckii ssp. bulgaricus and L. helveticus in co-culture was significant higher than those of single culture.

  16. Ascorbic acid stabilization of Re-188- and I-131-radiolabeled peptides for radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Guhlke, S.; Sartor, J.; Bender, H.; Biersack, H.J. [Bonn Univ. (Germany). Dept. of Nuclear Medicine; Zamora, P.O. [Bonn Univ. (Germany). Dept. of Nuclear Medicine]|[RhoMed Inc., Albuquerque, NM (United States); Knapp, F.F. Jr. [Oak Ridge National Lab. (ORNL), TN (United States). Nuclear Medicine Group; Rhodes, B.A. [RhoMed Inc., Albuquerque, NM (United States)

    1997-12-31

    Re-188 labeled RC-160 [ cyclic NH{sub 2}-(D)-Phe-Cys-Tyr-(D)-Trp-Lys-Val-Cys-Trp-NH{sub 2} ] cyclic NH is a radiolabeled somatostatin analog which is being explored for its potential as a local/regionally administered radiotherapeutic agent targeting somatostatin-receptor-positive tumors. The stability of {sup 188}Re-RC-160 towards radiolytic effects is a prerequisite for the success of such an approach. High radiation flux was found to result in radiolysis of the peptide, but addition of ascorbic acid to preparations of RC-160 and also somatostatin-14 was found to stabilize these peptides minimizing these radiolytic effects. Subsequent to ascorbic acid stabilization, {sup 188}Re-RC-160 was determined in vitro and in vivo to bind to somatostatin-receptor-positive cells (NCI-H69 human small cell lung carcinoma) but not to receptor-negative cells (Raji, Burkitt`s lymphoma). The comparative binding of Re-188 labeled RC-160 or CTOP [ cyclic NH{sub 2}-(D)-Phe-CysTyr-(D)-Trp-Orn-Thr-Pen-Thr-ol], a {mu}-opiod-receptor antagonist used as a negative control compound, was also determined in vitro and in vivo using NCI-H69 cells as targets. {sup 188}Re-RC-160 demonstrated a higher amount of net binding in vitro and in vivo compared to {sup 188}ReCTOP. (orig.)

  17. Modulation of antimicrobial host defense peptide gene expression by free fatty acids.

    Directory of Open Access Journals (Sweden)

    Lakshmi T Sunkara

    Full Text Available Routine use of antibiotics at subtherapeutic levels in animal feed drives the emergence of antimicrobial resistance. Development of antibiotic-alternative approaches to disease control and prevention for food animals is imperatively needed. Previously, we showed that butyrate, a major species of short-chain fatty acids (SCFAs fermented from undigested fiber by intestinal microflora, is a potent inducer of endogenous antimicrobial host defense peptide (HDP genes in the chicken (PLoS One 2011, 6: e27225. In the present study, we further revealed that, in chicken HD11 macrophages and primary monocytes, induction of HDPs is largely in an inverse correlation with the aliphatic hydrocarbon chain length of free fatty acids, with SCFAs being the most potent, medium-chain fatty acids moderate and long-chain fatty acids marginal. Additionally, three SCFAs, namely acetate, propionate, and butyrate, exerted a strong synergy in augmenting HDP gene expression in chicken cells. Consistently, supplementation of chickens with a combination of three SCFAs in water resulted in a further reduction of Salmonella enteritidis in the cecum as compared to feeding of individual SCFAs. More importantly, free fatty acids enhanced HDP gene expression without triggering proinflammatory interleukin-1β production. Taken together, oral supplementation of SCFAs is capable of boosting host immunity and disease resistance, with potential for infectious disease control and prevention in animal agriculture without relying on antibiotics.

  18. Comparison between Procalcitonin, Brain Natriuretic Peptide, and Uric Acid in Children with Cardiomyopathy and Controls

    Directory of Open Access Journals (Sweden)

    Noor Mohammad Noori

    2015-01-01

    Full Text Available Objective. This study was performed to determine the level of procalcitonin, Brain Natriuretic Peptide (BNP, and uric acid in children with cardiomyopathy in comparison with controls and the association with echocardiographic findings. Methods. The levels of BNP, procalcitonin, and serum uric acid were measured and the amounts of biomarkers compared with echocardiographic findings. Results. In this study mean age of participants was the same (p=0.321. The majority of echocardiographic indices in left and right heart have different means in case and controls (p<0.05. Means of BNP, procalcitonin, and uric acid were 213.814 ± 309.601, 9.326 ± 3.881, and 6.846 ± 1.814 for case group and 2.76 ± 1.013, 1.851 ± 1.466, and 3.317 ± 0.924 for control (p<0.001, respectively. In the patients group there was relationship of Ross classification with BNP (χ2 = 15.845, p<0.05 and with age (χ2 = 8.946, p<0.05. For uric acid and procalcitonin no significant relationships were observed. Conclusions. procalcitonin, uric acid, and BNP had significant relationship with many echocardiographic findings in participants. For patients, procalcitonin did not show correlation. The severity of illness based on the Ross classification showed significant correlation with BNP level and age in patients.

  19. 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.

  20. Testing the limits of rational design by engineering pH sensitivity into membrane active peptides

    Science.gov (United States)

    Wiedman, Gregory

    2015-01-01

    In this work, we sought to rationally design membrane active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane active peptides, pHlip and GALA. We created two hybrid sequences, MelP5_Δ4 and MelP5_Δ6 by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macro-molecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH-sensitivity into its sequence. We conclude that the properties of pHlip, GALA and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field. PMID:25572997

  1. Conformations of helical Aib peptides containing a pair of L-amino acid and D-amino acid.

    Science.gov (United States)

    Demizu, Yosuke; Yabuki, Yu-U; Doi, Mitsunobu; Sato, Yukiko; Tanaka, Masakazu; Kurihara, Masaaki

    2012-07-01

    A pair of L-leucine (L-Leu) and D-leucine (D-Leu) was incorporated into a-aminoisobutyric acid (Aib) peptide segments. Thedominant conformations of four hexapeptides, Boc-L-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe (1a), Boc-D-Leu-Aib-Aib-Aib-Aib-L-Leu-OMe(1b), Boc-Aib-Aib-L-Leu-L-Leu-Aib-Aib-OMe (2a), and Boc-Aib-Aib-D-Leu-L-Leu-Aib-Aib-OMe (2b), were investigated by IR,¹H NMR, CD spectra, and X-ray crystallographic analysis. All peptides 1a,b and 2a,b formed 3₁₀-helical structures in solution. X-ray crystallographic analysis revealed that right-handed (P) 3₁₀-helices were present in 1a and 1b and a mixture of right-handed(P) and left-handed (M) 3₁₀-helices was present in 2b in their crystalline states. PMID:22619002

  2. Absorption of Amino Acids and Peptides in a Child with a Variant of Hartnup Disease and Coexistent Coeliac Disease

    Science.gov (United States)

    Tarlow, M. J.; Seakins, J. W. T.; Lloyd, June K.; Matthews, D. M.; Cheng, B.; Thomas, A. J.

    1972-01-01

    A child with a variant of Hartnup disease and co-existent coeliac disease is described. Oral tolerance tests with L-histidine, L-tyrosine, and glycyl-L-tyrosine, and in vitro uptake studies on a small intestinal biopsy with L-histidine and glycyl-L-histidine, showed impaired absorption of the free amino acids, and showed that absorption of tyrosine and mucosal uptake of histidine was better from the dipeptides than from the free amino acids. This supports the hypothesis that the intestinal mucosa can take up small peptides intact, and that the peptide uptake mechanism is not involved in the intestinal defect of Hartnup disease. PMID:5086513

  3. Self-assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids.

    Science.gov (United States)

    Subbalakshmi, Chilukuri; Manorama, Sunkara V; Nagaraj, Ramakrishnan

    2012-05-01

    The morphology of structures formed by the self-assembly of short N-terminal t-butyloxycarbonyl (Boc) and C-terminal methyl ester (OMe) protected and Boc-deprotected hydrophobic peptide esters was investigated. We have observed that Boc-protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc-Ile-Ile-OMe, Boc-Phe-Phe-Phe-Ile-Ile-OMe and Boc-Trp-Ile-Ile-OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc-Leu-Ile-Ile-OMe and H-Leu-Ile-Ile-OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self-assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well-defined tertiary structures, upon removal of the Boc group, only H-Phe-Phe-Phe-Ile-Ile-OMe had the ability to adopt β-structure. Our results indicate that hydrophobic interaction is a very important determinant for self-assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self-assembly. PMID:22431418

  4. Composition of free and peptide-bound amino acids in beef chuck, loin, and round cuts.

    Science.gov (United States)

    Wu, G; Cross, H R; Gehring, K B; Savell, J W; Arnold, A N; McNeill, S H

    2016-06-01

    Meat is a food for humans. However, beef consumption in the United States has steadily declined by >14% over the past decade due to a variety of factors, including insufficient knowledge of animal protein. This study quantified all proteinogenic AA as well as nutritionally and physiologically significant nonproteinogenic AA and small peptides in beef cuts from 3 subprimals (chuck, round, and loin). Beef carcasses ( = 10) were selected at 3 commercial packing plants in the United States. Retail-cut samples were analyzed for the nitrogenous substances after acid, alkaline, or enzymatic hydrolysis and after deproteinization. In these chuck, round, and loin cuts, total amounts of glutamate (free plus peptide bound) were the highest (69-75 mg/g dry weight) followed by lysine, leucine, arginine, and glutamine in descending order. This is the first study to determine aspartate, asparagine, glutamate, and glutamine in meat proteins of any animal species. In all the beef samples evaluated, glutamine was the most abundant free AA (4.0-5.7 mg/g dry weight) followed by taurine, alanine, glutamate, and β-alanine. Additionally, samples from all beef cuts had high concentrations of anserine, carnosine, and glutathione, which were 2.8 to 3.7, 15.2 to 24.2, and 0.68 to 0.79 mg/g dry weight, respectively. Beef top loin steaks appear to provide higher protein nutrition values than top round steaks and under blade roasts, but all are excellent sources of proteinogenic AA as well as antioxidant AA and peptides to improve human growth, development, and health. Our findings may help guide future decisions regarding human and animal nutrition. PMID:27285936

  5. Amino Acid and Peptide Utilization Profiles of the Fluoroacetate-Degrading Bacterium Synergistetes Strain MFA1 Under Varying Conditions.

    Science.gov (United States)

    Leong, Lex E X; Denman, Stuart E; Hugenholtz, Philip; McSweeney, Christopher S

    2016-02-01

    Synergistetes strain MFA1 is an asaccharolytic ruminal bacterium isolated based on its ability to degrade fluoroacetate, a plant toxin. The amino acid and peptide requirements of the bacterium were investigated under different culturing conditions. The growth of strain MFA1 and its fluoroacetate degradation rate were enhanced by peptide-rich protein hydrolysates (tryptone and yeast extract) compared to casamino acid, an amino acid-rich protein hydrolysate. Complete utilization and preference for arginine, asparagine, glutamate, glycine, and histidine as free amino acids from yeast extract were observed, while the utilization of serine, threonine, and lysine in free form and peptide-bound glutamate was stimulated during growth on fluoroacetate. A predominant peptide in yeast extract preferentially utilized by strain MFA1 was partially characterized by high-liquid performance chromatography-mass spectrometry as a hepta-glutamate oligopeptide. Similar utilization profiles of amino acids were observed between the co-culture of strain MFA1 with Methanobrevibacter smithii without fluoroacetate and pure strain MFA1 culture with fluoroacetate. This suggests that growth of strain MFA1 could be enhanced by a reduction of hydrogen partial pressure as a result of hydrogen removal by a methanogen or reduction of fluoroacetate.

  6. Effect of the amino acid composition of cyclic peptides on their self-assembly in lipid bilayers.

    Science.gov (United States)

    Danial, Maarten; Perrier, Sébastien; Jolliffe, Katrina A

    2015-02-28

    The effect of amino acid composition on the formation of transmembrane channels in lipid bilayers upon self-assembly of alt-(L,D)-α-cyclic octapeptides has been investigated. Cyclic peptides comprising D-leucine, alternating with different combinations of L-azidolysine, L-lysine(Alloc), L-lysine and L-tryptophan were synthesized and the size of pores formed via self-assembly of these molecules in lipid bilayers was elucidated using large unilamellar vesicle fluorescence assays and dynamic light scattering. Pore formation was examined in large unilamellar vesicles made up of egg yolk phosphatidylcholine or Escherichia coli total lipid extract. From these analyses, we have established that cyclic peptides with charged side chains form large pores while those with neutral side chains form unimeric pores. Furthermore, the cyclic peptides that consist of non-symmetric amino acid configurations possess a higher membrane activity than the cyclic peptides with a symmetric amino acid configuration. In addition, we have found that peptide amphiphilicity plays a vital role in selective partitioning between bilayers that consist of egg yolk phosphatidylcholine and those comprised of E. coli total lipid extract. These results suggest that selective transbilayer channel formation via self-assembly may be a viable alternative for many applications that currently use more expensive, multistep synthesis methods. PMID:25566760

  7. Structural and dynamic evolution of the amphipathic N-terminus diversifies enzyme thermostability in the glycoside hydrolase family 12.

    Science.gov (United States)

    Jiang, Xukai; Chen, Guanjun; Wang, Lushan

    2016-08-21

    Understanding the molecular mechanism underlying protein thermostability is central to the process of efficiently engineering thermostable cellulases, which can provide potential advantages in accelerating the conversion of biomass into clean biofuels. Here, we explored the general factors that diversify enzyme thermostability in the glycoside hydrolase family 12 (GH12) using comparative molecular dynamics (MD) simulations coupled to a bioinformatics approach. The results indicated that protein stability is not equally distributed over the whole structure: the N-terminus is the most thermal-sensitive region of the enzymes with a β-sandwich architecture and it tends to lose its secondary structure during the course of protein unfolding. Furthermore, we found that the total interaction energy within the N-terminus is appreciably correlated with enzyme thermostability. Interestingly, the internal interactions within the N-terminus are organized in a special amphipathic pattern in which a hydrophobic packing cluster and a hydrogen bonding cluster lie at the two ends of the N-terminus. Finally, bioinformatics analysis demonstrated that the amphipathic pattern is highly conserved in GH12 and besides that, the evolution of the amino acids in the N-terminal region is an inherent mechanism underlying the diversity of enzyme thermostability. Taken together, our results demonstrate that the N-terminus is generally the structure that determines enzyme thermostability in GH12, and thereby it is also an ideal engineering target. The dynameomics study of a protein family can give a general view of protein functions, which will offer a wide range of applications in future protein engineering. PMID:27425569

  8. Anti-biofilm activity of ultrashort cinnamic acid peptide derivatives against medical device-related pathogens.

    Science.gov (United States)

    Laverty, Garry; McCloskey, Alice P; Gorman, Sean P; Gilmore, Brendan F

    2015-10-01

    The threat of antimicrobial resistance has placed increasing emphasis on the development of innovative approaches to eradicate multidrug-resistant pathogens. Biofilm-forming microorganisms, for example, Staphylococcus epidermidis and Staphylococcus aureus, are responsible for increased incidence of biomaterial infection, extended hospital stays and patient morbidity and mortality. This paper highlights the potential of ultrashort tetra-peptide conjugated to hydrophobic cinnamic acid derivatives. These peptidomimetic molecules demonstrate selective and highly potent activity against resistant biofilm forms of Gram-positive medical device-related pathogens. 3-(4-Hydroxyphenyl)propionic)-Orn-Orn-Trp-Trp-NH2 displays particular promise with minimum biofilm eradication concentration (MBEC) values of 125 µg/ml against methicillin sensitive (ATCC 29213) and resistant (ATCC 43300) S. aureus and activity shown against biofilm forms of Escherichia coli (MBEC: 1000 µg/ml). Kill kinetics confirms complete eradication of established 24-h biofilms at MBEC with 6-h exposure. Reduced cell cytotoxicity, relative to Gram-positive pathogens, was proven via tissue culture (HaCaT) and haemolysis assays (equine erythrocytes). Existing in nature as part of the immune response, antimicrobial peptides display great promise for exploitation by the pharmaceutical industry in order to increase the library of available therapeutic molecules. Ultrashort variants are particularly promising for translation as clinical therapeutics as they are more cost-effective, easier to synthesise and can be tailored to specific functional requirements based on the primary sequence allowing factors such as spectrum of activity to be varied. PMID:26310860

  9. Diphenylarsinic acid increased the synthesis and release of neuroactive and vasoactive peptides in rat cerebellar astrocytes.

    Science.gov (United States)

    Negishi, Takayuki; Takahashi, Masaki; Matsunaga, Yuki; Hirano, Seishiro; Tashiro, Tomoko

    2012-06-01

    An incident of poisoning occurred in Japan in 2003 when high-level contamination with arsenic, mainly diphenylarsinic acid (DPAA), was found in well water. People using this water particularly experienced cerebellar symptoms. In the present study, we investigated the adverse effects of DPAA on the cerebellum in vitro and in vivo to understand the biological mechanisms that cause cerebellar symptoms. Comprehensive gene expression analyses in primary cultured ratcerebellar cells exposed to 10 μM DPAA for 24 hours indicated significant alterations in the mRNA expression of genes encoding antioxidative stress proteins (heme oxigenase 1 and heat shock protein72) and neuroactive and vasoactive peptides (neuropeptide Y, adrenomedullin, monocyte chemoattractant protein 1, and fibroblast growth factor 2). Further analyses of proteins revealed that cultured cerebellar astrocytes expressed these antioxidative stress proteins and peptides in response to exposure to DPAA. In addition, these adverseeffects were also observed in the cerebellum exposed in vivo to DPAA (100 mg/L) for 21 days. These results suggested that cerebellarastrocytes irregularly secrete neuroactive and vasoactive peptidesagainst DPAA-induced oxidative stress, which leads to abnormal neural functions and disrupted cerebellar autoregulation dynamics and results in the onset of cerebellar symptoms.

  10. Evidence for extensive non-endocytotic translocation of peptide nucleic acids across mammalian plasma membranes.

    Science.gov (United States)

    Oehlke, Johannes; Turner, Yvonne; Pritz, Stephan; Bienert, Michael

    2011-09-01

    The ability of peptide nucleic acids (PNA) to enter and to cross filter-grown MDCK, HEK and CHO cells was studied by means of a protocol based on capillary electrophoresis combined with laser-induced fluorescence detection. The used approach avoided possible errors encountered in protocols based on confocal laserscanning microscopy and FACS analysis. In contradiction to the commonly anticipated unability of PNA to cross biomembranes, extensive translocation of unmodified PNA into and across the investigated cell types was found. The transport mode comprised a variety of energy dependent and -independent as well as temperature sensitive mechanisms being probably destined to natural substrates and hijacked by PNA. The presented results suggest active as well as passive export mechanisms rather than poor penetration into cells to be responsible for the only weak biological activity of unmodified PNA.

  11. Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers

    DEFF Research Database (Denmark)

    Berthold, Peter R; Shiraishi, Takehiko; Nielsen, Peter E

    2010-01-01

    Peptide nucleic acid (PNA) is potentially an attractive antisense and antigene agent for which more efficient cellular delivery systems are still warranted. The cationic polymer polyethylenimine (PEI) is commonly used for cellular transfection of DNA and RNA complexes, but is not readily applicable...... for PNA due to the (inherent) charge neutrality of PNA. However, PEI could function as an efficient scaffold for PNA via chemical conjugation. Accordingly, we modified PEI with the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (with and without a PEG...... moiety) and further reacted this with a cysteine PNA. The level of modification was determined spectrophotometrically with high accuracy, and the PNA transfection efficiency of the conjugates was evaluated in an antisense luciferase splice-correction assay using HeLa pLuc705 cells. We find that PEI...

  12. DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

    Science.gov (United States)

    Gaylord, Brent S.; Heeger, Alan J.; Bazan, Guillermo C.

    2002-08-01

    The light-harvesting properties of cationic conjugated polymers are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, "real-time" DNA detection. Signal transduction is controlled by hybridization of the neutral PNA probe and the negative DNA target. Electrostatic interactions bring the hybrid complex and cationic polymer within distances required for Förster energy transfer. Conjugated polymer excitation provides fluorescein emission >25 times higher than that obtained by exciting the dye, allowing detection of target DNA at concentrations of 10 pM with a standard fluorometer. A simple and highly sensitive assay with optical amplification that uses the improved hybridization behavior of PNA/DNA complexes is thus demonstrated.

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

    DEFF Research Database (Denmark)

    Birkedal, Henrik; Nielsen, Peter E

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine...... interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression...... suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair...

  14. Enhanced lubrication on tissue and biomaterial surfaces through peptide-mediated binding of hyaluronic acid

    Science.gov (United States)

    Singh, Anirudha; Corvelli, Michael; Unterman, Shimon A.; Wepasnick, Kevin A.; McDonnell, Peter; Elisseeff, Jennifer H.

    2014-10-01

    Lubrication is key for the efficient function of devices and tissues with moving surfaces, such as articulating joints, ocular surfaces and the lungs. Indeed, lubrication dysfunction leads to increased friction and degeneration of these systems. Here, we present a polymer-peptide surface coating platform to non-covalently bind hyaluronic acid (HA), a natural lubricant in the body. Tissue surfaces treated with the HA-binding system exhibited higher lubricity values, and in vivo were able to retain HA in the articular joint and to bind ocular tissue surfaces. Biomaterials-mediated strategies that locally bind and concentrate HA could provide physical and biological benefits when used to treat tissue-lubricating dysfunction and to coat medical devices.

  15. Sequence-selective targeting of duplex DNA by peptide nucleic acids

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Sequence-selective gene targeting constitutes an attractive drug-discovery approach for genetic therapy, with the aim of reducing or enhancing the activity of specific genes at the transcriptional level, or as part of a methodology for targeted gene repair. The pseudopeptide DNA mimic peptide...... nucleic acid (PNA) can recognize duplex DNA with high sequence specificity and affinity in triplex, duplex and double-duplex invasive modes or non-invasive triplex modes. Novel PNA modification has improved the affinity for DNA recognition via duplex invasion, double-duplex invasion and triplex...... recognition considerably. Such modifications have also resulted in new approaches to targeted gene repair and sequence-selective double-strand cleavage of genomic DNA....

  16. Quantification of glycated N-terminal peptide of hemoglobin using derivatization for multiple functional groups of amino acids followed by liquid chromatography/tandem mass spectrometry.

    Science.gov (United States)

    Sakaguchi, Yohei; Kinumi, Tomoya; Yamazaki, Taichi; Takatsu, Akiko

    2016-02-01

    A novel method of amino acid analysis using derivatization of multiple functional groups (amino, carboxyl, and phenolic hydroxyl groups) was applied to measure glycated amino acids in order to quantify glycated peptides and evaluate the degree of glycation of peptide. Amino and carboxyl groups of amino acids were derivatized with 1-bromobutane so that the hydrophobicities and basicities of the amino acids, including glycated amino acids, were improved. These derivatized amino acids could be detected with high sensitivity using LC-MS/MS. In this study, 1-deoxyfructosyl-VHLTPE and VHLTPE, which are N-terminal peptides of the β-chains of hemoglobin, were selected as target compounds. After reducing the peptide sample solution with sodium borohydride, the obtained peptides were hydrolyzed with hydrochloric acid. The released amino acids were then derivatized with 1-bromobutane and analyzed with LC-MS/MS. The derivatized amino acids, including glycated amino acids, could be separated using an octadecyl silylated silica column and good sharp peaks were detected. We show a confirmatory experiment that the proposed method can be applied to evaluate the degree of glycation of peptides, using mixtures of glycated and non-glycated peptide.

  17. 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 nanosi...

  18. Isolation, Characterization, and Synthesis of the Barrettides: Disulfide-Containing Peptides from the Marine Sponge Geodia barretti.

    Science.gov (United States)

    Carstens, Bodil B; Rosengren, K Johan; Gunasekera, Sunithi; Schempp, Stefanie; Bohlin, Lars; Dahlström, Mia; Clark, Richard J; Göransson, Ulf

    2015-08-28

    Two disulfide-containing peptides, barrettides A (1) and B (2), from the cold-water marine sponge Geodia barretti are described. Those 31 amino acid residue long peptides were sequenced using mass spectrometry methods and structurally characterized using NMR spectroscopy. The structure of 1 was confirmed by total synthesis using the solid-phase peptide synthesis approach that was developed. The two peptides were found to differ only at a single position in their sequence. The three-dimensional structure of 1 revealed that these peptides possess a unique fold consisting of a long β-hairpin structure that is cross-braced by two disulfide bonds in a ladder-like arrangement. The peptides are amphipathic in nature with the hydrophobic and charged residues clustered on separate faces of the molecule. The barrettides were found not to inhibit the growth of either Escherichia coli or Staphylococcus aureus but displayed antifouling activity against barnacle larvae (Balanus improvisus) without lethal effects in the concentrations tested. PMID:26222779

  19. 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...... intestinal adaptation in weanling pigs. During the first 6 d after weaning, piglets were intragastrically infused once daily with either deionized water (control), chenodeoxycholic acid (CDC; 60 mg/kg body weight), or β-sitoesterol (BSE; 100 mg/kg body weight). Infusing CDC increased plasma GLP-2 (P ....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...

  20. 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

  1. How mono-valent cations bend peptide turns and a first-principles database of amino acids and dipeptides

    Science.gov (United States)

    Baldauf, Carsten; Ropo, Matti; Blum, Volker; Scheffler, Matthias

    2014-10-01

    In this contribution we detail our efforts to investigate the structural effects of cations binding to peptides and amino acids. We perform first-principles studies employing long-range dispersion-corrected approximate density-functional theory and compare to gas-phase experiments.

  2. Alanine Esters of Enterococcal Lipoteichoic Acid Play a Role in Biofilm Formation and Resistance to Antimicrobial Peptides

    OpenAIRE

    Fabretti, Francesca; Theilacker, Christian; Baldassarri, Lucilla; Kaczynski, Zbigniew; Kropec, Andrea; Holst, Otto; Huebner, Johannes

    2006-01-01

    Enterococcus faecalis is among the predominant causes of nosocomial infections. Surface molecules like d-alanine lipoteichoic acid (LTA) perform several functions in gram-positive bacteria, such as maintenance of cationic homeostasis and modulation of autolytic activities. The aim of the present study was to evaluate the effect of d-alanine esters of teichoic acids on biofilm production and adhesion, autolysis, antimicrobial peptide sensitivity, and opsonic killing. A deletion mutant of the d...

  3. Enzymatic recycling of ascorbic acid from dehydroascorbic acid by glutathione-like peptides in the extracellular loops of aminergic G-protein coupled receptors.

    Science.gov (United States)

    Root-Bernstein, Robert; Fewins, Jenna; Rhinesmith, Tyler; Koch, Ariana; Dillon, Patrick F

    2016-07-01

    The intracellular recycling of ascorbic acid from dehydroascorbic acid by the glutathione-glutathione reductase system has been well-characterized. We propose that extracellular recycling of ascorbic acid is performed in a similar manner by cysteine-rich, glutathione-like regions of the first and second extracellular loops of some aminergic receptors including adrenergic, histaminergic, and dopaminergic receptors. Previous research in our laboratory demonstrated that ascorbic acid binds to these receptors at a site on their first or second extracellular loops, significantly enhancing ligand activity, and apparently recycling hundreds of times their own concentration of ascorbate in an enzymatic fashion. In this study, we have synthesized 25 peptides from the first and second extracellular loops of aminergic and insulin receptors and compared them directly to glutathione for their ability to prevent the oxidation of ascorbate and to regenerate ascorbate from dehydroascorbic acid. Peptide sequences that mimic glutathione in containing a cysteine and a glutamic acid-like amino acid also mimic glutathione activity in effects and in kinetics. Some (but not all) peptide sequences that contain one or more methionines instead of cysteine can significantly retard the oxidation of ascorbic acid but do not recycle it from dehydroascorbate into ascorbate. Peptides lacking both cysteines and methionines uniformly failed to alter significantly ascorbate or dehydroascorbate oxidation or reduction. We believe that this is the first proof that receptors may carry out both ligand binding and enzymatic activity extracellularly. Our results suggest the existence of a previously unknown extracellular system for recycling ascorbate. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26749062

  4. Testing the limits of rational design by engineering pH sensitivity into membrane-active peptides.

    Science.gov (United States)

    Wiedman, Gregory; Wimley, William C; Hristova, Kalina

    2015-04-01

    In this work, we sought to rationally design membrane-active peptides that are triggered by low pH to form macromolecular-sized pores in lipid bilayers. Such peptides could have broad utility in biotechnology and in nanomedicine as cancer therapeutics or drug delivery vehicles that promote release of macromolecules from endosomes. Our approach to rational design was to combine the properties of a pH-independent peptide, MelP5, which forms large pores allowing passage of macromolecules, with the properties of two pH-dependent membrane-active peptides, pHlip and GALA. We created two hybrid sequences, MelP5_Δ4 and MelP5_Δ6, by using the distribution of acidic residues on pHlip and GALA as a guide to insert acidic amino acids into the amphipathic helix of MelP5. We show that the new peptides bind to lipid bilayers and acquire secondary structure in a pH-dependent manner. The peptides also destabilize bilayers in a pH-dependent manner, such that lipid vesicles release the small molecules ANTS/DPX at low pH only. Thus, we were successful in designing pH-triggered pore-forming peptides. However, no macromolecular release was observed under any conditions. Therefore, we abolished the unique macromolecular poration properties of MelP5 by introducing pH sensitivity into its sequence. We conclude that the properties of pHlip, GALA, and MelP5 are additive, but only partially so. We propose that this lack of additivity is a limitation in the rational design of novel membrane-active peptides, and that high-throughput approaches to discovery will be critical for continued progress in the field.

  5. 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.

  6. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    Directory of Open Access Journals (Sweden)

    Nattawut Yotapan

    2014-09-01

    Full Text Available DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

  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

    2013-02-07

    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.

  8. Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

    Science.gov (United States)

    Pelc, Rebecca S; McClure, Jennifer C; Kaur, Simran J; Sears, Khandra T; Rahman, M Sayeedur; Ceraul, Shane M

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria's ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.

  9. Application of peptide nucleic acids containing azobenzene self-assembled electrochemical biosensors in detecting DNA sequences

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Hybridization of peptide nucleic acids probe containing azobenzene (NH2-TNT4, N-PNAs) with DNA was performed by covalently immobilizing of NH2-TNT4 in sequence on the 3-mercaptopropionic acid self-assembled monolayer modified gold electrode with the helps of N-(3-dimethylaminopropy1)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the hybrid was coded as N-PNAs/DNA. Using [Fe(CN)6]4-/3- (1:1) as the electrochemical indicator, the electrochemical properties of the N-PNAs self-assembled monolayer (N-PNAs-SAMs) and N-PNAs/DNA hybridization system under the conditions of before and after UV light irradiation were characterized with cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectra (EIS). Results showed that the redox currents decreased with the increase of irradiation time, suggesting that the ability of the charge transfer on the electrode surface was weakened and the conformation of hybrid system had been changed, and the control of PNAs/DNA hybridization could be realized by UV light irradiation.

  10. Whey peptide-based enteral diet attenuated elastase-induced emphysema with increase in short chain fatty acids in mice

    OpenAIRE

    Tomoda, Koichi; Kubo, Kaoru; Dairiki, Kazuo; Yamaji, Taketo; Yamamoto, Yoshifumi; Nishii, Yasue; Nakamura, Atsuhiro; Yoshikawa, Masanori; Hamada, Kaoru; Kimura, Hiroshi

    2015-01-01

    Background Systemic inflammation is present in chronic obstructive pulmonary disease (COPD). A whey peptide-based enteral diet reduce inflammation in patients with COPD, but its effect on COPD development has not been determined. On the other hand, it is known that short chain fatty acids (SCFAs), which are produced by micro-flora in the gut, attenuates bronchial asthma in mice model. Methods Mice with elastase-induced emphysema were fed with 1 of 3 diets (control diet, whey peptide-based ent...

  11. Identification and binding mechanism of phage displayed peptides with specific affinity to acid-alkali treated titanium.

    Science.gov (United States)

    Sun, Yuhua; Tan, Jing; Wu, Baohua; Wang, Jianxin; Qu, Shuxin; Weng, Jie; Feng, Bo

    2016-10-01

    Acid-alkali treatment is one of means widely used for preparing bioactive titanium surfaces. Peptides with specific affinity to titanium surface modified by acid-alkali two-steps treatment were obtained via phage display technology. Out of the eight new unique peptides, titanium-binding peptide 54 displayed by monoclonal M13 phage at its pIII coat protein (TBP54-M13 phage) was proved to have higher binding affinity to the substrate. The binding interaction occurred at the domain from phenylalanine at position 1 to arginine at position 6 in the sequences of TBP54 (FAETHRGFHFSF) mainly via the reaction of these residues with the Ti surface. Together the coordination and electrostatic interactions controlled the specific binding of the phage to the substrate. The binding affinity was dependent on the surface basic hydroxyl group content. In addition, the phage showed a different interaction way with the Ti surface without acid-alkali treatment along with an impaired affinity. This study could provide more understanding of the interaction mechanism between the selected peptide and its specific substrate, and develop a promising method for the biofunctionalization of titanium. PMID:27371890

  12. Organization and Structure of Branched Amphipathic Oligopeptide Bilayers.

    Science.gov (United States)

    Jia, Zhiguang; Whitaker, Susan K; Tomich, John M; Chen, Jianhan

    2016-09-27

    A class of self-assembling branched amphiphilic peptide capsules (BAPCs) was recently developed that could serve as a new drug delivery vehicle. BAPCs can encapsulate solutes up to ∼12 kDa during assembly, are unusually stable, and are readily taken up by cells with low cytotoxicity. Coarse-grained simulations have supported that BAPCs are defined by bilayers that resemble those formed by diacyl phospholipids. Here, atomistic simulations were performed to characterize the structure and organization of bilayers formed by three branched amphiphilic peptides (BAPs): bis(Ac-FLIVIGSII)-K-K4-CO-NH2, bis(Ac-CHA-LIVIGSII)-K-K4-CO-NH2, and bis(Ac-FLIVI)-K-K4-CO-NH2. The results show BAPs form a network of intra- and intermolecular backbone hydrogen bonds within the same leaflet in addition to hydrophobic side-chain interactions. The terminal residues of two leaflets form an interdigitation region locking two leaflets together. The phenyl groups in bis(Ac-FLIVIGSII)-K-K4-CO-NH2 and bis(Ac-FLIVI)-K-K4-CO-NH2 are tightly packed near the bilayer center but do not formed ordered structures with specific π-π stacking. Replacing phenyl groups with the cyclohexane side chain only slightly increases the level of disorder in bilayer structures and thus should not significantly affect the stability, consistent with experimental results on bis(Ac-CHA-LIVIGSII)-K-K4-CO-NH2 BAPCs. Self-assembly simulations further suggest that leaflet interdigitation likely occurs at early stages of BAPC formation. Atomistic simulations also reveal that the BAPC bilayers are highly permeable to water. This prediction was validated using fluorescence measurements of encapsulated self-quenching dye upon transferring BAPCs to buffers with different salt concentrations. Improved understanding of the organization and structure of BAPC bilayers at the atomic level will provide a basis for future rational modifications of BAP sequence to improve BAPC properties as a new class of delivery vehicle. PMID

  13. Development of Novel Radiogallium-Labeled Bone Imaging Agents Using Oligo-Aspartic Acid Peptides as Carriers

    OpenAIRE

    Kazuma Ogawa; Atsushi Ishizaki; Kenichiro Takai; Yoji Kitamura; Tatsuto Kiwada; Kazuhiro Shiba; Akira Odani

    2013-01-01

    (68)Ga (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 (68)Ga-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...

  14. 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

  15. Detection of antimicrobial (poly)peptides with acid urea polyacrylamide gel electrophoresis followed by Western immunoblot.

    Science.gov (United States)

    Porter, Edith; Valore, Erika V; Anouseyan, Rabin; Salzman, Nita H

    2015-01-01

    Antimicrobial (poly)peptides (AMPs) are ancient key effector molecules of innate host defense and have been identified in mammals, insects, plants, and even fungi (Nakatsuji and Gallo, J Invest Dermatol, 132: 887-895, 2012). They exhibit a cationic net charge at physiological pH and are rich in hydrophobic amino acids (Dufourc et al., Curr Protein Pept Sci, 13: 620-631, 2012). Their mode of action has been best investigated in bacteria. When assuming secondary structure the cationic and hydrophobic amino acids are sequestered creating a bipartitioned molecule in which the cationic amino acids mediate initial electrostatic interaction with the negatively charged bacterial surface and the hydrophobic amino acids mediate embedding into the bacterial membranes followed by a multitude of effects interfering with bacterial viability (Nicolas, FEBS J, 276: 6483-6496, 2009; Padovan et al., Curr Protein Pept Sci, 11: 210-219, 2010). However, immunomodulatory, antitumor, and other effects have been added to the ever increasing list of AMP functions (Pushpanathan et al., Int J Pept, 2013: 675391, 2013). Several classes of AMPs have been distinguished based on structure, namely anti-parallel beta-sheet, alpha-helical, circular, as well as disulfide bridge connectivity (Bond and Khalid, Protein Pept Lett, 17: 1313-1327, 2010). Many of the AMPs undergo posttranslational modification including further proteolysis. Biochemical analysis at the protein level is of great interest for a wide range of scientists and important when studying host-pathogen interaction, for example Salmonella invasion of the small intestine. Acid-urea polyacrylamide gel electrophoresis (AU-PAGE) followed by Western immunoblotting is an important tool for the identification and quantification of cationic AMPs. The protocol for these procedures outlined here describes, in detail, the necessary steps; including pouring the AU-gels, preparing the test samples, performing the electrophoretic separation and

  16. Targeting the S1 and S3 subsite of trypsin with unnatural cationic amino acids generates antimicrobial peptides with potential for oral administration.

    Science.gov (United States)

    Karstad, Rasmus; Isaksen, Geir; Wynendaele, Evelien; Guttormsen, Yngve; De Spiegeleer, Bart; Brandsdal, Bjørn-Olav; Svendsen, John Sigurd; Svenson, Johan

    2012-07-26

    This study investigates how the S1 and S3 site of trypsin can be challenged with cationic amino acid analogues to yield active antimicrobial peptides with stability toward tryptic degradation. It is shown that unnatural analogues can be incorporated to generate stable peptides with maintained bioactivity to allow for a potential oral uptake. Selected peptides were studied using isothermal calorimetry and computational methods. Both stable and unstable peptides were found to bind stoichiometrically to trypsin with dissociation constants ranging 2-60 μM, suggesting several different binding modes. The stability of selected peptides was analyzed in whole organ extracts and the incorporation of homoarginine and 2-amino-(3-guanidino)propanoic acid resulted in a 14- and 50-fold increase in duodenal stability. In addition, a 40- and 70-fold increase in stomach stability is also reported. Overall, these results illustrate how the incorporation of cationic side chains can be employed to generate bioactive peptides with significant systemic stability.

  17. 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

    2016-08-01

    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. PMID:27457924

  18. An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide.

    Science.gov (United States)

    Guan, Yihong; Zhu, Qinfang; Huang, Delai; Zhao, Shuyi; Jan Lo, Li; Peng, Jinrong

    2015-01-01

    The molecular weight (MW) of a protein can be predicted based on its amino acids (AA) composition. However, in many cases a non-chemically modified protein shows an SDS PAGE-displayed MW larger than its predicted size. Some reports linked this fact to high content of acidic AA in the protein. However, the exact relationship between the acidic AA composition and the SDS PAGE-displayed MW is not established. Zebrafish nucleolar protein Def is composed of 753 AA and shows an SDS PAGE-displayed MW approximately 13 kDa larger than its predicted MW. The first 188 AA in Def is defined by a glutamate-rich region containing ~35.6% of acidic AA. In this report, we analyzed the relationship between the SDS PAGE-displayed MW of thirteen peptides derived from Def and the AA composition in each peptide. We found that the difference between the predicted and SDS PAGE-displayed MW showed a linear correlation with the percentage of acidic AA that fits the equation y = 276.5x - 31.33 (x represents the percentage of acidic AA, 11.4% ≤ x ≤ 51.1%; y represents the average ΔMW per AA). We demonstrated that this equation could be applied to predict the SDS PAGE-displayed MW for thirteen different natural acidic proteins. PMID:26311515

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

    OpenAIRE

    Young Chon Choi; Ki Rok, Kwon; Suk Ho, Choi

    2006-01-01

    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...

  20. Viroporin potential of the lentivirus lytic peptide (LLP domains of the HIV-1 gp41 protein

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2007-11-01

    Full Text Available Abstract Background Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41 contains potential amphipathic α-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP -1, -2, and -3. Results Peptides corresponding to the LLP domains (from a clade B virus partition into lipid membranes, fold into α-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity. Conclusion These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs.

  1. Core–Shell Nanoparticle-Based Peptide Therapeutics and Combined Hyperthermia for Enhanced Cancer Cell Apoptosis

    OpenAIRE

    Shah, Birju P.; Pasquale, Nicholas; De, Gejing; Tan, Tao; Ma, Jianjie; Lee, Ki-Bum

    2014-01-01

    Mitochondria-targeting peptides have garnered immense interest as potential chemotherapeutics in recent years. However, there is a clear need to develop strategies to overcome the critical limitations of peptides, such as poor solubility and the lack of target specificity, which impede their clinical applications. To this end, we report magnetic core–shell nanoparticle (MCNP)-mediated delivery of a mitochondria-targeting pro-apoptotic amphipathic tail-anchoring peptide (ATAP) to malignant bra...

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

    Science.gov (United States)

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

    2004-07-01

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

  3. 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

  4. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    Science.gov (United States)

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications.

  5. Peptide nucleic acids arrest the growth of gastric cancer cells SGC7901

    Institute of Scientific and Technical Information of China (English)

    王宽; 张岂凡; 王锡山; 薛英威; 庞达; 傅松滨

    2004-01-01

    Background Peptide nucleic acid (PNA) has many characteristics useful in molecular biology. This paper described an effective way to raise the cell ingestion rate of PNA so as to kill gastric cancer cells.Methods Heteroduplexes of PNAs and oligonucleotides, wrapped by Lipofectamine 2000, were used to infect SGC7901 cells. The inhibitive effect of heteroduplexes was evaluated by analyzing cell clone forming and cell growth rate. Telomerase activity of SGC7901 cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and silver staining assay.Results PNAs showed a dose-dependent inhibition of cell proliferation. The percentage of proliferation inhibition was 99.4% after 7 days; the rate of cloning inhibition was 98.2% after 8 days;whereas for oligonucleotide groups, at the same concentration, the percentages were 50. 1% and 67. 5% respectively. Antisense PNA-DNA-Lipofectamine 2000 group (AP-D-L group) exhibited significantly different percentages from the control groups (P<0.05). The test result indicated that telomerase activity of the AP-D-L group was inhibited (P<0.05). At the same time, the impact on cell morphology was observed.Conclusions The results showed that PNAs are potent antisense reagents. The telomeraseassociated therapies are very promising for the treatment of malignant tumours.

  6. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    Science.gov (United States)

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications. PMID:26394062

  7. A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

    Science.gov (United States)

    Chen, Tai-Long; Chang, John Wen-Cheng; Hsieh, Jia-Juan; Cheng, Hsin-Yi; Chiou, Chiuan-Chian

    Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing. PMID:27566656

  8. Boswellic acids target the human immune system-modulating antimicrobial peptide LL-37.

    Science.gov (United States)

    Henkel, Arne; Tausch, Lars; Pillong, Max; Jauch, Johann; Karas, Michael; Schneider, Gisbert; Werz, Oliver

    2015-12-01

    The antimicrobial peptide LL-37 is the sole member of the human cathelicidin family with immune system-modulating properties and roles in autoimmune disease development. Small molecules able to interact with LL-37 and to modulate its functions have not been described yet. Boswellic acids (BAs) are pentacyclic triterpene acids that are bioactive principles of frankincense extracts used as anti-inflammatory remedies. Although various anti-inflammatory modes of action have been proposed for BAs, the pharmacological profile of these compounds is still incompletely understood. Here, we describe the identification of human LL-37 as functional target of BAs. In unbiased target fishing experiments using immobilized BAs as bait and human neutrophils as target source, LL-37 was identified as binding partner assisted by MALDI-TOF mass spectrometry. Thermal stability experiments using circular dichroism spectroscopy confirm direct interaction between BAs and LL-37. Of interest, this binding of BAs resulted in an inhibition of the functionality of LL-37. Thus, the LPS-neutralizing properties of isolated LL-37 were inhibited by 3-O-acetyl-β-BA (Aβ-BA) and 3-O-acetyl-11-keto-β-BA (AKβ-BA) in a cell-free limulus amoebocyte lysate assay with EC50=0.2 and 0.8 μM, respectively. Also, LL-37 activity was inhibited by these BAs in LL-37-enriched supernatants of stimulated neutrophils or human plasma derived from stimulated human whole blood. Together, we reveal BAs as inhibitors of LL-37, which might be a relevant mechanism underlying the anti-inflammatory properties of BAs and suggests BAs as suitable chemical tools or potential agents for intervention with LL-37 and related disorders. PMID:26361729

  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. Synthesis of a chiral amino acid with bicyclo[1.1.1]pentane moiety and its incorporation into linear and cyclic antimicrobial peptides.

    Science.gov (United States)

    Pritz, Stephan; Pätzel, Michael; Szeimies, Günter; Dathe, Margitta; Bienert, Michael

    2007-06-01

    The synthesis of the lipophilic chiral amino acid 1 bearing the bicyclo[1.1.1]pentane moiety is described. Linear and cyclic hexapeptides of the type Arg-Arg-Xaa-Yaa-Arg-Phe containing 1 instead of one or two tryptophan residues are prepared by solid phase peptide synthesis and the antimicrobial and hemolytic activity of the peptides obtained are discussed.

  11. A Comparison of the Effects of Amide and Acid Groups at the C-Terminus on the Collision-Induced Dissociation of Deprotonated Peptides

    Science.gov (United States)

    Bokatzian-Johnson, Samantha S.; Stover, Michele L.; Dixon, David A.; Cassady, Carolyn J.

    2012-09-01

    The dissociative behavior of peptide amides and free acids was explored using low-energy collision-induced dissociation and high level computational theory. Both positive and negative ion modes were utilized, but the most profound differences were observed for the deprotonated species. Deprotonated peptide amides produce a characteristic cm-2 - product ion (where m is the number of residues in the peptide) that is either absent or in low abundance in the analogous peptide acid spectrum. Peptide acids show an enhanced formation of cm-3 -; however, this is not generally as pronounced as cm-2 - production from amides. The most notable occurrence of an amide-specific product ion is for laminin amide (YIGSR-NH2) and this case was investigated using several modified peptides. Mechanisms involving 6- and 9-membered ring formation were proposed, and their energetic properties were investigated using G3(MP2) molecular orbital theory calculations. For example, with C-terminal deprotonation of pentaglycine amide, formation of cm-2 - and a 6-membered ring diketopiperazine neutral requires >31.6 kcal/mol, which is 26.1 kcal/mol less than the analogous process involving the peptide acid. The end group specific fragmentation of peptide amides in the negative ion mode may be useful for identifying such groups in proteomic applications.

  12. Hyaluronic Acid-Based Nanogels Produced by Microfluidics-Facilitated Self-Assembly Improves the Safety Profile of the Cationic Host Defense Peptide Novicidin

    DEFF Research Database (Denmark)

    Water, Jorrit J; Kim, YongTae; Maltesen, Morten J;

    2015-01-01

    peptide loading of 36 ± 4%. The nanogels exhibited good colloidal stability under different ionic strength conditions and allowed complete release of the peptide over 14 days. Furthermore, self-assembly of novicidin with hyaluronic acid into nanogels significantly improved the safety profile at least five...

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

    International Nuclear Information System (INIS)

    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

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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 emphasised. (author)

  16. 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.

  17. Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

    Science.gov (United States)

    Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

    2013-01-01

    White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH. PMID:23391931

  18. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

    OpenAIRE

    Kim, Ki-Hyun; Nielsen, Peter E.; Glazer, Peter M.

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable ...

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

    OpenAIRE

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

    2014-01-01

    Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple helix forming peptide nucleic acids (PNAs) that bind in the major grov...

  20. Measurement and modeling of acid dissociation constants of tri-peptides containing Glu, Gly, and His using potentiometry and generalized multiplicative analysis of variance.

    Science.gov (United States)

    Khoury, Rima Raffoul; Sutton, Gordon J; Hibbert, D Brynn; Ebrahimi, Diako

    2013-02-28

    We report pK(a) values with measurement uncertainties for all labile protons of the 27 tri-peptides prepared from the amino acids glutamic acid (E), glycine (G) and histidine (H). Each tri-peptide (GGG, GGE, GGH, …, HHH) was subjected to alkali titration and pK(a) values were calculated from triplicate potentiometric titrations data using HyperQuad 2008 software. A generalized multiplicative analysis of variance (GEMANOVA) of pK(a) values for the most acidic proton gave the optimum model having two terms, an interaction between the end amino acids plus an isolated main effect of the central amino acid.

  1. Enrichment of ACE inhibitory peptides in navy bean (Phaseolus vulgaris) using lactic acid bacteria.

    Science.gov (United States)

    Rui, Xin; Wen, Delan; Li, Wei; Chen, Xiaohong; Jiang, Mei; Dong, Mingsheng

    2015-02-01

    The present study was conducted to explore a novel strategy to enhance angiotensin I-converting enzyme (ACE) inhibitory activities of navy bean by preparation of navy bean milk (NBM) which was then subjected to fermentation of four lactic acid bacteria (LAB) strains, namely, Lactobacillus bulgaricus, Lactobacillus helveticus MB2-1, Lactobacillus plantarum B1-6, and Lactobacillus plantarum 70810. With the exception of L. helveticus MB2-1, the other three selected strains had good growth performances in NBM with viable counts increased to log 8.30-8.39 cfu ml(-1) during 6 h of fermentation, and thus were selected for the following investigations. Protein contents of NBM significantly reduced when treated with L. bulgaricus and L. plantarum B1-6, and the electrophoresis patterns showed the preferable proteins for LAB strains to hydrolyze were α- and β-type phaseolins, whereas γ-type phaseolin was resistant to hydrolysis. RP-HPLC analysis demonstrated all fermented NBM had higher intensities of peaks with retention times between 2.5 and 3.5 min indicative of formation of small peptides. All fermented NBM showed higher ACE inhibitory activity compared to the unfermented ones, for which 2 h, 3 h, and 5 h were found to be the optimum fermentation periods for respectively L. plantarum 70810, L. plantarum B1-6 and L. bulgaricus, with IC50 values of 109 ± 5.1, 108 ± 1.1, and 101 ± 2.2 μg protein ml(-1). The subsequent in vitro gastrointestinal simulation afforded all fermented extracts reduced IC50 values and the extracts fermented by L. plantarum B1-6 exerted the lowest IC50 value of 21 ± 2.1 μg protein ml(-1). The research has broadened our knowledge bases on the effect of LAB fermentation on the degradation of navy bean proteins and the capacity to release ACE inhibitory peptides. The approach was promising to obtain probiotic products with potential to serve as functional ingredients targeting hypertension. PMID:25536445

  2. Enrichment of ACE inhibitory peptides in navy bean (Phaseolus vulgaris) using lactic acid bacteria.

    Science.gov (United States)

    Rui, Xin; Wen, Delan; Li, Wei; Chen, Xiaohong; Jiang, Mei; Dong, Mingsheng

    2015-02-01

    The present study was conducted to explore a novel strategy to enhance angiotensin I-converting enzyme (ACE) inhibitory activities of navy bean by preparation of navy bean milk (NBM) which was then subjected to fermentation of four lactic acid bacteria (LAB) strains, namely, Lactobacillus bulgaricus, Lactobacillus helveticus MB2-1, Lactobacillus plantarum B1-6, and Lactobacillus plantarum 70810. With the exception of L. helveticus MB2-1, the other three selected strains had good growth performances in NBM with viable counts increased to log 8.30-8.39 cfu ml(-1) during 6 h of fermentation, and thus were selected for the following investigations. Protein contents of NBM significantly reduced when treated with L. bulgaricus and L. plantarum B1-6, and the electrophoresis patterns showed the preferable proteins for LAB strains to hydrolyze were α- and β-type phaseolins, whereas γ-type phaseolin was resistant to hydrolysis. RP-HPLC analysis demonstrated all fermented NBM had higher intensities of peaks with retention times between 2.5 and 3.5 min indicative of formation of small peptides. All fermented NBM showed higher ACE inhibitory activity compared to the unfermented ones, for which 2 h, 3 h, and 5 h were found to be the optimum fermentation periods for respectively L. plantarum 70810, L. plantarum B1-6 and L. bulgaricus, with IC50 values of 109 ± 5.1, 108 ± 1.1, and 101 ± 2.2 μg protein ml(-1). The subsequent in vitro gastrointestinal simulation afforded all fermented extracts reduced IC50 values and the extracts fermented by L. plantarum B1-6 exerted the lowest IC50 value of 21 ± 2.1 μg protein ml(-1). The research has broadened our knowledge bases on the effect of LAB fermentation on the degradation of navy bean proteins and the capacity to release ACE inhibitory peptides. The approach was promising to obtain probiotic products with potential to serve as functional ingredients targeting hypertension.

  3. Molecular mechanism of acid-catalyzed hydrolysis of peptide bonds using a model compound.

    Science.gov (United States)

    Pan, Bin; Ricci, Margaret S; Trout, Bernhardt L

    2010-04-01

    The stability of peptide bonds is a critical aspect of biological chemistry and therapeutic protein applications. Recent studies found elevated nonenzymatic hydrolysis in the hinge region of antibody molecules, but no mechanism was identified. As a first step in providing a mechanistic interpretation, this computational study examines the rate-determining step of the hydrolytic reaction of a peptide bond under acidic pH by a path sampling technique using a model compound N-MAA. Most previous computational studies did not include explicit water molecules, whose effects are significant in solution chemistry, nor did they provide a dynamic picture for the reaction process in aqueous conditions. Because no single trajectory can be used to describe the reaction dynamics due to fluctuations at finite temperatures, a variant version of the transition path sampling technique, the aimless shooting algorithm, was used to sample dynamic trajectories and to generate an ensemble of transition trajectories according to their statistical weights in the trajectory space. Each trajectory was computed as the time evolution of the molecular system using the Car-Parrinello molecular dynamics technique. The likelihood maximization procedure and its modification were used in extracting dynamically relevant degrees of freedom in the system, and approximations of the reaction coordinate were compared. Its low log-likelihood score and poor p(B) histogram indicate that the C-O distance previously assumed as the reaction coordinate for the rate-determining step is inadequate in describing the dynamics of the reaction. More than one order parameter in a candidate set including millions of geometric quantities was required to produce a convergent reaction coordinate model; its involvement of many degrees of freedom suggests that this hydrolytic reaction step is very complex. In addition to affecting atoms directly involved in bond-making and -breaking processes, the water network also has

  4. Cationic cell-penetrating peptides induce ceramide formation via acid sphingomyelinase: implications for uptake.

    NARCIS (Netherlands)

    Verdurmen, W.P.R.; Thanos, M.; Ruttekolk, I.R.R.; Gulbins, E.; Brock, R.E.

    2010-01-01

    Cationic cell-penetrating peptides (CPP) are receiving increasing attention as molecular transporters of membrane-impermeable molecules. Import of cationic CPP occurs both via endocytosis and - at higher peptide concentrations - in an endocytosis-independent manner via localized regions of the plasm

  5. Synthetic amphibian peptides and short amino-acids derivatives against planktonic cells and mature biofilm of Providencia stuartii clinical strains.

    Science.gov (United States)

    Ostrowska, Kinga; Kamysz, Wojciech; Dawgul, Małgorzata; Różalski, Antoni

    2014-01-01

    Over the last decade, the growing number of multidrug resistant strains limits the use of many of the currently available chemotherapeutic agents. Furthermore, bacterial biofilm, due to its complex structure, constitutes an effective barrier to conventional antibiotics. The in vitro activities of naturally occurring peptide (Citropin 1.1), chemically engineered analogue (Pexiganan), newly-designed, short amino-acid derivatives (Pal-KK-NH2, Pal-KKK-NH2, Pal-RRR-NH2) and six clinically used antimicrobial agents (Gatifloxacin, Ampicilin, Cefotaxime, Ceftriaxone, Cefuroxime and Cefalexin) were investigated against planktonic cells and mature biofilm of multidrug-resistant Providencia stuartii strains, isolated from urological catheters. The MICs, MBCs values were determined by broth microdilution technique. Inhibition of biofilm formation by antimicrobial agents as well as biofilm susceptibility assay were tested using a surrogate model based on the Crystal Violet method. The antimicrobial activity of amino-acids derivatives and synthetic peptides was compared to that of clinically used antibiotics. For planktonic cells, MICs of peptides and antibiotics ranged between 1 and 256 μg/ml and 256 and ≥ 2048 μg/ml, respectively. The MBCs values of Pexiganan, Citropin 1.1 and amino-acids derivatives were between 16 and 256 μg/ml, 64 and 256 μg/ml and 16 and 512 μg/ml, respectively. For clinically used antibiotics the MBCs values were above 2048 μg/ml. All of the tested peptides and amino-acids derivatives, showed inhibitory activity against P. stuartii biofilm formation, in relation to their concentrations. Pexiganan and Citropin 1.1 in concentration range 32 and 256 μg/ml caused both strong and complete suppression of biofilm formation. None of the antibiotics caused complete inhibition of biofilm formation process. The biofilm susceptibility assay verified the extremely poor antibiofilm activity of conventional antibiotics compared to synthetic peptides. The

  6. 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.

  7. 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.

  8. THE APPLICATION OF PEPTIDE NUCLEIC ACID PROBES FOR RAPID DETECTION AND ENUMERATION OF EUBACTERIA, STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA IN RECREATIONAL BEACHES OF S. FLORIDA. (R828830)

    Science.gov (United States)

    A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, S...

  9. Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation

    OpenAIRE

    Kang MJ; Park SH; Kang MH; Park MJ; Choi YW

    2013-01-01

    Myung Joo Kang, Sang Han Park, Mean Hyung Kang, Min Jung Park, Young Wook Choi College of Pharmacy, Chung-Ang University, Seoul, Korea Background: A novel dual ligand–modified liposome, folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier (FP-Lipo), was designed to overcome the nonselectivity of conventional penetrating peptide-tagged nanoparticulates and to provide the advantage of selective targeting of the folic acid receptor, which is frequently overexpressed on ep...

  10. Novel approaches to tumor imaging in mice: pre targeting with radiolabeled peptide nucleic acid

    International Nuclear Information System (INIS)

    Full text.Since targeting of tumour by conventional methods is not consistently favorable, we have considered pre targeting with separate administrations of anti tumour antibody and radiolabel. As an alternative to streptavidin and biotin for this application, we earlier considered single stranded peptide nucleic acid (PNA) bound to an irrelevant protein administered first and allowed to diffuse non specifically into tumour. This was followed later by the administration of 99 m Tc labeled complementary PNA. We now report on the first studies with PNA conjugated anti tumour antibody to allow specific binding. PNA was conjugated to the NRLU-10 IgG antibody while the complementary PNA (amine derivatized) was labeled with ((m Tc using MAG3. LS174T tumour-bearing nude mice received IV 200 ug of the PNA-antibody conjugate and 20 h later, received IV 100 ug (130 uCl) of 99m Tc- complementary PNA. Animals were imaged and sacrificed 5 h later. Because of rapid clearance, at sacrifice all tissue levels of 99 m Tc were low, the highest being kidneys at about 4%ID/gm. Tumour uptake was 0.55%ID/gm for the study animals vs. 0. 13 for controls and tumour/muscle ratios were 9.8 vs. 3.6 respectively. These values represent a 2.5-fold improvement in localization over the nonspecific study. The whole body images also reflected the superior targeting of study vs. control animals. We conclude that single-stranded PNAs should be a useful alternative to streptavidin and biotin for pre targeting studies

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

    Science.gov (United States)

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

    2015-03-15

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

  12. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    Science.gov (United States)

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

  13. Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2012-01-01

    splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA...... that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP...

  14. 2D-Qsar for 450 types of amino acid induction peptides with a novel substructure pair descriptor having wider scope

    Directory of Open Access Journals (Sweden)

    Osoda Tsutomu

    2011-11-01

    Full Text Available Abstract Background Quantitative structure-activity relationships (QSAR analysis of peptides is helpful for designing various types of drugs such as kinase inhibitor or antigen. Capturing various properties of peptides is essential for analyzing two-dimensional QSAR. A descriptor of peptides is an important element for capturing properties. The atom pair holographic (APH code is designed for the description of peptides and it represents peptides as the combination of thirty-six types of key atoms and their intermediate binding between two key atoms. Results The substructure pair descriptor (SPAD represents peptides as the combination of forty-nine types of key substructures and the sequence of amino acid residues between two substructures. The size of the key substructures is larger and the length of the sequence is longer than traditional descriptors. Similarity searches on C5a inhibitor data set and kinase inhibitor data set showed that order of inhibitors become three times higher by representing peptides with SPAD, respectively. Comparing scope of each descriptor shows that SPAD captures different properties from APH. Conclusion QSAR/QSPR for peptides is helpful for designing various types of drugs such as kinase inhibitor and antigen. SPAD is a novel and powerful descriptor for various types of peptides. Accuracy of QSAR/QSPR becomes higher by describing peptides with SPAD.

  15. Permeation of membranes by the neutral form of amino acids and peptides: relevance to the origin of peptide translocation

    Science.gov (United States)

    Chakrabarti, A. C.; Deamer, D. W.; Miller, S. L. (Principal Investigator)

    1994-01-01

    The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10(5) slower than facilitated inward transport across biological membranes. This suggest that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10(10) times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g. for certain signal sequences, toxins and thylakoid proteins) in vivo.

  16. Similar peptides from two beta cell autoantigens, proinsulin and glutamic acid decarboxylase, stimulate T cells of individuals at risk for insulin-dependent diabetes.

    OpenAIRE

    Rudy, G; N. Stone; Harrison, L C; Colman, P. G.; McNair, P; Brusic, V.; French, M. B.; Honeyman, M. C.; Tait, B.; Lew, A M

    1995-01-01

    BACKGROUND: Insulin (1) and glutamic acid decarboxylase (GAD) (2) are both autoantigens in insulin-dependent diabetes mellitus (IDDM), but no molecular mechanism has been proposed for their association. We have identified a 13 amino acid peptide of proinsulin (amino acids 24-36) that bears marked similarity to a peptide of GAD65 (amino acids 506-518) (G. Rudy, unpublished). In order to test the hypothesis that this region of similarity is implicated in the pathogenesis of IDDM, we assayed T c...

  17. 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

    2015-05-01

    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.

  18. Nasal delivery of peptides using powder carriers based on starch/poly(acrylic acid)

    OpenAIRE

    Pringels, E

    2006-01-01

    As peptides are not suitable for oral administration, they are generally administered parenterally. Due to the disadvantages associated with parenteral delivery alternative routes of administration (buccal, nasal, pulmonal, ocular, transdermal, rectal and vaginal route) have been investigated. In the present work, the nasal route was selected as an alternative route for peptide delivery. Despite the advantages related to nasal administration, the bioavailability remains low. In Chapter 1 the ...

  19. Solid Phase Chemical Synthesis and Structure - Activity Study of Brevinin - 2R and Analogues as Antimicrobial Peptides

    Directory of Open Access Journals (Sweden)

    Hashem Yaghoubi

    2015-10-01

    Full Text Available Background: Brevinin-2R, as 25 amino acids peptide of the skin of Rana ridibunda frog, possesses potent antimicrobial and low hemolytic activity. It has an N-terminal hydrophilic region and a C-terminal loop that is delineated by an intra-disulfide bridge. In our study, Brevinin-2R and its diastereomer as well  as its  cyclic  analogue  were  synthesized  and  characterized  in  order  to investigate its structural features and biological implications.Methods: MIC determination is based on the recommended classical method of national comittee for labratory safety standard (NCLSS and standard by Hancock With some change on cationic peptides. In this study All bacterial strains were obtained from Industrial-Scientific Research center.Results: Both analogues showed lower antimicrobial activities compared to Brevinin-2R. In spite of Brevinin-2R peptide which shows low hemolytic activity, these analogues failed to show any hemolytic activity even at higherconcentrations (up to 400 µ g/ml. Based on proteolytic stability measurements,diastereomer and cyclic analogues displayed 90% and 60% residual antimicrobial activity, respectively, while antimicrobial activity of Brevinin-2R was 20%. The CD analysis revealed that amphipathic α-helical conformation of the synthesized peptides is involved in antimicrobial effects.Conclusion: CD studies and HPLC based measurement of retention time using a reverse phase column indicated that the Brevinin-2R can form an amphipathic loop  resulting  in  an  enhanced  hydrophobicity.  The  hemolytic  activity  ofBrevinin-2R and its analogues appeared to correlate with the retention time aswell as the α-helicity. Accordingly, it seems that the combination of incorporating of D-amino acids into lytic peptides and their cyclization may result in developing new antimicrobial peptides with improved properties for treating infectious diseases.

  20. Structural features of peptoid-peptide hybrids in lipid-water interfaces

    DEFF Research Database (Denmark)

    Uggerhøj, Lars Erik; Munk, Jens K; Hansen, Paul R;

    2014-01-01

    The inclusion of peptoid monomers into antimicrobial peptides (AMPs) increases their proteolytic resistance, but introduces conformational flexibility (reduced hydrogen bonding ability and cis/trans isomerism). We here use NMR spectroscopy to answer how the insertion of a peptoid monomer influenc...... interaction of the amphipathic helices flanking the peptoid monomer and the side chain properties of the peptoid and its preceding residue....

  1. Effects of hydrophobicity on the antifungal activity of alpha-helical antimicrobial peptides.

    NARCIS (Netherlands)

    Jiang, Z.; Kullberg, B.J.; Lee, H van der; Vasil, A.I.; Hale, J.D.; Mant, C.T.; Hancock, R.E.; Vasil, M.L.; Netea, M.G.; Hodges, R.S.

    2008-01-01

    We utilized a series of analogs of D-V13K (a 26-residue amphipathic alpha-helical antimicrobial peptide, denoted D1) to compare and contrast the role of hydrophobicity on antifungal and antibacterial activity to the results obtained previously with Pseudomonas aeruginosa strains. Antifungal activity

  2. 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...

  3. Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from alpha-fetoprotein.

    Science.gov (United States)

    MacColl, R; Eisele, L E; Stack, R F; Hauer, C; Vakharia, D D; Benno, A; Kelly, W C; Mizejewski, G J

    2001-10-01

    A 34-amino-acid peptide has been chemically synthesized based on a sequence from human alpha-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt-peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible d-d transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the d-d molar extinction coefficient showed that the metal ion-ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)- and Co(II)-peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60 degrees C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution

  4. Structure-based rational design of peptide hydroxamic acid inhibitors to target tumor necrosis factor-α converting enzyme as potential therapeutics for hepatitis.

    Science.gov (United States)

    Wu, Dan; Gu, Qiuhong; Zhao, Ning; Xia, Fei; Li, Zhiwei

    2015-12-01

    The human tumor necrosis factor-α converting enzyme (TACE) has recently been raised as a new and promising therapeutic target of hepatitis and other inflammatory diseases. Here, we reported a successful application of the solved crystal structure of TACE complex with a peptide-like ligand INN for rational design of novel peptide hydroxamic acid inhibitors with high potency and selectivity to target and inhibit TACE. First, the intermolecular interactions between TACE catalytic domain and INN were characterized through an integrated bioinformatics approach, with which the key substructures of INN that dominate ligand binding were identified. Subsequently, the INN molecular structure was simplified to a chemical sketch of peptide hydroxamic acid compound, which can be regarded as a linear tripeptide capped by a N-terminal carboxybenzyl group (chemically protective group) and a C-terminal hydroxamate moiety (coordinated to the Zn(2+) at TACE active site). Based on the sketch, a virtual combinatorial library containing 180 peptide hydroxamic acids was generated, from which seven samples were identified as promising candidates by using a knowledge-based protein-peptide affinity predictor and were then tested in vitro with a standard TACE activity assay protocol. Consequently, three designed peptide hydroxamic acids, i.e. Cbz-Pro-Ile-Gln-hydroxamic acid, Cbz-Leu-Ile-Val-hydroxamic acid and Cbz-Phe-Val-Met-hydroxamic acid, exhibited moderate or high inhibitory activity against TACE, with inhibition constants Ki of 36 ± 5, 510 ± 46 and 320 ± 26 nM, respectively. We also examined the structural basis and non-bonded profile of TACE interaction with a designed peptide hydroxamic acid inhibitor, and found that the inhibitor ligand is tightly buried in the active pocket of TACE, forming a number of hydrogen bonds, hydrophobic forces and van der Waals contacts at the interaction interface, conferring both stability and specificity for TACE-inhibitor complex

  5. Microwave-assisted reaction of peptide formation by amino acid with phosphate: Exploration of the most possible channels for the origin of life

    Institute of Scientific and Technical Information of China (English)

    HU; Rong; TIAN; Jinping

    2006-01-01

    Microwave-assisted reaction of peptide formation by amino acids with phosphate was studied. The results showed that the products were a mixture of peptides containing dipeptide, octapeptides and cyclic peptides, which could be obtained in a short time. Polyphosphate was also produced synchronously by the intermolecular condensation of phosphate. The polymerization degree reached 99% (pyrophosphate 64%, trimetaphosphate 35%) after 2 h at 200℃ under microwave irradiation. The intermediates of the mixed anhydrides formed by the intermolecular condensation of phosphates and glycin were determined by ESI-MS. Peptides were also produced by the reaction of amino acids with trimetaphosphate in aqueous solution. The conversion degree of valine reached 46.5% even at room temperature. The cyclic process of peptide formation and phosphate polymerization, regeneration and utilization in amino acids-phosphate system under microwave irradiation was detected and proved. Peptides could be continually formed only by inputting energy into this system. The above recycle may be the most possible process for primitive peptide formation in the origin of life.

  6. 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

    Cysteine (Cys) oxidation is a crucial post-translational modification (PTM) associated with redox signaling and oxidative stress. As Cys is highly reactive to oxidants it forms a range of post-translational modifications, some that are biologically reversible (e.g. disulfides, Cys sulfenic acid......) 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......-translational modification (pKa Cys-SO3H negative" selection) followed...

  7. Interaction of Peptide Transporter 1 With D-Glucose and L-Glutamic Acid; Possible Involvement of Taste Receptors.

    Science.gov (United States)

    Arakawa, Hiroshi; Ohmachi, Taichi; Ichiba, Kiko; Kamioka, Hiroki; Tomono, Takumi; Kanagawa, Masahiko; Idota, Yoko; Hatano, Yasuko; Yano, Kentaro; Morimoto, Kaori; Ogihara, Takuo

    2016-01-01

    We investigated the influence of sweet and umami (savory) tastants on the intestinal absorption of cephalexin (CEX), a substrate of peptide transporter 1 (PEPT1, SLC15A1) in rats. After oral administration of glucose or mannitol to rats, CEX was administered together with a second dose of glucose or mannitol. Western blot analysis indicated that expression of PEPT1 in rat jejunum membrane was decreased by glucose, compared to mannitol. Furthermore, the maximum plasma concentration (Cmax) of orally administered CEX was reduced by glucose compared to mannitol. The effect of glucose was diminished by nifedipine, a L-type Ca(2+) channel blocker. We also found that Cmax of orally administered CEX was reduced by treatment with L-glutamic acid, compared to D-glutamic acid. Thus, excessive intake of glucose and L-glutamic acid may impair oral absorption of PEPT1 substrates. PMID:26852864

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

    Science.gov (United States)

    Wang, Yongjun; Wang, Ling; Yang, Huali; Xiao, Haoliang; Farooq, Athar; Liu, Zhonghua; Hu, Min; Shi, Xiaoliu

    2016-01-01

    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. PMID:27128941

  9. 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.

  10. Self-assembled multicompartment liquid crystalline lipid carriers for protein, peptide, and nucleic acid drug delivery.

    Science.gov (United States)

    Angelova, Angelina; Angelov, Borislav; Mutafchieva, Rada; Lesieur, Sylviane; Couvreur, Patrick

    2011-02-15

    Lipids and lipopolymers self-assembled into biocompatible nano- and mesostructured functional materials offer many potential applications in medicine and diagnostics. In this Account, we demonstrate how high-resolution structural investigations of bicontinuous cubic templates made from lyotropic thermosensitive liquid-crystalline (LC) materials have initiated the development of innovative lipidopolymeric self-assembled nanocarriers. Such structures have tunable nanochannel sizes, morphologies, and hierarchical inner organizations and provide potential vehicles for the predictable loading and release of therapeutic proteins, peptides, or nucleic acids. This Account shows that structural studies of swelling of bicontinuous cubic lipid/water phases are essential for overcoming the nanoscale constraints for encapsulation of large therapeutic molecules in multicompartment lipid carriers. For the systems described here, we have employed time-resolved small-angle X-ray scattering (SAXS) and high-resolution freeze-fracture electronic microscopy (FF-EM) to study the morphology and the dynamic topological transitions of these nanostructured multicomponent amphiphilic assemblies. Quasi-elastic light scattering and circular dichroism spectroscopy can provide additional information at the nanoscale about the behavior of lipid/protein self-assemblies under conditions that approximate physiological hydration. We wanted to generalize these findings to control the stability and the hydration of the water nanochannels in liquid-crystalline lipid nanovehicles and confine therapeutic biomolecules within these structures. Therefore we analyzed the influence of amphiphilic and soluble additives (e.g. poly(ethylene glycol)monooleate (MO-PEG), octyl glucoside (OG), proteins) on the nanochannels' size in a diamond (D)-type bicontinuous cubic phase of the lipid glycerol monooleate (MO). At body temperature, we can stabilize long-living swollen states, corresponding to a diamond cubic phase

  11. 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

    Previous studies suggested that gastrin-releasing peptide (a neuropeptide found in rat oxyntic mucosa) and oxyntomodulin (a glucagon-containing peptide of mammalian gut) could directly affect the acid secretion of the parietal cells. We therefore studied their effect on gastric acid production in...... 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.......Previous studies suggested that gastrin-releasing peptide (a neuropeptide found in rat oxyntic mucosa) and oxyntomodulin (a glucagon-containing peptide of mammalian gut) could directly affect the acid secretion of the parietal cells. We therefore studied their effect on gastric acid production...... 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...

  12. 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.

  13. Biophysical and morphological studies on the dual interaction of non-octarepeat prion protein peptides with copper and nucleic acids.

    Science.gov (United States)

    Chaves, Juliana A P; Sanchez-López, Carolina; Gomes, Mariana P B; Sisnande, Tháyna; Macedo, Bruno; de Oliveira, Vanessa End; Braga, Carolina A C; Rangel, Luciana P; Silva, Jerson L; Quintanar, Liliana; Cordeiro, Yraima

    2014-08-01

    Conversion of prion protein (PrP) to an altered conformer, the scrapie PrP (PrP(Sc)), is a critical step in the development of transmissible spongiform encephalopathies. Both Cu(II) and nucleic acid molecules have been implicated in this conversion. Full-length PrP can bind up to six copper ions; four Cu(II) binding sites are located in the octarepeat domain (residues 60-91), and His-96 and His-111 coordinate two additional copper ions. Experimental evidence shows that PrP binds different molecules, resulting in diverse cellular signaling events. However, there is little information about the interaction of macromolecular ligands with Cu(II)-bound PrP. Both RNA and DNA sequences can bind PrP, and this interaction results in reciprocal conformational changes. Here, we investigated the interaction of Cu(II) and nucleic acids with amyloidogenic non-octarepeat PrP peptide models (comprising human PrP residues 106-126 and hamster PrP residues 109-149) that retain His-111 as the copper-anchoring residue. The effect of Cu(II) and DNA or RNA sequences in the aggregation, conformation, and toxicity of PrP domains was investigated at low and neutral pH. Circular dichroism and EPR spectroscopy data indicate that interaction of the PrP peptides with Cu(II) and DNA occurs at pH 7. This dual interaction induces conformational changes in the peptides, modulating their aggregation, and affecting the morphology of the aggregated species, resulting in different cytotoxic effects. These results provide new insights into the role of Cu(II) and nucleic acid sequences in the structural conversion and aggregation of PrP, which are both critical events related to prion pathogenesis.

  14. {{text{C}}_{α }} - {text{C}} Bond Cleavage of the Peptide Backbone in MALDI In-Source Decay Using Salicylic Acid Derivative Matrices

    Science.gov (United States)

    Asakawa, Daiki; Takayama, Mitsuo

    2011-07-01

    The use of 5-formylsalicylic acid (5-FSA) and 5-nitrosalicylic acid (5-NSA) as novel matrices for in-source decay (ISD) of peptides in matrix-assisted laser desorption/ionization (MALDI) is described. The use of 5-FSA and 5-NSA generated a- and x-series ions accompanied by oxidized peptides [M - 2 H + H]+. The preferential formation of a- and x-series ions was found to be dependent on the hydrogen-accepting ability of matrix. The hydrogen-accepting ability estimated from the ratio of signal intensity of oxidized product [M - 2 H + H]+ to that of non-oxidized protonated molecule [M + H]+ of peptide was of the order 5-NSA > 5-FSA > 5-aminosalicylic acid (5-ASA) ≒ 2,5-dihydroxyl benzoic acid (2,5-DHB) ≒ 0. The results suggest that the hydrogen transfer reaction from peptide to 5-FSA and 5-NSA occurs during the MALDI-ISD processes. The hydrogen abstraction from peptides results in the formation of oxidized peptides containing a radical site on the amide nitrogen with subsequent radical-induced cleavage at the {{{C}}_{α }} - {{C}} bond, leading to the formation of a- and x-series ions. The most significant feature of MALDI-ISD with 5-FSA and 5-NSA is the specific cleavage of the {{{C}}_{α }} - {{C}} bond of the peptide backbone without degradation of side-chain and post-translational modifications (PTM). The matrix provides a useful complementary method to conventional MALDI-ISD for amino acid sequencing and site localization of PTMs in peptides.

  15. Absolute rate constants for the reaction of hypochlorous acid with protein side chains and peptide bonds

    DEFF Research Database (Denmark)

    Pattison, D I; Davies, Michael Jonathan

    2001-01-01

    , absolute second-order rate constants for the reactions of HOCl with protein side chains, model compounds, and backbone amide (peptide) bonds have been determined at physiological pH values. The reactivity of HOCl with potential reactive sites in proteins is summarized by the series: Met (3.8 x 10(7) M(-1......) x s(-1)) > backbone amides (10-10(-3) M(-1) x s(-1)) > Gln(0.03 M(-1) x s(-1)) approximately Asn (0.03 M(-1) x s(-1)). The rate constants for reaction of HOCl with backbone amides (peptide bonds) vary by 4 orders of magnitude with uncharged peptide bonds reacting more readily with HOCl than those...

  16. T-scale as a novel vector of topological descriptors for amino acids and its application in QSARs of peptides

    Science.gov (United States)

    Tian, Feifei; Zhou, Peng; Li, Zhiliang

    2007-03-01

    In this paper, a new topological descriptor T-scale is derived from principal component analysis (PCA) on the collected 67 kinds of structural and topological variables of 135 amino acids. Applying T-scale to three peptide panels as 58 angiotensin-converting enzyme (ACE) inhibitors, 20 thromboplastin inhibitors (TI) and 28 bovine lactoferricin-(17-31)-pentadecapeptides (LFB), the resulting QSAR models, constructed by partial least squares (PLS), are all superior to reference reports, with correlative coefficient r2 and cross-validated q2 of 0.845, 0.786; 0.996, 0.782 (0.988, 0.961); 0.760, 0.627, respectively.

  17. Short Peptide Nucleic Acids Bind Strongly to Homopurine Tract of Double Helical RNA at pH 5.5

    OpenAIRE

    Li, Ming; Zengeya, Thomas; Rozners, Eriks

    2010-01-01

    The important role that non-coding RNA plays in cell biology makes it an attractive target for molecular recognition. However, the discovery of small molecules that bind double helical RNA selectively and may serve as biochemical probes and potential drug leads has been relatively slow. Herein, we show that peptide nucleic acids, as short as six nucleobases, bind very strongly (Ka > 107) and sequence selectively to a homopurine tract of double helical RNA at pH 5.5. The isothermal titration c...

  18. A Peptide Mimetic of 5-Acetylneuraminic Acid-Galactose Binds with High Avidity to Siglecs and NKG2D.

    Directory of Open Access Journals (Sweden)

    Laura L Eggink

    Full Text Available We previously identified several peptide sequences that mimicked the terminal sugars of complex glycans. Using plant lectins as analogs of lectin-type cell-surface receptors, a tetravalent form of a peptide with the sequence NPSHPLSG, designated svH1C, bound with high avidity to lectins specific for glycans with terminal 5-acetylneuraminic acid (Neu5Ac-galactose (Gal/N-acetylgalactosamine (GalNAc sequences. In this report, we show by circular dichroism and NMR spectra that svH1C lacks an ordered structure and thus interacts with binding sites from a flexible conformation. The peptide binds with high avidity to several recombinant human siglec receptors that bind preferentially to Neu5Ac(α2,3Gal, Neu5Ac(α2,6GalNAc or Neu5Ac(α2,8Neu5Ac ligands. In addition, the peptide bound the receptor NKG2D, which contains a lectin-like domain that binds Neu5Ac(α2,3Gal. The peptide bound to these receptors with a KD in the range of 0.6 to 1 μM. Binding to these receptors was inhibited by the glycoprotein fetuin, which contains multiple glycans that terminate in Neu5Ac(α2,3Gal or Neu5Ac(α2,6Gal, and by sialyllactose. Binding of svH1C was not detected with CLEC9a, CLEC10a or DC-SIGN, which are lectin-type receptors specific for other sugars. Incubation of neuraminidase-treated human peripheral blood mononuclear cells with svH1C resulted in binding of the peptide to a subset of the CD14+ monocyte population. Tyrosine phosphorylation of siglecs decreased dramatically when peripheral blood mononuclear cells were treated with 100 nM svH1C. Subcutaneous, alternate-day injections of svH1C into mice induced several-fold increases in populations of several types of immune cells in the peritoneal cavity. These results support the conclusion that svH1C mimics Neu5Ac-containing sequences and interacts with cell-surface receptors with avidities sufficient to induce biological responses at low concentrations. The attenuation of inhibitory receptors suggests that svH1C

  19. Structural location determines functional roles of the basic amino acids of KR-12, the smallest antimicrobial peptide from human cathelicidin LL-37.

    Science.gov (United States)

    Mishra, Biswajit; Epand, Raquel F; Epand, Richard M; Wang, Guangshun

    2013-11-14

    Cationic antimicrobial peptides are recognized templates for developing a new generation of antimicrobials to combat superbugs. Human cathelicidin LL-37 is an essential host defense molecule in human innate immunity. Previously, we identified KR-12 as the smallest antibacterial peptide of LL-37. KR-12 has a narrow activity spectrum since it is active against Gram-negative Escherichia coli but not Gram-positive Staphylococcus aureus. The functional roles of the basic amino acids of KR-12, however, have not yet been elucidated. An alanine scan of cationic amino acids of KR-12 provided evidence for their distinct roles in the activities of the peptides. Bacterial killing and membrane permeation experiments indicate that the R23A and K25A mutants, as well as the lysine-to-arginine mutant, were more potent than KR-12. Another three cationic residues (K18, R19, and R29) of KR-12, which are located in the hydrophilic face of the amphiphathic helix, appeared to be more important in clustering anionic lipids or hemolysis than R23 and K25 in the interfacial region. While the loss of interfacial R23 or K25 reduced peptide helicity, underscoring its important role in membrane binding, the overall increase in peptide activity of KR-12 could be ascribed to the increased peptide hydrophobicity that outweighed the role of basic charge in this case. In contrast, the mutations of interfacial R23 or K25 reduced peptide bactericidal activity of GF-17, an overlapping, more hydrophobic and potent peptide also derived from LL-37. Thus, the hydrophobic context of the peptide determines whether an alanine substitution of an interfacial basic residue increases or decreases membrane permeation and peptide activity.

  20. Proinsulin C-peptide antagonizes the profibrotic effects of TGF-beta1 via up-regulation of retinoic acid and HGF-related signaling pathways.

    Science.gov (United States)

    Hills, Claire E; Willars, Gary B; Brunskill, Nigel J

    2010-04-01

    Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy. PMID:20197308

  1. Stoichiometric inhibition of amyloid beta-protein aggregation with peptides containing alternating alpha,alpha-disubstituted amino acids.

    Science.gov (United States)

    Etienne, Marcus A; Aucoin, Jed P; Fu, Yanwen; McCarley, Robin L; Hammer, Robert P

    2006-03-22

    We have prepared two peptides based on the hydrophobic core (Lys-Leu-Val-Phe-Phe) of amyloid beta-protein (Abeta) that contain alpha,alpha-disubstituted amino acids at alternating positions, but differ in the positioning of the oligolysine chain (AMY-1, C-terminus; AMY-2, N-terminus). We have studied the effects of AMY-1 and AMY-2 on the aggregation of Abeta and find that, at stoichiometric concentrations, both peptides completely stop Abeta fibril growth. Equimolar mixtures of AMY-1 and Abeta form only globular aggregates as imaged by scanning force microscopy and transmission electron microscopy. These samples show no signs of protofibrillar or fibrillar material even after prolonged periods of time (4.5 months). Also, 10 mol % of AMY-1 prevents Abeta self-assembly for long periods of time; aged samples (4.5 months) show only a few protofibrillar or fibrillar aggregates. Circular dichroism spectroscopy of equimolar mixtures of AMY-1 and Abeta show that the secondary structure of the mixture changes over time and progresses to a predominantly beta-sheet structure, which is consistent with the design of these inhibitors preferring a sheet-like conformation. Changing the position of the charged tail on the peptide, AMY-2 interacts with Abeta differently in that equimolar mixtures form large ( approximately 1 mum) globular aggregates which do not progress to fibrils, but precipitate out of solution. The differences in the aggregation mediated by the two peptides is discussed in terms of a model where the inhibitors act as cosurfactants that interfere with the native ability of Abeta to self-assemble by disrupting hydrophobic interactions either at the C-terminus or N-terminus of Abeta. PMID:16536517

  2. Isolation and amino acid sequence of crustacean hyperglycemic hormone precursor-related peptides.

    Science.gov (United States)

    Tensen, C P; Verhoeven, A H; Gaus, G; Janssen, K P; Keller, R; Van Herp, F

    1991-01-01

    The crustacean hyperglycemic hormone (CHH) is synthesized as part of a larger preprohormone in which the sequence of CHH is N-terminally flanked by a peptide for which the name CPRP (CHH precursor-related peptide) is proposed. Both CHH and CPRP are present in the sinus gland, the neurohemal organ of neurosecretory cells located in the eyestalk of decapod crustaceans. This paper describes the isolation and sequence analysis of CPRPs isolated from sinus glands of the crab Carcinus maenas, the crayfish Orconectes limosus and the lobster Homarus americanus. The published sequence of "peptide H" isolated from the land crab, Cardisoma carnifex, has now been recognized as a CPRP in this species. Sequence comparison reveals a high level of identity for the N-terminal region (residues 1-13) between all four peptides, while identity in the C-terminal domain is high between lobster and crayfish CPRP on the one hand, and between both crab species on the other. Conserved N-terminal residues include a putative monobasic processing site at position 11, which suggests that CPRP may be a biosynthetic intermediate from which a potentially bioactive decapeptide can be derived.

  3. Triazole-linked glycosyl amino acids and peptides : synthesis, scope and applications

    NARCIS (Netherlands)

    Kuijpers, Brian Hubert Margaretha

    2008-01-01

    Naturally occurring glycosylated peptides play an important role in various biological processes and are therefore interesting lead molecules for the preparation of new therapeutic drugs.Synthesis of these natural glycopeptides is frequently hampered by the sensitivity of the natural glycosidic link

  4. Amino acid microsequencing of internal tryptic peptides of heme-regulated eukaryotic initiation factor 2 alpha subunit kinase: homology to protein kinases.

    Science.gov (United States)

    Chen, J J; Pal, J K; Petryshyn, R; Kuo, I; Yang, J M; Throop, M S; Gehrke, L; London, I M

    1991-01-01

    We have purified the heme-regulated eukaryotic initiation factor 2 alpha subunit (eIF-2 alpha) kinase (HRI) from rabbit reticulocytes for amino acid microsequencing. This kinase is a single 92-kDa polypeptide and migrates in perfect alignment with 32P-labeled HRI on SDS/PAGE. Its functions of binding ATP and of autophosphorylation and eIF-2 alpha phosphorylation are inhibited by hemin. The amino acid sequences of three tryptic peptides of HRI have been obtained. A search of the data base of the National Biomedical Research Foundation reveals that these amino acid sequences are unique and that two of these three sequences show homology to protein kinases. HRI peptide P-52 contains Asp-Phe-Gly, which is the most highly conserved short stretch of amino acids in catalytic domain VII of protein kinases. HRI peptide P-74 contains the conserved amino acid residues Asp-(Met)-Tyr-Ser-(Val)-Gly-Val found in catalytic domain IX of protein kinases [Hanks, S. K., Quinn, A. M. & Hunter, T. (1988) Science 241, 42-52]. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. Synthetic HRI peptide P-74 is a very potent inhibitor of eIF-2 alpha phosphorylation by HRI. Since little is known about the function of conserved domain IX, P-74 peptide may be useful in elucidating the role of this domain of protein kinases. Images PMID:1671169

  5. Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics.

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    Full Text Available BACKGROUND: Cathelicidins are a family of antimicrobial peptides acting as multifunctional effector molecules of innate immunity, which are firstly found in mammalians. Recently, several cathelicidins have also been found from chickens and fishes. No cathelicidins from other non-mammalian vertebrates have been reported. PRINCIPAL FINDINGS: In this work, a cathelicidin-like antimicrobial peptide named cathelicidin-BF has been purified from the snake venoms of Bungarus fasciatus and its cDNA sequence was cloned from the cDNA library, which confirm the presence of cathelicidin in reptiles. As other cathelicidins, the precursor of cathelicidin-BF has cathelin-like domain at the N terminus and carry the mature cathelicidin-BF at the C terminus, but it has an atypical acidic fragment insertion between the cathelin-like domain and the C-terminus. The acidic fragment is similar to acidic domains of amphibian antimicrobial precursors. Phylogenetic analysis revealed that the snake cathelicidin had the nearest evolution relationship with platypus cathelicidin. The secondary structure of cathelicidin-BF investigated by CD and NMR spectroscopy in the presence of the helicogenic solvent TFE is an amphipathic alpha-helical conformation as many other cathelicidins. The antimicrobial activities of cathelicidin BF against forty strains of microorganisms were tested. Cathelicidin-BF efficiently killed bacteria and some fungal species including clinically isolated drug-resistance microorganisms. It was especially active against Gram-negative bacteria. Furthermore, it could exert antimicrobial activity against some saprophytic fungus. No hemolytic and cytotoxic activity was observed at the dose of up to 400 microg/ml. Cathelicidin-BF could exist stably in the mice plasma for at least 2.5 hours. CONCLUSION: Discovery of snake cathelicidin with atypical structural and functional characterization offers new insights on the evolution of cathelicidins. Potent, broad

  6. A novel vector of topological and structural information for amino acids and its QSAR applications for peptides and analogues

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A new descriptor, called vector of topological and structural information for coded and noncoded amino acids (VTSA), was derived by principal component analysis (PCA) from a matrix of 66 topological and structural variables of 134 amino acids. The VTSA vector was then applied into two sets of peptide quantitative structure-activity relationships or quantitative sequence-activity modelings (QSARs/ QSAMs). Molded by genetic partial least squares (GPLS), support vector machine (SVM), and immune neural network (INN), good results were obtained. For the datasets of 58 angiotensin converting en-zyme inhibitors (ACEI) and 89 elastase substrate catalyzed kinetics (ESCK) , the R2, cross-validation R2, and root mean square error of estimation (RMSEE) were as follows: ACEI, R2cu≥0.82, Q2cu≥0.77, Ermse≤0.44 (GPLS+SVM); ESCK, R2cu≥0.84, Q2cu≥0.82, Ermse≤0.20 (GPLS+INN), respectively.

  7. A novel vector of topological and structural information for amino acids and its QSAR applications for peptides and analogues

    Institute of Scientific and Technical Information of China (English)

    LI ZhiLiang; CHEN GuoHua; LU FengLin; LU TingTing; LI GenRong; SHU Mao; SUN JiaYing; YANG ShanBin; MEI Hu; ZHANG MengJun; ZHOU Ping; WU ShiRong

    2008-01-01

    A new descriptor, called vector of topological and structural information for coded and noncoded amino acids (VTSA), was derived by principal component analysis (PCA) from a matrix of 66 topological and structural variables of 134 amino acids. The VTSA vector was then applied into two sets of peptide quantitative structure-activity relationships or quantitative sequence-activity modelings (QSARs/ QSAMs). Molded by genetic partial least squares (GPLS), support vector machine (SVM), and immune neural network (INN), good results were obtained. For the datasets of 58 angiotensin converting enzyme inhibitors (ACEI) and 89 elastase substrate catalyzed kinetics (ESCK), the R2, cross-validation R2, and root mean square error of estimation (RMSEE) were as follows: ACEI, R2cu≥0.82, Q2cu≥0.77, Ermse≤ 0.44 (GPLS+SVM); ESCK, R2cu≥0.84, Q2cu≥0.82, Ermse≤0.20 (GPLS+INN), respectively.

  8. The role of citric acid in oral peptide and protein formulations: relationship between calcium chelation and proteolysis inhibition.

    Science.gov (United States)

    Welling, Søren H; Hubálek, František; Jacobsen, Jette; Brayden, David J; Rahbek, Ulrik L; Buckley, Stephen T

    2014-04-01

    The excipient citric acid (CA) has been reported to improve oral absorption of peptides by different mechanisms. The balance between its related properties of calcium chelation and permeation enhancement compared to a proteolysis inhibition was examined. A predictive model of CA's calcium chelation activity was developed and verified experimentally using an ion-selective electrode. The effects of CA, its salt (citrate, Cit) and the established permeation enhancer, lauroyl carnitine chloride (LCC) were compared by measuring transepithelial electrical resistance (TEER) and permeability of insulin and FD4 across Caco-2 monolayers and rat small intestinal mucosae mounted in Ussing chambers. Proteolytic degradation of insulin was determined in rat luminal extracts across a range of pH values in the presence of CA. CA's capacity to chelate calcium decreased ~10-fold for each pH unit moving from pH 6 to pH 3. CA was an inferior weak permeation enhancer compared to LCC in both in vitro models using physiological buffers. At pH 4.5 however, degradation of insulin in rat luminal extracts was significantly inhibited in the presence of 10mM CA. The capacity of CA to chelate luminal calcium does 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 bioavailability since this is a weak property of CA.

  9. Anxiolytic-Like Effect of a Salmon Phospholipopeptidic Complex Composed of Polyunsaturated Fatty Acids and Bioactive Peptides

    Science.gov (United States)

    Belhaj, Nabila; Desor, Frédéric; Gleizes, Céline; Denis, Frédéric M.; Arab-Tehrany, Elmira; Soulimani, Rachid; Linder, Michel

    2013-01-01

    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. PMID:24177675

  10. N-Terminal Fatty Acid Substitution Increases the Leishmanicidal Activity of CA(1-7)M(2-9), a Cecropin-Melittin Hybrid Peptide

    Science.gov (United States)

    Chicharro, Cristina; Granata, Cesare; Lozano, Rosario; Andreu, David; Rivas, Luis

    2001-01-01

    In order to improve the leishmanicidal activity of the synthetic cecropin A-melittin hybrid peptide CA(1-7)M(2-9) (KWKLFKKIGAVLKVL-NH2), a systematic study of its acylation with saturated linear fatty acids was carried out. Acylation of the Nɛ-7 lysine residue led to a drastic decrease in leishmanicidal activity, whereas acylation at lysine 1, in either the α or the ɛ NH2 group, increased up to 3 times the activity of the peptide against promastigotes and increased up to 15 times the activity of the peptide against amastigotes. Leishmanicidal activity increased with the length of the fatty acid chain, reaching a maximum for the lauroyl analogue (12 carbons). According to the fast kinetics, dissipation of membrane potential, and parasite membrane permeability to the nucleic acid binding probe SYTOX green, the lethal mechanism was directly related to plasma membrane permeabilization. PMID:11502512

  11. Single amino acid mutation in alpha-helical peptide affect second harmonic generation hyperpolarizability

    Science.gov (United States)

    Wei, Jing; Wang, Jin-Yun; Zhang, Min-Yi; Chai, Guo-Liang; Lin, Chen-Sheng; Cheng, Wen-Dan

    2013-01-01

    We investigate the effect of side chain on the first-order hyperpolarizability in α-helical polyalanine peptide with the 10th alanine mutation (Acetyl(ala)9X(ala)7NH2). Structures of various substituted peptides are optimized by ONIOM (DFT: AM1) scheme, and then linear and nonlinear optical properties are calculated by SOS//CIS/6-31G∗ method. The polarizability and first-order hyperpolarizability increase obviously only when 'X' represents phenylalanine, tyrosine and tryptophan. We also discuss the origin of nonlinear optical response and determine what caused the increase of first-order hyperpolarizability. Our results strongly suggest that side chains containing benzene, phenol and indole have important contributions to first-order hyperpolarizability.

  12. Hydrophobic amino acids in the hinge region of the 5A apolipoprotein mimetic peptide are essential for promoting cholesterol efflux by the ABCA1 transporter.

    Science.gov (United States)

    Sviridov, Denis O; Andrianov, Alexander M; Anishchenko, Ivan V; Stonik, John A; Amar, Marcelo J A; Turner, Scott; Remaley, Alan T

    2013-01-01

    The bihelical apolipoprotein mimetic peptide 5A effluxes cholesterol from cells and reduces inflammation and atherosclerosis in animal models. We investigated how hydrophobic residues in the hinge region between the two helices are important in the structure and function of this peptide. By simulated annealing analysis and molecular dynamics modeling, two hydrophobic amino acids, F-18 and W-21, in the hinge region were predicted to be relatively surface-exposed and to interact with the aqueous solvent. Using a series of 5A peptide analogs in which F-18 or W-21 was changed to either F, W, A, or E, only peptides with hydrophobic amino acids in these two positions were able to readily bind and solubilize phospholipid vesicles. Compared with active peptides containing F or W, peptides containing E in either of these two positions were more than 10-fold less effective in effluxing cholesterol by the ABCA1 transporter. Intravenous injection of 5A in C57BL/6 mice increased plasma-free cholesterol (5A: 89.9 ± 13.6 mg/dl; control: 38.7 ± 4.3 mg/dl (mean ± S.D.); P < 0.05) and triglycerides (5A: 887.0 ± 172.0 mg/dl; control: 108.9 ± 9.9 mg/dl; P < 0.05), whereas the EE peptide containing E in both positions had no effect. Finally, 5A increased cholesterol efflux approximately 2.5-fold in vivo from radiolabeled macrophages, whereas the EE peptide was inactive. These results provide a rationale for future design of therapeutic apolipoprotein mimetic peptides and provide new insights into the interaction of hydrophobic residues on apolipoproteins with phospholipids in the lipid microdomain created by the ABCA1 transporter during the cholesterol efflux process.

  13. 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.

  14. Production of carrier-peptide conjugates using chemically reactive unnatural amino acids

    Energy Technology Data Exchange (ETDEWEB)

    Young, Travis; Schultz, Peter G.

    2015-08-18

    Provided are methods of making carrier polypeptide that include incorporating a first unnatural amino acid into a carrier polypeptide variant, incorporating a second unnatural amino acid into a target polypeptide variant, and reacting the first and second unnatural amino acids to produce the conjugate. Conjugates produced using the provided methods are also provided. In addition, orthogonal translation systems in methylotrophic yeast and methods of using these systems to produce carrier and target polypeptide variants comprising unnatural amino acids are provided.

  15. 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.

    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

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

    Directory of Open Access Journals (Sweden)

    Gülin Güler-Gane

    Full Text Available 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

  17. 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.

  18. Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Zachar, Vladimir; Nielsen, P.E.;

    1997-01-01

    We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs) was investi......We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs...

  19. Fatty acid and peptide profiles in plasma membrane and membrane rafts of PUFA supplemented RAW264.7 macrophages.

    Directory of Open Access Journals (Sweden)

    Julia Schumann

    Full Text Available The eukaryotic cell membrane possesses numerous complex functions, which are essential for life. At this, the composition and the structure of the lipid bilayer are of particular importance. Polyunsaturated fatty acids may modulate the physical properties of biological membranes via alteration of membrane lipid composition affecting numerous physiological processes, e.g. in the immune system. In this systematic study we present fatty acid and peptide profiles of cell membrane and membrane rafts of murine macrophages that have been supplemented with saturated fatty acids as well as PUFAs from the n-3, the n-6 and the n-9 family. Using fatty acid composition analysis and mass spectrometry-based peptidome profiling we found that PUFAs from both the n-3 and the n-6 family have an impact on lipid and protein composition of plasma membrane and membrane rafts in a similar manner. In addition, we found a relation between the number of bis-allyl-methylene positions of the PUFA added and the unsaturation index of plasma membrane as well as membrane rafts of supplemented cells. With regard to the proposed significance of lipid microdomains for disease development and treatment our study will help to achieve a targeted dietary modulation of immune cell lipid bilayers.

  20. Protective Effect of Cod (Gadus macrocephalus) Skin Collagen Peptides on Acetic Acid-Induced Gastric Ulcer in Rats.

    Science.gov (United States)

    Niu, Huina; Wang, Zhicong; Hou, Hu; Zhang, Zhaohui; Li, Bafang

    2016-07-01

    This research was performed to explore the protective effect of cod skin collagen peptides (CCP) on gastric ulcer induced by acetic acid. The CCP were fractionated into low molecular CCP (LMCCP, Mw 3 kDa). In HMCCP and LMCCP, glycine of accounted for about one-third of the total amino acids without cysteine and tryptophan, and hydrophobic amino acids accounted for about 50%. After 21 d CCP treatment (60 or 300 mg/kg, p.o./daily), the healing effects on acetic acid-induced gastric ulcers were evaluated by macroscopic measure, microscopic measure, and immune histochemistry. Moreover, the expression levels of the growth factors, such as vascular endothelial growth factor, epidermal growth factor, transforming growth factor β1 (TGFβ1), and the heat shock protein 70 (HSP70) was detected. The results showed that both LMCCP and HMCCP could significantly decrease the ulcer areas and promote the healing of the lesions. They also could improve the levels of hexosamine, glutathione, superoxide dismutase, and glutathione peroxidase, and reduce the content of malondialdehyde and inducible nitric oxide synthase. In addition, the expression level of TGFβ1 gene and HSP70 mRNA was significantly improved by the treatment. It suggested that CCP could be able to improve symptoms of gastric ulcer and probably be used in the treatment of gastric ulcer. PMID:27219644

  1. Antimicrobial Peptide from the Wild Bee Hylaeus signatus Venom and Its Analogues: Structure-Activity Study and Synergistic Effect with Antibiotics.

    Science.gov (United States)

    Nešuta, Ondřej; Hexnerová, Rozálie; Buděšínský, Miloš; Slaninová, Jiřina; Bednárová, Lucie; Hadravová, Romana; Straka, Jakub; Veverka, Václav; Čeřovský, Václav

    2016-04-22

    Venoms of hymenopteran insects have attracted considerable interest as a source of cationic antimicrobial peptides (AMPs). In the venom of the solitary bee Hylaeus signatus (Hymenoptera: Colletidae), we identified a new hexadecapeptide of sequence Gly-Ile-Met-Ser-Ser-Leu-Met-Lys-Lys-Leu-Ala-Ala-His-Ile-Ala-Lys-NH2. Named HYL, it belongs to the category of α-helical amphipathic AMPs. HYL exhibited weak antimicrobial activity against several strains of pathogenic bacteria and moderate activity against Candida albicans, but its hemolytic activity against human red blood cells was low. We prepared a set of HYL analogues to evaluate the effects of structural modifications on its biological activity and to increase its potency against pathogenic bacteria. This produced several analogues exhibiting significantly greater activity compared to HYL against strains of both Staphylococcus aureus and Pseudomonas aeruginosa even as their hemolytic activity remained low. Studying synergism of HYL peptides and conventional antibiotics showed the peptides act synergistically and preferentially in combination with rifampicin. Fluorescent dye propidium iodide uptake showed the tested peptides were able to facilitate entrance of antibiotics into the cytoplasm by permeabilization of the outer and inner bacterial cell membrane of P. aeruginosa. Transmission electron microscopy revealed that treatment of P. aeruginosa with one of the HYL analogues caused total disintegration of bacterial cells. NMR spectroscopy was used to elucidate the structure-activity relationship for the effect of amino acid residue substitution in HYL. PMID:26998557

  2. pACC1 peptide loaded chitosan nanoparticles induces apoptosis via reduced fatty acid synthesis in MDA-MB-231 cells

    Science.gov (United States)

    Kaliaperumal, Jagatheesh; Hari, Natarajan; Pavankumar, Padarthi; Elangovan, Namasivayam

    2016-06-01

    The development of formulations with therapeutic peptides has been restricted to poor cell penetration and in this attempt; we developed pACC1 peptide loaded chitosan nanoparticles. The prepared nanoparticles were characterized with FT-IR, XRD, SEM and TEM. In addition, the suitable formulation was evaluated for hemocompatibility, plasma stability and embryo toxicity using Danio rerio embryo model. The results showed that pACC1 peptide loaded chitosan nanoparticles were compatible with plasma. They possess sustained release pattern and also found to be safe up to 300 mg/L in embryo toxicity tests. Cytotoxicity assays with MDA-MB-231 cell lines suggested that, pACC1 peptide loaded chitosan nanoparticles were capable of enhanced cellular penetration and reduced palmitic acid content, which was confirmed by H1 NMR. Hence, these nanoparticles could be employed as excellent adjuvant therapeutics while treating solid tumors with multi-drug resistance.

  3. Induction of severe experimental autoimmune neuritis with a synthetic peptide corresponding to the 53-78 amino acid sequence of the myelin P2 protein.

    Science.gov (United States)

    Rostami, A; Gregorian, S K; Brown, M J; Pleasure, D E

    1990-12-01

    We generated a synthetic peptide (SP-26), corresponding to the amino acid residues 53-78 of bovine P2 protein, which induced severe clinical and pathological characteristics of experimental autoimmune neuritis (EAN) in Lewis rats. Lymph node cell populations from SP-26-immunized rats elicited a proliferative response to the peptide and to the P2 protein. After 16 cycles of antigen stimulation with the peptide, the SP-26 T cell line shows a decreased response to P2, but not to SP-26. Fluorescence-activated cell sorter (FACS) analysis of a SP-26 T cell line indicated the majority of cells to be of CD4+ CD8-. This report demonstrates that the synthetic peptide SP-26 can induce severe EAN in Lewis rats in a dose-dependent manner. Furthermore, specific T cell lines reactive to SP-26 can be generated from the lymph nodes of SP-26-immunized rats. PMID:1699975

  4. Role of enthalpy-entropy compensation interactions in determining the conformational propensities of amino acid residues in unfolded peptides.

    Science.gov (United States)

    Toal, Siobhan E; Verbaro, Daniel J; Schweitzer-Stenner, Reinhard

    2014-02-01

    The driving forces governing the unique and restricted conformational preferences of amino acid residues in the unfolded state are still not well understood. In this study, we experimentally determine the individual thermodynamic components underlying intrinsic conformational propensities of these residues. Thermodynamic analysis of ultraviolet-circular dichroism (UV-CD) and (1)H NMR data for a series of glycine capped amino acid residues (i.e., G-x-G peptides) reveals the existence of a nearly exact enthalpy-entropy compensation for the polyproline II-β strand equilibrium for all investigated residues. The respective ΔHβ, ΔSβ values exhibit a nearly perfect linear relationship with an apparent compensation temperature of 295 ± 2 K. Moreover, we identified iso-equilibrium points for two subsets of residues at 297 and 305 K. Thus, our data suggest that within this temperature regime, which is only slightly below physiological temperatures, the conformational ensembles of amino acid residues in the unfolded state differ solely with respect to their capability to adopt turn-like conformations. Such iso-equilibria are rarely observed, and their existence herein indicates a common physical origin behind conformational preferences, which we are able to assign to side-chain dependent backbone solvation. Conformational effects such as differences between the number of sterically allowed side chain rotamers can contribute to enthalpy and entropy but not to the Gibbs energy associated with conformational preferences. Interestingly, we found that alanine, aspartic acid, and threonine are the only residues which do not share these iso-equilbiria. The enthalpy-entropy compensation discovered as well as the iso-equilbrium and thermodynamics obtained for each amino acid residue provide a new and informative way of identifying the determinants of amino acid propensities in unfolded and disordered states.

  5. Studies on N-terminal glycation of peptides in hypoallergenic infant formulas: quantification of alpha-N-(2-furoylmethyl) amino acids.

    Science.gov (United States)

    Penndorf, Ilka; Biedermann, Daniela; Maurer, Sarah V; Henle, Thomas

    2007-02-01

    To obtain information about the extent of the early Maillard reaction between the N-termini of peptides and lactose, alpha-N-(2-furoylmethyl) amino acids (FMAAs) were quantified together with epsilon-N-(2-furoylmethyl)lysine (furosine) in acid hydrolyzates of hypoallergenic infant formulas, conventional infant formulas, and human milk samples using RP-HPLC with UV-detection. FMAAs are formed during acid hydrolysis of peptide-bound N-terminal Amadori products (APs), and furosine is formed from the Amadori products of peptide-bound lysine. Unambiguous identification was achieved by means of LC/MS and UV-spectroscopy using independently prepared reference material. The extent of acid-induced conversion of APs to FMAAs was studied by RP-HPLC with chemiluminescent nitrogen detection (CLND). Depending on the corresponding alpha-N-lactulosyl amino acid, between 6.0% and 18.1% of FMAAs were formed during hydrolysis for 23 h at 110 degrees C in 8 N HCl. From epsilon-N-lactulosyllysine, 50% furosine is formed under these conditions. Whereas furosine was detectable in all assayed samples, five different FMAAs, alpha-FM-Lys, alpha-FM-Ala, alpha-FM-Val, alpha-FM-Ile, and alpha-FM-Leu, were exclusively detected in acid hydrolyzates of hypoallergenic infant formulas in amounts ranging from 35 to 396 mumol/100 g protein. Taking the conversion factors into account, modification of N-terminal amino acids in peptides by reducing carbohydrates was between 0.3% and 8.4%. This has to be considered within the discussion concerning the nutritional quality of peptide-containing foods.

  6. Separation of small molecular peptides with the same amino acid composition but different sequences by high performance liquid chromatography-electrospray ionization-mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Peptidomics has emerged as a new discipline in recent years. Mass spectrometry (MS) is the most universal and efficient tool for structure identification of proteins and peptides. However,there is a limitation for the identification of peptides with the same amino acid composition but different se-quences because these peptides have identical mass spectra of molecular ions. This paper presents a high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method for the separation of small molecular peptides with the same amino acid composition but dif-ferent sequences. Two tripeptides of Gly-Ser-Phe and Gly-Phe-Ser were used as a model sample. The separation behavior has been investigated and the separation conditions have been optimized. Under the optimum conditions,good repeatability was achieved. The developed method could provide a helpful reference for the separation of other peptides with the same amino acid composition but different sequences in the study of proteomics and peptidomics.

  7. Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates.

    Science.gov (United States)

    Elshan, N G R Dayan; Patek, Renata; Vagner, Josef; Mash, Eugene A

    2014-11-01

    Europium chelates conjugated with peptide ligands are routinely used as probes for conducting in vitro binding experiments. The presence of unchelated Eu ions in these formulations gives high background luminescence and can lead to poor results in binding assays. In our experience, the reported methods for purification of these probes do not achieve adequate removal of unchelated metal ions in a reliable manner. In this work, a xylenol orange-based assay for the quantification of unchelated metal ions was streamlined and used to determine levels of metal ion contamination as well as the success of metal ion removal on attempted purification. We compared the use of Empore chelating disks and Chelex 100 resin for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Both purification methods gave complete and selective removal of the contaminant metal ions. However, Empore chelating disks were found to give much higher recoveries of the probes under the conditions used. Related to the issue of probe recovery, we also describe a significantly more efficient method for the synthesis of one such probe using Rink amide AM resin in place of Tentagel S resin.

  8. Amino Acid-Dependent Attenuation of Toll-like Receptor Signaling by Peptide-Gold Nanoparticle Hybrids.

    Science.gov (United States)

    Yang, Hong; Fung, Shan-Yu; Xu, Shuyun; Sutherland, Darren P; Kollmann, Tobias R; Liu, Mingyao; Turvey, Stuart E

    2015-07-28

    Manipulation of immune responsiveness using nanodevices provides a potential approach to treat human diseases. Toll-like receptor (TLR) signaling plays a central role in the pathophysiology of many acute and chronic human inflammatory diseases, and pharmacological regulation of TLR responses is anticipated to be beneficial in many of these inflammatory conditions. Here we describe the discovery of a unique class of peptide-gold nanoparticle hybrids that exhibit a broad inhibitory activity on TLR signaling, inhibiting signaling through TLRs 2, 3, 4, and 5. As exemplified using TLR4, the nanoparticles were found to inhibit both arms of TLR4 signaling cascade triggered by the prototypical ligand, lipopolysaccharide (LPS). Through structure-activity relationship studies, we identified the key chemical components of the hybrids that contribute to their immunomodulatory activity. Specifically, the hydrophobicity and aromatic ring structure of the amino acids on the peptides were essential for modulating TLR4 responses. This work enhances our fundamental understanding of the role of nanoparticle surface chemistry in regulating innate immune signaling, and identifies specific nanoparticle hybrids that may represent a unique class of anti-inflammatory therapeutics for human inflammatory diseases.

  9. Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1.

    Science.gov (United States)

    Cao, Zhen; Wu, Xue-Fei; Peng, Yan; Zhang, Rui; Li, Na; Yang, Jin-Yi; Zhang, Shu-Qin; Zhang, Wan-Qin; Zhao, Jie; Li, Shao

    2015-11-01

    Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine.

  10. Design of an α-helical antimicrobial peptide with improved cell-selective and potent anti-biofilm activity.

    Science.gov (United States)

    Zhang, Shi-Kun; Song, Jin-Wen; Gong, Feng; Li, Su-Bo; Chang, Hong-Yu; Xie, Hui-Min; Gao, Hong-Wei; Tan, Ying-Xia; Ji, Shou-Ping

    2016-01-01

    AR-23 is a melittin-related peptide with 23 residues. Like melittin, its high α-helical amphipathic structure results in strong bactericidal activity and cytotoxicity. In this study, a series of AR-23 analogues with low amphipathicity were designed by substitution of Ala1, Ala8 and Ile17 with positively charged residues (Arg or Lys) to study the effect of positively charged residue distribution on the biological viability of the antimicrobial peptide. Substitution of Ile17 on the nonpolar face with positively charged Lys dramatically altered the hydrophobicity, amphipathicity, helicity and the membrane-penetrating activity against human cells as well as the haemolytic activity of the peptide. However, substitution on the polar face only slightly affected the peptide biophysical properties and biological activity. The results indicate that the position rather than the number of positively charged residue affects the biophysical properties and selectivity of the peptide. Of all the analogues, A(A1R, A8R, I17K), a peptide with Ala1-Arg, Ala8-Arg and Ile17-Lys substitutions, exhibited similar bactericidal activity and anti-biofilm activity to AR-23 but had much lower haemolytic activity and cytotoxicity against mammalian cells compared with AR-23. Therefore, the findings reported here provide a rationalization for peptide design and optimization, which will be useful for the future development of antimicrobial agents. PMID:27271216

  11. Modulation of Backbone Flexibility for Effective Dissociation of Antibacterial and Hemolytic Activity in Cyclic Peptides.

    Science.gov (United States)

    Oddo, Alberto; Thomsen, Thomas T; Britt, Hannah M; Løbner-Olesen, Anders; Thulstrup, Peter W; Sanderson, John M; Hansen, Paul R

    2016-08-11

    Bacterial resistance to antibiotic therapy is on the rise and threatens to evolve into a worldwide emergency: alternative solutions to current therapies are urgently needed. Cationic amphipathic peptides are potent membrane-active agents that hold promise as the next-generation therapy for multidrug-resistant infections. The peptides' behavior upon encountering the bacterial cell wall is crucial, and much effort has been dedicated to the investigation and optimization of this amphipathicity-driven interaction. In this study we examined the interaction of a novel series of nine-membered flexible cyclic AMPs with liposomes mimicking the characteristics of bacterial membranes. Employed techniques included circular dichroism and marker release assays, as well as microbiological experiments. Our analysis was aimed at correlating ring flexibility with their antimicrobial, hemolytic, and membrane activity. By doing so, we obtained useful insights to guide the optimization of cyclic antimicrobial peptides via modulation of their backbone flexibility without loss of activity. PMID:27563396

  12. High efficacy of a 20 amino acid peptide of the acidic ribosomal protein P0 against the cattle tick, Rhipicephalus microplus.

    Science.gov (United States)

    Rodríguez-Mallon, Alina; Encinosa, Pedro E; Méndez-Pérez, Lídice; Bello, Yamil; Rodríguez Fernández, Rafmary; Garay, Hilda; Cabrales, Ania; Méndez, Luis; Borroto, Carlos; Estrada, Mario Pablo

    2015-06-01

    Current strategies to control cattle ticks use integrated control programs (ICP) that include vaccination. Reduction in the use of chemicals and in the cost of tick control, the delay or elimination of acaricide resistance and the decreasing of environmental pollution are the advantages of using these programs. This integrated program is potentially applicable to all genotypes of chemical resistant ticks. However, the problem here is to improve the efficacy of anti-tick vaccines. The P0 protein is a structural component of the ribosome of all organisms. We have identified an immunogenic region of ribosomal protein P0 from Rhipicephalus spp. ticks that is not very conserved compared to the orthologous protein in their hosts. A synthetic 20 amino acid peptide from this sequence was effective as a vaccine against Rhipicephalus sanguineus infestations in an immunization and challenge experiment using rabbits. In this paper, the same peptide used as vaccine against the cattle tick Rhipicephalus Boophilus microplus shows a significant diminution in the number of engorged females recovered, in the weight of females and the weight of egg masses. The number of eggs hatched was also significantly reduced for the vaccinated group, with an overall effectivity for the antigen pP0 of 96%. These results, together with the conserved sequence of the P0 peptide among ticks, suggest that this antigen could be a good broad spectrum vaccine candidate. It would be expected to be active against many species of ticks and thus has promise in an ICP for effective control of ticks and thereby to improve the efficiency and productivity of the livestock industry. PMID:25958782

  13. Design of antimicrobial peptides conjugated biodegradable citric acid derived hydrogels for wound healing.

    Science.gov (United States)

    Xie, Zhiwei; Aphale, Nikhil V; Kadapure, Tejaswi D; Wadajkar, Aniket S; Orr, Sara; Gyawali, Dipendra; Qian, Guoying; Nguyen, Kytai T; Yang, Jian

    2015-12-01

    Wound healing is usually facilitated by the use of a wound dressing that can be easily applied to cover the wound bed, maintain moisture, and avoid bacterial infection. In order to meet all of these requirements, we developed an in situ forming biodegradable hydrogel (iFBH) system composed of a newly developed combination of biodegradable poly(ethylene glycol) maleate citrate (PEGMC) and poly(ethylene glycol) diacrylate (PEGDA). The in situ forming hydrogel systems are able to conform to the wound shape in order to cover the wound completely and prevent bacterial invasion. A 2(k) factorial analysis was performed to examine the effects of polymer composition on specific properties, including the curing time, Young's modulus, swelling ratio, and degradation rate. An optimized iFBH formulation was achieved from the systematic factorial analysis. Further, in vitro biocompatibility studies using adult human dermal fibroblasts (HDFs) confirmed that the hydrogels and degradation products are not cytotoxic. The iFBH wound dressing was conjugated and functionalized with antimicrobial peptides as well. Evaluation against bacteria both in vitro and in vivo in rats demonstrated that the peptide-incorporated iFBH wound dressing offered excellent bacteria inhibition and promoted wound healing. These studies indicated that our in situ forming antimicrobial biodegradable hydrogel system is a promising candidate for wound treatment.

  14. Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging

    Science.gov (United States)

    Pipkorn, Rüdiger; Wiessler, Manfred; Waldeck, Waldemar; Hennrich, Ute; Nokihara, Kiyoshi; Beining, Marcel; Braun, Klaus

    2012-01-01

    Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB) gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized. PMID:22211082

  15. Improved Synthesis Strategy for Peptide Nucleic Acids (PNA appropriate for Cell-specific Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Rüdiger Pipkorn, Manfred Wiessler, Waldemar Waldeck, Ute Hennrich, Kiyoshi Nokihara, Marcel Beining, Klaus Braun

    2012-01-01

    Full Text Available Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized.

  16. Adsorption of biomedical coating molecules, amino acids, and short peptides on magnetite (110)

    Science.gov (United States)

    Aschauer, Ulrich; Selloni, Annabella

    2015-07-01

    Superparamagnetic iron oxide nanoparticles for biomedical applications are usually coated with organic molecules to form a steric barrier against agglomeration. The stability of these coatings is well established in the synthesis medium but is more difficult to assess in physiological environment. To obtain a first theoretical estimate of their stability in such an environment, we perform density functional theory calculations of the adsorption of water, polyvinyl alcohol (PVA) and polyethylene glycol (PEG) coating molecules, as well as the monomer and dimer of glycine as a prototype short peptide, on the (110) surface of magnetite (Fe3O4) in vacuo. Our results show that PVA binds significantly stronger to the surface than both PEG and glycine, while the difference between the latter two is quite small. Depending on the coverage, the water adsorption strength is intermediate between PVA and glycine. Due to its strongly interacting OH side groups, PVA is likely to remain bound to the surface in the presence of short peptides. This stability will have to be further assessed by molecular dynamics in the solvated state for which the present work forms the basis.

  17. 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 (R

  18. Hyaluronic acid hydrogels with IKVAV peptides for tissue repair and axonal regeneration in an injured rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Y T [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing, 100084 (China); Tian, W M [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing, 100084 (China); Yu, X [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing, 100084 (China); Cui, F Z [Biomaterials Laboratory, Department of Materials Science and Engineering, Tsinghua University, Beijing, 100084 (China); Hou, S P [Beijing Institute of Neuroscience, Capital University of Medical Sciences, Beijing, 100054 (China); Xu, Q Y [Beijing Institute of Neuroscience, Capital University of Medical Sciences, Beijing, 100054 (China); Lee, In-Seop [Institute of Physics and Applied Physics, and Atomic-scale Surface Science Research Center, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2007-09-15

    A biocompatible hydrogel of hyaluronic acid with the neurite-promoting peptide sequence of IKVAV was synthesized. The characterization of the hydrogel shows an open porous structure and a large surface area available for cell interaction. Its ability to promote tissue repair and axonal regeneration in the lesioned rat cerebrum is also evaluated. After implantation, the polymer hydrogel repaired the tissue defect and formed a permissive interface with the host tissue. Axonal growth occurred within the microstructure of the network. Within 6 weeks the polymer implant was invaded by host-derived tissue, glial cells, blood vessels and axons. Such a hydrogel matrix showed the properties of neuron conduction. It has the potential to repair tissue defects in the central nervous system by promoting the formation of a tissue matrix and axonal growth by replacing the lost tissue.

  19. Production of Bioactive Peptides from Soybean Meal by Solid State Fermentation with Lactic Acid Bacteria and Protease

    Directory of Open Access Journals (Sweden)

    Naifu Wang

    2014-10-01

    Full Text Available In this study, soybean meal was first solid state fermented with different strains of Lactic Acid Bacteria (LAB. Among the strains used, Lactobacillus plantarum Lp6 was selected for further studies because of its highest Degree of Hydrolysis (DH of protein (2.49±0.08% in soybean meal after 72 h fermentation. Soybean meal fermented with L. plantarum Lp6 can also improve its DPPH radical scavenging and Angiotensin Converting Enzyme (ACE inhibitory activities. The addition of protease into soybean meal during the fermentation resulted in lowered IC50 of DPPH radical scavenging and ACE inhibitory activities, indicating more bioactive peptides were produced during fermentation. Molecular weight distribution analysis revealed the Extracts from Fermented Soybean Meal (EFSM was mainly composed of oligopeptides. These results indicated that soybean meal fermented with L. plantarum Lp6 and protease could be an easy and cheap method to produce functional food.

  20. 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...... that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric...... hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta...

  1. Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage.

    Science.gov (United States)

    Sugiyama, Toru; Kuwata, Keiko; Imamura, Yasutada; Demizu, Yosuke; Kurihara, Masaaki; Takano, Masashi; Kittaka, Atsushi

    2016-01-01

    This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules. PMID:27373637

  2. Influence of Amino Acid Compositions and Peptide Profiles on Antioxidant Capacities of Two Protein Hydrolysates from Skipjack Tuna (Katsuwonus pelamis Dark Muscle

    Directory of Open Access Journals (Sweden)

    Chang-Feng Chi

    2015-04-01

    Full Text Available Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis dark muscle was investigated. Dark muscles from skipjack tuna were hydrolyzed using five separate proteases, including pepsin, trypsin, Neutrase, papain and Alcalase. Two hydrolysates, ATH and NTH, prepared using Alcalase and Neutrase, respectively, showed the strongest antioxidant capacities and were further fractionated using ultrafiltration and gel filtration chromatography. Two fractions, Fr.A3 and Fr.B2, isolated from ATH and NTH, respectively, showed strong radical scavenging activities toward 2,2-diphenyl-1-picrylhydrazyl radicals (EC50 1.08% ± 0.08% and 0.98% ± 0.07%, hydroxyl radicals (EC50 0.22% ± 0.03% and 0.48% ± 0.05%, and superoxide anion radicals (EC50 1.31% ± 0.11% and 1.56% ± 1.03% and effectively inhibited lipid peroxidation. Eighteen peptides from Fr.A3 and 13 peptides from Fr.B2 were isolated by reversed-phase high performance liquid chromatography, and their amino acid sequences were determined. The elevated antioxidant activity of Fr.A3 might be due to its high content of hydrophobic and aromatic amino acid residues (181.1 and 469.9 residues/1000 residues, respectively, small molecular sizes (3–6 peptides, low molecular weights (524.78 kDa, and amino acid sequences (antioxidant score 6.11. This study confirmed that a smaller molecular size, the presence of hydrophobic and aromatic amino acid residues, and the amino acid sequences were the key factors that determined the antioxidant activities of the proteins, hydrolysates and peptides. The results also demonstrated that the derived hydrolysates and fractions from skipjack tuna (K. pelamis dark muscles could prevent oxidative reactions and might be useful for food preservation and medicinal purposes.

  3. Exploring the activity space of peptides binding to diverse SH3 domains using principal property descriptors derived from amino acid rotamers.

    Science.gov (United States)

    He, Ping; Wu, Wei; Yang, Kang; Jing, Tao; Liao, Ke-Long; Zhang, Wei; Wang, Hai-Dong; Hua, Xing

    2011-01-01

    Although there were intensive works addressed on multivariate extraction of the informative components from numerous physicochemical parameters of amino acids in isolated state, the various conformational behaviors of amino acids in complicated biological context have long been underappreciated in the field of quantitative structure-activity relationship (QSAR). In this work, the amino acid rotamers, which were derived from statistical survey of protein crystal structures, were used to reproduce the conformational variety of amino acid side-chains in real condition. In this procedure, these rotamers were superposed into a nx x ny x nz lattice and an artificial probe was employed to detect four kinds of nonbonding field potentials (i.e., electrostatic, steric, hydrophobic, and hydrogen bonds) at each lattice point using a Gaussian-type potential function; the generated massive data were then subjected to a principal component analysis (PCA) treatment to obtain a set of few, informative amino acid descriptors. We used this set of descriptors, that we named principal property descriptors derived from amino acid rotamers (PDAR), to characterize over 13,000 peptides with known binding affinities to 10 types of SH3 domains. Genetic algorithm/ partial least square regression (GA/PLS) modeling and Monte Carlo cross-validation (MCCV) demonstrated that the correlation between the PDAR descriptors and the binding affinities of peptides are comparable with or even better than previously published models. Furthermore, from the PDAR-based QSAR models we concluded that the core motif of peptides, particularly the electrostatic property, hydrophobicity, and hydrogen bond at residue positions P3, P2, and/or P0, contribute significantly to the hAmph SH3 domain-peptide binding, whereas two ends of the peptides, such as P6, P4, P-4, and P5, only play a secondary role in the binding.

  4. Synthesis of Peptides from α- and β-Tubulin Containing Glutamic Acid Side-Chain Linked Oligo-Glu with Defined Length

    Directory of Open Access Journals (Sweden)

    Werner Tegge

    2010-01-01

    Full Text Available Side-chain oligo- and polyglutamylation represents an important posttranslational modification in tubulin physiology. The particular number of glutamate units is related to specific regulatory functions. In this work, we present a method for the synthesis of building blocks for the Fmoc synthesis of peptides containing main chain glutamic acid residues that carry side-chain branching with oligo-glutamic acid. The two model peptide sequences CYEEVGVDSVEGEG-E(E-EEGEEY and CQDATADEQG-E(E-FEEEEGEDEA from the C-termini of mammalian α1- and β1-tubulin, respectively, containing oligo-glutamic acid side-chain branching with lengths of 1 to 5 amino acids were assembled in good yield and purity. The products may lead to the generation of specific antibodies which should be important tools for a more detailed investigation of polyglutamylation processes.

  5. 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

    2016-01-01

    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. PMID:27087493

  6. The mycosubtilin synthetase of Bacillus subtilis ATCC6633 : A multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase

    NARCIS (Netherlands)

    Duitman, EH; Hamoen, LW; Rembold, M; Venema, G; Seitz, H; Saenger, W; Bernhard, F; Reinhardt, R; Schmidt, M; Ullrich, C; Stein, T; Leenders, F; Vater, J

    1999-01-01

    Bacillus subtilis strain ATCC6633 has been identified as a producer of mycosubtilin, a potent antifungal peptide antibiotic. Mycosubtilin, which belongs to the iturin family of lipopeptide antibiotics, is characterized by a p-amino fatty acid moiety linked to the circular heptapeptide Asn-Tyr-Asn-Cl

  7. Influence of Amino Acid Compositions and Peptide Profiles on Antioxidant Capacities of Two Protein Hydrolysates from Skipjack Tuna (Katsuwonus pelamis) Dark Muscle

    OpenAIRE

    Chang-Feng Chi; Fa-Yuan Hu; Bin Wang; Zhong-Rui Li; Hong-Yu Luo

    2015-01-01

    Influence of amino acid compositions and peptide profiles on antioxidant capacities of two protein hydrolysates from skipjack tuna (Katsuwonus pelamis) dark muscle was investigated. Dark muscles from skipjack tuna were hydrolyzed using five separate proteases, including pepsin, trypsin, Neutrase, papain and Alcalase. Two hydrolysates, ATH and NTH, prepared using Alcalase and Neutrase, respectively, showed the strongest antioxidant capacities and were further fractionated using ultrafiltration...

  8. 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)

    Behrens, C; Harrit, N; 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...

  9. Folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier for enhanced intracellular drug delivery to cancer cells: conformational characterization and in vitro cellular uptake evaluation

    Directory of Open Access Journals (Sweden)

    Kang MJ

    2013-03-01

    Full Text Available Myung Joo Kang, Sang Han Park, Mean Hyung Kang, Min Jung Park, Young Wook Choi College of Pharmacy, Chung-Ang University, Seoul, Korea Background: A novel dual ligand–modified liposome, folic acid-tethered Pep-1 peptide-conjugated liposomal nanocarrier (FP-Lipo, was designed to overcome the nonselectivity of conventional penetrating peptide-tagged nanoparticulates and to provide the advantage of selective targeting of the folic acid receptor, which is frequently overexpressed on epithelial cancer cells. Methods: FP-Lipo was prepared by a sequential process of formation of a maleimide-derivatized small unilamellar vesicle, postinsertion of distearoyl phosphatidyl ethanolamine-polyethylene glycol 2000–folate to the vesicle, and Pep-1 peptide conjugation via thiol-maleimide linkage. Conformational and physical characteristics of the FP-Lipo nanocarriers were investigated for the extent of Pep-1 peptide and folic acid on the surface, vesicle size, and zeta potential. In vitro cellular uptake behaviors of the novel carrier containing a fluorescein dextran isothiocyanate probe were examined by spectrophotometry or by confocal laser scanning microscopy. Results: A novel nanocarrier bearing approximately 750 folate ligands and 100 penetrating peptides per vesicle was successfully prepared. The physical properties were as follows: 140 nm in size; 5 mV in zeta potential; less than 0.3 in polydispersity index. An in vitro cellular uptake study revealed that the FP-Lipo nanocarrier system exhibited more than twofold enhanced translocation into the folic acid receptor–positive HeLa cells compared with the single Pep-1 peptide–modified liposome. Meanwhile, its cellular association and internalization into the folic acid receptor–negative normal HaCaT cells was comparable with that of Pep-1 peptide–modified liposome. Conclusion: An advanced dual ligand-modified liposome is potentially useful for the treatment of folic acid receptor

  10. 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. PMID:27552222

  11. Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments.

    Science.gov (United States)

    Csapó, E; Majláth, Z; Juhász, Á; Roósz, B; Hetényi, A; Tóth, G K; Tajti, J; Vécsei, L; Dékány, I

    2014-11-01

    The interaction between kynurenic acid (KYNA) and two peptide fragments (ca. 30 residues) of Human Glutamate Receptor 201-300 (GluR1) using surface plasmon resonance (SPR) spectroscopy was investigated. Because of the medical interest in the neuroscience, GluR1 is one of the important subunits of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). AMPARs are ionotoropic glutamate receptors, which are mediating fast synaptic transmission and are crucial for plasticity in the brain. On the other hand, KYNA has been suggested to have neuroprotective activity and it has been considered for apply in therapy in certain neurobiological disorders. In this article the adsorption of the GluR1201-230 and GluR1231-259 peptides were studied on gold biosensor chip. The peptides were chemically bonded onto the gold surface via thiol group of L-cysteine resulted in the formation of peptide monolayer on the SPR chip surface. Because the GluR1231-259 peptide does not contain L-cysteine the Val256 was replaced by Cys256. The cross sectional area and the surface orientation of the studied peptides were determined by SPR and theoretical calculations (LOMETS) as well. The binding capability of KYNA on the peptide monolayer was studied in the concentration range of 0.1-5.0 mM using 150 mM NaCl ionic strength at pH 7.4 (±0.02) in phosphate buffer solutions. In order to determine the binding enthalpy the experiments were carried out between +10°C and +40°C. The heat of adsorption was calculated by using adsorption isotherms at different surface loading of KYNA on the SPR chip.

  12. From a pro-apoptotic peptide to a lytic peptide: One single residue mutation.

    Science.gov (United States)

    Zhou, Xi-Rui; Zhang, Qiang; Tian, Xi-Bo; Cao, Yi-Meng; Liu, Zhu-Qing; Fan, Ruru; Ding, Xiu-Fang; Zhu, Zhentai; Chen, Long; Luo, Shi-Zhong

    2016-08-01

    Further discovery and design of new anticancer peptides are important for the development of anticancer therapeutics, and study on the detailed acting mechanism and structure-function relationship of peptides is critical for anticancer peptide design and application. In this study, a novel anticancer peptide ZXR-1 (FKIGGFIKKLWRSKLA) derived from a known anticancer peptide mauriporin was developed, and a mutant ZXR-2 (FKIGGFIKKLWRSLLA) with only one residue difference at the 14th position (Lys→Leu) was also engineered. Replacement of the lysine with leucine made ZXR-2 more potent than ZXR-1 in general. Even with only one residue mutation, the two peptides displayed distinct anticancer modes of action. ZXR-1 could translocate into cells, target on the mitochondria and induce cell apoptosis, while ZXR-2 directly targeted on the cell membranes and caused membrane lysis. The variance in their acting mechanisms might be due to the different amphipathicity and positive charge distribution. In addition, the two Ile-Leu pairs (3-10 and 7-14) in ZXR-2 might also play a role in improving its cytotoxicity. Further study on the structure-function relationship of the two peptides may be beneficial for the design of novel anticancer peptides and peptide based therapeutics. PMID:27207743

  13. Peptide coupling between amino acids and the carboxylic acid of a functionalized chlorido-gold(I)-phosphane.

    Science.gov (United States)

    Kriechbaum, Margit; List, Manuela; Himmelsbach, Markus; Redhammer, Günther J; Monkowius, Uwe

    2014-10-01

    We have developed a protocol for the direct coupling between methyl ester protected amino acids and the chlorido-gold(I)-phosphane (p-HOOC(C6H4)PPh2)AuCl. By applying the EDC·HCl/NHS strategy (EDC·HCl = N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide hydrochloride, NHS = N-hydroxysuccinimide), the methyl esters of l-phenylalanine, glycine, l-leucine, l-alanine, and l-methionine are coupled with the carboxylic acid of the gold complex in moderate to good yields (62-88%). All amino acid tagged gold complexes were characterized by (1)H and (13)C NMR spectroscopy and high-resolution mass spectrometry. As corroborated by measurement of the angle of optical rotation, no racemization occurred during the reaction. The molecular structure of the leucine derivative was determined by single-crystal X-ray diffraction. In the course of developing an efficient coupling protocol, the acyl chlorides (p-Cl(O)C(C6H4)PPh2)AuX (X = Cl, Br) were also prepared and characterized. PMID:25203269

  14. A plausible simultaneous synthesis of amino acids and simple peptides on the primordial Earth.

    Science.gov (United States)

    Parker, Eric T; Zhou, Manshui; Burton, Aaron S; Glavin, Daniel P; Dworkin, Jason P; Krishnamurthy, Ramanarayanan; Fernández, Facundo M; Bada, Jeffrey L

    2014-07-28

    Following his seminal work in 1953, Stanley Miller conducted an experiment in 1958 to study the polymerization of amino acids under simulated early Earth conditions. In the experiment, Miller sparked a gas mixture of CH4, NH3, and H2O, while intermittently adding the plausible prebiotic condensing reagent cyanamide. For unknown reasons, an analysis of the samples was not reported. We analyzed the archived samples for amino acids, dipeptides, and diketopiperazines by liquid chromatography, ion mobility spectrometry, and mass spectrometry. A dozen amino acids, 10 glycine-containing dipeptides, and 3 glycine-containing diketopiperazines were detected. Miller's experiment was repeated and similar polymerization products were observed. Aqueous heating experiments indicate that Strecker synthesis intermediates play a key role in facilitating polymerization. These results highlight the potential importance of condensing reagents in generating diversity within the prebiotic chemical inventory.

  15. 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.

  16. Vibrational analysis of amino acids and short peptides in aqueous media. V. The effect of the disulfide bridge on the structural features of the peptide hormone somatostatin-14.

    Science.gov (United States)

    Hernández, Belén; Carelli, Claude; Coïc, Yves-Marie; De Coninck, Joël; Ghomi, Mahmoud

    2009-09-24

    To emphasize the role played by the S-S bridge in the structural features of somatostatin-14 (SST-14), newly recorded CD and Raman spectra of this cyclic peptide and its open analogue obtained by Cys-->Ser substitution are presented. CD spectra of both peptides recorded in aqueous solutions in the 100-500 microM concentration range are strikingly similar. They reveal principally that random conformers constitute the major population in both peptides. Consequently, the S-S bridge has no structuring effect at submillimolar concentrations. In methanol, the CD spectrum of somatostatin-14 keeps globally the same spectral shape as that observed in water, whereas its open analogue presents a major population of helical conformers. Raman spectra recorded as a function of peptide concentration (5-20 mM) and also in the presence of 150 mM NaCl provide valuable conformational information. All Raman spectra present a mixture of random and beta-hairpin structures for both cyclic and open peptides. More importantly, the presence or the absence of the disulfide bridge does not seem to influence considerably different populations of secondary structures within this range of concentrations. CD and Raman data obtained in the submillimolar and millimolar ranges of concentrations, respectively, lead us to accept the idea that SST-14 monomers aggregate upon increasing concentration, thus stabilizing beta-hairpin conformations in solution. However, even at high concentrations, random conformers do not disappear. Raman spectra of SST-14 also reveal a concentration effect on the flexibility of the S-S linkage and consequently on that of its cyclic part. In conclusion, although the disulfide linkage does not seem to markedly influence the SST-14 conformational features in aqueous solutions, its presence seems to be necessary to ensure the flexibility of the cyclic part of this peptide and to maintain its closed structure in lower dielectric constant environments.

  17. Replacement of the N-terminal tyrosine residue in opioid peptides with 3-(2,6-dimethyl-4-carbamoylphenyl)propanoic acid (Dcp) results in novel opioid antagonists.

    Science.gov (United States)

    Lu, Yixin; Lum, Tze Keong; Leow Augustine, Yoon Wui; Weltrowska, Grazyna; Nguyen, Thi M-D; Lemieux, Carole; Chung, Nga N; Schiller, Peter W

    2006-08-24

    3-(2,6-Dimethyl-4-carbamoylphenyl)propanoic acid (Dcp), a 2',6'-dimethyltyrosine analogue containing a carbamoyl group in place of the hydroxyl function and lacking the amino group, was synthesized. The replacement of Tyr1 in an enkephalin analogue and in dynorphin A(1-11)-NH2 with Dcp resulted in the first opioid peptide-derived antagonists that do not contain a phenolic hydroxyl group at the 1-position residue. The cyclic peptide Dcp-c[D-Cys-Gly-Phe(pNO2)-D-Cys]NH2 represents a novel, potent mu opioid antagonist. PMID:16913729

  18. 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

  19. 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.

  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)

    E2, along with Erns 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, 818CPIGWTGVIEC828, 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 818CPIGWTGVIEC828 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. A metallothionein mimetic peptide protects neurons against kainic acid-induced excitotoxicity

    DEFF Research Database (Denmark)

    Sonn, Katrin; Pankratova, Stanislava; Korshunova, Irina;

    2010-01-01

    the neuroprotective effect of EmtinB in the in vitro and in vivo models of kainic acid (KA)-induced neurotoxicity. We show that EmtinB passes the blood-brain barrier and is detectable in plasma for up to 24 hr. Treatment with EmtinB significantly attenuates seizures in C57BL/6J mice exposed to moderate (20 mg...

  3. Differential binding of monomethylarsonous acid compared to arsenite and arsenic trioxide with zinc finger peptides and proteins.

    Science.gov (United States)

    Zhou, Xixi; Sun, Xi; Mobarak, Charlotte; Gandolfi, A Jay; Burchiel, Scott W; Hudson, Laurie G; Liu, Ke Jian

    2014-04-21

    Arsenic is an environmental toxin that enhances the carcinogenic effect of DNA-damaging agents, such as ultraviolet radiation and benzo[a]pyrene. Interaction with zinc finger proteins has been shown to be an important molecular mechanism for arsenic toxicity and cocarcinogenesis. Arsenicals such as arsenite, arsenic trioxide (ATO), and monomethylarsonous acid (MMA(III)) have been reported to interact with cysteine residues of zinc finger domains, but little is known about potential differences in their selectivity of interaction. Herein we analyzed the interaction of arsenite, MMA(III), and ATO with C2H2, C3H1, and C4 configurations of zinc fingers using UV-vis, cobalt, fluorescence, and mass spectrometry. We observed that arsenite and ATO both selectively bound to C3H1 and C4 zinc fingers, while MMA(III) interacted with all three configurations of zinc finger peptides. Structurally and functionally, arsenite and ATO caused conformational changes and zinc loss on C3H1 and C4 zinc finger peptide and protein, respectively, whereas MMA(III) changed conformation and displaced zinc on all three types of zinc fingers. The differential selectivity was also demonstrated in zinc finger proteins isolated from cells treated with these arsenicals. Our results show that trivalent inorganic arsenic compounds, arsenite and ATO, have the same selectivity and behavior when interacting with zinc finger proteins, while methylation removes the selectivity. These findings provide insights on the molecular mechanisms underlying the differential effects of inorganic versus methylated arsenicals, as well as the role of in vivo arsenic methylation in arsenic toxicity and carcinogenesis.

  4. Determination of aromatic and sulfur-containing amino acids, peptides, and proteins using high-performance liquid chromatography with photolytic electrochemical detection

    Energy Technology Data Exchange (ETDEWEB)

    Dou, Lin; Krull, I.S. (Northeastern Univ., Boston, MA (USA))

    1990-12-01

    Aromatic amino acids, sulfur-containing amino acids, peptides containing such constituents, and proteins can now be detected in high-performance liquid chromatography by the use of on-line, postcolumn, continuous photolytic derivatization with electrochemical (HPLC-h{nu}-EC) detection. The overall approach is a very simple, reproducible, rapid, and fully automatable approach for the determination of certain amino acids, peptides, and proteins with excellent selectivity, sensitivity, and linearities of response. Dual-electrode response ratios, lamp-on/lamp-off behavior, and chromatographic capacity factors all contribute to the enhanced selectivity of the overall HPLC-h{nu}-EC determination for these particular classes of bioorganics and biopolymers. The analytical figures of merit, chromatography detection, and method validation approaches have all be optimally derived and demonstrated reproducible. Applications of the basic methodology to real-world samples are demonstrated and validated.

  5. Salt-resistant short antimicrobial peptides.

    Science.gov (United States)

    Mohanram, Harini; Bhattacharjya, Surajit

    2016-05-01

    Antimicrobial peptides (AMPs) are promising leads for the development of antibiotics against drug resistant bacterial pathogens. However, in vivo applications of AMPs remain obscure due to salt and serum mediated inactivation. The high cost of chemical synthesis of AMPs also impedes potential clinical application. Consequently, short AMPs resistant toward salt and serum inactivation are desirable for the development of peptide antibiotics. In this work, we designed a 12-residue amphipathic helical peptide RR12 (R-R-L-I-R-L-I-L-R-L-L-R-amide) and two Trp containing analogs of RR12 namely RR12Wpolar (R-R-L-I-W-L-I-L-R-L-L-R-amide), and RR12Whydro (R-R-L-I-R-L-W-L-R-L-L-R-amide). Designed peptides demonstrated potent antibacterial activity; MIC ranging from 2 to 8 μM, in the presence of sodium chloride (150 mM and 300 mM). Antibacterial activity of these peptides was also detected in the presence of human serum. Designed peptides, in particular RR12 and RR12Whydro, were only poorly hemolytic. As a mode of action; these peptides demonstrated efficient permeabilization of bacterial cell membrane and lysis of cell structure. We further investigated interactions of the designed peptides with lipopolysaccharide (LPS), the major component of the outer membrane permeability barrier of Gram-negative bacteria. Designed peptides adopted helical conformations in complex with LPS. Binding of peptides with LPS has yielded dissociation the aggregated structures of LPS. Collectively, these designed peptides hold ability to be developed for salt-resistant antimicrobial compounds. Most importantly, current work provides insights for designing salt-resistant antimicrobial peptides. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 345-356, 2016. PMID:26849911

  6. Isolation and characterization of a new class of amphipathic biopolymers capable of self-assembly from aqueous media

    Energy Technology Data Exchange (ETDEWEB)

    Martin, G.G.; Cannon, G.C.; McCormick, C.L. [Univ. of Southern Mississippi, Hattiesburg, MS (United States)

    1996-10-01

    Extensive research is being done in many laboratories to investigate the role of synthetic hydrophobically-modified polymers and amphipathic proteins for their potential in phase-transfer, sequestration, and elimination of polluting hydrocarbons and surfactants. Our laboratory has begun a research program which is aimed at the development of a new class of environmentally benign biomaterials using the amphipathic proteins termed {open_quotes}hydrophobins{close_quotes} and an associated polysaccharide, schizophyllan. These biopolymers can stabilize oil dispersions, attach strongly to polyethylene and polytetrafluoroethylene surfaces rendering them hydrophilic, and can self-assemble into a stable, flexible membrane. Preliminary experiments in our laboratory and others have demonstrated the immense technological potential of this class of biomaterials for surface modification of membranes and coatings, fouling resistance, controlled delivery, protective encapsulation, and drag reduction.

  7. Induction of porcine host defense peptide gene expression by short-chain fatty acids and their analogs.

    Directory of Open Access Journals (Sweden)

    Xiangfang Zeng

    Full Text Available Dietary modulation of the synthesis of endogenous host defense peptides (HDPs represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections. However, HDP regulation by dietary compounds such as butyrate is species-dependent. To examine whether butyrate could induce HDP expression in pigs, we evaluated the expressions of a panel of porcine HDPs in IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and primary monocytes in response to sodium butyrate treatment by real-time PCR. We revealed that butyrate is a potent inducer of multiple, but not all, HDP genes. Porcine β-defensin 2 (pBD2, pBD3, epididymis protein 2 splicing variant C (pEP2C, and protegrins were induced markedly in response to butyrate, whereas pBD1 expression remained largely unaltered in any cell type. Additionally, a comparison of the HDP-inducing efficacy among saturated free fatty acids of different aliphatic chain lengths revealed that fatty acids containing 3-8 carbons showed an obvious induction of HDP expression in IPEC-J2 cells, with butyrate being the most potent and long-chain fatty acids having only a marginal effect. We further investigated a panel of butyrate analogs for their efficacy in HDP induction, and found glyceryl tributyrate, benzyl butyrate, and 4-phenylbutyrate to be comparable with butyrate. Identification of butyrate and several analogs with a strong capacity to induce HDP gene expression in pigs provides attractive candidates for further evaluation of their potential as novel alternatives to antibiotics in augmenting innate immunity and disease resistance of pigs.

  8. Cleavage of peptide bonds bearing ionizable amino acids at P1 by serine proteases with hydrophobic S1 pocket

    International Nuclear Information System (INIS)

    Research highlights: → Large pK shifts in ionizable groups when buried in the protein interior. → Substrate dependent shifts in pH optimum for serine proteases. → Lys side chain is a stronger acid in serine protease S1 pocket than Asp side chain. -- Abstract: Enzymatic hydrolysis of the synthetic substrate succinyl-Ala-Ala-Pro-Xxx-pNA (where Xxx = Leu, Asp or Lys) catalyzed by bovine chymotrypsin (CHYM) or Streptomyces griseus protease B (SGPB) has been studied at different pH values in the pH range 3-11. The pH optima for substrates having Leu, Asp, and Lys have been found to be 7.5-8.0, 5.5-6.0, and ∼10, respectively. At the normally reported pH optimum (pH 7-8) of CHYM and SGPB, the substrate with Leu at the reactive site is more than 25,000-fold more reactive than that with Asp. However, when fully protonated, Asp is nearly as good a substrate as Leu. The pK values of the side chains of Asp and Lys in the hydrophobic S1 pocket of CHYM and SGPB have been calculated from pH-dependent hydrolysis data and have been found to be about 9 for Asp and 7.4 and 9.7 for Lys for CHYM and SGPB, respectively. The results presented in this communication suggest a possible application of CHYM like enzymes in cleaving peptide bonds contributed by acidic amino acids between pH 5 and 6.

  9. A family of helminth molecules that modulate innate cell responses via molecular mimicry of host antimicrobial peptides.

    Science.gov (United States)

    Robinson, Mark W; Donnelly, Sheila; Hutchinson, Andrew T; To, Joyce; Taylor, Nicole L; Norton, Raymond S; Perugini, Matthew A; Dalton, John P

    2011-05-01

    Over the last decade a significant number of studies have highlighted the central role of host antimicrobial (or defence) peptides in modulating the response of innate immune cells to pathogen-associated ligands. In humans, the most widely studied antimicrobial peptide is LL-37, a 37-residue peptide containing an amphipathic helix that is released via proteolytic cleavage of the precursor protein CAP18. Owing to its ability to protect against lethal endotoxaemia and clinically-relevant bacterial infections, LL-37 and its derivatives are seen as attractive candidates for anti-sepsis therapies. We have identified a novel family of molecules secreted by parasitic helminths (helminth defence molecules; HDMs) that exhibit similar biochemical and functional characteristics to human defence peptides, particularly CAP18. The HDM secreted by Fasciola hepatica (FhHDM-1) adopts a predominantly α-helical structure in solution. Processing of FhHDM-1 by F. hepatica cathepsin L1 releases a 34-residue C-terminal fragment containing a conserved amphipathic helix. This is analogous to the proteolytic processing of CAP18 to release LL-37, which modulates innate cell activation by classical toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). We show that full-length recombinant FhHDM-1 and a peptide analogue of the amphipathic C-terminus bind directly to LPS in a concentration-dependent manner, reducing its interaction with both LPS-binding protein (LBP) and the surface of macrophages. Furthermore, FhHDM-1 and the amphipathic C-terminal peptide protect mice against LPS-induced inflammation by significantly reducing the release of inflammatory mediators from macrophages. We propose that HDMs, by mimicking the function of host defence peptides, represent a novel family of innate cell modulators with therapeutic potential in anti-sepsis treatments and prevention of inflammation.

  10. Racemic synthesis and solid phase peptide synthesis application of the chimeric valine/leucine derivative 2-amino-3,3,4-trimethyl-pentanoic acid.

    Science.gov (United States)

    Pelà, M; Del Zoppo, L; Allegri, L; Marzola, E; Ruzza, C; Calo, G; Perissutti, E; Frecentese, F; Salvadori, S; Guerrini, R

    2014-07-01

    The synthesis of non natural amino acid 2-amino-3,3,4-trimethyl-pentanoic acid (Ipv) ready for solid phase peptide synthesis has been developed. Copper (I) chloride Michael addition, followed by a Curtius rearrangement are the key steps for the lpv synthesis. The racemic valine/leucine chimeric amino acid was then successfully inserted in position 5 of neuropeptide S (NPS) and the diastereomeric mixture separated by reverse phase HPLC. The two diastereomeric NPS derivatives were tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPS receptor where they behaved as partial agonist and pure antagonist.

  11. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs: Synthesis and DNA Binding.

    Directory of Open Access Journals (Sweden)

    Yuliya Kirillova

    Full Text Available New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA.

  12. Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

    OpenAIRE

    Pessione, Enrica; Cirrincione, Simona

    2016-01-01

    Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the ma...

  13. Metal-amino acid (or peptide)-nucleoside (or related bases) ternary complexes

    Energy Technology Data Exchange (ETDEWEB)

    Terron, A.; Fiol, J.J.; Herrero, L.A.; Garcia-Raso, A. [Departament de Quimica. Universitat de les Illes Balears. Palma de Mallorca. (Spain); Apella, M.C. [Cerela Centro de Referencia de Lactobacilos, Tucaman, Argentina (Antigua and Barbuda); Caubet, A.; Moreno, V. [Departament de Quimica Inorganica. Universitat de Barcelona. Barcelona (Spain)

    1997-05-01

    The knowledge of simultaneous metal ion interaction with proteins and nucleic acids is one of the most exciting subjects inside the Inorganic Biochemistry. In the last years, several groups have published articles on the synthesis and characterization of ternary complexes bringing relevant data on the structure and stability of metallo biomolecules. In this short review, the last contributions found in the literature are collected. Comments on the factors influencing the behaviour and stability of these systems are offered. (Author) 100 refs.

  14. Amphipathic antenna of an inward rectifier K+ channel responds to changes in the inner membrane leaflet.

    Science.gov (United States)

    Iwamoto, Masayuki; Oiki, Shigetoshi

    2013-01-01

    Membrane lipids modulate the function of membrane proteins. In the case of ion channels, they bias the gating equilibrium, although the underlying mechanism has remained elusive. Here we demonstrate that the N-terminal segment (M0) of the KcsA potassium channel mediates the effect of changes in the lipid milieu on channel gating. The M0 segment is a membrane-anchored amphipathic helix, bearing positively charged residues. In asymmetric membranes, the M0 helix senses the presence of negatively charged phospholipids on the inner leaflet. Upon gating, the M0 helix revolves around the axis of the helix on the membrane surface, inducing the positively charged residues to interact with the negative head groups of the lipids so as to stabilize the open conformation (i.e., the "roll-and-stabilize model"). The M0 helix is thus a charge-sensitive "antenna," capturing temporary changes in lipid composition in the fluidic membrane. This unique type of sensory device may be shared by various types of membrane proteins.

  15. 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.

  16. The high resolution structure of tyrocidine A reveals an amphipathic dimer.

    Science.gov (United States)

    Loll, Patrick J; Upton, Elizabeth C; Nahoum, Virginie; Economou, Nicoleta J; Cocklin, Simon

    2014-05-01

    Tyrocidine A, one of the first antibiotics ever to be discovered, is a cyclic decapeptide that binds to membranes of target bacteria, disrupting their integrity. It is active against a broad spectrum of Gram-positive organisms, and has recently engendered interest as a potential scaffold for the development of new drugs to combat antibiotic-resistant pathogens. We present here the X-ray crystal structure of tyrocidine A at a resolution of 0.95Å. The structure reveals that tyrocidine forms an intimate and highly amphipathic homodimer made up of four beta strands that associate into a single, highly curved antiparallel beta sheet. We used surface plasmon resonance and potassium efflux assays to demonstrate that tyrocidine binds tightly to mimetics of bacterial membranes with an apparent dissociation constant (K(D)) of 10 μM, and efficiently permeabilizes bacterial cells at concentrations equal to and below the K(D). Using variant forms of tyrocidine in which the fluorescent probe p-cyano-phenylalanine had been inserted on either the polar or apolar face of the molecule, we performed fluorescence quenching experiments, using both water-soluble and membrane-embedded quenchers. The quenching results, together with the structure, strongly support a membrane association model in which the convex, apolar face of tyrocidine's beta sheet is oriented toward the membrane interior, while the concave, polar face is presented to the aqueous phase.

  17. 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

    2016-03-01

    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. PMID:26978374

  18. Deep Subseafloor Fungi as an Untapped Reservoir of Amphipathic Antimicrobial Compounds

    Directory of Open Access Journals (Sweden)

    Marion Navarri

    2016-03-01

    Full Text Available 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.

  19. Importance of Large Intestine in Regulating Bile Acids and Glucagon-Like Peptide-1 in Germ-Free Mice.

    Science.gov (United States)

    Selwyn, Felcy Pavithra; Csanaky, Iván L; Zhang, Youcai; Klaassen, Curtis D

    2015-10-01

    It is known that 1) elevated serum bile acids (BAs) are associated with decreased body weight, 2) elevated glucagon-like peptide-1 (GLP-1) levels can decrease body weight, and 3) germ-free (GF) mice are resistant to diet-induced obesity. The purpose of this study was to test the hypothesis that a lack of intestinal microbiota results in more BAs in the body, resulting in increased BA-mediated transmembrane G protein-coupled receptor 5 (TGR5) signaling and increased serum GLP-1 as a mechanism of resistance of GF mice to diet-induced obesity. GF mice had 2- to 4-fold increased total BAs in the serum, liver, bile, and ileum. Fecal excretion of BAs was 63% less in GF mice. GF mice had decreased secondary BAs and increased taurine-conjugated BAs, as anticipated. Surprisingly, there was an increase in non-12α-OH BAs, namely, β-muricholic acid, ursodeoxycholic acid (UDCA), and their taurine conjugates, in GF mice. Further, in vitro experiments confirmed that UDCA is a primary BA in mice. There were minimal changes in the mRNA of farnesoid X receptor target genes in the ileum (Fibroblast growth factor 15, small heterodimer protein, and ileal bile acid-binding protein), in the liver (small heterodimer protein, liver receptor homolog-1, and cytochrome P450 7a1), and BA transporters (apical sodium dependent bile acid transporter, organic solute transporter α, and organic solute transporter β) in the ileum of GF mice. Surprisingly, there were marked increases in BA transporters in the large intestine. Increased GLP-1 levels and gallbladder size were observed in GF mice, suggesting activation of TGR5 signaling. In summary, the GF condition results in increased expression of BA transporters in the colon, resulting in 1) an increase in total BA concentrations in tissues, 2) a change in BA composition to favor an increase in non-12α-OH BAs, and 3) activation of TGR5 signaling with increased gallbladder size and GLP-1.

  20. Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

    OpenAIRE

    Topham, Christopher M.; Smith, Jeremy C.

    2006-01-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the bindin...

  1. 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)

    OpenAIRE

    Choi, Chang Bon; Kwon, Hana; Kim, Sung Il; Yang, Un Mok; Lee, Ju Hwan; Park, Eun Kyu

    2016-01-01

    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 Sunflo...

  2. 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.

  3. Interaction of some hydrophobic amino acids, peptides, and protein with aqueous 3-chloro-1,2-propanediol and 3-chloro-1-propanol: Biophysical studies

    Energy Technology Data Exchange (ETDEWEB)

    Keswani, Neelam [Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076 (India); Kishore, Nand, E-mail: nandk@chem.iitb.ac.i [Department of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400 076 (India)

    2011-04-15

    Research highlights: Thermodynamic properties of amino acids, peptides and protein determined in solution. The solvents chosen were 3-chloropropan-1-ol and 3-chloropropan-1,2-diol. {yields}The results enabled understanding the interactions quantitatively in these systems affecting the protein stability. Fine details of interactions provided in-depth analysis. - Abstract: The apparent molar volume V{sub 2,{phi},} apparent molar isentropic compressibility K{sub S,2,{phi},} and heat of dilution (q) of aqueous glycine, alanine, {alpha}-amino butyric acid, valine, leucine, diglycine, triglycine, and hen egg white lysozyme have been determined in aqueous solutions of 3-chloropropano-1-ol and 3-chloropropan-1,2-diol solutions at T = 298.15 K. These data have been used to calculate the infinite dilution standard partial molar volume V{sub 2,m}{sup 0}, partial molar isentropic compressibility K{sub S,2,m}{sup 0}, and enthalpy of dilution {Delta}{sub dil}H{sup o} of the amino acids and peptides in aqueous 3-chloropropano-1-ol and 3-chloropropan-1,2-diol, and the standard partial molar quantities of transfer of the amino acids and peptides to the aqueous alcohol and diol solutions. The linear correlation of V{sub 2,m}{sup 0} for a homologous series of amino acids has been utilized to calculate the contribution of the charged end groups (NH{sub 3}{sup +},COO{sup -}), CH{sub 2} group and other alkyl chains of the amino acids to the values of V{sub 2,m}{sup 0}. The results on the standard partial molar volumes of transfer, compressibility and enthalpy of dilution from water to aqueous alcohol and diol solutions have been correlated and interpreted in terms of ion-polar, ion-hydrophobic, and hydrophobic-hydrophobic group interactions. The heat of dilution of these amino acids, peptides, and hen egg white lysozyme measured in aqueous solutions of 3-chloropropano-1-ol and 3-chloropropan-1,2-diol by using isothermal titration calorimetry along with the volumetric, compressibility

  4. Effect of Fluorescent Labels on Peptide and Amino Acid Sample Dimensionality in Two Dimensional nLC × μFFE Separations.

    Science.gov (United States)

    Geiger, Matthew; Bowser, Michael T

    2016-02-16

    Multidimensional separations present a unique opportunity for generating the high peak capacities necessary for the analysis of complex biological mixtures. We have coupled nano liquid chromatography with micro free flow electrophoresis (nLC × μFFE) to produce high peak capacity separations of peptide and amino acid mixtures. Currently, μFFE largely relies on laser-induced fluorescence (LIF) detection. We have demonstrated that the choice of fluorescent label significantly affects the fractional coverage and peak capacity of nLC × μFFE separations of peptides and amino acids. Of the labeling reagents assessed, Chromeo P503 performed the best for nLC × μFFE separations of peptides. A nLC × μFFE analysis of a Chromeo P503-labeled BSA tryptic digest produced a 2D separation that made effective use of the available separation space (48%), generating a corrected peak capacity of 521 in a 5 min separation window (104 peaks/min). nLC × μFFE separations of NBD-F-labeled peptides produced similar fractional coverage and peak capacity, but this reagent was able to react with multiple reaction sites, producing an unnecessarily complex analyte mixture. NBD-F performed the best for nLC × μFFE separations of amino acids. NBD-F-labeled amino acids produced a 2D separation that covered 36% of the available separation space, generating a corrected peak capacity of 95 in a 75 s separation window (76 peaks/min). Chromeo P503 and Alexa Fluor 488-labeled amino acids were not effectively separated in the μFFE dimension, giving 2D separations with poor fractional coverage and peak capacity. PMID:26757484

  5. Dual Role of Hydrophobic Racemic Thioesters of α-Amino Acids in the Generation of Isotactic Peptides and Co-peptides in Water; Implications for the Origin of Homochirality

    Science.gov (United States)

    Illos, Roni A.; Clodic, Gilles; Bolbach, Gerard; Weissbuch, Isabelle; Lahav, Meir

    2010-02-01

    Thioesters of α-amino acids are considered as plausible monomers for the generation of the primeval peptides. DL-Leucine-thioethyl esters (LeuSEt), where the L-enantiomer was tagged with deuterium atoms, undergo polycondensation in water or in bicarbonate or imidazole buffer solutions to yield mainly heterochiral (atactic) peptides and diketopiperazine, as analyzed by MALDI-TOF and ESI mass-spectrometry. In variance, when polymerization of DL(d10) -Leu, first activated with N,N'-carbonyldiimidazole, then initiated with ethanethiol or with DL(d3) -LeuSEt yielded a library of peptides up to 30 detectable residues where those of homochiral sequence (isotactic) are the dominant diastereoisomers. At these conditions, racemic β-sheets are formed and operate as stereoselective templates in the process of chain-elongation. Isotopic L: L(d10)-Leu co-peptides were obtained in the polymerization of L(d10)-Leu with L-LeuSEt. By contrast, mixtures of oligo- D-Leu and oligo- L(d10)-Leu were obtained in the polymerization of mixtures of D-LeuSEt with activated L(d10)-Leu. Isotactic co-peptides containing Leu and Val residues were formed in the polymerization of mixtures of activated DL(d8)-Val with DL(d3) -LeuSEt in water, implying that the racemic β-sheets exert regio-enantio-selection but not chemo-selection. A reaction pathway is suggested, where LeuSEt operates both as initiator of the reaction as well as a multimer.

  6. 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.

  7. 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.

  8. Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii.

    Science.gov (United States)

    Zhang, Xiaofeng; Wu, Shan; Li, Ke; Shuai, Jiangbing; Dong, Qiang; Fang, Weihuan

    2012-07-01

    A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.

  9. Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium.

    Science.gov (United States)

    McNeer, Nicole Ali; Anandalingam, Kavitha; Fields, Rachel J; Caputo, Christina; Kopic, Sascha; Gupta, Anisha; Quijano, Elias; Polikoff, Lee; Kong, Yong; Bahal, Raman; Geibel, John P; Glazer, Peter M; Saltzman, W Mark; Egan, Marie E

    2015-01-01

    Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here we use triplex-forming peptide nucleic acids and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep-sequencing reveals negligible off-target effects in partially homologous sites. Intranasal delivery of nanoparticles in CF mice produces changes in the nasal epithelium potential difference assay, consistent with corrected CFTR function. Also, gene correction is detected in the nasal and lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects. PMID:25914116

  10. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids.

    Science.gov (United States)

    Kim, Ki-Hyun; Nielsen, Peter E; Glazer, Peter M

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences by Watson-Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules. PMID:17977869

  11. Investigation on natural diets of larval marine animals using peptide nucleic acid-directed polymerase chain reaction clamping.

    Science.gov (United States)

    Chow, Seinen; Suzuki, Sayaka; Matsunaga, Tadashi; Lavery, Shane; Jeffs, Andrew; Takeyama, Haruko

    2011-04-01

    The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected. PMID:20535520

  12. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    Science.gov (United States)

    Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

  13. 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.

  14. Molecular dynamics investigation of the influence of anionic and zwitterionic interfaces on antimicrobial peptides' structure: implications for peptide toxicity and activity

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Kaznessis, Yiannis N

    2006-01-01

    Molecular dynamics simulations of three related helical antimicrobial peptides have been carried out in zwitterionic diphosphocholine (DPC) micelles and anionic sodiumdodecylsulfate (SDS) micelles. These systems can be considered as model mammalian and bacterial membrane interfaces, respectively...... properties. Based on the simulations, we argue that secondary structure stability often leads to toxic properties. We also propose that G10 and T7 operate by the carpet mechanism of cell lysis. Toxicity of peptides operating by the carpet mechanism can be attenuated by reducing the peptide helical content...... amphipathic peptide structures, which bind weakly to the micelle. Simulations in SDS were carried out to compare the influence of membrane electrostatics on peptide structure. All three peptides bound strongly to SDS, and retained helical form. This corresponds well with their equally potent antibacterial...

  15. Simulations and analysis of the Raman scattering and differential Raman scattering/Raman optical activity (ROA) spectra of amino acids, peptides and proteins in aqueous solution

    DEFF Research Database (Denmark)

    Jalkanen, Karl J.; Nieminen, R. M.; Bohr, Jakob

    2000-01-01

    The Raman and Raman optical activity (ROA) spectra of amino acids and small peptides in aqueous solution have been simulated by density functional theory and restricted Hartree/Fock methods. The treatment of the aqueous environment in treated in two ways. The water molecules in the first hydration...... shell which strongly interact with the molecule are treated explicitly while the waters in the bulk are treated by a continuum model. The structures are optimized and the harmonic force elds are calculated. The derivatives needed to simulate the Raman and ROA intensities are calculated from first...... principles. The simulated Raman and ROA spectra have been compared to recently meassured spectra on amino acids and peptides. The simulations and understanding from them are used to interpret the Raman and ROA spectra of proteins. A comparison to vibrational absorption (VA) and vibrational circular dichroism...

  16. Abiogenic Syntheses of Lipoamino Acids and Lipopeptides and their Prebiotic Significance

    Science.gov (United States)

    Sproul, Gordon

    2015-12-01

    Researchers have formed peptide bonds under a variety of presumed prebiotic conditions. Here it is proposed that these same conditions would have also formed amide bonds between fatty acids and amino acids, producing phosphate-free amphipathic lipoamino acids and lipopeptides. These compounds are known to form vesicles and are ubiquitous in living organisms. They could represent molecules that provided protection by membranes as well as possibilities for proto-life metabolism . It is here demonstrated that when a fatty acid is heated with various amino acids, optimally in the presence of suitable salts or minerals, lipoamino acids are formed. Magnesium and potassium carbonates as well as iron (II) sulfide are found to be particularly useful in these reactions. In this manner N-lauroylglycine, N-lauroylalanine, N-stearoylalanine and several other lipoamino acids have been synthesized. Similarly, when glycylglycine was heated with lauric acid in the presence of magnesium carbonate, the lipopeptide N-lauroylglycylglycine was formed. Such compounds are proposed to have been critical precursors to the development of life.

  17. 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.

  18. A conserved amphipathic helix in the N-terminal regulatory region of the papillomavirus E1 helicase is required for efficient viral DNA replication.

    Science.gov (United States)

    Morin, Geneviève; Fradet-Turcotte, Amélie; Di Lello, Paola; Bergeron-Labrecque, Fanny; Omichinski, James G; Archambault, Jacques

    2011-06-01

    The papillomavirus E1 helicase, with the help of E2, assembles at the viral origin into a double hexamer that orchestrates replication of the viral genome. The N-terminal region (NTR) of E1 is essential for DNA replication in vivo but dispensable in vitro, suggesting that it has a regulatory function. By deletion analysis, we identified a conserved region of the E1 NTR needed for efficient replication of viral DNA. This region is predicted to form an amphipathic α-helix (AH) and shows sequence similarity to portions of the p53 and herpes simplex virus (HSV) VP16 transactivation domains known as transactivation domain 2 (TAD2) and VP16C, which fold into α-helices upon binding their target proteins, including the Tfb1/p62 (Saccharomyces cerevisiae/human) subunit of general transcription factor TFIIH. By nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we found that a peptide spanning the E1 AH binds Tfb1 on the same surface as TAD2/VP16C and with a comparable affinity, suggesting that it does bind as an α-helix. Furthermore, the E1 NTRs from several human papillomavirus (HPV) types could activate transcription in yeast, and to a lesser extent in mammalian cells, when fused to a heterologous DNA-binding domain. Mutation of the three conserved hydrophobic residues in the E1 AH, analogous to those in TAD2/VP16C that directly contact their target proteins, decreased transactivation activity and, importantly, also reduced by 50% the ability of E1 to support transient replication of DNA in C33A cells, at a step following assembly of the E1-E2-ori preinitiation complex. These results demonstrate the existence of a conserved TAD2/VP16C-like AH in E1 that is required for efficient replication of viral DNA. PMID:21450828

  19. Influence of the amount of co-infused amino acids on post-therapeutic potassium levels in peptide receptor radionuclide therapy

    OpenAIRE

    Werner, Rudolf A; Lapa, Constantin; Bluemel, Christina; Lückerath, Katharina; Schirbel, Andreas; Strate, Alexander; Buck, Andreas K.; Herrmann, Ken

    2014-01-01

    Background Peptide receptor radionuclide therapy (PRRT) is routinely used for advanced or metastasized neuroendocrine tumours (NET). To prevent nephrotoxicity, positively charged amino acids (AA) are co-infused. The aim of this study was to correlate the risk for therapy-related hyperkalaemia with the total amount of AA infused. Methods Twenty-two patients undergoing PRRT with standard activities of 177Lu-DOTATATE/-TOC were monitored during two following treatment cycles with co-i...

  20. Dietary protein effects on growth performance, carcass traits and expression of selected jejunal peptide and amino acid transporters in broiler chickens

    OpenAIRE

    A Corzo; II RE Loar; MT Kidd; SC Burgess

    2011-01-01

    The effect of dietary protein on growth, carcass traits and some specific intestinal intestinal peptide and amino acid transporters in broiler chickens was studied. Birds received a common pre-test diet, and were subsequently fed either a standard positive control diet (PC) or a reduced CP diet (RCP) from 21 to 42 d of age. Growth was negatively impacted with feeding of RCP as manifested by an increase in feed consumption and feed conversion ratio. Carcass traits also showed evidence of negat...

  1. Short-Chain Fatty Acids Stimulate Glucagon-Like Peptide-1 Secretion via the G-Protein-Coupled Receptor FFAR2

    OpenAIRE

    Tolhurst, Gwen; Heffron, Helen; Lam, Yu Shan; Parker, Helen E; Habib, Abdella M; Diakogiannaki, Eleftheria; Cameron, Jennifer; Grosse, Johannes; Reimann, Frank; Gribble, Fiona M.

    2012-01-01

    Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of “indigestible” prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the ...

  2. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    OpenAIRE

    Chin, Joanna Y; Kuan, Jean Y.; Lonkar, Pallavi S.; Krause, Diane S.; Seidman, Michael M.; Peterson, Kenneth R.; Nielsen, Peter E.; Kole, Ryszard; Glazer, Peter M.

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the hu...

  3. Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent,diethylenetriamine pentaacetic acid gadolinium

    Institute of Scientific and Technical Information of China (English)

    GUO You-min; LIU Min; YANG Jun-le; GUO Xiao-juan; WANG Si-cen; DUAN Xiao-yi; WANG Peng

    2007-01-01

    Background The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance (MR) contrast agent. Cell penetrating peptide (CPP) is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium (Gd-DTPA) in molecular imaging in vitro. Methods Fluorescein-5-isothiocyanate (FITC) and Gd-DTPA respectively labeled with CPP (FITC-CPP, Gd-DTPA-CPP) were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T1WI signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide reduction assay (MTT). Results The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry (FITC-CPP MW=2163.34, Gd-DTPA-CPP MW=2285.99), were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T1 weighted imaging (T1WI) signal, and that the T1WI signal intensity was increasing in a time-dependent manner (r=0.972, P=0.001), while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different (P=0.225). By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant (F=0.006, P=1.000) effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group. Conclusions The newly constructed CPP based on polyarginine can translocate cells by carrying FITC

  4. Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion

    Energy Technology Data Exchange (ETDEWEB)

    Yu, I.-M.; Holdaway, H.A.; Chipman, P.R.; Kuhn, R.J.; Rossmann, M.G.; Chen, J.; Purdue

    2010-07-27

    Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.

  5. Oleic acid and glucose regulate glucagon-like peptide 1 receptor expression in a rat pancreatic ductal cell line

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Leshuai W.; McMahon Tobin, Grainne A.; Rouse, Rodney L., E-mail: rodney.rouse@fda.hhs.gov

    2012-10-15

    The glucagon-like peptide 1 receptor (GLP1R) plays a critical role in glucose metabolism and has become an important target for a growing class of drugs designed to treat type 2 diabetes. In vitro studies were designed to investigate the effect of the GLP1R agonist, exenatide (Ex4), in “on-target” RIN-5mF (islet) cells as well as in “off-target” AR42J (acinar) and DSL-6A/C1 (ductal) cells in a diabetic environment. Ex4 increased islet cell proliferation but did not affect acinar cells or ductal cells at relevant concentrations. A high caloric, high fat diet is a risk factor for impaired glucose tolerance and type-2 diabetes. An in vitro Oleic acid (OA) model was used to investigate the effect of Ex4 in a high calorie, high fat environment. At 0.1 and 0.4 mM, OA mildly decreased the proliferation of all pancreatic cell types. Ex4 did not potentiate the inhibitory effect of OA on cell proliferation. Akt phosphorylation in response to Ex4 was diminished in OA-treated ductal cells. GLP1R protein detected by western blot was time and concentration dependently decreased after glucose stimulation in OA-treated ductal cells. In ductal cells, OA treatment altered the intracellular localization of GLP1R and its co-localization with early endosome and recycling endosomes. Chloroquine (lysosomal inhibitor), N-acetyl-L-cysteine (reactive oxygen species scavenger) and wortmannin (a phosphatidylinositol-3-kinase inhibitor), fully or partially, rescued GLP1R protein in OA-pretreated, glucose-stimulated ductal cells. The impact of altered regulation on phenotype/function is presently unknown. However, these data suggest that GLP1R regulation in ductal cells can be altered by a high fat, high calorie environment. -- Highlights: ► Exenatide did not inhibit islet, acinar or ductal cell proliferation. ► GLP1R protein decreased after glucose stimulation in oleic acid-treated ductal cells. ► Oleic acid treatment altered localization of GLP1R with early and recycling

  6. Laccase mediated-synthesis of hydroxycinnamoyl-peptide from ferulic acid and carnosine.

    Science.gov (United States)

    Aljawish, Abdulhadi; Chevalot, Isabelle; Madad, Nidal; Paris, Cédric; Muniglia, Lionel

    2016-06-10

    Carnosine (CAR) dipeptide was functionalized with ferulic acid (FA) as substrate using laccase from Myceliophtora thermophila as biocatalyst. The enzymatic reaction was performed in aqueous medium under mild conditions (pH 7.5, 30°C) as an eco-friendly procedure. Results showed that this enzymatic process led to the synthesis of two new derivatives (P1, P2), from the coupling between CAR and FA derived products. Conditions allowing a high production of P1, P2 derivatives were determined with an optimal ratio of (FA: CAR) of (1:1.6) at optimal time reaction of 8h. Under these optimal conditions, the coupling between CAR and FA-products was demonstrated, resulting in the decrease of -NH2 groups (almost 50%) as quantified via derivatization. Due to the presence of FA in the structure of these new derivatives, they exhibited higher hydrophobic property than carnosine. Structural analyses by mass spectrometry showed that P1 and P2 (FA-CAR) derivatives exhibited the same molecular mass (MM 770g/mol) containing one CAR-molecule and three FA-molecules but with different chemical structures. Furthermore, these derivatives presented improved antioxidant (almost 10 times) and anti-proliferative (almost 18 times) properties in comparison with CAR. Moreover, P1 derivative exhibited higher antioxidant and anti-proliferative activities than P2 derivative, which confirmed the different structures of P1 and P2. These results suggested that the oxidized phenols coupling with carnosine is a promising process to enhance the CAR-properties. PMID:27084055

  7. Cloning of a crustin-like, single whey-acidic-domain, antibacterial peptide from the haemocytes of the European lobster, Homarus gammarus, and its response to infection with bacteria.

    Science.gov (United States)

    Hauton, C; Brockton, V; Smith, V J

    2006-03-01

    Degenerate PCR was used to isolate a 221-base pair nucleotide sequence of a new crustin-like antibacterial peptide from the haemocytes of the European lobster, Homarus gammarus. Rapid amplification of cDNA ends was used to extend the sequence to determine the complete open reading frame and un-translated regions. The inferred amino acid sequence of this peptide was found to be similar to crustin-like peptides isolated for several species of shrimp as well as the shore crab, Carcinus maenas. The sequence also contains a single-whey-acidic protein (WAP) domain, similar to novel antibacterial single-whey-acidic domain (SWD) peptides that have been recently described in the tiger shrimp, Penaeus monodon, and the Pacific white shrimp, Litopenaeus vannamei. Real-time PCR was used to analyse the expression of the gene coding for this peptide. The gene is up regulated after inoculation with the Gram-positive lobster pathogen Aerococcus viridans var. homari but down regulated after inoculation with the Gram-negative bacteria Listonella anguillarum. Phylogenetic analysis of this new peptide shows that it is most related to other antimicrobial crustin peptides and that the crustins are only distantly related to the antibacterial SWD peptides recently described. PMID:16144710

  8. Surface aggregation of urinary proteins and aspartic acid-rich peptides on the faces of calcium oxalate monohydrate investigated by in situ force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Weaver, M L; Qiu, S R; Hoyer, J R; Casey, W H; Nancollas, G H; De Yoreo, J J

    2008-05-28

    The growth of calcium oxalate monohydrate in the presence of Tamm-Horsfall protein (THP), osteopontin (OPN), and the 27-residue synthetic peptides (DDDS){sub 6}DDD and (DDDG){sub 6}DDD [where D = aspartic acid and X = S (serine) or G (glycine)] was investigated via in situ atomic force microscopy (AFM). The results show that these three growth modulators create extensive deposits on the crystal faces. Depending on the modulator and crystal face, these deposits can occur as discrete aggregates, filamentary structures, or uniform coatings. These proteinaceous films can lead to either the inhibition or increase of the step speeds (with respect to the impurity-free system) depending on a range of factors that include peptide or protein concentration, supersaturation and ionic strength. While THP and the linear peptides act, respectively, to exclusively increase and inhibit growth on the (-101) face, both exhibit dual functionality on the (010) face, inhibiting growth at low supersaturation or high modulator concentration and accelerating growth at high supersaturation or low modulator concentration. Based on analyses of growth morphologies and dependencies of step speeds on supersaturation and protein or peptide concentration, we argue for a picture of growth modulation that accounts for the observations in terms of the strength of binding to the surfaces and steps and the interplay of electrostatic and solvent-induced forces at crystal surface.

  9. On the activation of bovine plasma factor XIII. Amino acid sequence of the peptide released by thrombin and the terminal residues of the subunit polypeptides.

    Science.gov (United States)

    Nakamura, S; Iwanaga, S; Suzuki, T

    1975-12-01

    A blood coagulation factor, Factor XIII, was highly purified from bovine fresh plasma by a method similar to those used for human plasma Factor XIII. The isolated Factor XIII consisted of two subunit polypeptides, a and b chains, with molecular weights of 79,000 +/- 2,000 and 75,000 +/- 2,000, respectively. In the conversion of Factor XIII to the active enzyme, Factor XIIIa, by bovine thrombin [EC 3.4.21.5], a peptide was liberated. This peptide, designated tentatively as "activation peptide," was isolated by gel-filtration on a Sephadex G-75 column. It contained a total of 37 amino acid residues with a masked N-terminal residue and C-terminal arginine. The whole amino acid sequence of "Activation peptide" was established by the dansyl-Edman method and standard enzymatic techniques, and the masked N-terminal residue was identified as N-acetylserine by using a rat liver acylamino acid-releasing enzyme. This enzyme specifically cleaved the N-acetylserylglutamyl peptide bond serine and the remaining peptide, which was now reactive to 1-dimethylamino-naphthalene-5-sulfonyl chloride. A comparison of the sequences of human and bovine "Activation peptide" revealed five amino acids replacements, Ser-3 to Thr; Gly-5 to Arg; Ile-14 to Val; Thr-18 to Asn, and Pro-26 to Leu. Another difference was the deletion of Leu-34 in the human peptide. Adsorption chromatography on a hydroxylapatite column in the presence of 0.1% sodium dodecyl sulfate was developed as a preparative procedure for the resolution of the two subunit polypeptides, a or a' chain and b chain, constituting the protein molecule of Factor XIII or Factor XIIIa. End group analyses on the isolated pure chains revealed that the structural change of Factor XIII during activation with thrombin occurs only in the N-terminal portion of the a chain, not in the N-terminal end of the b chain or in the C-terminal ends of the a and b chains. From these results, it was concluded that the activation of bovine plasma Factor XIII

  10. A Study of Bioactivity of Corn Peptides with Low Molecular Weight Ⅱ: Effect on Plasma Free Amino Acid Concentrations in Rats

    Institute of Scientific and Technical Information of China (English)

    XU Li; ZHANG Li-qiang; WU Xiao-xia; WANG Na; ZHANG Xue-zhong

    2003-01-01

    The effects of the ingestion of corn peptides with a low molecular weight(LMCP) prepared from zein on some plasma free amino acid concentrations in rats that had taken ethanol were investigated. LMCP(1.0 g/kg body weight) in 15% ethanol(10 mL/kg body weight) was given to Wister rats by intragastrical administration. The amino acid analysis showed that the concentrations of alanine, leucine, and proline in the plasma reached their maximum levels at 30 min for the LMCP-intake group. They are 582.39, 99.60 and 272.51 μg/L, respectively. But in the control group, the plasma free amino acid levels were not changed obviously. Therefore, LMCP could cause an increase in concentration of some free amino acids such as alanine, leucine and proline etc. in plasma of the rats that have taken ethanol.

  11. 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

  12. Synthesis and characterization of antigenic influenza A M2e protein peptide-poly(acrylic) acid bioconjugate and determination of toxicity in vitro.

    Science.gov (United States)

    Kilinc, Yasemin Budama; Akdeste, Zeynep Mustafaeva; Koc, Rabia Cakir; Bagirova, Melahat; Allahverdiyev, Adil

    2014-01-01

    The influenza A virus is a critical public health problem that causes epidemics and pandemics, and occurs widely all over the world. Various vaccines against the virus have not provided a solution to the problem. Different approaches, particularly M2e peptide-based vaccines, are available for developing universal vaccines against influenza A. However, it is important to select a suitable carrier to obtain an effective vaccine. Accordingly, studies on the usage of various carriers are ongoing. Particularly, polymer-based carriers have gained importance due to both drug delivery and adjuvant effects. Therefore, bioconjugate of the M2e protein peptide from the influenza A virus covalent bonded with poly(acrylic) acid was synthesized in our study for the first time. The characterization was performed using size-exclusion chromatography and fluorescence spectroscopy; subsequently, it was found that the bioconjugate of the examined lower doses (0.05 and 0.5 mg/ml) have no toxic effects on human cell lines. These results suggest that, in the future, the poly(acrylic) acid bioconjugate of the M2e peptide should be studied in vivo for universal vaccine development against the influenza A virus. PMID:25482080

  13. Repair of rat cranial bone defect by using bone morphogenetic protein-2-related peptide combined with microspheres composed of polylactic acid/polyglycolic acid copolymer and chitosan.

    Science.gov (United States)

    Li, Jingfeng; Jin, Lin; Wang, Mingbo; Zhu, Shaobo; Xu, Shuyun

    2015-07-08

    The effects of the transplanted bone morphogenetic protein-2 (BMP2) -related peptide P24 and rhBMP2 combined with poly(lactic-co-glycolic acid) (PLGA)/chitosan (CS) microspheres were investigated in promoting the repair of rat cranial bone defect. Forty white rats were selected and equally divided into four groups (group A: 1 μg of rhBMP2/PLGA/CS composite; group B: 3 mg of P24/PLGA/CS composite; group C: 0.5 μg of rhBMP2 + 1.5 mg of P24/PLGA/CS composite; group D: blank PLGA/CS material), and rat cranial bone defect models with a diameter of 5 mm were established. The materials were transplanted to the cranial bone defects. The animals were sacrificed on weeks 6 and 12 post-operation. Radiographic examinations (x-ray imaging and 3D CT scanning) and histological evaluations were performed. The repaired areas of cranial bone defects were measured, and the osteogenetic abilities of various materials were compared. Cranial histology, imaging, and repaired area measurements showed that the osteogenetic effects at two time points (weeks 6 and 12) in group C were better than those in groups A and B. The effects in groups A and B were similar. Group D achieved the worst repair effect of cranial bone defects, where a large number of fibrous connective tissues were observed. The PLGA/CS composite microspheres loaded with rhBMP2 and P24 had optimal concrescence and could mutually increase their osteogenesis capability. rhBMP2 + P24/PLGA/CS composite is a novel material for bone defect repair with stable activity to induce bone formation.

  14. Conformational Changes and Association of Membrane-Interacting Peptides in Myelin Membrane Models: A Case of the C-Terminal Peptide of Proteolipid Protein and the Antimicrobial Peptide Melittin.

    Science.gov (United States)

    Appadu, Ashtina; Jelokhani-Niaraki, Masoud; DeBruin, Lillian

    2015-11-25

    Model membranes composed of various lipid mixtures can segregate into liquid-ordered (Lo) and liquid-disordered (Ld) phases. In this study, lipid vesicles composed of mainly Lo or Ld phases as well as complex lipid systems representing the cytosolic leaflet of the myelin membrane were characterized by fluorescence resonance energy transfer with a donor/acceptor pair that preferentially partitioned into Lo or Ld phases, respectively. The fluidity of the lipid systems containing >30% cholesterol was modulated in the presence of the amphipathic peptide melittin. With all the studied lipid systems, melittin attained an α-helical conformation as determined by CD spectroscopy and attained varying degrees of membrane association and penetration as determined by intrinsic Trp fluorescence. The other protein domain utilized was a putative amphipathic helical peptide derived from the cytosolic C-terminal sequence of proteolipid protein (PLP) which is the most abundant protein in the myelin membrane. The C-terminal PLP peptide transitioned from a random coil to an α-helix in the presence of trifluoroethanol. Upon interacting with each of lipid vesicle system, the PLP peptide also folded into a helix; however, at high concentrations of the peptide with fluid lipid systems, associated helices transmuted into a β-sheet conformer. The membrane-associated aggregation of the cytosolic C-termini could be a mechanism by which the transmembrane PLP multimerizes in the myelin membrane. PMID:26561987

  15. Antimicrobial peptides: a review of how peptide structure impacts antimicrobial activity

    Science.gov (United States)

    Soares, Jason W.; Mello, Charlene M.

    2004-03-01

    Antimicrobial peptides (AMPs) have been discovered in insects, mammals, reptiles, and plants to protect against microbial infection. Many of these peptides have been isolated and studied exhaustively to decipher the molecular mechanisms that impart protection against infectious bacteria, fungi, and viruses. Unfortunately, the molecular mechanisms are still being debated within the scientific community but valuable clues have been obtained through structure/function relationship studies1. Biophysical studies have revealed that cecropins, isolated from insects and pigs, exhibit random structure in solution but undergo a conformational change to an amphipathic α-helix upon interaction with a membrane surface2. The lack of secondary structure in solution results in an extremely durable peptide able to survive exposure to high temperatures, organic solvents and incorporation into fibers and films without compromising antibacterial activity. Studies to better understand the antimicrobial action of cecropins and other AMPs have provided insight into the importance of peptide sequence and structure in antimicrobial activities. Therefore, enhancing our knowledge of how peptide structure imparts function may result in customized peptide sequences tailored for specific applications such as targeted cell delivery systems, novel antibiotics and food preservation additives. This review will summarize the current state of knowledge with respect to cell binding and antimicrobial activity of AMPs focusing primarily upon cecropins.

  16. Differential effect of arachidonic acid on the vasoactive intestinal peptide receptor/effector system in rat prostatic epithelium during sexual maturation.

    Science.gov (United States)

    Carmena, M J; Hueso, C; Solano, R M; Prieto, J C

    1992-01-01

    The effects of alterations in the membrane lipid environment on vasoactive intestinal peptide (VIP) binding and VIP-stimulated cyclic AMP accumulation have been analyzed by arachidonic acid treatment of prostatic epithelial cells from rats at puberty and maturity, two critical developmental periods with characteristic lipidic and androgenic statuses. Treating cells with 0.1 mM arachidonic acid for 15 min at 37 degrees C increased the affinity of VIP receptors and the potency of the neuropeptide (up to five times) in the formation of cyclic AMP at maturity, but not at puberty. The average plasma membrane fluidity (as measured by fluorescence polarization of diphenylhexatriene) remained unmodified after arachidonic acid treatment of cells. The modifications observed in mature rats were specific for the VIP receptor/effector system, since cyclic AMP stimulation by isoproterenol or forskolin was not affected by cell treatment with arachidonic acid. These results are compatible with the existence of a particular lipidic microdomain surrounding the VIP receptor in the cell membrane that would be altered by exposure to arachidonic acid (either directly or through conversion of arachidonic acid to its metabolites, as suggested by experiments on inhibition of the arachidonic acid cascade). This would make it possible for the activation of protein kinase C to phosphorylate VIP receptors in cells from mature rats, but not in those from pubertal animals with a very different membrane lipid composition (as suggested by the corresponding values of membrane fluidity and transition temperature).

  17. Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To investigate the effects of anti-sense peptide nucleic acids (PNAs) targeting Ki-67gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations (1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L). The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and 3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide (ASODNs)targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs (16.9±0.7) was significantly inhibited as compared with that of the control groups (28.6±0.4) (P<0.01). The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs (42.1±2.2)was significantly reduced when compared with that of the control groups (83.6±1.4) (P<0.01). Proliferation of 786-0 cells treated with anti-sense PNAs (20.7±1.5) was significantly inhibited as compared with that of the control groups (58.6±1.4) (P<0.01). The apoptosis rate of 786-0 cells treated with anti-sense PNAs (28.7±2.3) was significantly increased higher compared with that of the control groups (13.8±1.0) (P<0.01). From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

  18. Atrial natriuretic peptide attenuates inflammatory responses on oleic acid-induced acute lung injury model in rats

    Institute of Scientific and Technical Information of China (English)

    ZHU Yao-bin; ZHANG Yan-bo; LIU Dong-hai; LI Xiao-feng; LIU Ai-jun; FAN Xiang-ming; QIAO Chen-hui

    2013-01-01

    Background An inflammatory response leading to organ dysfunction and failure continues to be a major problem after injury in many clinical conditions such as sepsis,severe burns,and trauma.It is increasingly recognized that atrial natriuretic peptide (ANP) possesses a broad range of biological activities,including effects on endothelial function and inflammation.A recent study has revealed that ANP exerts anti-inflammatory effects.In this study we tested the effects of human ANP (hANP) on lung injury in a model of oleic acid (OA)-induced acute lung injury (ALl) in rats.Methods Rats were randomly assigned to three groups (n=6 in each group).Rats in the control group received a 0.9% solution of NaCl (1 ml.kg1.h-1) by continuous intravenous infusion,after 30 minutes a 0.9% solution of NaCl (1 ml/kg) was injected intravenously,and then the 0.9% NaCl infusion was restarted.Rats in the ALl group received a 0.9% NaCl solution (1 ml·kg-1·h-1) intravenous infusion,after 30 minutes OA was injected intravenously (0.1 ml/kg),and then the 0.9% NaCl infusion was restarted.Rats in the hANP-treated ALI group received a hANP (0.1μg·kg-1·min-1) infusion,after 30 minutes OA was injected intravenously (0.1 ml/kg),and then the hANP infusion was restarted.The anti-inflammation effects of hANP were evaluated by histological examination and determination of serum cytokine levels.Results Serum intedeukin (IL)-1β,IL-6,IL-10 and tumor necrosis factor (TNF) α were increased in the ALI group at six hours.The levels of all factors were significantly lower in the hANP treated rats (P <0.005).Similarly,levels of IL-1β,IL-6,IL-10 and TNF-α were higher in the lung tissue in the ALI group at six hours.hANP treatment significantly reduced the levels of these factors in the lungs (P <0.005).Histological examination revealed marked reduction in interstitial congestion,edema,and inflammation.Conclusion hANP can attenuate inflammation in an OA-induced lung injury in rat model.

  19. Folic Acid-Targeted and Cell Penetrating Peptide-Mediated Theranostic Nanoplatform for High-Efficiency Tri-Modal Imaging-Guided Synergistic Anticancer Phototherapy.

    Science.gov (United States)

    Li, Na; Li, Tingting; Liu, Chen; Ye, Shiyi; Liang, Jiangong; Han, Heyou

    2016-05-01

    A novel nanomaterial with precisely-defined size and shape, biocompatible composition, and excellent stability, which can integrate multi modal targeted imaging and therapy into a single system for visualized therapeutics, has recently attracted significant research interest. Here, we developed a multifunctional nanoplatform based on silica-coated 4-mercaptobenzoic acid-modified gold nanorods (Au NRs) decorated with gold nanoclusters rich in the photosensitizer Ce6 (Au-Ce6 NCs). The nanoparticles also comprised folic acid and cell penetrating peptide molecules anchored on the surface, obtaining the Au@SiO2@Au-cell penetrating peptide nanocomposite. The Au-Ce6 NCs enhanced the photophysical stability, provided numerous bonding sites and offered a large surface-area and interior space to achieve a high drug loading efficiency (up to 55%). The anchored folic acid and cell penetrating peptide synergistically enhanced the efficiency of uptake of nanocomposites by HeLa cells (up to 70.7%) and improved therapeutic efficacy. The nanocomposite also has good water-solubility, excellent biocompatibility, and long-term stability against illumination and exposure to pH 3-12, thus facilitating their bioapplications in cancer theranostics. Here, the nanocomposite was established for high-resolution and noninvasive tri-modal surface-enhanced Raman spectrum/dark-field/fluorescence imaging-guided high-efficiency synergistic photodynamic/photothermal therapy of cancer. Our studies demonstrate that the multifunctional nanocomposite has the potential as a novel and sensitive contrast agent for complementary and synergistic theranostics in the clinic. PMID:27305812

  20. Effect of pH, urea, peptide length, and neighboring amino acids on alanine alpha-proton random coil chemical shifts.

    Science.gov (United States)

    Carlisle, Elizabeth A; Holder, Jessica L; Maranda, Abby M; de Alwis, Adamberage R; Selkie, Ellen L; McKay, Sonya L

    2007-01-01

    Accurate random coil alpha-proton chemical shift values are essential for precise protein structure analysis using chemical shift index (CSI) calculations. The current study determines the chemical shift effects of pH, urea, peptide length and neighboring amino acids on the alpha-proton of Ala using model peptides of the general sequence GnXaaAYaaGn, where Xaa and Yaa are Leu, Val, Phe, Tyr, His, Trp or Pro, and n = 1-3. Changes in pH (2-6), urea (0-1M), and peptide length (n = 1-3) had no effect on Ala alpha-proton chemical shifts. Denaturing concentrations of urea (8M) caused significant downfield shifts (0.10 +/- 0.01 ppm) relative to an external DSS reference. Neighboring aliphatic residues (Leu, Val) had no effect, whereas aromatic amino acids (Phe, Tyr, His and Trp) and Pro caused significant shifts in the alanine alpha-proton, with the extent of the shifts dependent on the nature and position of the amino acid. Smaller aromatic residues (Phe, Tyr, His) caused larger shift effects when present in the C-terminal position (approximately 0.10 vs. 0.05 ppm N-terminal), and the larger aromatic tryptophan caused greater effects in the N-terminal position (0.15 ppm vs. 0.10 C-terminal). Proline affected both significant upfield (0.06 ppm, N-terminal) and downfield (0.25 ppm, C-terminal) chemical shifts. These new Ala correction factors detail the magnitude and range of variation in environmental chemical shift effects, in addition to providing insight into the molecular level interactions that govern protein folding.

  1. Development of a platform for nasal delivery of peptides and vaccines using powder carriers based on starch/poly(acrylic) acid

    OpenAIRE

    Coucke, Delphine

    2009-01-01

    The interest in and importance of systemic drug delivery via the nasal route have expanded in recent decades since nasal administration offers an interesting alternative for the conventional oral and parenteral drug delivery routes. In this thesis, the nasal route was selected as an alternative route for peptide and vaccine delivery. A spray-dried combination of maize starch (Amioca®) and a cross-linked acrylic acid-based polymer (Carbopol® 974P) was used as powder carrier to amplify the ...

  2. Localization of an O-glycosylated site in the recombinant barley alpha-amylase 1 produced in yeast and correction of the amino acid sequence using matrix-assisted laser desorption/ionization mass spectrometry of peptide mixtures

    DEFF Research Database (Denmark)

    Andersen, Jens S.; Søgaard, M; Svensson, B;

    1994-01-01

    , and analyzed directly by MALDI-MS. Based on the three mass spectrometric peptide maps, an error in the sequence deduced from cDNA, resulting in a mass difference of 28 Da, was located to a sequence stretch of 5 amino acid residues; furthermore, a dihexose substituent was identified on Thr410. Subsequent Edman...... degradation of two selected peptides isolated from the endoproteinase Lys-C digest corrected the sequence to be Val instead of Ala in position 284 and confirmed the O-glycosylation. These results demonstrate that the direct peptide mixture analysis by MALDI-MS is a rapid and sensitive method for protein......Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of peptide mixtures was used to characterize recombinant barley alpha-amylase 1, produced in yeast. Three peptide mixtures were generated by cleavage with CNBr, digestion with endoproteinase Lys-C and Asp-N, respectively...

  3. ANTIMICROBIAL PEPTIDES: AN EFFECTIVE ALTERNATIVE FOR ANTIBIOTIC THERAPY

    Directory of Open Access Journals (Sweden)

    KK PULICHERLA

    2013-01-01

    Full Text Available Extensive use of classical antibiotics has led to the growing emergence of many resistant strains of pathogenic bacteria. Evidence has suggested that cationic antimicrobial peptides (AMP’s are of greatest potential to represent a new class of antibiotics. These peptides have a good scope in current antibiotic research. During the past two decades several AMPs have been isolated from a wide variety of animals (both vertebrates and invertebrates, and plants as well as from bacteria and fungi. These are relatively small (<10kDa, cationic and amphipathic peptides of variable length, sequence and structure. These peptides exhibit broad-spectrum activity against a wide range of microorganisms including gram-positive and gram-negative bacteria, protozoa, yeast, fungi and viruses. Most of these peptides are believed to act by disrupting the plasma membrane leading to the lysis of the cell. Antimicrobial peptides encompass a wide variety of structural motifs such as α -helical peptides, β -sheet peptides, looped peptides and extended peptides. Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs in recombinant bacterial expression systems which were produced by cloning. This article aims to review in brief the sources of antimicrobial peptides, diversity in structural features, mode of action, production strategies and insight into the current data on their antimicrobial activity followed by a brief comment on the peptides that have entered clinical trials.

  4. The intrinsic antimicrobial activity of citric acid-coated manganese ferrite nanoparticles is enhanced after conjugation with the antifungal peptide Cm-p5

    Science.gov (United States)

    Lopez-Abarrategui, Carlos; Figueroa-Espi, Viviana; Lugo-Alvarez, Maria B; Pereira, Caroline D; Garay, Hilda; Barbosa, João ARG; Falcão, Rosana; Jiménez-Hernández, Linnavel; Estévez-Hernández, Osvaldo; Reguera, Edilso; Franco, Octavio L; Dias, Simoni C; Otero-Gonzalez, Anselmo J

    2016-01-01

    Diseases caused by bacterial and fungal pathogens are among the major health problems in the world. Newer antimicrobial therapies based on novel molecules urgently need to be developed, and this includes the antimicrobial peptides. In spite of the potential of antimicrobial peptides, very few of them were able to be successfully developed into therapeutics. The major problems they present are molecule stability, toxicity in host cells, and production costs. A novel strategy to overcome these obstacles is conjugation to nanomaterial preparations. The antimicrobial activity of different types of nanoparticles has been previously demonstrated. Specifically, magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Finally, an antifungal peptide (Cm-p5) from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae) was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity. PMID:27563243

  5. The intrinsic antimicrobial activity of citric acid-coated manganese ferrite nanoparticles is enhanced after conjugation with the antifungal peptide Cm-p5.

    Science.gov (United States)

    Lopez-Abarrategui, Carlos; Figueroa-Espi, Viviana; Lugo-Alvarez, Maria B; Pereira, Caroline D; Garay, Hilda; Barbosa, João Arg; Falcão, Rosana; Jiménez-Hernández, Linnavel; Estévez-Hernández, Osvaldo; Reguera, Edilso; Franco, Octavio L; Dias, Simoni C; Otero-Gonzalez, Anselmo J

    2016-01-01

    Diseases caused by bacterial and fungal pathogens are among the major health problems in the world. Newer antimicrobial therapies based on novel molecules urgently need to be developed, and this includes the antimicrobial peptides. In spite of the potential of antimicrobial peptides, very few of them were able to be successfully developed into therapeutics. The major problems they present are molecule stability, toxicity in host cells, and production costs. A novel strategy to overcome these obstacles is conjugation to nanomaterial preparations. The antimicrobial activity of different types of nanoparticles has been previously demonstrated. Specifically, magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Finally, an antifungal peptide (Cm-p5) from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae) was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity. PMID:27563243

  6. Moving Away from the Reference Genome: Evaluating a Peptide Sequencing Tagging Approach for Single Amino Acid Polymorphism Identifications in the Genus Populus

    Energy Technology Data Exchange (ETDEWEB)

    Abraham, Paul E [ORNL; Adams, Rachel M [ORNL; Tuskan, Gerald A [ORNL; Hettich, Robert {Bob} L [ORNL

    2013-01-01

    The genetic diversity across natural populations of the model organism, Populus, is extensive, containing a single nucleotide polymorphism roughly every 200 base pairs. When deviations from the reference genome occur in coding regions, they can impact protein sequences. Rather than relying on a static reference database to profile protein expression, we employed a peptide sequence tagging (PST) approach capable of decoding the plasticity of the Populus proteome. Using shotgun proteomics data from two genotypes of P. trichocarpa, a tag-based approach enabled the detection of 6,653 unexpected sequence variants. Through manual validation, our study investigated how the most abundant chemical modification (methionine oxidation) could masquerade as a sequence variant (AlaSer) when few site-determining ions existed. In fact, precise localization of an oxidation site for peptides with more than one potential placement was indeterminate for 70% of the MS/MS spectra. We demonstrate that additional fragment ions made available by high energy collisional dissociation enhances the robustness of the peptide sequence tagging approach (81% of oxidation events could be exclusively localized to a methionine). We are confident that augmenting fragmentation processes for a PST approach will further improve the identification of single amino acid polymorphism in Populus and potentially other species as well.

  7. The intrinsic antimicrobial activity of citric acid-coated manganese ferrite nanoparticles is enhanced after conjugation with the antifungal peptide Cm-p5

    Science.gov (United States)

    Lopez-Abarrategui, Carlos; Figueroa-Espi, Viviana; Lugo-Alvarez, Maria B; Pereira, Caroline D; Garay, Hilda; Barbosa, João ARG; Falcão, Rosana; Jiménez-Hernández, Linnavel; Estévez-Hernández, Osvaldo; Reguera, Edilso; Franco, Octavio L; Dias, Simoni C; Otero-Gonzalez, Anselmo J

    2016-01-01

    Diseases caused by bacterial and fungal pathogens are among the major health problems in the world. Newer antimicrobial therapies based on novel molecules urgently need to be developed, and this includes the antimicrobial peptides. In spite of the potential of antimicrobial peptides, very few of them were able to be successfully developed into therapeutics. The major problems they present are molecule stability, toxicity in host cells, and production costs. A novel strategy to overcome these obstacles is conjugation to nanomaterial preparations. The antimicrobial activity of different types of nanoparticles has been previously demonstrated. Specifically, magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Finally, an antifungal peptide (Cm-p5) from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae) was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity.

  8. Cyclisation Increases the Stability of the Sea Anemone Peptide APETx2 but Decreases Its Activity at Acid-Sensing Ion Channel 3

    Directory of Open Access Journals (Sweden)

    Lachlan D. Rash

    2012-07-01

    Full Text Available APETx2 is a peptide isolated from the sea anemone Anthopleura elegantissima. It is the most potent and selective inhibitor of acid-sensing ion channel 3 (ASIC3 and it is currently in preclinical studies as a novel analgesic for the treatment of chronic inflammatory pain. As a peptide it faces many challenges in the drug development process, including the potential lack of stability often associated with therapeutic peptides. In this study we determined the susceptibility of wild-type APETx2 to trypsin and pepsin and tested the applicability of backbone cyclisation as a strategy to improve its resistance to enzymatic degradation. Cyclisation with either a six-, seven- or eight-residue linker vastly improved the protease resistance of APETx2 but substantially decreased its potency against ASIC3. This suggests that either the N- or C-terminus of APETx2 is involved in its interaction with the channel, which we confirmed by making N- and C-terminal truncations. Truncation of either terminus, but especially the N-terminus, has detrimental effects on the ability of APETx2 to inhibit ASIC3. The current work indicates that cyclisation is unlikely to be a suitable strategy for stabilising APETx2, unless linkers can be engineered that do not interfere with binding to ASIC3.

  9. The Design and Construction of K11: A Novel α-Helical Antimicrobial Peptide

    Directory of Open Access Journals (Sweden)

    Huang Jin-Jiang

    2012-01-01

    Full Text Available Amphipathic α-helical antimicrobial peptides comprise a class of broad-spectrum agents that are used against pathogens. We designed a series of antimicrobial peptides, CP-P (KWKSFIKKLTSKFLHLAKKF and its derivatives, and determined their minimum inhibitory concentrations (MICs against Pseudomonas aeruginosa, their minimum hemolytic concentrations (MHCs for human erythrocytes, and the Therapeutic Index (MHC/MIC ratio. We selected the derivative peptide K11, which had the highest therapeutic index (320 among the tested peptides, to determine the MICs against Gram-positive and Gram-negative bacteria and 22 clinical isolates including Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Klebsiella pneumonia. K11 exhibited low MICs (less than 10 μg/mL and broad-spectrum antimicrobial activity, especially against clinically isolated drug-resistant pathogens. Therefore, these results indicate that K11 is a promising candidate antimicrobial peptide for further studies.

  10. Novel and Convenient Method for the Preparation of Phosphonate Peptides and Phosphonamidate Peptides

    Institute of Scientific and Technical Information of China (English)

    XU Jia-Xi; FU Nan-Yan; GAO Yuan-He; ZHNAG Qi-Han; DUAN Li-Fang

    2003-01-01

    @@ Phosphonate and phosphonamidate peptides are phosphorus analogues of natural peptides. They have been great used as stable mimetics of tetrahedral transition states as enzyme inhibitors and as haptens for catalytic antibody research in recent years. Although several methods are available for the preparation of phosphonate peptides and phosphonamidate peptides, all of them use phosphonic acid derivatives as starting materials. The overall yields from the synthesis of phosphonic acid derivatives to desired peptides are not satisfactory in most cases.

  11. An amphipathic alpha-helix controls multiple roles of brome mosaic virus protein 1a in RNA replication complex assembly and function.

    Directory of Open Access Journals (Sweden)

    Ling Liu

    2009-03-01

    Full Text Available Brome mosaic virus (BMV protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase (2a(Pol and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic alpha-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a-ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a-membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2a(Pol accumulation. The second class of helix A mutations not only maintains efficient 1a-membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2a(Pol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.

  12. 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).

    Science.gov (United States)

    Choi, Chang Bon; Kwon, Hana; Kim, Sung Il; Yang, Un Mok; Lee, Ju Hwan; Park, Eun Kyu

    2016-02-01

    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. PMID:26732444

  13. Positively charged templates for labeling internalizing antibodies: comparison of N-succinimidyl 5-iodo-3-pyridinecarboxylate and the D-amino acid peptide KRYRR

    Energy Technology Data Exchange (ETDEWEB)

    Foulon, Catherine F.; Welsh, Philip C.; Bigner, Darell D.; Zalutsky, Michael R. E-mail: zalut001@mc.duke.edu

    2001-10-01

    Receptor-mediated internalization of monoclonal antibodies (mAbs), such as those specific for the epidermal growth factor receptor variant III (EGFRvIII), can lead to rapid loss of radioactivity from the target cell. In the current study, the anti-EGFRvIII mAb L8A4 was radioiodinated using two methods -N-succinimidyl 5-iodo-3-pyridinecarboxylate (SIPC) and via a D-amino acid peptide LysArgTyrArgArg (D-KRYRR). Paired-label internalization assays performed on EGFRvIII-expressing U87{delta}EGFR cells in vitro demonstrated that labeling L8A4 using D-KRYRR resulted in significantly higher retention of radioiodine in the intracellular compartment. In athymic mice with D256 human glioma xenografts, tumor uptake was similar for both labeling methods through 24 hr. However, an up to fourfold higher tumor retention was observed for mAb labeled with the D-amino acid peptide at later time points. Radiation absorbed dose calculations based on these biodistribution data indicated that L8A4 labeled using D-KRYRR exhibited better tumor-to-normal-organ radiation dose ratios, suggesting that this labeling method may be of particular value for labeling internalizing mAbs.

  14. Bio-Inspired Nitrile Hydration by Peptidic Ligands Based on L-Cysteine, L-Methionine or L-Penicillamine and Pyridine-2,6-dicarboxylic Acid

    Directory of Open Access Journals (Sweden)

    Cillian Byrne

    2014-12-01

    Full Text Available Nitrile hydratase (NHase, EC 4.2.1.84 is a metalloenzyme which catalyses the conversion of nitriles to amides. The high efficiency and broad substrate range of NHase have led to the successful application of this enzyme as a biocatalyst in the industrial syntheses of acrylamide and nicotinamide and in the bioremediation of nitrile waste. Crystal structures of both cobalt(III- and iron(III-dependent NHases reveal an unusual metal binding motif made up from six sequential amino acids and comprising two amide nitrogens from the peptide backbone and three cysteine-derived sulfur ligands, each at a different oxidation state (thiolate, sulfenate and sulfinate. Based on the active site geometry revealed by these crystal structures, we have designed a series of small-molecule ligands which integrate essential features of the NHase metal binding motif into a readily accessible peptide environment. We report the synthesis of ligands based on a pyridine-2,6-dicarboxylic acid scaffold and L-cysteine, L-S-methylcysteine, L-methionine or L-penicillamine. These ligands have been combined with cobalt(III and iron(III and tested as catalysts for biomimetic nitrile hydration. The highest levels of activity are observed with the L-penicillamine ligand which, in combination with cobalt(III, converts acetonitrile to acetamide at 1.25 turnovers and benzonitrile to benzamide at 1.20 turnovers.

  15. Inhibition of amyloid fibril formation of human amylin by N-alkylated amino acid and alpha-hydroxy acid residue containing peptides

    NARCIS (Netherlands)

    Rijkers, DTS; Hoppener, JWM; Posthuma, G; Lips, CJM; Liskamp, RMJ

    2002-01-01

    Amyloid deposits are formed as a result of uncontrolled aggregation of (poly)peptides or proteins. Today several diseases are known, for example Alzheimer's disease, Creutzfeldt-Jakob disease, mad cow disease, in which amyloid formation is involved. Amyloid fibrils are large aggregates of beta-pleat

  16. Loop VIII/IX of the Na+-citrate transporter CitS of Klebsiella pneumoniae folds into an amphipathic surface helix

    NARCIS (Netherlands)

    Sobczak, [No Value; Lolkema, JS; Sobczak, Iwona

    2005-01-01

    The sodium ion-dependent citrate transporter CitS of Klebsiella pneumoniae is a member of, the 2-hydroxycarboxylate transporter (2HCT) family whose members transport divalent citrate in symport with two sodium ions. Profiles of the hydrophobic moment suggested the presence of an amphipathic helical

  17. The role of MscL amphipathic N terminus indicates a blueprint for bilayer-mediated gating of mechanosensitive channels.

    Science.gov (United States)

    Bavi, Navid; Cortes, D Marien; Cox, Charles D; Rohde, Paul R; Liu, Weihong; Deitmer, Joachim W; Bavi, Omid; Strop, Pavel; Hill, Adam P; Rees, Douglas; Corry, Ben; Perozo, Eduardo; Martinac, Boris

    2016-01-01

    The bacterial mechanosensitive channel MscL gates in response to membrane tension as a result of mechanical force transmitted directly to the channel from the lipid bilayer. MscL represents an excellent model system to study the basic biophysical principles of mechanosensory transduction. However, understanding of the essential structural components that transduce bilayer tension into channel gating remains incomplete. Here using multiple experimental and computational approaches, we demonstrate that the amphipathic N-terminal helix of MscL acts as a crucial structural element during tension-induced gating, both stabilizing the closed state and coupling the channel to the membrane. We propose that this may also represent a common principle in the gating cycle of unrelated mechanosensitive ion channels, allowing the coupling of channel conformation to membrane dynamics. PMID:27329693

  18. Induction of Porcine Host Defense Peptide Gene Expression by Short-Chain Fatty Acids and Their Analogs

    OpenAIRE

    Zeng, Xiangfang; Sunkara, Lakshmi T; Jiang, Weiyu; Bible, Megan; Carter, Scott; Ma, Xi; Qiao, Shiyan; Zhang, Guolong

    2013-01-01

    Dietary modulation of the synthesis of endogenous host defense peptides (HDPs) represents a novel antimicrobial approach for disease control and prevention, particularly against antibiotic-resistant infections. However, HDP regulation by dietary compounds such as butyrate is species-dependent. To examine whether butyrate could induce HDP expression in pigs, we evaluated the expressions of a panel of porcine HDPs in IPEC-J2 intestinal epithelial cells, 3D4/31 macrophages, and primary monocytes...

  19. Amyloid-Beta Peptide, Oxidative Stress and Inflammation in Alzheimer's Disease: Potential Neuroprotective Effects of Omega-3 Polyunsaturated Fatty Acids

    OpenAIRE

    S. C. Dyall

    2010-01-01

    Alzheimer's disease is the most common form of dementia in the elderly and is a progressive neurodegenerative disorder characterised by a decline in cognitive function and also profound alterations in mood and behaviour. The pathology of the disease is characterised by the presence of extracellular amyloid peptide deposits and intracellular neurofibrillary tangles in the brain. Although many hypotheses have been put forward for the aetiology of the disease, increased inflammation and oxidativ...

  20. A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids.

    Science.gov (United States)

    Maity, Debabrata; Jiang, Juanjuan; Ehlers, Martin; Wu, Junchen; Schmuck, Carsten

    2016-05-01

    A cationic molecular peptide beacon NAP1 functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences. NAP1 most likely binds in a folded form into the minor groove of ds-DNA, which results in a remarkable change in its fluorescence properties. As NAP1 exhibits quite low cytotoxicity, it can also be used for imaging of nuclear DNA in cells. PMID:27071707

  1. A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids.

    Science.gov (United States)

    Maity, Debabrata; Jiang, Juanjuan; Ehlers, Martin; Wu, Junchen; Schmuck, Carsten

    2016-05-01

    A cationic molecular peptide beacon NAP1 functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences. NAP1 most likely binds in a folded form into the minor groove of ds-DNA, which results in a remarkable change in its fluorescence properties. As NAP1 exhibits quite low cytotoxicity, it can also be used for imaging of nuclear DNA in cells.

  2. Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Lactobacillus spp. in milk samples

    OpenAIRE

    Machado, António; Almeida, Carina; Carvalho, Ana; Boyen, Filip; Haesebrouck, Freddy; L. R. Rodrigues; Cerca, Nuno; Azevedo, N. F.

    2013-01-01

    Lactobacillus species constitute one of the dominant and beneficial bacteria in our body and are used in developed countries as a microbial adjuvant. Identification of these probiotic bacteria is traditionally performed by culture-based techniques. However, such methods are very time-consuming and can give inaccurate results, especially when Lactobacillus is present in mixed bacterial complex communities. Our study aimed to accurately identify Lactobacillus spp. using a novel Peptide Nucleic ...

  3. 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.

  4. Affinity-Based Screening of Tetravalent Peptides Identifies Subtype-Selective Neutralizers of Shiga Toxin 2d, a Highly Virulent Subtype, by Targeting a Unique Amino Acid Involved in Its Receptor Recognition.

    Science.gov (United States)

    Mitsui, Takaaki; Watanabe-Takahashi, Miho; Shimizu, Eiko; Zhang, Baihao; Funamoto, Satoru; Yamasaki, Shinji; Nishikawa, Kiyotaka

    2016-09-01

    Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), can be classified into two subgroups, Stx1 and Stx2, each consisting of various closely related subtypes. Stx2 subtypes Stx2a and Stx2d are highly virulent and linked with serious human disorders, such as acute encephalopathy and hemolytic-uremic syndrome. Through affinity-based screening of a tetravalent peptide library, we previously developed peptide neutralizers of Stx2a in which the structure was optimized to bind to the B-subunit pentamer. In this study, we identified Stx2d-selective neutralizers by targeting Asn16 of the B subunit, an amino acid unique to Stx2d that plays an essential role in receptor binding. We synthesized a series of tetravalent peptides on a cellulose membrane in which the core structure was exactly the same as that of peptides in the tetravalent library. A total of nine candidate motifs were selected to synthesize tetravalent forms of the peptides by screening two series of the tetravalent peptides. Five of the tetravalent peptides effectively inhibited the cytotoxicity of Stx2a and Stx2d, and notably, two of the peptides selectively inhibited Stx2d. These two tetravalent peptides bound to the Stx2d B subunit with high affinity dependent on Asn16. The mechanism of binding to the Stx2d B subunit differed from that of binding to Stx2a in that the peptides covered a relatively wide region of the receptor-binding surface. Thus, this highly optimized screening technique enables the development of subtype-selective neutralizers, which may lead to more sophisticated treatments of infections by Stx-producing EHEC. PMID:27382021

  5. Dicyclopropylmethyl peptide backbone protectant.

    Science.gov (United States)

    Carpino, Louis A; Nasr, Khaled; Abdel-Maksoud, Adel Ali; El-Faham, Ayman; Ionescu, Dumitru; Henklein, Peter; Wenschuh, Holger; Beyermann, Michael; Krause, Eberhard; Bienert, Michael

    2009-08-20

    The N-dicyclopropylmethyl (Dcpm) residue, introduced into amino acids via reaction of dicyclopropylmethanimine hydrochloride with an amino acid ester followed by sodium cyanoborohydride or triacetoxyborohydride reduction, can be used as an amide bond protectant for peptide synthesis. Examples which demonstrate the amelioration of aggregation effects include syntheses of the alanine decapeptide and the prion peptide (106-126). Avoidance of cyclization to the aminosuccinimide followed substitution of Fmoc-(Dcpm)Gly-OH for Fmoc-Gly-OH in the assembly of sequences containing the sensitive Asp-Gly unit.

  6. Molecular dynamics simulations of the helical antimicrobial peptide ovispirin-1 in a zwitterionic dodecylphosphocholine micelle

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Kaznessis, Yiannis N

    2005-01-01

    We have carried out a 40-ns all-atom molecular dynamics simulation of the helical antimicrobial peptide ovispirin-1 (OVIS) in a zwitterionic diphosphocholine (DPC) micelle. The DPC micelle serves as an economical and effective model for a cellular membrane owing to the presence of a choline...... headgroup, which resembles those of membrane phospholipids. OVIS, which was initially placed along a micelle diameter, diffuses out to the water-DPC interface, and the simulation stabilizes to an interface-bound steady state in 40 ns. The helical content of the peptide marginally increases in the process....... The final conformation, orientation, and the structure of OVIS are in excellent agreement with the experimentally observed properties of the peptide in the presence of lipid bilayers composed of 75% zwitterionic lipids. The amphipathic peptide binds to the micelle with its hydrophobic face buried...

  7. Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

    Science.gov (United States)

    Oehlke, J; Birth, P; Klauschenz, E; Wiesner, B; Beyermann, M; Oksche, A; Bienert, M

    2002-08-01

    The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

  8. 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 aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid...... antioxidant activity in these fractions.......The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid...... 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...

  9. Role of peptide bond in the realization of biological activity of short peptides.

    Science.gov (United States)

    Khavinson, V Kh; Tarnovskaya, S I; Lin'kova, N S; Chervyakova, N A; Nichik, T E; Elashkina, E V; Chalisova, N I

    2015-02-01

    We performed a comparative analysis of biological activity of Lys-Glu peptide and its amino acid constituents. It was established that Lys-Glu stimulated proliferation of splenic cells in organotypic culture, while the mixture of glutamic acid and lysine inhibited culture growth. Using the method of molecular docking, we showed that glutamic acid, lysine, and Lys-Glu peptide can interact with different DNA sequences. The energy of interaction and the most beneficial localization of glutamic acid, lysine, and Lys-Glu peptide in DNA molecule was calculated. We demonstrated the interaction of the peptide and amino acids with DNA along the minor groove. The energy of DNA interaction with the peptide is higher than with individual amino acids. The peptide bonds increase the interaction of Lys-Glu peptide with DNA, which potentiates the biological effect on cell proliferation in organotypic culture of splenic cells.

  10. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    DEFF Research Database (Denmark)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S;

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...... cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor...

  11. Inhibitory effect of the carnosine-gallic acid synthetic peptide on MMP-2 and MMP-9 in human fibrosarcoma HT1080 cells.

    Science.gov (United States)

    Kim, Sung-Rae; Eom, Tae-Kil; Byun, Hee-Guk

    2014-09-01

    Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that degrade extracellular matrix components and play important roles in a variety of biological and pathological processes such as malignant tumor metastasis and invasion. In this study, we constructed carnosine-gallic acid peptide (CGP) to identify a better MMP inhibitor than carnosine. The inhibitory effects of CGP on MMP-2 and MMP-9 were investigated in the human fibrosarcoma (HT1080) cell line. As a result, CGP significantly decreased MMP-2 and MMP-9 expression levels without a cytotoxic effect. Moreover, CGP may inhibit migration and invasion in HT1080 cells through the urokinase plasminogen activator (uPA)-uPA receptor signaling pathways to inhibit MMP-2 and MMP-9. Based on these results, it appears that CGP may play an important role in preventing and treating several MMP-2 and MMP-9-mediated health problems such as metastasis. PMID:24956509

  12. Synthesis of Biotin Linkers with the Activated Triple Bond Donor [p-(N-propynoylaminotoluic Acid] (PATA for Efficient Biotinylation of Peptides and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Martina Jezowska

    2012-11-01

    Full Text Available Biotin is an important molecule for modern biological studies including, e.g., cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition, commonly referred to as “click chemistry”. As we reported recently, the activated triple bond donor p-(N-propynoylaminotoluic acid (PATA gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.

  13. Tuning hydrophobicity of highly cationic tetradecameric Gramicidin S analogues using adamantane amino acids

    NARCIS (Netherlands)

    Knijnenburg, A.D.; Kapoerchan, V.V.; Spalburg, E.; Neeling, A.J. de; Mars-Groenendijk, R.H.; Noort, D.; Marel, G.A. van der; Overkleeft, H.S.; Overhand, M.

    2010-01-01

    Ring extended Gramicidin S analogues containing adamantane amino acids and six cationic residues were designed and evaluated. Systematic replacement of the hydrophobic residues with adamantane amino acids resulted in a small set of compounds with varying amphipathic character. It was found that the

  14. Impact of single-dose application of TGF-β, copper peptide, stanozolol and ascorbic acid in hydrogel on midline laparatomy wound healing in a diabetic mouse model.

    Science.gov (United States)

    Konerding, Moritz A; Ziebart, Thomas; Wolloscheck, Tanja; Wellmann, Axel; Ackermann, Maximilian

    2012-08-01

    Despite numerous advances and improvements in surgical techniques the incidence of incisional hernias after laparotomy remains high. The aim of this study was to investigate possible effects of single application of ascorbic acid, stanozolol, a synthetic anabolic steroid, copper peptide and transforming growth factor-β (TGF-β) on laparotomy wound healing in an incisional wound model in diabetic mice. After diabetes induction with streptozotozin in Balb-c mice, midline laparatomies were carried out. Closure of the linea alba was followed by single-dose application of the agents dissolved in a hydrogel before skin closure. The functional outcome was assessed in terms of maximum tensile strength. In addition, vessel densities, collagen contents and proliferation, were measured. The breaking strength of the skin 14 days after surgery was significantly higher in ascorbic acid (ΑΑ)-treated incisional wounds, whereas the other agents did not show a significantly better functional outcome. No significant differences were seen in vessel densities. Collagen type III contents was higher in the ΑΑ-treated animals, whereas the percentage of Ki67-positive nuclei was lower compared to the other groups. These data underline the positive effect of topically applied ascorbic acid in wound healing. PMID:22614259

  15. Defect formation of lytic peptides in lipid membranes and their influence on the thermodynamic properties of the pore environment

    CERN Document Server

    Oliynyk, V; Heimburg, T; Oliynyk, Vitaliy; Kaatze, Udo; Heimburg, Thomas

    2006-01-01

    We present an experimental study of the pore formation processes of small amphipathic peptides in model phosphocholine lipid membranes. We used atomic force microscopy to characterize the spatial organization and structure of alamethicin- and melittin- induced defects in lipid bilayer membranes and the influence of the peptide on local membrane properties. Alamethicin induced holes in gel DPPC membranes were directly visualized at different peptide concentrations. We found that the thermodynamic state of lipids in gel membranes can be influenced by the presence of alamethicin such that nanoscopic domains of fluid lipids form close to the peptide pores, and that the elastic constants of the membrane are altered in their vicinity. Melittin-induced holes were visualized in DPPC and DLPC membranes at room temperature in order to study the influence of the membrane state on the peptide induced hole formation. Also differential scanning calorimetry was used to investigate the effect of alamethicin on the lipid memb...

  16. Structural and functional characterization of hBD-1(Ser35), a peptide deduced from a DEFB1 polymorphism.

    Science.gov (United States)

    Circo, Raffaella; Skerlavaj, Barbara; Gennaro, Renato; Amoroso, Antonio; Zanetti, Margherita

    2002-04-26

    beta-Defensins are mammalian antimicrobial peptides that share a unique disulfide-bonding motif of six conserved cysteines. An intragenic polymorphism of the DEFB1 gene that changes a highly conserved Cys to Ser in the peptide coding region has recently been described. The deduced peptide cannot form three disulfide bonds, as one of the cysteines is unpaired. We have determined the cysteine connectivities of a corresponding synthetic hBD-1(Ser35) peptide, investigated the structure by circular dichroism spectroscopy, and assayed the in vitro antimicrobial activity. Despite a different arrangement of the disulfides, hBD-1(Ser35) proved as active as hBD-1 against the microorganisms tested. This activity likely depends on the ability of hBD-1(Ser35) to adopt an amphipathic conformation in hydrophobic environment, similar to the wild type peptide, as suggested by CD spectroscopy. PMID:12054642

  17. Noninvasive tagging of proteins with an inorganic chromophore. Selectivity of chloro(terpyridine)platinum(II) toward amino acids, peptides, and cytochromes c

    Energy Technology Data Exchange (ETDEWEB)

    Brothers, H.M. II; Kostic, N.M.

    1988-05-18

    The complex (Pt(trpy)Cl)/sup +/ exhibits unexpected selectivity toward amino acid side chains in cytochromes c from Candida krusei and bakers' yeast. Although kinetic studies with amino acids and peptides as entering ligands prove this complex to be completely selective toward thiol over imidazole, His-33 and His-39 residues (in both proteins) are labeled with greater yields than the Cys-102 residue (in the bakers' yeast protein). The binding sites are determined by peptide mapping and other methods. The Pt(trpy)/sup 2+/ tags are stable, and the protein derivatives are separated by cation-exchange chromatography. The (Pt(trpy)His)/sup 2+/ and (Pt(trpy)Cys)/sup +/ chromophores are easily detected and quantitated owing to their characteristic and strong UV-vis bands. Spectroscopic and electrochemical measurements show that labeling with the new reagent does not alter the structural and redox properties of the cytochromes c. The unexpected outcome of the protein labeling indicates that, contrary to the common assumption, Cys-102 is not exposed at the protein surface. Modification of this residue with various organic reagents and dimerization of the protein must be accompanied by a perturbation of the conformation, which makes Cys-102 accessible to the reagent or to another molecule of the protein. These predictions from the labeling study are confirmed subsequently by the crystallographic study of the iso-1 cytochrome c from bakers' yeast. The inorganic complex (Pt(trpy)Cl)/sup +/ differs from the other reagents for protein modification by its noninvasiveness, a property that may well render it useful as a probe of the protein surface. 59 refs., 2 figs., 4 tabs.

  18. CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure.

    Science.gov (United States)

    Schalle, J; Kämpfer, U; Schürch, S; Kuhn-Nentwig, L; Haeberli, S; Nentwig, W

    2001-09-01

    CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys6-Cys21, Cys13-Cys30, Cys20-Cys48, Cys32-Cys46). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (omega-AGA-IVA, omega-AGA-IVB, mu-AGA-I and mu-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail. PMID:11693532

  19. Conformational properties of 1,4- and 1,5-substituted 1,2,3-triazole amino acids – building units for peptidic foldamers

    KAUST Repository

    Kann, Nina

    2015-01-01

    © The Royal Society of Chemistry 2015. Peptidic foldamers have recently emerged as a novel class of artificial oligomers with properties and structural diversity similar to that of natural peptides, but possessing additional interesting features granting them great potential for applications in fields from nanotechnology to pharmaceuticals. Among these, foldamers containing 1,4- and 1,5-substitued triazole amino acids are easily prepared via the Cu- and Ru-catalyzed click reactions and may offer increased side chain variation, but their structural capabilities have not yet been widely explored. We here describe a systematic analysis of the conformational space of the two most important basic units, the 1,4-substitued (4Tzl) and the 1,5-substitued (5Tzl) 1,2,3-triazole amino acids, using quantum chemical calculations and NMR spectroscopy. Possible conformations of the two triazoles were scanned and their potential minima were located using several theoretical approaches (B3LYP/6-311++G(2d,2p), ωB97X-D/6-311++G(2d,2p), M06-2X/6-311++G(2d,2p) and MP2/6-311++G(2d,2p)) in different solvents. BOC-protected versions of 4Tzl and 5Tzl were also prepared via one step transformations and analyzed by 2D NOESY NMR. Theoretical results show 9 conformers for 5Tzl derivatives with relative energies lying close to each other, which may lead to a great structural diversity. NMR analysis also indicates that conformers preferring turn, helix and zig-zag secondary structures may coexist in solution. In contrast, 4Tzl has a much lower number of conformers, only 4, and these lack strong intraresidual interactions. This is again supported by NMR suggesting the presence of both extended and bent conformers. The structural information provided on these building units could be employed in future design of triazole foldamers. This journal is

  20. Bile acid-regulated peroxisome proliferator-activated receptor-α (PPARα) activity underlies circadian expression of intestinal peptide absorption transporter PepT1/Slc15a1.

    Science.gov (United States)

    Okamura, Ayako; Koyanagi, Satoru; Dilxiat, Adila; Kusunose, Naoki; Chen, Jia Jun; Matsunaga, Naoya; Shibata, Shigenobu; Ohdo, Shigehiro

    2014-09-01

    Digested proteins are mainly absorbed as small peptides composed of two or three amino acids. The intestinal absorption of small peptides is mediated via only one transport system: the proton-coupled peptide transporter-1 (PepT1) encoded from the soluble carrier protein Slc15a1. In mammals, intestinal expression of PepT1/Slc15a1 oscillates during the daily feeding cycle. Although the oscillation in the intestinal expression of PepT1/Slc15a1 is suggested to be controlled by molecular components of circadian clock, we demonstrated here that bile acids regulated the oscillation of PepT1/Slc15a1 expression through modulating the activity of peroxisome proliferator-activated receptor α (PPARα). Nocturnally active mice mainly consumed their food during the dark phase. PPARα activated the intestinal expression of Slc15a1 mRNA during the light period, and protein levels of PepT1 peaked before the start of the dark phase. After food intake, bile acids accumulated in intestinal epithelial cells. Intestinal accumulated bile acids interfered with recruitment of co-transcriptional activator CREB-binding protein/p300 on the promoter region of Slc15a1 gene, thereby suppressing PPARα-mediated transactivation of Slc15a1. The time-dependent suppression of PPARα-mediated transactivation by bile acids caused an oscillation in the intestinal expression of PepT1/Slc15a1 during the daily feeding cycle that led to circadian changes in the intestinal absorption of small peptides. These findings suggest a molecular clock-independent mechanism by which bile acid-regulated PPARα activity governs the circadian expression of intestinal peptide transporter.

  1. The mechanism of gentisic acid-induced relaxation of the guinea pig isolated trachea: the role of potassium channels and vasoactive intestinal peptide receptors

    Directory of Open Access Journals (Sweden)

    J.F. Cunha

    2001-03-01

    Full Text Available We examined some of the mechanisms by which the aspirin metabolite and the naturally occurring metabolite gentisic acid induced relaxation of the guinea pig trachea in vitro. In preparations with or without epithelium and contracted by histamine, gentisic acid caused concentration-dependent and reproducible relaxation, with mean EC50 values of 18 µM and Emax of 100% (N = 10 or 20 µM and Emax of 92% (N = 10, respectively. The relaxation caused by gentisic acid was of slow onset in comparison to that caused by norepinephrine, theophylline or vasoactive intestinal peptide (VIP. The relative rank order of potency was: salbutamol 7.9 > VIP 7.0 > gentisic acid 4.7 > theophylline 3.7. Gentisic acid-induced relaxation was markedly reduced (24 ± 7.0, 43 ± 3.9 and 78 ± 5.6% in preparations with elevated potassium concentration in the medium (20, 40 or 80 mM, respectively. Tetraethylammonium (100 µM, a nonselective blocker of the potassium channels, partially inhibited the relaxation response to gentisic acid, while 4-AP (10 µM, a blocker of the voltage potassium channel, inhibited gentisic acid-induced relaxation by 41 ± 12%. Glibenclamide (1 or 3 µM, at a concentration which markedly inhibited the relaxation induced by the opener of ATP-sensitive K+ channels, levcromakalim, had no effect on the relaxation induced by gentisic acid. Charybdotoxin (0.1 or 0.3 µM, a selective blocker of the large-conductance Ca2+-activated K+ channels, caused rightward shifts (6- and 7-fold of the gentisic acid concentration-relaxation curve. L-N G-nitroarginine (100 µM, a NO synthase inhibitor, had no effect on the relaxant effect of gentisic acid, and caused a slight displacement to the right in the relaxant effect of the gentisic acid curve at 300 µM, while methylene blue (10 or 30 µM or ODQ (1 µM, the inhibitors of soluble guanylate cyclase, all failed to affect gentisic acid-induced relaxation. D-P-Cl-Phe6,Leu17[VIP] (0.1 µM, a VIP receptor antagonist

  2. Structural characterizations of fusion peptide analogs of influenza virus hemagglutinin. Implication of the necessity of a helix-hinge-helix motif in fusion activity.

    Science.gov (United States)

    Hsu, Chun-Hua; Wu, Shih-Hsiung; Chang, Ding-Kwo; Chen, Chinpan

    2002-06-21

    Infection by enveloped viruses initially involves membrane fusion between viral and host cell membranes. The fusion peptide plays a crucial role in triggering this reaction. To clarify how the fusion peptide exerts this specific function, we carried out biophysical studies of three fusion peptide analogs of influenza virus hemagglutinin HA2, namely E5, G13L, and L17A. E5 exhibits an activity similar to the native fusion peptide, whereas G13L and L17A, which are two point mutants of the E5 analog, possess much less fusion activity. Our CD data showed that the conformations of these three analogs in SDS micelles are pH-dependent, with higher alpha-helical contents at acidic pH. Tryptophan fluorescence emission experiments indicated that these three analogs insert deeper into lipid bilayers at acidic pH. The three-dimensional structure of the E5 analog in SDS micelles at pH 4.0 revealed that two segments, Leu(2)-Glu(11) and Trp(14)-Ile(18), form amphipathic helical conformations, with Gly(12)-Gly(13) forming a hinge. The hydrophobic residues in the N- and C-terminal helices form a hydrophobic cluster. At neutral pH, however, the C-terminal helix of Trp(14)-Ile(18) reduces dramatically, and the hydrophobic core observed at acidic pH is severely disrupted. We suggest that the disruption of the C-terminal helix renders the E5 analog fusion-inactive at neutral pH. Furthermore, the decrease of the hinge and the reduction of fusion activity in G13L reveal the importance of the hinge in fusion activity. Also, the decrease in the C-terminal helix and the reduction of fusion activity in L17A demonstrates the importance of the C-terminal helix in fusion activity. Based on these biophysical studies, we propose a model that illustrates the structural change of the HA2 fusion peptide analog and explains how the analog interacts with the lipid bilayer at different pH values.

  3. 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.

    Science.gov (United States)

    Narverud, Ingunn; Myhrstad, Mari C W; Herzig, Karl-Heinz; Karhu, Toni; Dahl, Tuva B; Halvorsen, Bente; Ulven, Stine M; Holven, Kirsten B

    2016-01-01

    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 (FAs) 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 FAs 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 2 weeks. Fasting and postprandial blood samples at 3 and 6 h after intake were analyzed. 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 FAs from different origin stimulates intestinal peptide release without any difference between the different fat compositions.

  4. 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

    Science.gov (United States)

    Narverud, Ingunn; Myhrstad, Mari C. W.; Herzig, Karl-Heinz; Karhu, Toni; Dahl, Tuva B.; Halvorsen, Bente; Ulven, Stine M.; Holven, Kirsten B.

    2016-01-01

    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 (FAs) 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 FAs 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 2 weeks. Fasting and postprandial blood samples at 3 and 6 h after intake were analyzed. 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 FAs from different origin stimulates intestinal peptide release without any difference between the different fat compositions. PMID:27630989

  5. 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.

    Science.gov (United States)

    Narverud, Ingunn; Myhrstad, Mari C W; Herzig, Karl-Heinz; Karhu, Toni; Dahl, Tuva B; Halvorsen, Bente; Ulven, Stine M; Holven, Kirsten B

    2016-01-01

    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 (FAs) 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 FAs 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 2 weeks. Fasting and postprandial blood samples at 3 and 6 h after intake were analyzed. 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 FAs from different origin stimulates intestinal peptide release without any difference between the different fat compositions. PMID:27630989

  6. Synthesis of C-linked carbo-β2-amino acids and β2-peptides: design of new motifs for left-handed 12/10- and 10/12-mixed helices.

    Science.gov (United States)

    Sharma, Gangavaram V M; Reddy, Nelli Yella; Ravi, Rapolu; Sreenivas, Bommagani; Sridhar, Gattu; Chatterjee, Deepak; Kunwar, Ajit C; Hofmann, Hans-Jörg

    2012-12-14

    C-linked carbo-β(2)-amino acids (β(2)-Caa), a new class of β-amino acid with a carbohydrate side chain having d-xylo configuration, were prepared from d-glucose. The main idea behind the design of the new β-amino acids was to move the steric strain of the bulky carbohydrate side chain from the Cβ- to the Cα-carbon atom and to explore its influence on the folding propensities in peptides with alternating (R)- and (S)-β(2)-Caas. The tetra- and hexapeptides derived were studied employing NMR (in CDCl(3)), CD, and molecular dynamics simulations. The β(2)-peptides of the present study form left-handed 12/10- and 10/12-mixed helices independent of the order of the alternating chiral amino acids in the sequence and result in a new motif. These results differ from earlier findings on β(3)-peptides of the same design, containing a carbohydrate side chain with d-xylo configuration, which form exclusively right-handed 12/10-mixed helices. Quantum chemical calculations employing ab initio MO theory suggest the side chain chirality as an important factor for the observed definite left- or right-handedness of the helices in the β(2)- and β(3)-peptides.

  7. Composite Membranes of Recombinant Silkworm Antimicrobial Peptide and Poly (L-lactic Acid) (PLLA) for biomedical application.

    Science.gov (United States)

    Li, Zhi; Liu, Xuan; Li, Yi; Lan, Xiqian; Leung, Polly Hangmei; Li, Jiashen; Li, Gang; Xie, Maobin; Han, Yanxia; Lin, Xiaofen

    2016-01-01

    Antimicrobial peptides, produced by innate immune system of hosts in response to invading pathogens, are capable of fighting against a spectrum of bacteria, viruses, fungi, parasites and cancer cells. Here, a recombinant silkworm AMP Bmattacin2 from heterologous expression is studied, indicating a broad spectrum of antibacterial activity and showing selective killing ability towards skin and colon cancer cells over their normal cell counterparts. For the purpose of biomedical application, the electrospinning fabrication technique is employed to load Bmattacin2 into PLLA nanofibrous membrane. In addition to a good compatibility with the normal cells, Bmattacin2 loaded nanofibrous membranes demonstrate instant antibacterial effects and sustained anticancer effects. The cancer cell and bacteria targeting dynamics of recombinant Bmattacin2 are investigated. With these characteristics, PLLA/Bmattacin2 composite membranes have a great potential for developing novel biomedical applications such as cancer therapies and wound healing treatments. PMID:27503270

  8. 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

  9. Effects of postprandial starvation on mRNA expression of endocrine-, amino acid and peptide transporter-, and metabolic enzyme-related genes in zebrafish (Danio rerio).

    Science.gov (United States)

    Tian, Juan; He, Gen; Mai, Kangsen; Liu, Chengdong

    2015-06-01

    The goal of this study was to systematically evaluate the molecular activities of endocrine-, amino acid and peptide transporters-, and metabolic enzyme-related genes in 35-day-old mixed-sex zebrafish (Danio rerio) after feeding . Zebrafish with initial body weights ranging from 9 to 11 mg were fasted for 384 h in a controlled indoor environment. Fish were sampled at 0, 3, 6, 12, 24, 48, 96, 192, and 384 h after fed. Overall, the present study results show that the regulatory mechanism that insulin-like growth factor I negative feedback regulated growth hormone is conserved in zebrafish, as it is in mammals, but that regulation of growth hormone receptors is highly intricate. Leptin and cholecystokinin are time-dependent negative feedback signals, and neuropeptide Y may be an important positive neuropeptide for food intake in zebrafish. The amino acid/carnitine transporters B(0,+) (ATB(0,+)) and broad neutral (0) amino acid transporter 1(B(0)AT1) mRNA levels measured in our study suggest that protein may be utilized during 24-96 h of fasting in zebrafish. Glutamine synthetase mRNA levels were downregulated, and glutamate dehydrogenase, alanine aminotransferase, aspartate transaminase, and trypsin mRNA levels were upregulated after longtime fasting in this study. The mRNA expression levels of fatty acid synthetase decreased significantly (P < 0.05), whereas those of lipoprotein lipase rapidly increased after 96 h of fasting. Fasting activated the expression of glucose synthesis genes when fasting for short periods of time; when fasting is prolonged, the mRNA levels of glucose breakdown enzymes and pentose phosphate shunt genes decreased. PMID:25805459

  10. Determination of binding capacity and adsorption enthalpy between Human Glutamate Receptor (GluR1) peptide fragments and kynurenic acid by surface plasmon resonance experiments. Part 2: Interaction of GluR1270-300 with KYNA.

    Science.gov (United States)

    Csapó, E; Bogár, F; Juhász, Á; Sebők, D; Szolomájer, J; Tóth, G K; Majláth, Z; Vécsei, L; Dékány, I

    2015-09-01

    In the course of our previous work, the interactions of two peptide fragments (GluR1201-230 and GluR1231-259) of human glutamate receptor (GluR1201-300) polypeptide with kynurenic acid (KYNA) were investigated by surface plasmon resonance (SPR) spectroscopy. Besides quantitation of the interactions, the enthalpies of binding of KYNA on certain peptide fragment-modified gold surfaces were also reported. In the present work, a third peptide fragment (GluR1270-300) of the glutamate receptor was synthesized and its interaction with KYNA was investigated by an SPR technique. This 31-membered peptide was chemically bonded onto a gold-coated SPR chip via a cysteine residue. The peptide-functionalized biosensor chip was analyzed by atomic force microscopy (AFM) and theoretical calculations were performed on the structure and dimensions of the peptide on the gold surface. In order to determine the isosteric heat of adsorption of the binding of KYNA on the peptide-functionalized gold thin film, SPR experiments were carried out between +10°C and +40°C. The results on the GluR1270-300-KYNA system were compared with the previously published binding parameters of the interactions of GluR1201-230 and GluR1231-259 with KYNA. The binding abilities of KYNA with all three peptide fragments immobilized on the gold surface were estimated by a molecular docking procedure and the binding free energies of these AMPA receptor subunits with KYNA were determined.

  11. Recent development of peptide self-assembly

    Institute of Scientific and Technical Information of China (English)

    Xiubo Zhao; Fang Pan; Jian R. Lu

    2008-01-01

    Amino acids are the building blocks to build peptides and proteins. Recent development in peptide synthesis has however enabled us to mimic this natural process by preparing various long and short peptides possessing different conformations and biological functions. The self-assembly of short designed peptides into molecular nanostructures is becoming a growing interest in nanobiotechnology. Self-assembled peptides exhibit several attractive features for applications in tissue regeneration, drug delivery, biological surface engineering as well as in food science, cosmetic industry and antibiotics. The aim of this review is to introduce the readers to a number of representative studies on peptide self-assembly.

  12. Binding of cationic peptides (KX)4K to DPPG bilayers. Increasing the hydrophobicity of the uncharged amino acid X drives formation of membrane bound β-sheets: A DSC and FT-IR study.

    Science.gov (United States)

    Hädicke, André; Blume, Alfred

    2016-06-01

    The binding of cationic peptides of the sequence (KX)4K to lipid vesicles of negatively charged dipalmitoyl-phosphatidylglycerol (DPPG) was investigated by differential scanning calorimetry (DSC) and temperature dependent Fourier-transformed infrared (FT-IR) spectroscopy. The hydrophobicity of the uncharged amino acid X was changed from G (glycine) over A (alanine), Abu (α-aminobutyric acid), V (valine) to L (leucine). The binding of the peptides caused an increase of the phase transition temperature (Tm) of DPPG by up to 20°C. The shift depended on the charge ratio and on the hydrophobicity of the amino acid X. Unexpectedly, the upward shift of Tm increased with increasing hydrophobicity of X. FT-IR spectroscopy showed a shift of the CH2 stretching vibrations of DPPG to lower frequency, particularly for bilayers in the liquid-crystalline phase, indicating an ordering of the hydrocarbon chains when the peptides were bound. Changes in the lipid C=O vibrational band indicated a dehydration of the lipid headgroup region after peptide binding. (KG)4K was bound in an unordered structure at all temperatures. All other peptides formed intermolecular antiparallel β-sheets, when bound to gel phase DPPG. However, for (KA)4K and (KAbu)4K, the β-sheets converted into an unordered structure above Tm. In contrast, the β-sheet structures of (KV)4K and (KL)4K remained stable even at 80°C when bound to the liquid-crystalline phase of DPPG. Strong aggregation of DPPG vesicles occurred after peptide binding. For the aggregates, we suggest a structure, where aggregated single β-sheets are sandwiched between opposing DPPG bilayers with a dehydrated interfacial region.

  13. The intrinsic antimicrobial activity of citric acid-coated manganese ferrite nanoparticles is enhanced after conjugation with the antifungal peptide Cm-p5

    Directory of Open Access Journals (Sweden)

    Lopez-Abarrategui C

    2016-08-01

    studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli or Gram-positive bacteria (Staphylococcus aureus. Finally, an antifungal peptide (Cm-p5 from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity. Keywords: nanoparticles, conjugation, antifungal, Cm-p5 peptide

  14. 肽核酸在分子生物学技术中的应用%Application of Peptide Nucleic Acid in Molecular Biotechnology

    Institute of Scientific and Technical Information of China (English)

    王建华; 郭泽琴

    2013-01-01

    Peptide nucleic acid ( PNA) is a nucleic acid mimic in which the deoxyribose phosphate backbone has been replaced by a pseudo-peptide polymer to which the nucleobases are linked. PNA forms complexes with DNA following the Watson-Crick base-pairing rules. The PNA-DNA complexes exhibited high thermal stability and mismatch sensitivity. The high stability of these hybrids has been explained by the absence of negative charges along the PNA backbone. Furthermore, PNA is resistant to biological degradation and can bind complementary RNA or DNA sequences with extraordinary high affinity and specificity. PNA possesses many of the properties desired for a good antisense agent or antigene drugs, such as the detection of a single base mutate in nucleic acids, the diagnosis and detection by PCR molecular beacon, quantitative analysis of fluorescence in situ hybridization, genechip and biosensor technology and so on. Thus, the above-mentioned applications of PNA in molecular biotechnology in recent years and the prospect of its application were summarized.%肽核酸(PNA)作为一种人工合成的核酸类似物,以中性的肽链酰胺2-氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余部分与DNA相同.PNA可通过Watson-Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构.与传统的DNA或RNA相比,PNA具有生物学稳定性高、杂交特异性强、杂合体的稳定性高和杂交速度快等明显优点,使PNA具有良好的物理化学性质和生物学特性,在检测目的核酸序列中单碱基突变、PCR基因分子诊断与检测、荧光原位杂交定量分析、基因芯片和生物传感器技术等调控水平和临床应用上有自己的特点.简要综述了近年来肽核酸在上述分子生物学技术中的运用以及应用前景的展望.

  15. Clinical Study on Treatment of Advanced Non-small Cell Lung Cancer by Arsenious Acid Combined with Tα-1 Thymus Peptide and Megestrol Acetate

    Institute of Scientific and Technical Information of China (English)

    郭岳峰; 焦智民

    2002-01-01

    Objective:To observe the therapeutic effect of arsenious acid combined with Tα-1 thymuspeptide and megestrol acetate on advanced non-small cell lung cancer. Methods: Nintey-two patients weredivided randomly into the treated group(n= 45) and the control group(n= 47). The treated group weretreated with arsenious acid combined with Tα-1 thymus peptide and megestrol acetate, and the controlgroup were treated with chemotherapy in the NP protocol. Results: (1) Therapeutic effect: In the 36 pa-tients of the treated group, 2 were evaluated as CR, 8 as PR, 9 as MR, 8 as SD and 9 as PD, the CR+PRrate being 27.7% (10/36), while in the 40 patients of the control group, the corresponding numbers were3, 10, 11, 9, 7 and 32.5% (13/40). Comparison between the CR+PR rate between the two groupsshowed insignificant difference (P>0.05). (2)Clinical benefit rate: The positive rate of Karnofsky scoresin the treated group and the control group was 44.4 % and 20.0 % respectively; and the positive rate ofbody weight in the two groups was 33.3 % and 12.5 % respectively, the difference between the two groupswas significant ( all P<0.05). (3)Changes of T- cell subsets and NK cell activity: CD4 and CD4/CD8 ra-tio after treatment in the treated group increased significantly (P<0.05), while in the control group,CD3, CD4, CD4/CD8 ratio and NK cell activity all lowered significantly (P<0.01). Comparison betweenthe two groups after treatment showed significant difference in CD4, CD4/CD8 ratio and NK cell activity,with those in the treated group all higher than those in the control group (P<0.01). (4)Adeverse-reac-tion: No serious adverse reaction was found in both two groups. (5)Median survival period: The treatedgroup was 30 weeks and that in the control group was 28.5 weeks, and the difference between the twogroups was insignificant (P>0.05). Conclusion: Arsenious acid combined with Tα-1 thymus peptide andmegestrol acetate was a relatively effective scheme with low toxicity in

  16. [The current concepts on the absorption of monosaccharides, amino acids and peptides in the mammalian small intestine].

    Science.gov (United States)

    Timofeeva, N M; Iezuitova, N N; Gromova, L V

    2000-01-01

    The review is mainly devoted to the development of ideas about absorption, or transport, of basic nutrients in the small intestine in humans and higher animal. The absorption processes have been characterized on the example of such substances, vital for organism, as carbohydrates and proteins. The review considers a molecular structure of transporters--protein molecules, which take part in a transfer of the products of lumenal and membrane digestion of carbohydrates (glucose, galactose, fructose) and proteins (amino acids, oligopeptides) across the enterocyte membranes. An information is presented about genetic disturbances of transport of certain amino acids during such diseases as Hartnup disease, cystinuria, and iminoglycineuria. PMID:11094795

  17. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    Science.gov (United States)

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.

  18. The use of sub-critical water hydrolysis for the recovery of peptides and free amino acids from food processing wastes. Review of sources and main parameters.

    Science.gov (United States)

    Marcet, Ismael; Álvarez, Carlos; Paredes, Benjamín; Díaz, Mario

    2016-03-01

    Food industry processing wastes are produced in enormous amounts every year, such wastes are usually disposed with the corresponding economical cost it implies, in the best scenario they can be used for pet food or composting. However new promising technologies and tools have been developed in the last years aimed at recovering valuable compounds from this type of materials. In particular, sub-critical water hydrolysis (SWH) has been revealed as an interesting way for recovering high added-value molecules, and its applications have been broadly referred in the bibliography. Special interest has been focused on recovering protein hydrolysates in form of peptides or amino acids, from both animal and vegetable wastes, by means of SWH. These recovered biomolecules have a capital importance in fields such as biotechnology research, nutraceuticals, and above all in food industry, where such products can be applied with very different objectives. Present work reviews the current state of art of using sub-critical water hydrolysis for protein recovering from food industry wastes. Key parameters as reaction time, temperature, amino acid degradation and kinetic constants have been discussed. Besides, the characteristics of the raw material and the type of products that can be obtained depending on the substrate have been reviewed. Finally, the application of these hydrolysates based on their functional properties and antioxidant activity is described. PMID:26831563

  19. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    Science.gov (United States)

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines. PMID:27430566

  20. Characterization and performance of short cationic antimicrobial peptide isomers.

    Science.gov (United States)

    Juba, Melanie; Porter, Devin; Dean, Scott; Gillmor, Susan; Bishop, Barney

    2013-07-01

    Cationic antimicrobial peptides (CAMPs) represent an ancient defense mechanism against invading bacteria, with peptides such as the cathelicidins being essential elements of vertebrate innate immunity. CAMPs are typically associated with broad-spectrum antimicrobial potency and limited bacterial resistance. The cathelicidin identified from the elapid snake Naja atra (NA-CATH) contains a semi-conserved repeated 11-residue motif (ATRA motif) with a sequence pattern consistent with formation of an amphipathic helical conformation. Short peptide amides (ATRA-1, -1A, -1P, and -2) generated based on the pair of ATRA motifs in NA-CATH exhibited varied antimicrobial potencies. The small size of the ATRA peptides, coupled with their varied antimicrobial performances, make them interesting models to study the impact various physico-chemical properties have on antimicrobial performance in helical CAMPs. Accordingly, the D- and L-enantiomers of the peptide ATRA-1A, which in earlier studies had shown both good antimicrobial performance and strong helical character, were investigated in order to assess the impact peptide stereochemistry has on antimicrobial performance and interaction with chiral membranes. The ATRA-1A isomers exhibit varied potencies against four bacterial strains, and their conformational properties in the presence of mixed zwitterionic/anionic liposomes are influenced by anionic lipid content. These studies reveal subtle differences in the properties of the peptide isomers. Differences are also seen in the abilities of the ATRA-1A isomers to induce liposome fusion/aggregation, bilayer rearrangement and lysing through turbidity studies and fluorescence microscopy. The similarities and differences in the properties of the ATRA-1A isomers could aid in efforts to develop D-peptide-based therapeutics using high-performing L-peptides as templates.

  1. Electromembrane extraction of peptides.

    Science.gov (United States)

    Balchen, Marte; Reubsaet, Léon; Pedersen-Bjergaard, Stig

    2008-06-20

    Rapid extraction of eight different peptides using electromembrane extraction (EME) was demonstrated for the first time. During an extraction time of 5 min, the model peptides migrated from a 500 microL aqueous acidic sample solution, through a thin supported liquid membrane (SLM) of an organic liquid sustained in the pores in the wall of a porous hollow fiber, and into a 25 microL aqueous acidic acceptor solution present inside the lumen of the hollow fiber. The driving force of the extraction was a 50 V potential sustained across the SLM, with the positive electrode in the sample and the negative electrode in the acceptor solution. The nature and the composition of the SLM were highly important for the EME process, and a mixture of 1-octanol and 15% di(2-ethylhexyl) phosphate was found to work properly. Using 1mM HCl as background electrolyte in the sample and 100 mM HCl in the acceptor solution, and agitation at 1050 rpm, enrichment up to 11 times was achieved. Recoveries were found to be dependent on the structure of the peptide, indicating that the polarity and the number of ionized groups were important parameters affecting the extraction efficiency. The experimental findings suggested that electromembrane extraction of peptides is possible and may be a valuable tool for future extraction of peptides. PMID:18479691

  2. Synthesis and Characterization of a Biodegradable Copolymer: RGD Peptide Modification of Poly (lactic acid-co-lysine)

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    The poly ( lactic acid- co-lysine ) was synthesized using IR and 1 H NMR to characterize the copolymer. And then the RGD modification copolymer RGD-PLAL was prepared. The contact angles were used to see the RGD modification occurrence. Also high molecular weight polymer was controlled to the reaction of polymerization of copolymer.

  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

    (P = .07). GLP-2 administration caused an approximately 15% reduction in pentagastrin-stimulated gastric acid and chloride secretion (P secretion but does not seem to have an influence on gastric...... emptying. The stimulation of glucagon secretion by GLP-2 may counteract the glucagonostatic effect of GLP-1. Changes in postprandial lipid excursions seem to reflect enhanced intestinal nutrient absorption during GLP-2 administration....

  4. FERONIA interacts with ABI2-type phosphatases to facilitate signaling cross-talk between abscisic acid and RALF peptide in Arabidopsis.

    Science.gov (United States)

    Chen, Jia; Yu, Feng; Liu, Ying; Du, Changqing; Li, Xiushan; Zhu, Sirui; Wang, Xianchun; Lan, Wenzhi; Rodriguez, Pedro L; Liu, Xuanming; Li, Dongping; Chen, Liangbi; Luan, Sheng

    2016-09-13

    Receptor-like kinase FERONIA (FER) plays a crucial role in plant response to small molecule hormones [e.g., auxin and abscisic acid (ABA)] and peptide signals [e.g., rapid alkalinization factor (RALF)]. It remains unknown how FER integrates these different signaling events in the control of cell growth and stress responses. Under stress conditions, increased levels of ABA will inhibit cell elongation in the roots. In our previous work, we have shown that FER, through activation of the guanine nucleotide exchange factor 1 (GEF1)/4/10-Rho of Plant 11 (ROP11) pathway, enhances the activity of the phosphatase ABA Insensitive 2 (ABI2), a negative regulator of ABA signaling, thereby inhibiting ABA response. In this study, we found that both RALF and ABA activated FER by increasing the phosphorylation level of FER. The FER loss-of-function mutant displayed strong hypersensitivity to both ABA and abiotic stresses such as salt and cold conditions, indicating that FER plays a key role in ABA and stress responses. We further showed that ABI2 directly interacted with and dephosphorylated FER, leading to inhibition of FER activity. Several other ABI2-like phosphatases also function in this pathway, and ABA-dependent FER activation required PYRABACTIN RESISTANCE (PYR)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR)-A-type protein phosphatase type 2C (PP2CA) modules. Furthermore, suppression of RALF1 gene expression, similar to disruption of the FER gene, rendered plants hypersensitive to ABA. These results formulated a mechanism for ABA activation of FER and for cross-talk between ABA and peptide hormone RALF in the control of plant growth and responses to stress signals. PMID:27566404

  5. Specific targeting of A54 homing peptide-functionalized dextran-g-poly(lactic-co-glycolic acid micelles to tumor cells

    Directory of Open Access Journals (Sweden)

    Situ JQ

    2015-01-01

    Full Text Available Jun-Qing Situ,1 Yi-Qing Ye,2 Xiu-Liang Zhu,3 Ri-Sheng Yu,3 Jian You,1 Hong Yuan,1 Fu-Qiang Hu,1 Yong-Zhong Du11College of Pharmaceutical Sciences, 2Women’s Hospital, 3Department of Radiology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, People’s Republic of ChinaAbstract: The delivery of chemotherapeutics into tumor cells is a fundamental knot for tumor-target therapy to improve the curative effect and avoid side effects. Here, A54 peptide-functionalized poly(lactic-co-glycolic acid-grafted dextran (A54-Dex-PLGA was synthesized. The synthesized A54-Dex-PLGA self-assembled to form micelles with a low critical micelle concentration of 16.79 µg·mL-1 and diameter of about 50 nm. With doxorubicin (DOX base as a model antitumor drug, the drug-encapsulation efficiency of DOX-loaded A54-Dex-PLGA micelles (A54-Dex-PLGA/DOX reached up to 75%. In vitro DOX release from the A54-Dex-PLGA/DOX was prolonged to 72 hours. The A54-Dex-PLGA micelles presented excellent internalization ability into hepatoma cells (BEL-7402 cell line and HepG2 cell line in vitro, and the cellular uptake of the micelles by the BEL-7402 cell line was specific, which was demonstrated by the blocking experiment. In vitro antitumor activity studies confirmed that A54-Dex-PLGA/DOX micelles suppressed tumor-cell (BEL-7402 cell growth more effectively than Dex-PLGA micelles. Furthermore, in vivo biodistribution testing demonstrated that the A54-Dex-PLGA micelles had a higher distribution ability to BEL-7402 tumors than that to HepG2 tumors.Keywords: homing peptide, polymeric micelles, doxorubicin, tumor-cell targeting

  6. Polyethylene glycol–polylactic acid nanoparticles modified with cysteine–arginine–glutamic acid–lysine–alanine fibrin-homing peptide for glioblastoma therapy by enhanced retention effect

    Directory of Open Access Journals (Sweden)

    Wu J

    2014-11-01

    Full Text Available Junzhu Wu,1,2,* Jingjing Zhao,1,3,* Bo Zhang,1 Yong Qian,1 Huile Gao,1 Yuan Yu,1 Yan Wei,1 Zhi Yang,1 Xinguo Jiang,1 Zhiqing Pang1 1Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, 2School of Pharmacy, Dali University, Xiaguan, 3School of Pharmacy, China Pharmaceutical University, Nanjing, People’s Republic of China *These authors contributed equally to this work Abstract: For a nanoparticulate drug-delivery system, crucial challenges in brain-glioblastoma therapy are its poor penetration and retention in the glioblastoma parenchyma. As a prevailing component in the extracellular matrix of many solid tumors, fibrin plays a critical role in the maintenance of glioblastoma morphology and glioblastoma cell differentiation and proliferation. We developed a new drug-delivery system by conjugating polyethylene glycol–polylactic acid nanoparticles (NPs with cysteine–arginine–glutamic acid–lysine–alanine (CREKA; TNPs, a peptide with special affinity for fibrin, to mediate glioblastoma-homing and prolong NP retention at the tumor site. In vitro binding tests indicated that CREKA significantly enhanced specific binding of NPs with fibrin. In vivo fluorescence imaging of glioblastoma-bearing nude mice, ex vivo brain imaging, and glioblastoma distribution demonstrated that TNPs had higher accumulation and longer retention in the glioblastoma site over unmodified NPs. Furthermore, pharmacodynamic results showed that paclitaxel-loaded TNPs significantly prolonged the median survival time of intracranial U87 glioblastoma-bearing nude mice compared with controls, Taxol, and NPs. These findings suggested that TNPs were able to target the glioblastoma and enhance retention, which is a valuable strategy for tumor therapy. Keywords: CREKA peptide, nanoparticles, retention effect, paclitaxel, glioblastoma

  7. Fluorogenic Tagging of Peptide and Protein 3-Nitrotyrosine with 4-(Aminomethyl)-benzenesulfonic Acid for Quantitative Analysis of Protein Tyrosine Nitration.

    Science.gov (United States)

    Sharov, Victor S; Dremina, Elena S; Galeva, Nadezhda A; Gerstenecker, Gary S; Li, Xiaobao; Dobrowsky, Rick T; Stobaugh, John F; Schöneich, Christian

    2010-01-01

    Protein 3-nitrotyrosine (3-NT) has been recognized as an important biomarker of nitroxidative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT continues to represent a challenge since in vivo it frequently does not accumulate on proteins in amounts detectable by quantitative analytical methods. Here, we describe a novel approach of fluorescent tagging and quantitation of peptide-bound 3-NT residues based on the selective reduction to 3-AT followed by reaction with 4-(amino-methyl)benzenesulfonic acid (ABS) in the presence of K(3)Fe(CN)(6) to form a highly fluorescent 2-phenylbenzoxazole product. Synthetic 3-NT peptide (0.005-1 μM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 μM K(3)Fe(CN)(6) in 0.1 M Na(2)HPO(4) buffer (pH 9.0) was converted with yields >95% to a single fluorescent product incorporating two ABS molecules per 3-NT residue, with fluorescence excitation and emission maxima at 360 ± 2 and 490 ± 2 nm, respectively, and a quantum yield of 0.77 ± 0.08, based on reverse-phase LC with UV and fluorescence detection, fluorescence spectroscopy and LC-MS-MS analysis. This protocol was successfully tested for quantitative analysis of in vitro Tyr nitration in a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from C2C12 cultured cells exposed to peroxynitrite, with a detection limit of ca. 1 pmol 3-NT by fluorescence spectrometry, and an apparent LOD of 12 and 40 pmol for nitropeptides alone or in the presence of 100 μg digested cell proteins, respectively. LC-MS-MS analysis of ABS tagged peptides revealed that the fluorescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration in proteomic studies. Fluorogenic tagging with ABS also can be instrumental for detection and visualization of protein 3-NT in LC and gel-based protein separations. PMID

  8. Toxicity assessment of repeated intravenous injections of arginine–glycine–aspartic acid peptide conjugated CdSeTe/ZnS quantum dots in mice

    Directory of Open Access Journals (Sweden)

    Wang YW

    2014-10-01

    Full Text Available You-Wei Wang, Kai Yang, Hong Tang, Dan Chen, Yun-Long Bai Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China Background: Nanotechnology-based near-infrared quantum dots (NIR QDs have many excellent optical properties, such as high fluorescence intensity, good fluorescence stability, and strong tissue-penetrating ability. Integrin αvß3 is highly and specifically expressed in tumor angiogenic vessel endothelial cells of almost all carcinomas. Recent studies have shown that NIR QDs linked to peptides containing the arginine–glycine–aspartic acid (RGD sequence (NIR QDs-RGD can specifically target integrin αvß3 expressed in endothelial cells of tumor angiogenic vessels in vivo, and they offer great potential for early cancer diagnosis, in vivo tumor imaging, and tumor individualized therapy. However, the toxicity profile of NIR QDs-RGD has not been reported. This study was conducted to investigate the toxicity of NIR QDs-RGD when intravenously administered to mice singly and repeatedly at the dose required for successful tumor imaging in vivo.Materials and methods: A NIR QDs-RGD probe was prepared by linking NIR QDs with the maximum emission wavelength of 800 nm (QD800 to the RGD peptide (QD800-RGD. QD800-RGD was intravenously injected to BALB/C mice once or twice (200 pmol equivalent of QD800 for each injection. phosphate-buffered saline solution was used as control. Fourteen days postinjection, toxicity tests were performed, including complete blood count (white blood cell, red blood cell, hemoglobin, platelets, lymphocytes, and neutrophils and serum biochemical analysis (total protein, albumin, albumin/globulin, aspartate aminotransferase, alanine aminotransferase, and blood urea nitrogen. The coefficients of liver, spleen, kidney, and lung weight to body weight were measured, as well as their oxidation and antioxidation indicators, including

  9. Self-assembling peptide and protein nanodiscs for studies of membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi

    of proteins encoded by the human genome. G-protein coupled receptors mediate the majority of hormone and neurotransmitter signals as well as being responsible for perception of light, smell and taste in the human body, and a number of Nobel prizes has been awarded based on their study. Structural...... membrane proteins. A minimalistic approach was tested where the ApoA1 protein was mimicked my small amphipathic helical peptides. The resulting discs were very similar to ApoA1 based discs in size and in their ability to stabilize incorporated membrane proteins. Furthermore, due to their enhanced dynamical...

  10. The helical structure of surfactant peptide KL4 when bound to POPC: POPG lipid vesicles.

    Science.gov (United States)

    Mills, Frank D; Antharam, Vijay C; Ganesh, Omjoy K; Elliott, Doug W; McNeill, Seth A; Long, Joanna R

    2008-08-12

    KL 4 is a 21-residue peptide employed as a functional mimic of lung surfactant protein B, which successfully lowers surface tension in the alveoli. A mechanistic understanding of how KL 4 affects lipid properties has proven elusive as the secondary structure of KL 4 in lipid preparations has not been determined at high resolution. The sequence of KL 4 is based on the C-terminus of SP-B, a naturally occurring helical protein that binds to lipid interfaces. The spacing of the lysine residues in KL 4 precludes the formation of a canonical amphipathic alpha-helix; qualitative measurements using Raman, CD, and FTIR spectroscopies have given conflicting results as to the secondary structure of the peptide as well as its orientation in the lipid environment. Here, we present a structural model of KL 4 bound to lipid bilayers based on solid state NMR data. Double-quantum correlation experiments employing (13)C-enriched peptides were used to quantitatively determine the backbone torsion angles in KL 4 at several positions. These measurements, coupled with CD experiments, verify the helical nature of KL 4 when bound to lipids, with (phi, psi) angles that differ substantially from common values for alpha-helices of (-60, -45). The average torsion angles found for KL 4 bound to POPC:POPG lipid vesicles are (-105, -30); this deviation from ideal alpha-helical structure allows KL 4 to form an amphipathic helix at the lipid interface. PMID:18636713

  11. Production of Bioactive Peptides from Soybean Meal by Solid State Fermentation with Lactic Acid Bacteria and Protease

    OpenAIRE

    Naifu Wang; Guowei Le; Yonghui Shi; Yuan Zeng

    2014-01-01

    In this study, soybean meal was first solid state fermented with different strains of Lactic Acid Bacteria (LAB). Among the strains used, Lactobacillus plantarum Lp6 was selected for further studies because of its highest Degree of Hydrolysis (DH) of protein (2.49±0.08%) in soybean meal after 72 h fermentation. Soybean meal fermented with L. plantarum Lp6 can also improve its DPPH radical scavenging and Angiotensin Converting Enzyme (ACE) inhibitory activities. The addition of protease into s...

  12. Pathway for Ca2+ influx into cells by trichosporin-B-VIa, an alpha-aminoisobutyric acid-containing peptide, from the fungus Trichoderma polysporum.

    Science.gov (United States)

    Tachikawa, E; Nogimori, K; Takahashi, S; Mizuma, K; Itoh, K; Kashimoto, T; Nagaoka, Y; Iida, A; Fujita, T

    1996-06-13

    Trichosporin (TS) -B-VIa, a fungal alpha-aminoisobutyric acid (Aib) -containing peptide consisting of 19 amino acid residues and a phenylalaninol, produced both 45Ca2+ influx into bovine adrenal chromaffin cells and catecholamine secretion from the cells. The secretion induced by TS-B-VIa at lower concentrations (2-5 microM) was completely dependent on the external Ca2+, while that induced by TS-B-VIa at higher concentrations (10-30 microM) was partly independent of the Ca2+. The concentration-response curves (2-5 microM) for the TS-B-VIa-induced Ca2+ influx and secretion correlated well. The TS-B-VIa (at 5 microM) -induced secretion was not antagonized by diltiazem, a blocker of L-type voltage-sensitive Ca2+ channels. The treatment of fura-2-loaded C6 glioma cells with TS-B-VIa (2-5 microM) led to an increase in the intracellular free Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner but the stimulatory effects of TS-B-VIa on [Ca2+]i were only slightly observed in Ca(2+)-free medium, indicating that TS-B-VIa causes Ca2+ influx from the external medium into the C6 cells. The TS-B-VIa-induced increase in [Ca2+]i in the C6 cells was not antagonized by diltiazem and by SK&F 96365, a novel blocker of receptor-mediated Ca2+ entry. High K+ increased neither [Ca2+]1 in the C6 cells nor Mn2+ influx into the cells, while TS-B-VIa increased Mn2+ influx. Also in other non-excitable cells, bovine platelets, similar results were obtained. These results strongly suggest that the mechanism of Ca2+ influx by TS-B-VIa at the lower concentrations is distinct from the event of Ca2+ influx through receptor-operated or L-type voltage-sensitive Ca2+ channels in both excitable cells (the chrornaffin cells) and non-excitable cells (the C6 cells and the platelets) and that TS-B-VIa per se may form Ca(2+)-permeable ion channels in biological membranes. On the other hand, the peptide at the higher concentrations seems to damage cell membranes. PMID:8679651

  13. Trichosporin-B-III, an alpha-aminoisobutyric acid-containing peptide, causes Ca(2+)-dependent catecholamine secretion from adrenal medullary chromaffin cells.

    Science.gov (United States)

    Tachikawa, E; Takahashi, S; Furumachi, K; Kashimoto, T; Iida, A; Nagaoka, Y; Fujita, T; Takaishi, Y

    1991-11-01

    We examined the effect of trichosporin-B-III, an alpha-aminoisobutyric acid-containing antibiotic peptide consisting of 19 amino acid residues and a phenylalaninol, on catecholamine secretion from cultured bovine adrenal chromaffin cells. Incubation of the cells with trichosporin-B-III (3-20 microM) caused an increase in the secretion of catecholamines. The secretion induced by trichosporin-B-III at low concentrations (3 and 5 microM) was completely dependent on external Ca2+, whereas that induced by higher concentrations (10 and 20 microM) was partly independent of Ca2+. Trichosporin-B-III at low concentration (5 microM) did not increase the release of lactate dehydrogenase, a marker enzyme of cytoplasm, from the cells. In contrast, the peptide at higher concentration (10 microM) increased the release of the enzyme. Trichosporin-B-III also caused both 45Ca2+ influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and 45Ca2+ influx behaved similarly in relation to trichosporin-B-III concentration (3-10 microM). The time courses of the increases in secretion, 45Ca2+ influx, and intracellular free Ca2+ concentration induced by trichosporin-B-III were also quite similar. Trichosporin-B-III-induced (at 5 microM) secretion was not affected by the elimination of Na+ from the incubation medium or by the addition of tetrodotoxin, a blocker of highly selective voltage-dependent Na+ channels, or hexamethonium, a blocker of nicotinic acetylcholine receptors. On the other hand, both diltiazem (2-200 microM) and nicardipine (1-200 microM), blockers of voltage-dependent Ca2+ channels, inhibited the secretion induced by trichosporin-B-III (5 microM) in a concentration-dependent manner. Trichosporin-B-III-induced (at 5 microM) secretion also was suppressed by the addition of Mn2+ (5 mM) to the medium. The diltiazem (20 microM) inhibition of trichosporin-B-III-induced (at 5 microM) secretion was reversed by

  14. Effects of 125I-Labeled Peptide Nuclear Acid Targeting Ki67 on the Growth of Implanted Human Renal Cell Carcinoma in Nude Mice

    Institute of Scientific and Technical Information of China (English)

    JiacunChen; JunnianZheng; SongWu; HaibiaoLai; XiaoqingSun; JunjieLiu

    2004-01-01

    OBJECTIVE To investigate the potential of 125I-labeled anti-sense peptide nucleic acids (125I-AS-PNAs) to inhibit the expression of the Ki-67 gene and growth of implanted human renal carcinoma cells in nude mice.METHODS Anti-sense peptide nucleic acids (AS-PNAs) targeting the Ki67 gene were synthesized and labeled with 125I by the Chloraseptine-T method. Drugs including PNAs and 125I-AS-PNAs capsulated by cationic lipid were directly injected into tumors in nude mice. The Ki67 expression in tumors was detected by an immunohistochemical technique and Western blot. The apoptosis of tumor cells was detected by a TUNEL assay. Tumor volumes were measured every 3 days and tumor suppression rates were calculated at 12 days after treatment. Control groups were treated with AS-PNA, MMPNAs (mismatch PNAs) and 125I-Na.RESULTS The Ki67 expression rate of tumors treated by 125I-AS-PNAs [(15.3±1.8)%] was lower than that treated by AS-PNAs [(23.0±2.4)%] (P<0.01). The Ki67 protein production rate of tumors treated by 125I-AS-PNAs [(43.6±3.5)%] was lower than that treated by AS-PNAs [(59.7±2.3)%] (P<0.01 ). The apoptosis rate of tumors treated by 125I-AS-PNAs [(40.3±2.4)%] was higher than that treated by AS-PNAs [(31.1 ±2.0)%] (P<0.01). The volume of tumors treated by 125I-AS-PNAs [(330.4±57.8) mm3 ]was smallerthan that treated by AS- PNAs [(513.2±64.2 ) mm3] (P<0.01 ).CONCLUSION 125I-AS-PNAs targeted against the Ki67 gene have a greater inhibitory effect on the expression of the Ki67 gene and a larger apoptotic action on human renal carcinoma cells and can more efficiently inhibit tumor growth than AS-PNAs. 125I-AS-PNAs targeting the Ki67 gene may be a promising anti-sense/anti-gene radiotherapy method for treating renal cell carcinoma.

  15. Core-shell nanoparticle-based peptide therapeutics and combined hyperthermia for enhanced cancer cell apoptosis.

    Science.gov (United States)

    Shah, Birju P; Pasquale, Nicholas; De, Gejing; Tan, Tao; Ma, Jianjie; Lee, Ki-Bum

    2014-09-23

    Mitochondria-targeting peptides have garnered immense interest as potential chemotherapeutics in recent years. However, there is a clear need to develop strategies to overcome the critical limitations of peptides, such as poor solubility and the lack of target specificity, which impede their clinical applications. To this end, we report magnetic core-shell nanoparticle (MCNP)-mediated delivery of a mitochondria-targeting pro-apoptotic amphipathic tail-anchoring peptide (ATAP) to malignant brain and metastatic breast cancer cells. Conjugation of ATAP to the MCNPs significantly enhanced the chemotherapeutic efficacy of ATAP, while the presence of targeting ligands afforded selective delivery to cancer cells. Induction of MCNP-mediated hyperthermia further potentiated the efficacy of ATAP. In summary, a combination of MCNP-mediated ATAP delivery and subsequent hyperthermia resulted in an enhanced effect on mitochondrial dysfunction, thus resulting in increased cancer cell apoptosis. PMID:25133971

  16. Primary structure of peptides and ion channels. Role of amino acid side chains in voltage gating of melittin channels.

    OpenAIRE

    Tosteson, M T; Alvarez, O; Hubbell, W; Bieganski, R M; Attenbach, C; Caporales, L H; Levy, J J; Nutt, R F; Rosenblatt, M.; Tosteson, D. C.

    1990-01-01

    Melittin produces a voltage-dependent increase in the conductance of planar lipid bilayers. The conductance increases when the side of the membrane to which melittin has been added (cis-side) is made positive. This paper reports observations on the effect of modifying two positively charged amino acid residues within the NH2-terminal region of the molecule: lysine at position 7 (K7), and the NH2-terminal glycine (G1). We have synthesized melittin analogues in which K7 is replaced by asparagin...

  17. Identification of IgE-binding peptide and critical amino acids of Jatropha curcas allergen involved in allergenic response.

    Science.gov (United States)

    Crespo, Livia Maia; de Oliveira, Natalia Deus; Damatta, Renato Augusto; do Nascimento, Viviane Veiga; Soares, Thais Pacheco; Machado, Olga Lima Tavares

    2016-01-01

    Increasing energy demand has spurred interest in the use of biofuels. Jatropha curcas (physic nut), an inedible oilseed, is a potential source of bioenergy. The seeds, however, contain allergens such as Jat c 1, a 2S albumin that can induce hypersensitivity reactions in humans and result in allergic diseases. Recent advances in identifying and characterizing plant allergens and, in particular, their immunoglobulin E (IgE)-binding epitopes have produced a wealth of information. We identified IgE-binding regions and the critical amino acids involved in the degranulation of mast cells and the release of histamine, preliminary steps for the prevention and treatment of this allergy. Four IgE-binding regions were identified in the sequence of Jat c 1. We identified and demonstrated the fundamental role of two glutamic acid residues in IgE binding. The sequence LEKQLEEGEVGS produces a random loop on the most exposed part of Jat c 1. This region is important to the stimulation of the allergic response. The possibility of using this information to produce vaccines and other pharmacological agents for allergy treatment is discussed. PMID:27119058

  18. Naturally evolved enhanced Cd tolerance of Dianthus carthusianorum L. is not related to accumulation of thiol peptides and organic acids.

    Science.gov (United States)

    Wójcik, Małgorzata; Dresler, Sławomir; Plak, Andrzej; Tukiendorf, Anna

    2015-05-01

    Two contrasting ecotypes of Dianthus carthusianorum L., metallicolous (M) and nonmetallicolous (NM), were cultivated in hydroponics at 0-50 μM Cd for 14 days to compare their Cd accumulation, sensitivity and tolerance mechanisms. While both ecotypes contained similar concentrations of Cd in the shoots and roots, the M ecotype was more Cd-tolerant (as measured by fresh weight production and root and leaf viability). Both ecotypes accumulated phytochelatins (PCs) in response to Cd with a higher amount thereof found in the NM ecotype. Concentrations of PCs remained unchanged with increasing Cd concentrations in the root tissues, but their content in the shoots increased. The addition of L-buthionine-sulfoximine (BSO) diminished glutathione (GSH) accumulation and arrested PC production, which increased the sensitivity to Cd of the NM, but not M ecotype. Organic acids (malate and citrate) as well as proline accumulation did not change significantly after Cd exposition and was at the same level in both ecotypes. The enhanced Cd tolerance of the M ecotype of D. carthusianorum cannot be explained in terms of restricted Cd uptake and differential production of PCs, organic acids or proline; some other mechanisms must be involved in its adaptation to the high Cd content in the environment.

  19. Naturally evolved enhanced Cd tolerance of Dianthus carthusianorum L. is not related to accumulation of thiol peptides and organic acids.

    Science.gov (United States)

    Wójcik, Małgorzata; Dresler, Sławomir; Plak, Andrzej; Tukiendorf, Anna

    2015-05-01

    Two contrasting ecotypes of Dianthus carthusianorum L., metallicolous (M) and nonmetallicolous (NM), were cultivated in hydroponics at 0-50 μM Cd for 14 days to compare their Cd accumulation, sensitivity and tolerance mechanisms. While both ecotypes contained similar concentrations of Cd in the shoots and roots, the M ecotype was more Cd-tolerant (as measured by fresh weight production and root and leaf viability). Both ecotypes accumulated phytochelatins (PCs) in response to Cd with a higher amount thereof found in the NM ecotype. Concentrations of PCs remained unchanged with increasing Cd concentrations in the root tissues, but their content in the shoots increased. The addition of L-buthionine-sulfoximine (BSO) diminished glutathione (GSH) accumulation and arrested PC production, which increased the sensitivity to Cd of the NM, but not M ecotype. Organic acids (malate and citrate) as well as proline accumulation did not change significantly after Cd exposition and was at the same level in both ecotypes. The enhanced Cd tolerance of the M ecotype of D. carthusianorum cannot be explained in terms of restricted Cd uptake and differential production of PCs, organic acids or proline; some other mechanisms must be involved in its adaptation to the high Cd content in the environment. PMID:25510617

  20. Biodiscovery of aluminum binding peptides

    Science.gov (United States)

    Adams, Bryn L.; Sarkes, Deborah A.; Finch, Amethist S.; Hurley, Margaret M.; Stratis-Cullum, Dimitra

    2013-05-01

    Cell surface peptide display systems are large and diverse libraries of peptides (7-15 amino acids) which are presented by a display scaffold hosted by a phage (virus), bacteria, or yeast cell. This allows the selfsustaining peptide libraries to be rapidly screened for high affinity binders to a given target of interest, and those binders quickly identified. Peptide display systems have traditionally been utilized in conjunction with organic-based targets, such as protein toxins or carbon nanotubes. However, this technology has been expanded for use with inorganic targets, such as metals, for biofabrication, hybrid material assembly and corrosion prevention. While most current peptide display systems employ viruses to host the display scaffold, we have recently shown that a bacterial host, Escherichia coli, displaying peptides in the ubiquitous, membrane protein scaffold eCPX can also provide specific peptide binders to an organic target. We have, for the first time, extended the use of this bacterial peptide display system for the biodiscovery of aluminum binding 15mer peptides. We will present the process of biopanning with macroscopic inorganic targets, binder enrichment, and binder isolation and discovery.

  1. Short communication: Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide.

    Science.gov (United States)

    Paul, Moushumi; Phillips, John G; Renye, John A

    2016-05-01

    An 8-AA (8mer) fragment (PFPEVFGK) of a known antihypertensive peptide derived from bovine αS1-casein (C12 antihypertensive peptide) was synthesized by microwave-assisted solid-phase peptide synthesis and purified by reverse phase HPLC. Its ability to inhibit angiotensin-converting enzyme (ACE) was assessed and compared with that of the parent 12mer peptide (FFVAPFPEVFGK) to determine the effect of truncating the sequence on overall hypotensive activity. The activity of the truncated 8mer peptide was found to be almost 1.5 times less active than that of the 12mer, with ACE-inhibiting IC50 (half-maximal inhibitory concentration) values of 108 and 69μM, for the 8mer and 12mer, respectively. Although the 8mer peptide is less active than the original 12mer peptide, its overall activity is comparable to activities reported for other small proteins that elicit physiological responses within humans. These results suggest that microbial degradation of the 12mer peptide would not result in a complete loss of antihypertensive activity if used to supplement fermented foods and that the stable 8mer peptide could have potential as a blood pressure-lowering agent for use in functional foods. PMID:26971162

  2. Short communication: Measuring the angiotensin-converting enzyme inhibitory activity of an 8-amino acid (8mer) fragment of the C12 antihypertensive peptide.

    Science.gov (United States)

    Paul, Moushumi; Phillips, John G; Renye, John A

    2016-05-01

    An 8-AA (8mer) fragment (PFPEVFGK) of a known antihypertensive peptide derived from bovine αS1-casein (C12 antihypertensive peptide) was synthesized by microwave-assisted solid-phase peptide synthesis and purified by reverse phase HPLC. Its ability to inhibit angiotensin-converting enzyme (ACE) was assessed and compared with that of the parent 12mer peptide (FFVAPFPEVFGK) to determine the effect of truncating the sequence on overall hypotensive activity. The activity of the truncated 8mer peptide was found to be almost 1.5 times less active than that of the 12mer, with ACE-inhibiting IC50 (half-maximal inhibitory concentration) values of 108 and 69μM, for the 8mer and 12mer, respectively. Although the 8mer peptide is less active than the original 12mer peptide, its overall activity is comparable to activities reported for other small proteins that elicit physiological responses within humans. These results suggest that microbial degradation of the 12mer peptide would not result in a complete loss of antihypertensive activity if used to supplement fermented foods and that the stable 8mer peptide could have potential as a blood pressure-lowering agent for use in functional foods.

  3. Expression of the Gene Encoding the Tetraploid of Carboxyl-terminal Peptide of β-hCG Containing Thirty-seven Amino Acid Residues in E. coli

    Institute of Scientific and Technical Information of China (English)

    王健; 沈卫英; 周清平; 申庆祥

    2000-01-01

    Objective This study was carried out to investigate the possible enhancement of immunogenicity of the carboxyl-terminal peptide of β-hCG which is made up of 37 amino acid residues (109~145) and contains the specific epitope (antigenic determinant) of hCG.Materials & Methods hCGβ-CTP37 tetraploid cDNA was constructed by linking four hCGβ-CTP37 cDNAs together. The product was then subcloned into the E. coli expression vector pQE60 to construct the expression vector pQE60/ (hCGβ-CTP37)4. Recombinant (hCGβ-CTP37 ) 4 was expressed in E. coil-X-blue.Results Western blot analysis showed that the tetraploid of hCGβ-CTP37 had an apparent molecular weight of 20 kD and had relatively stronger anti-hCG antibody-binding activity compared with the diploid from.Conclusion The tetraploid of hCGβ-CTP37 may be a more potent immunogen for raising anti-hCG vaccines for fertility regulation or suppression of tumor.

  4. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

    Directory of Open Access Journals (Sweden)

    Liselotte Hardy

    Full Text Available Bacterial vaginosis (BV, a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1% and a specificity of 89.4% (95% confidence interval: 76.1% - 96% of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

  5. SERUM CONCENTRATIONS OF HYALURONIC ACID, PROCOLLAGEN TYPE Ⅲ NH2-TERMINAL PEPTIDE, AND LAMININ IN PATIENTS WITH CHRONIC CONGESTIVE HEART FAILURE

    Institute of Scientific and Technical Information of China (English)

    Gang Li; Qing-bo Yan; Liang-ming Wei

    2006-01-01

    Objective To explore the role of serum fibrotic indices including hyaluronic acid (HA), procollagen type ⅢNH2-terminal peptide (PCⅢP), and laminin (LN) in assessing the severity of myocardial fibrosis in chronic congestive heart failure (CHF).Methods Serum levels of HA, PCⅢP, and LN in 39 patients with CHF [ 14 with New York Heart Association (NYHA) functional class Ⅱ, 21 with class Ⅲ, 4 with class Ⅳ] and in 46 patients with NYHA functional class Ⅰ were assessed by radioimmunoassay.Results The serum concentrations of HA, PCⅢP, and LN were 359.75 ± 84. 59 μg/L, 77.88±24. 67μg/L,86.73±23.90 μg/L in CHF group, and 211.60±54. 80μg/L, 64.82±23.99 μg/L, 82.26±23.98μg/L in NYHA functional class Ⅰ group, respectively. The HA level was significantly higher in CHF patients as compared with NYHA functional class Ⅰ group (P<0.05 ). However, no difference was found in the levels of PCⅢP and LN between CHF group and NYHA functional class Ⅰ group. The serum HA concentration was negatively correlated with left ventricular ejection fraction (r=-0.71, P<0.05 ).Conclusion Serum HA level may act as an indicator for myocardial fibrosis.

  6. 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...... cancer gene in JAR cells. METHODS: We screened 10 different 15 mer PNAs targeting intron2 at both the 5;- and the 3;-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3;-splice site of intron3 with a complementarity of 4 bases to intron......3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT). RESULTS: We show that several of these PNAs effectively...

  7. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

    Science.gov (United States)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S; Krause, Diane S; Seidman, Michael M; Peterson, Kenneth R; Nielsen, Peter E; Kole, Ryszard; Glazer, Peter M

    2008-09-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells. PMID:18757759

  8. Antimicrobial Peptides from Plants

    Science.gov (United States)

    Tam, James P.; Wang, Shujing; Wong, Ka H.; Tan, Wei Liang

    2015-01-01

    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. PMID:26580629

  9. 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.

  10. Electron transfer in peptides.

    Science.gov (United States)

    Shah, Afzal; Adhikari, Bimalendu; Martic, Sanela; Munir, Azeema; Shahzad, Suniya; Ahmad, Khurshid; Kraatz, Heinz-Bernhard

    2015-02-21

    In this review, we discuss the factors that influence electron transfer in peptides. We summarize experimental results from solution and surface studies and highlight the ongoing debate on the mechanistic aspects of this fundamental reaction. Here, we provide a balanced approach that remains unbiased and does not favor one mechanistic view over another. Support for a putative hopping mechanism in which an electron transfers in a stepwise manner is contrasted with experimental results that support electron tunneling or even some form of ballistic transfer or a pathway transfer for an electron between donor and acceptor sites. In some cases, experimental evidence suggests that a change in the electron transfer mechanism occurs as a result of donor-acceptor separation. However, this common understanding of the switch between tunneling and hopping as a function of chain length is not sufficient for explaining electron transfer in peptides. Apart from chain length, several other factors such as the extent of the secondary structure, backbone conformation, dipole orientation, the presence of special amino acids, hydrogen bonding, and the dynamic properties of a peptide also influence the rate and mode of electron transfer in peptides. Electron transfer plays a key role in physical, chemical and biological systems, so its control is a fundamental task in bioelectrochemical systems, the design of peptide based sensors and molecular junctions. Therefore, this topic is at the heart of a number of biological and technological processes and thus remains of vital interest.

  11. 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.

  12. Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jeremy C [ORNL; Topham, Christopher [University of Heidelberg

    2007-01-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like base pair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNADNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs.

  13. Orientation preferences of backbone secondary amide functional groups in peptide nucleic acid complexes: quantum chemical calculations reveal an intrinsic preference of cationic D-amino acid-based chiral PNA analogues for the P-form.

    Science.gov (United States)

    Topham, Christopher M; Smith, Jeremy C

    2007-02-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNA.DNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs. PMID:17071666

  14. 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.

  15. A novel artificial nerve graft for repairing long-distance sciatic nerve defects: a self-assembling peptide nanofiber scaffold-containing poly(lactic-co-glycolic acid) conduit

    OpenAIRE

    Wang, Xianghai; Pan, Mengjie; Wen, Jinkun; Tang, Yinjuan; Hamilton, Audra D.; Li, Yuanyuan; Qian, Changhui; Liu, Zhongying; Wu, Wutian; Guo, Jiasong

    2014-01-01

    In this study, we developed a novel artificial nerve graft termed self-assembling peptide nanofiber scaffold (SAPNS)-containing poly(lactic-co-glycolic acid) (PLGA) conduit (SPC) and used it to bridge a 10-mm-long sciatic nerve defect in the rat. Retrograde tracing, behavioral testing and histomorphometric analyses showed that compared with the empty PLGA conduit implantation group, the SPC implantation group had a larger number of growing and extending axons, a markedly increased diameter of...

  16. Effect of acid treatment on extracting collagen peptide fromsturgeon skin by enzymatic hydrolysis%酸处理对酶法提取鲟鱼皮胶原多肽的影响

    Institute of Scientific and Technical Information of China (English)

    刘培勇; 李丁宁; 刘亮; 王燕; 刘良忠; 谭贝妮

    2012-01-01

    以人工养殖的鲟鱼鱼皮为研究对象,通过单因素实验比较乳酸、柠檬酸、乙酸对酶法提取鲟鱼皮胶原多肽的影响,并考察了酸溶胀浓度、溶胀固液比、溶胀时间及溶胀温度对其的影响。在此基础上利用正交实验对提取鲟鱼皮胶原蛋白肽的酸处理工艺进行优化。结果表明,采用溶胀酸为乳酸,在质量浓度为3%,溶胀固液比为1∶5,溶胀时间为2h,溶胀温度为30℃的条件下,鲟鱼皮胶原蛋白肽的水解度为43.58%,短肽得率为55.84%,水解度比不经酸溶胀的要高出23.08%。%As artificial breeding sturgeon skin for study,the lactic acid,citric acid and acetic acid for effects of extracting sturgeon skin collagen peptide by enzymatic method were investigated by single-factor experiment,and the swelling acid concentration,solid-liquid ratio,swelling time and swelling temperature for its influence were studied.On this basis,the optimal acid treatment process of extracting sturgeon skin collagen peptide by enzymatic method was obtained by orthogonal experiment.The results showed that as lactic acid for swelling acid,acid concentration 3%,swelling solid-liquid ratio 1:5,swelling time 2h and swelling temperature 30℃,the hydrolysis degree of sturgeon skin collagen peptide by enzymatic method was 43.58%,and the rate of obtained short peptide was 55.84%,whose hydrolysis degree increased 23.08% compared to without acid treatment.

  17. 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

    2015-12-01

    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.

  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...... improve biomolecular recognition by synthetic nucleic acid analogues. Circular dichroism (CD) measurements showed no distortion of the duplex structure by the incorporated peptide chains while studies in human serum indicated superior stability of the POCs compared to LNA/DNA mixmers and unmodified DNA...

  19. Fabrication of Odor Sensor Using Peptide

    Science.gov (United States)

    Hotokebuchi, Yuta; Hayashi, Kenshi; Toko, Kiyoshi; Chen, Ronggang; Ikezaki, Hidekazu

    We report fabrication of an odor sensor using peptides. Peptides were designed to acquire the specific reception for a target odor molecule. Au surface of the sensor electrode was coated by the designed peptide using the method of self assembled monolayers (SAMs). Functionalized Au surfaces by the peptides were confirmed by ellipsometry and cyclic voltammetry. The odorants of vanillin, phenethyl alcohol and hexanol were discriminated by QCM sensor with the peptide surface. Moreover, we verified specific interaction between amino acid (Trp) and vanillin by fluorescence assay.

  20. Vibrational analysis of amino acids and short peptides in hydrated media. VIII. Amino acids with aromatic side chains: L-phenylalanine, L-tyrosine, and L-tryptophan.

    Science.gov (United States)

    Hernández, Belén; Pflüger, Fernando; Adenier, Alain; Kruglik, Sergei G; Ghomi, Mahmoud

    2010-11-25

    Four out of the 20 natural α-amino acids (α-AAs) contain aromatic rings in their side chains. In a recent paper (J. Phys. Chem. B 2010, 114, 9072-9083), we have analyzed the structural and vibrational features of l-histidine, one of the potent elements of this series. Here, we report on the three remaining members of this family, i.e., l-phenylalanine, l-tyrosine, and l-tryptophan. Their solution (H(2)O and D(2)O) Raman scattering and Fourier transform infrared absorption attenuated total reflection (FT-IR ATR) spectra were measured at room temperature from the species corresponding to those existing at physiological conditions. Because of the very low water solubility of tyrosine, special attention was paid to avoid any artifact concerning the report of the vibrational spectra corresponding to nondissolved powder of this AA in aqueous solution. Finally, we could obtain for the first time the Raman and FT-IR spectra of tyrosine at very low concentration (2.3 mM) upon long accumulation time. To clarify this point, those vibrational spectra of tyrosine recorded either in the solid phase or in a heterogeneous state, where dissolved and nondissolved species of this AA coexist in aqueous solution, are also provided as Supporting Information . To carry out a discussion on the general geometrical and vibrational behavior of these AAs, we resorted to quantum mechanical calculations at the DFT/B3LYP/6-31++G* level, allowing (i) determination of potential energy surfaces of these AAs in a continuum solvent as a function of the torsion angles χ(1) and χ(2), defining the conformation of each aromatic side chain around C(α)-C(β) and C(β)-C(γ) bonds, respectively; (ii) analysis of geometrical features of the AAs surrounded by clusters of n explicit (n = 5-7) water molecules interacting with the backbone and aromatic rings; and (iii) assignment of the observed vibrational modes by means of the theoretical data provided by the lowest energy conformers of explicitly